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United Arab Emirates University Scholarworks@UAEU Dissertations Electronic Theses and Dissertations Winter 12-2013 CHARACTERIZING THE GENETIC BASES OF AUTOSOMAL RECESSIVE DISORDERS Nadia Awni Akawi Follow this and additional works at: http://scholarworks.uaeu.ac.ae/all_dissertations Part of the Medicine and Health Sciences Commons Recommended Citation Akawi, Nadia Awni, "CHARACTERIZING THE GENETIC BASES OF AUTOSOMAL RECESSIVE DISORDERS" (2013). Dissertations. Paper 33. This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at Scholarworks@UAEU. It has been accepted for inclusion in Dissertations by an authorized administrator of Scholarworks@UAEU. United Arab Emirates University College of Medicine and Health Sciences CHARACTERIZING THE GENETIC BASES OF AUTOSOMAL RECESSIVE DISORDERS Nadia Awni Akawi This dissertation is submitted in partial fulfillment of the requirements for the Doctorate of Philosophy in Genetics Under the direction of Professors Bassam R. Ali and Lihadh Al-Gazali December 2013 DECLARATION OF ORIGINAL WORK PAGE I, Nadia Awni Akawi, the undersigned, a student at the United Arab Emirates University (UAEU) and the author of the dissertation titled “CHARACTERIZING THE GENETIC BASES OF AUTOSOMAL RECESSIVE DISORDERS”, hereby solemnly declare that this dissertation is an original work done and prepared by me under the guidance of the Professors Bassam R. Ali and Lihadh Al-Gazali, in the College of Medicine and Health Sciences at UAEU. This work has not been previously formed as the basis for the award of any degree, diploma or similar title at this or any other university. The materials borrowed from other sources and included in my dissertation have been properly acknowledged. Student’s Signature……………………. ii Date………………… Copyright © 2013 by Nadia Awni Akawi All Rights Reserved iii Approved by Advisory Committee: 1) Advisor …………………………………………………………. Title……………………………………………………………… Department……………………………………………………... Institution……………………………………………………….. Signature………………………………Date………………….. 2) Co-advisor ……………………………………………………… Title………………………………………………………………. Department……………………………………………………… Institution………………………………………………………… Signature………………………………Date…………………... 3) Member ………………………………………………………… Title……………………………………………………………… Department…………………………………………………….. Institution……………………………………………………….. Signature………………………………Date………………….. 4) Member ………………………………………………………… Title……………………………………………………………… Department…………………………………………………….. Institution……………………………………………………….. Signature………………………………Date………………….. iv PhD Examining Committee: 1) Advisor …………………………………………………………. Title……………………………………………………………… Department…………………………………………………….. Institution……………………………………………………….. Signature………………………………Date…………………. 2) Co-advisor …………………………………………………….. Title……………………………………………………………… Department…………………………………………………….. Institution……………………………………………………….. Signature………………………………Date………………….. 3) Member ………………………………………………………… Title……………………………………………………………… Department…………………………………………………….. Institution……………………………………………………….. Signature………………………………Date…………………. 4) External Examiner ……………………………………………. Title……………………………………………………………… Department…………………………………………………….. Institution……………………………………………………….. Signature………………………………Date…………………. v Accepted by Dean of the College of Medicine and Health Sciences ……………………… Signature……………………………… Date………………... Dean of the College of Graduate Studies……………………………………….. Signature……………………………… Copy…...of…… vi Date…………………. ABSTRACT Autosomal recessive disorders have devastating effects on patients and their families. Elucidating the genetic bases of such disorders is essential to improve their clinical outcome and for implementing effective prevention programs. In this dissertation, the genetic bases of seven autosomal recessive disorders in consanguineous families were investigated using homozygosity mapping followed by candidate genes or whole-exome sequencing. Consequently, novel genomic loci and novel mutations have been revealed. The mutations underlying Silver-Russell syndrome in three families were found in OBSL1 and CUL7 genes, known to cause 3-M syndrome. In addition, the mutations identified in COL11A1 in two families were the first to link this gene with fibrochondrogenesis. Furthermore, the mutations detected in JAM3 in unrelated families confirmed the importance of this gene in maintaining blood vessels integrity. While, splicing defects in LINS and TTC23 genes provided evidence on the possible involvement of these genes in human cognition. Novel mutations in POMGNT1 and PRG4 have been found in families affected by congenital muscular dystrophy and camptodactyly-arthropathy-coxa-vara-pericarditis syndrome, respectively. Finally, a neonatal progeroid syndrome has been mapped to a novel single locus on chromosome19p13.3-13.2. These findings contributed significantly to our understanding of the studied disorders and their underlying molecular mechanisms. vii ACKNOWLEDGMENTS Firstly, I wish to thank God Almighty for his provision, protection and guidance. I would like to express my gratitude to the United Arab Emirates University for funding my PhD. I also give my deepest thanks and appreciation to all the patients and their families for participating in this research. My heartfelt special thanks go to my advisors Prof. Bassam Ali and Prof. Lihadh Al-Gazali. I am very grateful for their guidance not only in my study but also in helping me to become a strong and successful person. Their patience, flexibility, knowledgeable insight and genuine caring enabled me to complete my research projects. I would like to thank Prof. Bassam Ali for giving me the opportunity to do my PhD research in his group and for coaching me in the fine art of scientific investigation and writing. My sincere gratitude extends to Prof. Lihadh Al-Gazali for introducing me to the world of gene identification and for recruiting and diagnosing all the investigated families described in this dissertation. I would like to thank the members of my dissertation committee for reviewing my work. I wish to acknowledge Prof. Josef Gleeson for his help in bioinformatics analysis and Prof.Geoff Woods for his help in STR genotyping. I would like to thank Prof. Christopher A. Walsh and Dr. Ganesh Mochida for his bioinformatics training. I acknowledge the help of Mr. Thachillath Pramathan in Fibroblasts culturing and Mr. Tariq S for his help in confocal microscopy. viii I would also like to express my gratitude to my lab colleagues who helped me and contributed to creating a very pleasant working atmosphere. In particular I thank Ms. Anne John for our great working relationship and for her patient assistance in all lab related problems, Miss Salma Ben-Salem for her thoughtful discussions and help with the STR genotyping. I would also like to thank my friend Dr. Satwat Hashmi for always being there for me. I am deeply indebted to my husband. You really deserve far more credit than I can ever give you. Without your strength, patience, and understanding, it would not have been possible for me to finish this dissertation. My most heartfelt thanks go to my parents, sisters and brothers. Your integrity and warmth provided a wonderful environment in which to grow. Without them, I would never have achieved what I have achieved today. ix DEDICATION I would like to dedicate this dissertation to my husband, Hatem and our children, Majd and Khaled I am forever indebted to you for your constant love and support which have enabled me to bring this work to completion x TABLE OF CONTENTS TITLE PAGE......................................................................................................i DECLARATION OF ORIGINAL WORK PAGE.................................................ii COPYRIGHT PAGE.........................................................................................iii SIGNATURE PAGE................................................................. ........................iv ABSTRACT........................................................................................... ..........vii ACKNOWLEDGMENTS.................................................................................viii DEDICATION....................................................................................................x TABLE OF CONTENTS...................................................................................xi LIST OF TABLES................................................................................... ........xix LIST OF FIGURES.........................................................................................xxi LIST OF PUBLICATIONS.............................................................................xxv CHAPTER 1: INTRODUCTION ................................................................... 1 1.1. Overview ............................................................................................ 2 1.1.1. The United Arab Emirates: country and population ........................ 2 1.1.2. Consanguinity and genetic diseases in the United Arab Emirates .. 3 1.1.3. Magnitude and burden of genetic disorders in the United Arab Emirates....................................................................................................... 5 1.1.4. Current genetic facilities in the United Arab Emirates ..................... 6 1.2. Statement of the problem................................................................... 8 1.3. Objectives ........................................................................................ 10 1.4. Literature review............................................................................... 11 1.4.1. Biological concepts ....................................................................... 11 1.4.1.1. Monogenic disorders ................................................................. 13 1.4.1.1.1. 1.4.2.1. Autosomal recessive disorders .............................................. 14 Why study rare autosomal recessive diseases?........................ 16 1.4.2.2. Approaches for the identification of recessive disease-causing genes and mutations.................................................................................. 20 1.4.2.2.1. 1.4.2.2.1.1. Homozygosity mapping .......................................................... 22 Principles of homozygosity mapping ................................... 23 1.4.2.2.1.1.1. Microsatellite markers ..........................................................25 1.4.2.2.1.1.2. Single nucleotide polymorphisms (SNPs) ........................... 26 1.4.2.2.1.2. Pitfalls of homozygosity mapping ........................................ 26 1.4.2.2.2. DNA sequencing .................................................................... 29 1.4.2.2.2.1. Early DNA sequencing technologies ................................... ...30 xi 1.4.2.2.2.2. Recent advances in DNA sequencing technologies............ 31 1.4.2.2.2.3. mapping The integration of DNA sequencing with homozygosity … ........................................................................................ 35 1.4.2.2.2.4. Whole-exome sequencing................................................... 36 1.4.2.2.2.4.1. The filtering strategy in whole-exome sequencing .............. 37 1.4.2.2.3. Selection of candidate genes and recognition of pathogenic mutations .. ............................................................................................ .39 1.4.3. Conclusion ……………………………………………….41 1.5. Potential contributions and limitations of the study .......................... 42 1.5.1. Limitations..................................................................................... 42 1.5.2. Potential contributions................................................................... 43 CHAPTER 2: GENERAL METHODS......................................................... 44 2.1. Families............................................................................................ 45 2.1.1. Consent and ethics approval ........................................................ 45 2.1.2. Families recruitment and patients assessment ............................. 45 2.2. Methods ........................................................................................... 45 2.2.1. DNA, RNA & protein extraction ..................................................... 45 2.2.1.1. DNA extraction .......................................................................... 45 2.2.1.2. RNA extraction .......................................................................... 46 2.2.1.3. Protein extraction....................................................................... 47 2.2.2. Polymerase chain reaction (PCR) amplification ............................ 47 2.2.2.1. Standard PCR ........................................................................... 47 2.2.2.2. Reverse transcription................................................................. 48 2.2.2.3. Real-time PCR........................................................................... 48 2.2.2.4. Site directed mutagenesis ......................................................... 49 2.2.3. Sequencing................................................................................... 50 2.2.3.1. Sanger sequencing.................................................................... 50 2.2.3.1.1. PCR products sequencing...................................................... 50 2.2.3.1.2. Plasmid sequencing ............................................................... 51 2.2.3.1.3. Whole-exome sequencing...................................................... 51 2.2.4. 2.2.4.1. Genotyping ................................................................................... 52 Genome wide SNP genotyping.................................................. 52 xii 2.2.4.2. 2.2.5. Microsatellite genotyping ........................................................... 53 Electrophoresis ............................................................................. 54 2.2.5.1. Agarose gel electrophoresis ...................................................... 54 2.2.5.2. Western blot analysis ................................................................ 54 2.2.6. Cellular studies ............................................................................. 55 2.2.6.1. Culture of human cell line .......................................................... 55 2.2.6.2. Transient transfection of cultured cells ...................................... 56 2.2.6.3. Confocal fluorescence microscopy ............................................ 56 2.2.6.4. Fibroblast culturing .................................................................... 57 2.2.7. Bioinformatics analysis ................................................................. 57 2.2.7.1. Website addresses for internet resources ................................. 57 2.2.7.2. Primer design ............................................................................ 57 2.2.7.3. Homozygosity mapping analysis ............................................... 58 2.2.7.4. Prioritization of genes and variants............................................ 58 2.2.7.5. Analysis of Sanger sequences and exome sequencing data .... 58 CHAPTER 3: RESULTS AND DISSCUSSION .......................................... 61 SECTION 1: Is autosomal recessive Silver–Russell syndrome a separate entity .......................................................................................................... 62 3.1.1. Background................................................................................... 63 3.1.1.1. Three M syndrome .................................................................... 63 3.1.1.1.1. Clinical features...................................................................... 63 3.1.1.1.2. Genetic heterogeneity ............................................................ 63 3.1.1.2. Silver–Russell syndrome ........................................................... 64 3.1.1.2.1. Clinical features...................................................................... 64 3.1.1.2.2. Genetic heterogeneity ............................................................ 65 3.1.2. The purpose of the study .............................................................. 65 3.1.3. Results.......................................................................................... 66 3.1.2.1. Clinical assessment of probands and families ........................... 66 3.1.2.1.1. Family A ................................................................................. 66 3.1.2.1.2. Family B ................................................................................. 67 3.1.2.1.3. Family C ................................................................................. 72 xiii 3.1.2.1.4. Family D ................................................................................. 74 3.1.2.2.1. Family A ................................................................................. 76 3.1.2.2.2. Family B ................................................................................. 80 3.1.2.2.3. Family C ................................................................................. 81 3.1.2.2.4. Family D ................................................................................. 90 3.1.4. Discussion .................................................................................... 93 3.1.4.1. The differential diagnosis of Three-M syndrome........................ 93 3.1.4.2. The Three-M syndrome pathogenesis....................................... 97 3.1.5. Conclusion .................................................................................. 100 SECTION 2: Unraveling the genetic defects and the pathophysiologic mechanism underlying fibrochondrogenesis in patients from the UAE .... 102 3.2.1. Background................................................................................. 103 3.2.2. The purpose of the study ............................................................ 104 3.2.3. Results........................................................................................ 104 3.2.3.1. Clinical phenotyping of UAE patients expanded the phenotype especially in survivors .............................................................................. 104 3.2.3.1.1. Family A (FA) ....................................................................... 104 3.2.3.1.2. Family B (FB) ....................................................................... 107 3.2.3.1.3. Family C (FC) ....................................................................... 110 3.2.3.2. Gene mapping and mutation analysis on UAE families with FBCG revealed homozygous two mutations in COL11A1 gene .............. 110 3.2.3.3. Functional studies on the c.3708+437T>G mutation confirm the predicted loss of function mechanism underlying FBCG.......................... 119 3.2.4. Discussion .................................................................................. 121 3.2.4.1. Clinical heterogeneity of type XI collagenopathies .................. 121 3.2.4.2. Genetic heterogeneity of FBCG............................................... 123 3.2.4.3. Phenotypic spectrum of COL11A1 mutations .......................... 123 3.2.4.4. Pathophysiological mechanisms underlying COL11A1 mutations …............................................................................................. 126 3.2.5. Conclusion .................................................................................. 128 SECTION 3: Delineation of the clinical, molecular and cellular aspects of several novel JAM3 mutations underlying the autosomal recessive xiv hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts ................................................................................. 130 3.3.1. Background................................................................................. 131 3.3.2. The purpose of the study ............................................................ 132 3.3.3. Results........................................................................................ 133 3.3.3.1. Clinical assessment of the studied families ............................. 133 3.3.3.1.1. Family 1................................................................................ 133 3.3.3.1.2. Family 2................................................................................ 136 3.3.3.1.3. Family 3................................................................................ 138 3.3.3.2. JAM3 mutational analysis ........................................................ 139 3.3.3.3. The p.C219Y mutation resulted in JAM3 trafficking defect ...... 140 3.3.4. Discussion .................................................................................. 150 3.3.4.1. The impact of JAM3 mutations on the protein product ............ 150 3.3.4.2. Clinical consequences of JAM3 deficiency .............................. 152 3.3.5. Conclusion .................................................................................. 155 SECTION 4: LINS and TTC23 are candidate genes for autosomal recessive intellectual disability ................................................................................. 156 3.4.1. Background................................................................................. 157 3.4.2. The purpose of the study ............................................................ 157 3.4.3. Results........................................................................................ 158 3.4.3.1. Clinical assessment of patients ............................................... 158 3.4.3.2. regions Genome-wide linkage analysis revealed four homozygous .. .............................................................................................. 161 3.4.3.3. Whole-exome sequencing identified two splicing mutations in LINS and TTC23 genes ........................................................................... 168 3.4.3.4. The two splicing mutations were confirmed to be deleterious at the mRNA level of both genes ................................................................. 168 To investigate the consequences of the molecular defect caused by the detected splicing mutation, reverse transcription-PCR was performed.... 168 3.4.3.5. 3.4.4. The impact of exon 5 skipping in LINS on the mRNA expression.. …............................................................................................. 174 Discussion .................................................................................. 181 xv 3.4.4.1. Lines/LINS is important for segment polarity in Drosophila and plays a putative dual role in WNT canonical pathway .............................. 183 3.4.4.2. 3.4.5. LINS in the cognitive pathways................................................ 189 Conclusions ................................................................................ 190 SECTION 5: Characterizing the genetic basis of a clinically heterogenous autosomal recessive congenital muscular dystrophy in a highly inbred Arab family ....................................................................................................... 192 3.5.1. Background................................................................................. 193 3.5.2. The purpose of the study ............................................................ 194 3.5.3. Results........................................................................................ 194 3.5.3.1. Clinical assessments of patients.............................................. 194 3.5.3.2. Molecular genetic investigations.............................................. 197 3.5.3.2.1. Homozygosity mapping analysis revealed multiple homozygous regions across the genome...................................................................... 197 3.5.3.2.2. Whole-exome sequencing revealed a novel nonsense mutation in POMGNT1 gene .................................................................................. 197 3.5.4. Discussion .................................................................................. 198 3.5.4.1. Genetic heterogeneity of CMD-dystroglycanopathy with brain and eye anomalies.......................................................................................... 205 3.5.4.2. Clinical heterogeneity of CMD-dystroglycanopathy with brain and eye anomalies.......................................................................................... 206 3.5.4.3. 3.5.5. Phenotypic spectrum of POMGNT1 mutations ........................ 206 Conclusions ................................................................................ 208 SECTION 6: A novel mutation in PRG4 gene underlying camptodactylyarthropathy-coxa vara-pericarditis syndrome with the possible expansion of the phenotype to include congenital cataract........................................... 209 3.6.1. Background................................................................................. 210 3.6.2. The purpose of the study ............................................................ 211 3.6.3. Results........................................................................................ 211 3.6.3.1. Clinical assessments ............................................................... 211 3.6.3.2. Molecular genetic investigation.................................................214 3.6.4. Discussion .................................................................................. 216 3.6.5. Conclusion ..................................................................................... 220 xvi SECTION 7: Mapping of an autosomal recessive progeroid syndrome with neonatal presentation and long survival to a single locus on chromosome 19p13.3-13.2............................................................................................ 221 3.7.1. Background................................................................................. 222 3.7.2. The purpose of the study ............................................................ 223 3.7.3. Results........................................................................................ 223 3.7.3.1. Clinical assessments of patients.............................................. 223 3.7.3.2. Molecular genetic investigation................................................ 229 3.7.3.2.1. results Sequencing of LMNA and ZMPSTE24 genes showed negative …………………………………………………………………....229 3.7.3.2.2. Genome-wide homozygosity mapping linked the condition to 19p13.3p13.2 ........................................................................................... 229 3.7.3.2.3. Whole-exome sequencing analysis failed to reveal a convincing causal mutation........................................................................................ 232 3.7.3.2.4. region Computational prioritizing of the genes within the mapped ….......................................................................................... 233 3.7.4. Discussion .................................................................................. 233 3.7.5. Conclusions ................................................................................ 236 CHAPTER 4: GENERAL CONCLUSIONS & FUTURE PERSPECTIVES 238 4.1. General conclusions....................................................................... 239 4.2. Future perspectives........................................................................ 244 4.2.1. Identifying the genetic bases of additional autosomal recessive disorders .................................................................................................. 244 4.2.2. Establishing pathogenicity of the identified genes and mutations245 REFERENCES ........................................................................................ 246 APPENDICES.......................................................................................... 281 Appendix A - (JAM3 cDNA Cloning Site and Associated Tags)............... 282 Appendix B - (All the Primers Used in This Dissertation). ........................ 283 Appendix C1- (Real Time-PCR COL11A1 Gene Expression Results)..... 292 Appendix C2- (Real Time-PCR COL11A1 Gene Expression Results)..... 293 Appendix E1- (Real Time-PCR LINS Gene Expression Results)............. 295 Appendix E2- (Real Time-PCR LINS Gene Expression Results)............. 296 xvii Appendix F- (The Alignment of the Three Known LINS Isoforms and Showing the Reported Homozygous Mutations) ...................................... 297 Appendix G- (Blocks of Shared Homozygosity between the Affected Individuals in the Progeoid Family) .......................................................... 298 xviii LIST OF TABLES Table 1-1……………………………………………………………………………18 The steroid-resistant nephrotic syndrome (SRNS) subtypes Table 1-2……………………………………………………………………………21 Fanconi anemia (FA) reported genes Table 2-1……………………………………………………………………………60 Web-based resources Table 3.1-1………………………………………………………………………….69 Clinical and radiological features of the studied patients compared to SRS and 3-M syndrome Table 3.1-2………………………………………………………………………….78 STR genotyping results for the homozygous region on chromosome 2 in family A Table 3.1-3………………………………………………………………………….82 Conservation of the 373-tryptophan (W) in the OBSL1 protein across species Table 3.1-4………………………………………………………………………….84 STR genotyping results for the homozygous region on chromosome 2 in family B Table 3.1-5………………………………………………………………………….85 STR genotyping results for the homozygous region on chromosome 6 in family B Table 3.1-6………………………………………………………………………….88 STR genotyping results for the homozygous region on chromosome 2 in family C Table 3.1-7………………………………………………………………………….92 Conservation of the 217-serine (S) and 218-arginine (R) in CUL7 protein across species Table 3.1-8………………………………………………………………………….94 Summary of genotyping and mutational screening results for all the recruited families Table 3.2-1………………………………………………………………………..106 The clinical and radiological features of the eight FBCG patients from three families from the UAE Table 3.2-2………………………………………………………………………..114 List of the twenty genes sequenced in this study. xix Table 3.2-3………………………………………………………………………..125 Comparison of the phenotypes associated with disease causing heterozygotes genotypes in COL11A1 gene Table 3.3-1………………………………………………………………………..135 Clinical features of hemorrhagic brain destruction-JAM3 type Table 3.3-2………………………………………………………………………..142 Summary of all the mutations detected in JAM3 Table 3.4-1………………………………………………………………………..162 Intervals of shared homozygosity between the two affected individuals of the studied family Table 3.4-2………………………………………………………………………..167 Summary metrics of all and novel variants identified by the exome sequencing Table 3.4-3………………………………………………………………………..186 The reported downstream target genes of lines in Drosophila Table 3.5-1………………………………………………………………………..200 Intervals of shared homozygosity between the two affected individuals of the studied family Table 3.5-2………………………………………………………………………..203 Summary metrics of all and novel variants identified by the whole-exome sequencing Table 3.6-1………………………………………………………………………..212 STR genotyping results for the two homozygous regions identified in the studied family Table 3.7-1………………………………………………………………………226 Clinical features of the three affected sibs in this report compared to Wiedemann-Rautenstrauch syndrome (WRS) xx LIST OF FIGURES Figure 1-1…………………………………………………………………………….4 Average rates of marriages between first cousins among various Arab populations Figure 1-2…………………………………………………………………………..15 Autosomal recessive mode of inheritance Figure 1-3…………………………………………………………………………..24 Illustration of homozygosity mapping approach for recessive disease genes identification Figure 1-4…………………………………………………………………………..27 Schematic representation of genome-wide SNP genotyping workflow Figure 1-5…………………………………………………………………………..31 Schematic representation of the Sanger sequencing workflow Figure 1-6…………………………………………………………………………..34 Schematic representation of massively parallel sequencing workflow Figure 1-7…………………………………………………………………………..38 Filtering steps of whole-exome sequencing data Figure 3.1-1………………………………………………………………………...68 Clinical features of some affected members from family A Figure 3.1-2………………………………………………………………………...70 Clinical features of some affected members from family B Figure 3.1-3………………………………………………………………………...73 Clinical data of some affected members from family C Figure 3.1-4………………………………………………………………………...75 Clinical features of some affected members from family D Figure 3.1-5………………………………………………………………………...77 Homozygosity mapping results in family A Figure 3.1-6………………………………………………………………………...79 The p.W373C mutation found in family A. Figure 3.1-7………………………………………………………………………...83 Homozygosity mapping results in family B Figure 3.1-8………………………………………………………………………...86 Mutation analysis results in family B xxi Figure 3.1-9………………………………………………………………………...87 Homozygosity mapping results in family C Figure 3.1-10……………………………………………………………………….89 Mutation analysis results in family C Figure 3.1-11……………………………………………………………………….91 Mutation analysis results in family D Figure 3.2-1……………………………………………………………………….105 Pedigree of family FA Figure 3.2-2……………………………………………………………………….108 Pedigree of family FB Figure 3.2-3……………………………………………………………………….109 Clinical features of Clinical features of some affected members from family FB Figure 3.2-4……………………………………………………………………….111 Clinical features of the affected child from family FC Figure 3.2-5……………………………………………………………………….112 Homozygosity mapping of the UAE families with FBCG Figure 3.2-6……………………………………………………………………….115 Mutation analysis results in family FA Figure 3.2-7……………………………………………………………………….116 A novel insertion of a 53 nucleotide between exon 48 and 49 of COL11A1 cDNA in family FB Figure 3.2-8……………………………………………………………………….117 Mutation analysis results for intron 48 of COL11A1 in family FB Figure 3.2-9……………………………………………………………………….118 Schematic representation for the genomic region encompassing exons E46 to E57 of the COL11A1 gene indicating the locations of the two detected mutations in the UAE families FA and FB Figure 3.2-10……………………………………………………………………...120 Expression analysis of the homozygous c.3708+437T>G mutation in COL11A1 gene Figure 3.3-1……………………………………………………………………….134 Pedigrees of the three studied families Figure 3.3-2……………………………………………………………………….137 Clinical features of some affected members in this study xxii Figure 3.3-3……………………………………………………………………….144 The identified JAM3 mutations Figure 3.3-4……………………………………………………………………….146 p.C219Y mutation disrupts normal intracellular JAM3 protein localization Figure 3.3-5……………………………………………………………………….147 Subcellular localization in HeLa cells of flag-tagged wild-type (WT) and C219Y mutant of JAM3 with different cellular confluency Figure 3.3-6……………………………………………………………………….148 Comparison between subcellular localization of WT and C219Y mutant of JAM3 Figure 3.3-7……………………………………………………………………….149 Subcellular localization of flag-tagged JAM3-WT and JAM3-C219Y in COS7 cells Figure 3.4-1……………………………………………………………………….160 Clinical Data of the studied family Figure 3.4-2……………………………………………………………………….163 Genome-wide homozygosity in the studied family Figure 3.4-3……………………………………………………………………….170 Integrative Genomics Viewer (IGV) visualization of homozygous mutation c.1219_1222+1delAAAGG in the LINS gene from exome data Figure 3.4-4……………………………………………………………………….171 Integrative Genomics Viewer (IGV) visualization of the second novel mutation detected in this study by whole exome sequencing Figure 3.4-5……………………………………………………………………….172 Sanger sequencing verification of the c.1219_1222+1delAAAGG mutation in the LINS gene Figure 3.4-6……………………………………………………………………….175 Exon skipping analysis by reverse transcription Figure 3.4-7……………………………………………………………………….176 Schematic diagram of the splicing defect seen in patients based on Sanger sequencing data of the cDNA Figure 3.4-8……………………………………………………………………….177 Proposed alternative splicing variants of LINS Figure 3.4-9……………………………………………………………………….178 Sequencing results of the TTC23 cDNA. xxiii Figure 3.4-10…………………………………………………………………….179 Expression analysis of the homozygous c.1219_1222+1delAAAGG mutation in the LINS gene Figure 3.4-11……………………………………………………………………...180 Conservation across species of the amino acids that are predicted to be deleted from LINS protein in the patients Figure 3.4-12……………………………………………………………………...188 Lines/LINS plays a putative dual role in WNT canonical pathway Figure 3.5-1……………………………………………………………………….197 Pedigree of the studied family Figure 3.5-2……………………………………………………………………….201 HomozygosityMapper view of the Genome wide homozygosity analysis results Figure 3.5-3……………………………………………………………………….202 Zoom in view into the homozygous region detected on chromosome 1 Figure 3.5-4……………………………………………………………………….204 Integrative Genomics Viewer (IGV) visualization of homozygous mutation c.1460 in POMGNT1 gene from exome data Figure 3.5-5……………………………………………………………………….205 Mutation analysis results in the studied family Figure 3.6-1……………………………………………………………………….213 Pedigree of the studied family Figure 3.6-2……………………………………………………………………….216 Representative DNA sequencing chromatograms displaying the c.1320dupC mutation of PRG4 Figure 3.6-3……………………………………………………………………….218 Schematic presentation of PRG4 protein domains showing the position of all the reported mutations Figure 3.7-1……………………………………………………………………….225 Pedigree of the affected family Figure 3.7-2……………………………………………………………………….228 Representative images of patient 1 Figure 3.7-3……………………………………………………………………….231 Representative images of patient 2 and 3 Figure 3.7-4……………………………………………………………………….232 Genome-wide genotyping results xxiv LIST OF PUBLICATIONS Akawi NA, Al-Jasmi F, Al-shamsi A, Ali BR* and Al-Gazali L*. 2013. LINS, a Modulator of the WNT Signaling Pathway, is Involved in Human Cognition. Orphanet J Rare Dis, 8(1):87. Akawi NA, Canpolat FE , White SM, Esquerra JQ, Sanchez MM, Gamundi MJ, Mochida GH, Walsh CA, Ali BR* and Al-Gazali L*. 2013. Delineation of the Clinical, Molecular and Cellular Aspects of Novel JAM3 Mutations Underlying the Autosomal Recessive Hemorrhagic Destruction of the Brain, Subependymal Calcification and Congenital Cataracts. Hum Mut, 34(3):498-505. Akawi NA, Ali BR and Al-Gazali L. 2013. A Progeroid Syndrome with Neonatal Presentation and Long Survival Maps to 19p13.3-13.2. Birth Defects Res A Clin Mol Teratol, 97(7):456-62. Akawi NA, Ali BR, Al-Gazali L. 2013. A response to Dr. Alzahrani's letter to the editor regarding the mechanism underlying fibrochondrogenesis. Gene, pii: S0378-1119(13)00927-X. Akawi NA, Ali BR, Al-Gazali L. 2012. Stüve-Wiedemann Syndrome and Related Bent Bone Dysplasias. Clin Genet, 82:12-21. Akawi NA, Al-Gazali L* and Ali BR*. 2012. Clinical and molecular analysis of UAE Fibrochondrogenesis patients expands the phenotype and reveals two COL11A1 homozygous null mutations. Clin Genet, 82:147-156. Akawi NA, Ali BR and Al-Gazali L. 2012. A novel mutation in PRG4 gene underlying camptodactyly-arthropathy-coxa vara-pericarditis syndrome with the possible expansion of the phenotype to include congenital cataract. Birth Defects Res A Clin Mol Teratol, 94:553-556. Akawi NA, Ali BR, Hamamy H , Al-Hadidy A & Al-Gazali L. 2011. Is Autosomal Recessive Silver-Russel Syndrome a separate entity or is it part of the 3-M Syndrome spectrum?. Am J Med Genet A, 155:1236-1245. Mochida GH, Ganesh V, Felie J, Gleason D, Hill RS, Clapham KR, Rakiec D, Tan W-H, Akawi NA, Al-Saffar M, Partlow JN, Tinschert S, Barkovich AJ, Ali B, Al-Gazali L & Walsh CA. 2010. A homozygous mutation in the tight junction protein JAM3 causes hemorrhagic destruction of the brain, subependymal calcification and congenital cataracts. Am J Hum Genet, 88:882-889. xxv CHAPTER 1: INTRODUCTION 1 1.1. 1.1.1. Overview The United Arab Emirates: country and population The United Arab Emirates (UAE) is a union of seven emirates located in the Middle East, bordering the Arabian Gulf, the Gulf of Oman, the Sultanate of Oman and the Kingdom of Saudi Arabia. It occupies an area of approximately 83,600 square kilometers (km) with 867 km of land boundaries with Oman (410 km) and Saudi Arabia (457 km) and 1,318 km of coastline stretches. The city of Abu Dhabi is the capital of the country and is located in the largest Emirate (Abu Dhabi) of the confederation. The UAE population is estimated to be around 8 million (http://www.worldbank.org/), with an annual growth rate of 3.055%, birth rate of 15.76 births/1,000 population (2012 est.) and Infant mortality rate of 11.59 deaths/1,000 live births (2012 est.) (http://www.cia.gov/library/publications/the-world-factbook/geos/ae.html). However, the growth rate of the UAE population is mainly due to the inflow of expatriates [Alwash and Abbas, 1999]. Of the total population, Emirati nationals constitute around 15% with other Arabs and Iranians constituting 23%, South Asians 50% with the rest being expatriates from European and East Asians countries [Al-Gazali and Ali, 2010]. UAE remains tribal in nature. Belonging to a tribe is still considered important, socially and economically. Therefore, to maintain this tribal structure, marriages occurred mainly between close relatives of the same tribe or even extended family [Heard-Bay, 1976; 1999]. However, intermixing with adjacent Arabs of the Arabian Peninsula and with the Persians as well as 2 the East Africans of the Omani Empire territories has occurred for decades [Abdulla, 1978]. In addition, continuous migrations to the UAE have been taking place for centuries from Baluchistan and other neighboring countries, of whom many have become UAE nationals [Al-Gazali and Ali, 2010]. Intermarriages between the local population and expatriates are limited and the majority of the local Bedouin population still forms tribal communities that are quite isolated. Therefore, the population of the UAE is fairly heterogeneous and ethnically diverse embracing several isolated subpopulations of different origins. 1.1.2. Consanguinity and genetic diseases in the United Arab Emirates Notably, Arab societies place a great emphasis on the role and importance of the family. Thus in addition to the tribal and clan endogamy, consanguineous marriages are customary in most Arab communities including the UAE (Figure 1-1) [Al-Gazali et al., 2006]. For instance, it was estimated that approximately 50% of all marriages among Emirati nationals are of the consanguineous type [Al-Gazali et al., 1995; Al-Gazali et al., 1997]. Of the total marriages, 26% were between first cousins, 3.5% were between double first cousins, 3.1% were between first cousins once removed, and 3% were between second cousins [Al-Gazali et al., 1997; Al-Gazali and Ali, 2010]. The national population of UAE is also characterized by large family size and high birth rates, with an average of five children per family [Al-Gazali et al., 2006]. Furthermore, women continue to reproduce after the age of 3 Figure 1-1. Average rates of marriages between first cousins among various Arab populations. (Adapted from Al-Gazali et al., 2006). 4 40 years and men into their 60s. This style contributes to the morbidity and mortality of newborns and leads to increases in the prevalence of genetic disorders and congenital malformations [Al-Gazali et al., 2006]. Several studies in the Arab world, including the UAE, directly compared the prevalence of major congenital defects in consanguineous and nonconsanguineous progeny [Al-Gazali et al., 1995; 1997; Abdulrazzaq et al., 1999; Bromiker et al., 2004; Dawodu et al., 2005; Al Hosani et al., 2005]. They found a higher frequency of congenital malformations among offspring of biologically related couples. 1.1.3. Magnitude and burden of genetic disorders in the United Arab Emirates The magnitude of genetic disorders and birth defects is relatively high given the small population size of the UAE [Christianson et al., 2006]. More than 300 genetic disorders have been diagnosed among the UAE national population, with rare and previously unreported syndromes and metabolic defects being especially apparent [Al-Gazali et al., 2005; Tadmouri et al., 2004; 2006] (www.cags.ac.ae). The majority of these genetic disorders are monogenic and recessively inherited, presumably due to increased consanguinity [Al-Gazali et al., 2005; Tadmouri et al., 2006]. The burden of genetic diseases is significant in the UAE [Al-Gazali and Ali, 2010]. For instance, congenital malformations were found to be the fourth cause of death in the country and half of these anomalies were caused by specific recessive mutations [Al-Gazali et al., 1995; 1997; Dawodu et al., 2000]. Furthermore, a substantial proportion of the physical and mental 5 handicaps in this country were also found to be inherited in a recessive manner [Al-Gazali and Ali, 2010]. For example, 92% of non-syndromic and 57% of syndromic childhood deafness assessed in the UAE were attributed to autosomal recessive genes [Al-Gazali, 1998]. Another study in the UAE has shown that 36 out of 38,046 births (about 1:1000) examined in the UAE had some type of skeletal dysplasia [Al-Gazali et al., 2003]. Out of these 36 cases, 18 were attributed to autosomal recessive genes [Al-Gazali et al., 2003]. 1.1.4. Current genetic facilities in the United Arab Emirates Although UAE has made a considerable progress in the healthcare sector, genetic disorders’ diagnosis and counseling remain challenging [AlGazali et al., 2005; Al-Gazali and Ali, 2010]. There are very limited diagnostic facilities for genetic and metabolic disorders. Most samples are currently being sent to diagnostic laboratories abroad. Genetic counseling in the available genetic centers is provided by a very small number of specialized clinical Geneticists who are not supported by genetic counselors, health visitors, or social workers. The first governmental Genetic service in UAE was specifically for thalassemia and other hemoglobinopathies and was established in Dubai by the Dubai Department of Health in 1989. This Genetic and thalassemia center is currently providing services for thalassemia patients from all over the UAE. Proper general clinical genetic service was established in Al-Ain in the College of Medicine and Health Sciences (CMHS) in 1990, at the United Arab 6 Emirates University (UAEU). This was supported by a small diagnostic Cytogenetic laboratory. This service, has evolved over the years and is currently the main genetic research and service facility in the country. In addition to this service there are 2 other genetic service providers, one is in Dubai at the Pediatric Department of Latifa hospital and the other one, is in Abu Dhabi under the Maternity and Child Health Department of the Ministry of Health and located in two Primary Health Care Centers supported by a small Cytogenetic laboratory. 7 1.2. Statement of the problem The magnitude and burden of genetic conditions on the UAE population and health care system are highly significant with many of the conditions being still of unknown causes. The overall aim of this study is to identify the genes and mutations underlying rare autosomal recessive disorders and congenital malformations in families residing in the UAE. The basic hypothesis is that the large family size, in conjunction with clan/tribe endogamy and high consanguinity rates in the UAE heterogeneous population increases the expression of autosomal recessive traits including disease phenotypes. That is because carrier rates of specific genetic defects are high within tribes or extended families compared to the general population, and therefore the probability of expression of autosomal recessive disorder is increased when both parents have some common ancestor(s). Thus, the demographic and other characteristics within the UAE subpopulations are suitable to undertake this type of project. Two main strategies were used in this study to elucidate the molecular defects in the investigated families with autosomal recessive disorders. Both approaches started by homozygosity mapping to identify the blocks of homozygosity that can be analyzed for linkage with the disease phenotype. If the detected homozygous regions were a few in number and small in terms of the number of genes and harbored some good candidates, then direct DNA sequencing for the candidate gene(s) was performed. However, to expedite the molecular bases of clinically heterogeneous genetic disorders with 8 apparently recessive mode of inheritance, whole-exome sequencing was carried out with the focus on the homozygous regions at the analysis stages. These strategies are explained in details in section 1.4.2.2 of the introduction. 9 1.3. Objectives The overall goal of this project was to elucidate the genes and/or mutations that are responsible for congenital autosomal recessive conditions. The aim is to expand the knowledge regarding the etiology of recessive conditions. The information will be useful to facilitate diagnosis, prevention and therapies. The specific objectives are to: Recruit inbred families from the residents of the UAE with established or deduced consanguinity producing multiple affected children exhibiting a congenital genetic disorder or malformation. Map the disease locus using genome-wide genotyping on all the available family members with the aim of identifying homozygous segments that are unique and only shared between the affected individuals. Identify disease causing gene(s) and/or mutation(s) by either direct DNA Sanger sequencing of the strong candidate genes within the mapped locus/loci or by performing whole-exome sequencing. Validate the pathogenicity of the identified candidate variants using bioinformatics tools and/or experimental functional assays. 10 1.4. Literature review 1.4.1. Biological concepts The human body is built up of trillions of cells that carry out all the biological functions including, but not limited to, providing structure and stability to providing energy and means for reproduction. Most cells in the body are generally specialized to perform specific biological functions. However, all nucleated somatic cells contain the same genetic information in the form of long chains of deoxyribonucleic acid (DNA). DNA is packaged with specific proteins into thread-like structures called chromosomes. These chromosomes are responsible for transferring genetic information from one generation to the next. Chromosomes are also responsible for the expression of the encoded proteins that perform the majority of the cellular biological functions. Humans have 23 pairs of chromosomes located in the nucleus of every nucleated cell. Out of the 23 pairs, there are 22 pairs of autosomes numbered from 1 to 22 and a pair of sex chromosomes (the X and Y chromosomes). The sex chromosomes determine the gender of the individual (male or female). Each chromosome is constructed of DNA tightly coiled many times around proteins called histones that support its structure. Individual chromosomes harbor hundreds to thousands of genes. Expression of individual genes is tightly regulated with each gene expressed at a specific developmental stage, with specific quantity and cellular circumstances. 11 Proteins are macromolecules consisting of one or more chains of amino acids linked together by peptide bonds and play fundamental roles in maintaining the structure and function of the organisms’ cells, tissues, and organs. The primary amino acids sequence of each protein is unique and differs from other proteins. This sequence is dedicated by the nucleotide sequence of their corresponding genes located on specific chromosomes. There are alternative forms of each gene that occur at the same locus on homologous chromosomes called alleles. Different combination of alleles may result in different observable phenotypic traits. A person may inherit two identical alleles of the same gene from either parents (homozygous) or two different alleles, one from each parent (heterozygous). DNA is composed of four types of nucleotides linked together by phosphodiester bonds with two anti-parallel complimentary strands forming the double helix. This arrangement is ensured by pairing adenine (A) with thymine (T) and cytosine (C) with guanine (G). In the coding parts of each gene, a three-letter sequence (codon) codes for a particular amino acid of a particular protein. Therefore, the sequence of those codons determines the sequence of the encoded protein. Any error in the DNA sequence of a gene is commonly termed as a mutation, especially when it is associated with a disease. Some of these mutations alter the amino acid sequence of the protein encoded by the gene, while others may affect its expression. If that altered protein is vital or crucial for the operations of the human body, a disease state may occur and is referred to as a “genetic disorder”. Genetic 12 diseases especially the conditions that are present from birth (congenital) can be caused by simple alterations such as single nucleotide substitutions (synonymous, non-synonymous or nonsense), deletions or insertions. 1.4.1.1. Monogenic disorders A monogenic or single gene disorder is a genetic condition exhibiting a Mendelian pattern of inheritance (dominant or recessive), and caused by mutations in a single gene occurring in all cells of the body. The nature of the disease depends on the biological function(s) performed by the mutated gene. Though individually relatively rare, monogenic disorders are responsible for a substantial loss of life worldwide [Penchaszadeh et al., 1999; Boycott et al., 2013]. The global prevalence of all monogenic diseases at birth is approximately 10/1000 live births [Penchaszadeh et al.,1999]. More than 7000 human diseases are now known to be caused by single gene defects [Bell et al., 2011; Brunham and Hayden, 2013; Boycott et al., 2013]. With significant advances in sequencing technologies the number of these diseases is growing at a rapid and unprecedented pace. Pathogenic mutations have been reported in around 5,000 human genes in the human gene mutation database [Brunham and Hayden, 2013]. However, genetic manipulations experiments in other organisms suggest that a much higher percentage of the known human genes (~22,000) might be associated with phenotypes [Brunham and Hayden, 2013]. For example, traditional genetic manipulations of yeast and mice genomes demonstrated that most of the genes in those organisms are associated with a noticeable phenotype 13 [Winzeler et al., 1999; Hillenmeyer et al., 2008; Ayadi et al., 2012; Brunham and Hayden, 2013]. Based on the chromosomal location of the mutated gene, monogenic disorders are categorized into sex-linked and autosomal. As the names suggest, sex-linked disorders are caused by mutations in genes located on the sex chromosomes (X and Y). These disorders are then called X-linked and Y-linked disorders. On the other hand, autosomal monogenic disorders result from errors in single genes located on one of the 22 pairs of autosomes. In diploid organisms like humans, each autosomal gene has two copies (called alleles) in the same individual with one allele inherited from the father and the other allele inherited from the mother. For autosomal recessive conditions to develop, mutations in the two alleles are essential whereas a mutation in only one allele is sufficient for the development of an autosomal dominant condition. 1.4.1.1.1. Autosomal recessive disorders Autosomal recessive disorders are monogenic disorders caused by mutations in a single autosomal gene. An affected child with a recessive disease must carry two mutated copies of a gene, one from each parent (Figure 1-2). In other words, to be affected by an autosomal recessive disorder, the person needs to have two defective alleles. The mutated alleles of a specific gene can be either identical (homozygous) or different (compound heterozygous). The parents of an affected child carry a single copy of the mutated gene and typically do not exhibit the disease phenotype 14 Figure 1-2. Autosomal recessive mode of inheritance. The main criteria for autosomal recessive inheritance are the following: 1) the parents of an affected child are obligate carriers of the recessive disease- causing allele, 2) the pathogenic recessive disease characteristically appears in one out of four children (the recurrence risk is 1:4 or 25% for each pregnancy), 3) there is a 2 in 4 chance that a child will inherit a single copy of the mutated gene and therefore be carrier, 4) males and females are equally likely to be affected. (http://www.discern-genetics.org/) Key Dominant allele Recessive allele Dominant normal trait Recessive pathogenic trait 15 and therefore are referred to as heterozygous carriers. The children of two carrier parents for a mutated gene have one in four chance of inheriting both defective copies and therefore exhibiting the autosomal recessive disorder. This risk (25% or 1 in 4) is the same for every pregnancy regardless of the outcome of previous pregnancies. One child out of two of the children of two carrier parents has the chance of being a healthy carrier by inheriting a single defective copy of that gene. One child out of four of their children has the chance of being neither affected nor a carrier and referred to as a normal child. The occurrence of the autosomal recessive disorder is not affected by the sex of the child. Of the 7028 disorders with suspected Mendelian mode of inheritance described in Online Mendelian Inheritance in Man database (OMIM), 4000 are autosomal recessive. Around 1139 have an established molecular basis and many disorders have yet to be identified [Bell et al., 2011]. 1.4.2. Identification of disease gene 1.4.2.1. Why study rare autosomal recessive diseases? Taking into account that the primary causes of many pediatric conditions remain elusive, it is likely that many of them are manifestations of rare recessive monogenic disorders [Hildebrandt et al., 2009]. For example, several studies have demonstrated that recessive mutations in the autosomal gene NPHS2 (OMIM*604766) are the molecular defects underlying up to 25% of all cases with steroid-resistant nephrotic syndrome (SRNS) in childhood 16 [Boute et al., 2000; Ruf et al. 2004; Hinkes et al., 2007; Hinkes et al., 2008]. SRNS is a heterogeneous group of monogenic disorders each caused by a different gene (Table 1-1). Therefore, identification of the causative genes and mutations for recessive disorders facilitates accurate diagnosis in the patients and carrier testing in their families which are prerequisites for providing effective genetic counseling. In addition, genetic diagnosis of recessive disease is of significant importance for patient management and some cases serves as a starting point for therapeutic interventions. This is exemplified in the treatable disorder acrodermatitis enteropathica (OMIM#201100) which is a rare autosomal recessive disorder of zinc deficiency [Kilic et al., 2012]. The condition is caused by mutations in the intestinal zinc-specific transporter encoded by SLC39A4 (OMIM*607059) eliminating its ability to transport zinc. Affected infants usually exhibit acrodermatitis, alopecia, and diarrhea. Once acrodermatitis enteropathica is correctly diagnosed, patients are simply treated with orally administered zinc sulphate [Coromilas et al., 2011]. Detailed studies have highlighted the importance of characterizing the mutations causing rare autosomal recessive disorder. For example, it has been demonstrated experimentally that some mutations can have drastically different consequences even when occurring at the same site. For instance, in-depth analysis of the molecular consequences of specific patient mutations in hyaline fibromatosis syndrome (OMIM#228600) revealed the necessity of personalized therapy. This condition is a rare autosomal recessive disorder 17 Table 1-1. The steroid-resistant nephrotic syndrome (SRNS) subtypes. Phenotype Nephrotic syndrome, type 2 Nephrotic syndrome, type 5 Nephrotic syndrome, type 3 Nephrotic syndrome, type 4 Nephrotic syndrome, type 6 Nephrotic syndrome, type 7 Nephrotic syndrome, type 8 Nephrotic syndrome, type 1 OMIM number 600995 614199 610725 256370 614196 615008 615244 256300 18 Gene NPHS2 NPHS5 NPHS3 NPHS4 NPHS6 NPHS7 NPHS8 NPHS1 OMIM number 604766 150325 608414 607102 600579 601440 601925 602716 caused by homozygous mutations in the gene encoding a protein involved in extracellular matrix homeostasis (ANTXR2; OMIM*608041). Yan et al [2013] were able to rescue ANTXR2 protein in patients carrying one base insertion but not in those carrying two base insertions at the same site of the gene by targeting the nonsense mediated decay pathway. Furthermore, studying the genetic basis of rare recessive disorders contributes to our understanding of gene functions and biological pathways underlying health and disease in general [Ropers, 2007]. The molecular information gained from investigating monogenic disorders has provided clues to the pathogenesis of common and complex diseases as well as therapies for them [Dietz, 2010]. Splitting complex disorders into many single, often monogenic, entities has significantly increased the chances for understanding the underlying pathogenetic mechanisms of those multifactorial disorders [Ropers, 2007]. This was mainly achieved by defining novel candidate genes that are part of the same pathway. For example, fanconi anemia (FA; OMIM#227650), the most common genomic instability syndrome, that is caused by recessive autosomal or X-linked mutation in one of 15 genes in the FA pathway listed in table 1-2. This pathway plays an important role in guarding the genome by coordinating a complex mechanism that joins proteins of three different DNA repair pathways in response to genotoxic insults [Deakyne and Mazin, 2011]. The pathway is a very well appreciated model system for several biological processes including DNA repair, cancer progression and protein ubiquitination [Moldovan and 19 D'Andrea, 2009]. The current knowledge of the FA pathway has been accumulating as a result of examining FA patients harboring homozygous mutations in the genes that encode the core complex of the pathway. The first FA gene to be cloned is FANCA being defective in more than 60% of FA cases [Moldovan and D'Andrea, 2009]. Mutations in 14 more genes encoding other core complex members were later identified in different FA patients (Table 1-2). Furthermore, although FA is a rare recessive disorder, understanding the functional role of the FA proteins and their interactions with other DNA damage response proteins provided broader opportunities for new cancer therapeutics [Jenkins et al., 2012]. 1.4.2.2. Approaches for the identification of recessive disease-causing genes and mutations Recessive disorders are most commonly seen in communities with higher rates of consanguineous marriages [Ropers, 2010]. Consanguineous marriages are a common practice in a number of communities worldwide including most Arab countries where marriages commonly occur between first cousins. Consanguinity increases the coefficient of inbreeding, which increases the likelihood of presence of pathogenic mutations in the homozygous state [Alkuraya et al., 2010]. This has made homozygosity mapping the strategy of choice for elucidating the genetic defects underlying autosomal recessive disorders in extended consanguineous families [Ropers, 2007; Hildebrandt et al., 2009]. Subsequently, gene identification and mutational screening is achieved either by candidate genes approach (the selection of the most likely disease-causing genes residing within the 20 Table 1-2. Fanconi anemia (FA) reported genes. Gene symbol OMIM number Chromosomal position Prevalence* FANCA 607139 16q24.3 66% ¥ FANCB 300514 Xp22.31 2% FANCC 227645 9q22.31 10% FANCD1 605724 13q12-13 2% FANCD2 227646 3q25-3 2% FANCE 600901 6p21-22 2% FANCF 603467 11p15 2% FANCG 602956 9p13 9% FANCI 609053 15q25-26 2% FANCJ 609054 17q22-24 2% FANCL 614083 2p16.1 0.2% FANCM 614087 14q21.3 0.2% FANCN 610832 16p12.1 2% FANCO 613390 17q22 NA FANCP 613951 16p13.3 NA *Moldovan and D'Andrea, 2009. ¥ The only X-linked gene of the FA genes. 21 homozygous regions) using Sanger sequencing or by massively parallel sequencing techniques [Hildebrandt et al., 2009]. A two steps approach (homozygosity mapping followed by sequencing) has proved to be the most robust strategy for unmasking numerous recessive mutations [Hildebrandt et al. 2009; Alkuraya et al., 2012]. Several hundred successful stories have been recently reported in the literature describing the identification of recessive disease causing genes and mutations in consanguineous families using the aforementioned two step approach [Tukel et al., 2010; Abou Jamra et al., 2011; Bloch-Zupan et al., 2011; Najmabadi et al., 2011; Dussaillant et al., 2012; Casey et al., 2012; Coussa et al. 2013, Grosch et al. 2013; Bonnefond et al., 2013; Capo-Chichi et al., 2013]. 1.4.2.2.1. Homozygosity mapping The homozygosity mapping concept was first recognized in 1953 by Smith [Smith, 1953]. However, in 1987, Lander and Botstein formulated a technique to search for homozygous regions by descent in consanguineous families and called it homozygosity mapping [Lander and Botstein, 1987]. Since then the homozygosity mapping technique has been used successfully for mapping autosomal recessive disorders in numerous consanguineous families worldwide [for example see, Faivre et al., 2003]. Currently, homozygosity mapping is considered to be one of the most powerful gene discovery approaches in the recent history of human genetics [Alkuraya et al., 2010]. 22 1.4.2.2.1.1. Principles of homozygosity mapping The main assumption in this approach is that the parents are related and therefore harbor pieces of identical DNA inherited from a common ancestor [Sheffield et al., 1995a]. Therefore, the homozygosity mapping approach largely relies on using consanguineous families, in which the parents are related, and have children exhibiting the same disease phenotype. It assumes that they inherited an identical homozygous duplicate of chromosomal DNA harboring a segment of identical markers or DNA polymorphisms (haplotype) including a homozygous disease-causing mutation. It also assumes that the recessive-disease causing mutation is “identical by descent” segregating to the affected children from a common ancestor through both parents. Subsequently, the mutated copies were joined together through the mating of the carrier parents in the affected children, causing the pathogenic phenotype (Figure 1-3). The homozygosity mapping approach scans the whole genome of the affected children and their families, using single nucleotide or short tandem repeat polymorphisms, to locate those homozygous blocks which are supposed to carry the disease-causing mutated gene. It is predicted that offspring of first cousin unions are homozygous in approximately 6% of their genomes. However, Woods et al [2006] elegantly demonstrated that offspring of first cousin unions from highly inbred consanguineous families are homozygous in about 11% of their genomes. In addition, the authors showed that these offspring carried around 20 homozygous segments each with a size of more than 3 cM. They also 23 Figure 1-3. Illustration of homozygosity mapping approach for recessive disease-genes identification. Chromosomal segment carrying a rare recessive mutation may segregate to a child from a common ancestor through the father and the mother if they were consanguineous. In that child, the combination of the two identical by decent chromosomal segments carrying the same mutation renders the child to be homozygous for that mutation. Although for each generation succession there is an opportunity for a crossing over between sister chromatids to occur in the parents' gametes, there is a high likelihood that in the affected child consecutive genetic markers surrounding the mutation will not have recombined and will be identical (homozygous) by descent. This segment of homozygous markers can be mapped leading to successful mapping of the disease gene. (http://autozygosity.org/). 24 observed in their study that the disease gene is usually located in the longest homozygous segment. 1.4.2.2.1.1.1. Microsatellite markers Microsatellite markers or short tandem repeats (STRs) polymorphisms are multiple repeats of di, tri or tetra nucleotides [Sheffield et al., 1995b]. They are extremely polymorphic, a characteristic that makes them useful as genetic markers to identify a specific chromosome or locus. The number of repeats within a microsatellite marker varies between individuals and in most occasions even between paired chromosomes in the same individual. They are mainly found in non-coding regions within the genome and are present approximately every 10-50 kilobases (kb). Typically, around 400 microsatellite markers are used in such mapping studies spaced at an average 10 kb density throughout the genome. In this technique, genomic DNA from each individual in a family is amplified using fluorescently labeled primers for each microsatellite marker. Different alleles for each marker are pooled together based on their sizes and labeling. Then, they are separated by size using an automated electrophoresis system. Finally, the electrophoresis results are analyzed and the signals are visualized using specific software. Many recessive disorders loci and genes have been mapped by scanning the whole genome using polymorphic microsatellite markers to track the blocks of homozygosity [for example see, Aligianis et al., 2005]. 25 1.4.2.2.1.1.2. Single nucleotide polymorphisms (SNPs) A single nucleotide polymorphism (SNP) is a variation at a single base pair in DNA. SNPs are the most abundant genetic variants and provide the most comprehensive resource for ascertaining genetic diversity. Millions of SNPs are distributed over the whole genome [Zhao et al., 2003; Puffenberger et al., 2004]. In addition, physically linked SNPs are co-inherited as a series of alleles in a pattern known as a haplotype [Puffenberger et al., 2004]. Therefore, currently SNPs are most commonly used in high-density highthroughput genotyping strategies for recessive disease-gene mapping. To do this, arrays that assess homozygosity or heterozygosity of millions of SNPs that cover the whole genome are employed. Many successful homozygosity mapping projects were achieved using the high-density SNP genotyping arrays (Figure 1-4) [for example see, Fabbro et al., 2011]. For example, the affymetrix genome-wide human SNP Array 6.0 contains more than 906,600 SNPs and more than 946,000 probes for the detection of copy number variations (CNVs). 1.4.2.2.1.2. Pitfalls of homozygosity mapping The majority of autosomal recessive genetic disorders worldwide are extremely rare where only a small number of cases are available [Ku et al., 2011]. Thus it is often very difficult to ascertain a sufficient number of families with the same phenotype to investigate using the homozygosity mapping approach. Moreover, small family size, which is the norm nowadays in many populations, is another problem in homozygosity mapping studies where only 26 Figure 1-4. Schematic representation of genome-wide SNP genotyping workflow. This method involves specific yet distinct enzymatic reactions to reproducibly amplify, label, and then capture the human genome on an array tailed with specific probes. 1) Genomic DNA is digested with restriction enzymes StyI/NSPI (6.0 array) that will cut genomic DNA at specific sites (black triangles). 2) The digested fragments are ligated to a common adaptor with T4 DNA ligase. 3) The fragments containing the adaptor undergo PCR using TITANIUM™Taq DNA polymerase and adaptor specific primer. 4) The amplicons are then fragmented by DNAse I enzyme. 5) The end of each DNA fragment is labeled using terminal deoxynucleotidyl transferase (TdT). 6) Capture all labeled DNA into a predesigned array containing a subset of allelespecific oligonucleotides (probes). This array will then be scanned by a detection system that records and interprets the hybridization signals. (http://www.affymetrix.com). 27 one or two affected children are available [Ku et al., 2011]. Therefore, for the very rare autosomal recessive disorders the challenge, even in highly consanguineous populations like the UAE, is to find enough families with children affected by the same phenotype [Romdhane et al., 2012]. In these cases it is sometimes difficult to find other families even if exhibiting similar phenotypes that carry mutations in the same gene making it challenging to prove pathogenicity. Although uncommon, sometimes these recessively inherited disorders in consanguineous families may be caused by compound heterozygous mutations. In this case, the disease locus may not be identifiable within a homozygous haplotype when performing a genome-wide scan for homozygous regions. This is exemplified in a study on a consanguineous family from Jordan exhibiting the autosomal recessive Karak syndrome (OMIM# 610217) described originally by Mubaidin et al [2003]. In this family, Morgan et al [2006] detected the same compound heterozygous disease-causing mutations in PLA2G6 gene (OMIM*603604) in the two affected siblings using direct DNA sequencing. The author genotyped the gene vicinity on chromosome 22 using microsatellite markers revealing that both affected children shared a common heterozygous haplotype surrounding the disease-causing gene that was different to their parents. Thus, in this family, anyone searching for homozygous regions using homozygosity mapping would have missed the disease locus as it was not contained within a homozygous region in the affected individuals. 28 The occurrence of allelic heterogeneity and/or locus heterogeneity within a consanguineous family is another challenge faced in some occasions when searching for homozygous stretches [Ku et al., 2011]. This phenomenon may occur when the recessive disorder segregated in that family is caused by different mutations in the same gene or in different genes respectively. For example, the mapping of Bardet-Biedl syndrome (BBS; OMIM#209900) in an extended consanguineous Lebanese family uncovered a complex pattern of mutations [Laurier et al., 2006]. One sibship of the pedigree was found to be carrying a homozygous mutation in the BBS2 gene (OMIM*606151) on chromosome 16. Other sibships of the extended pedigree carried a homozygous mutation in another gene called BBS10 (OMIM*610148) located on chromosome 12. Interestingly, a single patient in the last sibship carried compound heterozygous mutations in the BBS10 gene causing the disease. The former observation challenged linkage analysis due to the expectation of a single locus and mutation, while the latter resulted in loss of homozygosity in the markers flanking the BBS10 gene and led to a false negative result. 1.4.2.2.2. DNA sequencing The proposal to map and sequence the entire human genome (~3 billion nucleotides) was initiated and started to be implemented in the mid1980s in the form of “the human genome project” [Lewin, 1987; Bentley, 2000]. To achieve this grand goal, substantial academic and commercial 29 efforts were instigated to develop sequencing platforms capable of delivering relatively fast, accurate and high-throughput DNA sequencing data. 1.4.2.2.2.1. Early DNA sequencing technologies Two DNA sequencing techniques were invented at the end of 1970's. These were the Maxam-Gilbert (chemical cleavage) method and the Sanger (or dideoxy) method [Gilbert and Maxam, 1973; Sanger et al., 1977]. The Maxam-Gilbert method involves radioactive labeling of one end of the DNA fragment to be sequenced [França et al., 2002]. Chemical treatment generates breaks at a small proportion of one or two of the four deoxynucleotides (dNTPs) in four separate reactions. These cleavages lead to the generation of a series of labeled fragments, from the radiolabelled end to the first 'cut' site in each molecule. The four reactions can then be analyzed by gel electrophoresis side by side for size separation and visualization. The Maxam-Gilbert approach is best suited for sequencing short oligonucleotides. The Sanger method on the other hand is based on chain termination by the incorporation of a dideoxynucleotide (ddNTP), which lacks the 3’hydroxyl group of a normal dNTP, during the DNA polymerase elongation reactions (Figure 1-5) [França et al., 2002; Shendure and Ji, 2008]. This produces DNA strands of varying lengths that can be separated by gel electrophoresis by their size depending on which base is at the end of each fragment. With the advent of PCR and automation of the Sanger sequencing technique each of the four ddNTPs was labeled with a different fluorescent dye, each of which emit light at different wavelength. These modifications 30 allowed longer reads of DNA strands (800-1000 nucleotides) including some entire genes. The automated Sanger sequencing allowed researchers to sequence the entire 3.2 billion nucleotides of the human genome which was completed in 2003 [Yavartanoo and Choi, 2013]. 1.4.2.2.2.2. Recent advances in DNA sequencing technologies Recent technical advances including automation and new chemistries have dramatically changed the speed and sequencing range from 1000 base sequencing to massively parallel sequencing of the whole-genome [Stranneheim and Lundeberg, 2012]. These advances resulted in massive and unparalleled reductions in the cost of DNA sequencing. Several Figure 1-5. Schematic representation of the Sanger sequencing workflow. In this protocol a specific part of the genome is amplified by PCR (step1) using target specific primers (Forward and Revers), deoxyribonucleotides (dNTPS; dC, dA, dT, dG) and Taq DNA polymerase; generating millions of copies of that particular sequence. After purification, this sequence is used as a template for a chain termination reaction (step2). This reaction is very much similar to normal PCR in reagents and cycling except for the use of single primer (either forward or reverse) instead of two and the addition of fluorescently labeled dideoxynucleotides (ddNTPs; ddC, ddA, ddT, ddG) in specific ratios. These ddNTPs lack the 3’-OH group found in normal dNTPs and thus unable to form 3’-5’ phosphodiester bonds with other dNTPs causing the DNA strand to be terminated when they are incorporated in the growing chain. This reaction generates DNA strands of varying length each ending with a differently labeled ddNTP. Then these strands are injected in a capillary where they migrate according to their sizes in an electrophoresis gel passing a laser beam which will excite the labeling dyes to emit different wave lengths collected by a digital camera. The signal then is translated into sequencing chromatograms showing each nucleotide within the initial sequence (~800 nucleotides) with a different color using specific software (step 3). (http://www.genomebc.ca/education/articles/sequencing/). 31 32 sequencing platforms that are currently in use have inherited many features of the Sanger sequencing [Stranneheim and Lundeberg, 2012]. This includes the use of polymerases for synthesis, modified nucleotides and fluorescent automated detection. However, the main difference compared to the Sanger method is that all the massively parallel sequencing platforms require the DNA of the whole genome to be clonally amplified forming a consensus template prior to sequencing [Ratan et al., 2013]. To achieve that, the genomic DNA sample is fragmented, ligated to an adaptor, amplified by PCR then captured, re-amplified, labeled and sequenced as illustrated in Figure 16 [Neiman et al., 2012]. These steps may vary depending on different platforms. Millions of short reads of the initial target are generated per run. The first massively parallel sequencing technology was released in 2005 [Stranneheim and Lundeberg, 2012]. This technology was based on the concept of sequencing-by-synthesis (SBS) or pyrosequencing that was first conceived in the mid of 1980’s [Melamede et al., 1985; Nyrén, 2007]. SBS is unlike the Sanger method, chain elongation can be resumed with the addition of nucleotides generating a light by enzymatic action which is recorded. The second and the most successful massively parallel sequencing platform to be commercialized was the Illumina platform based on a reversible dye terminator SBS approach [Shendure and Ji, 2008; Stranneheim and Lundeberg, 2012]. In this chemistry, the incorporation of each labeled terminator in the DNA template is detected by fluorescence imaging of the surface of a flow cell. Then this labeled terminator is 33 Figure 1-6. Schematic representation of massively parallel sequencing workflow. The general working flow of this approach is the following: 1) the whole genomic DNA is randomly fragmented by different methods such as sonication and enzymatic digestion, 2) ligation of the generated fragments to a specific adaptor, 3) DNA template is clonally amplified by PCR to produce higher yields of the DNA library, 4) capture or hybridize the prepared DNA library on flow cell surface then re-amplify it again, 4) the labeling and detection steps usually occur simultaneously in the genome analyzer machine. These steps may vary depending on different platforms. (http://www.illumina.com/). 34 chemically removed, generating an extendable base that is ready for a new round of sequencing. More recently, several other massively parallel sequencing approaches have been developed revolutionizing the sequencing technology and consequently genetic research [for review see, Stranneheim and Lundeberg, 2012]. 1.4.2.2.2.3. Sanger The integration of DNA sequencing with homozygosity mapping sequencing has complemented homozygosity mapping successfully in the identification of recessive disease-causing genes and mutations within homozygous regions for several years [for example, Barnes et al., 2013]. However, mapping approaches in consanguineous families would often identify multiple or too large homozygous regions encompassing substantial numbers of candidate genes [Dussaillant et al., 2012]. This made the identification of disease-causing genes by candidate genes screening strategy using Sanger sequencing lengthy, laborious, and expensive. Thereby, too many accurately mapped autosomal recessive disorders remained unsolved for a long time [for examples, Tukel et al., 2005; Tétreault et al., 2006]. This has changed in the recent years due to the development of massively parallel sequencing technologies and their efficient integration with homozygosity mapping to accelerate the investigation of recessive disorders in consanguineous families [Ku et al., 2011]. Currently with the falling costs of high throughput sequencing technologies, whole-exome sequencing has become the most commonly used approach for recessive disease gene discovery [Bamshad et al., 2011; Gilissen et al., 2012]. 35 1.4.2.2.2.4. Whole-exome sequencing Whole-exome sequencing (WES) is massively parallel sequencing of all the exons of all the protein-coding genes in the human genome [Bamshad et al., 2011]. The entire exome encompasses less than 2% of the 3.2 billion bases of the human genome [Oetting, 2011]. Hence, it is possible to get deep coverage sequencing with relatively few reads which is necessary for the detection of rare variants underlying recessive disorders [Bamshad et al., 2011; Carr et al., 2013]. Moreover, it is well accepted that the majority of genetic variants that underlie Mendelian disorders disrupt exonic sequences where they can cause deleterious effects [Botstein et al., 2003; Gilissen et al., 2011]. Sequencing the genes’ exons should include the flanking splice sites in which there is high functional variation. Therefore, the whole-exome represents an enriched portion of the genome that can be used to search for variants with large effect sizes. Nevertheless, WES technique has important limitations that should be kept in mind when this approach is used to study recessive disorders. These limitations include the inability to detect intronic mutations, structural or CNVs which cause substantial number of recessive disorders. Despite the advances in sequencing technologies, some exons residing in repetitive or high CG rich areas of the genome are not well covered in the WES chips [for example see, Bloch-Zupan et al., 2011]. 36 1.4.2.2.2.4.1. The filtering strategy in whole-exome sequencing The main challenge of WES is to identify the disease-causing variant among the substantial number of variants detected in each exome. Therefore, a filtering strategy to reduce the initial number of detected variants is essential to identify the causal mutation (Figure 1-7) [for review see, Gilissen et al., 2012]. Approximately 50,000 variants are detected from each sequenced exome [Gilissen et al., 2010; Becker et al., 2011]. Based on the number of reads for each variant (reading depth) some variants can be excluded (usually if < five reads) [Gilissen et al., 2012]. Filtering all the detected variants against available variation databases such as dbSNP, 1000 Genome project and exome variants database reduces their number substantially. This exclusion is based on the assumption that for a very rare recessive disorder, the disease causing mutation is unique in patients or at least very rare in the general population. In addition, intronic and synonymous mutations that do not alter the protein sequence can be filtered out, based on the main assumption of exome sequencing approach that the disease-causing mutation is most likely located within the protein-coding regions of the genome. The most significant reduction follows from excluding heterozygous variants based on parental consanguinity which implies that the causal mutation is inherited from a common ancestor and thus found in a homozygous state in patients. This step typically reduces the number of potential candidate mutations to 150-500 of novel non-synonymous, nonsense, indels, or splice-site variants [Stitziel et al., 2011]. To identify the 37 Figure 1-7. Filtering steps of whole-exome sequencing data. A substantial number of variants are identified in every exome sequencing run. Therefore, in order to reduce this number and to identify the recessive disease causing mutation we have to filter this data in the following order: 1) exclude all common variants (frequency>5%), 2) exclude variants that will not alter the protein sequence (intronic and synonymous), 3) exclude heterozygous variants, 4) exclude variants that are not detected in all the affected siblings exhibiting similar phenotype, 4) exclude variants that are not located in the pre-mapped disease homozygous region(s). (http://www.goldenhelix.com/). All variants detected in an exome Few variants 38 causative mutation among these variants traditional mapping strategies such as homozygosity mapping have been adapted for WES. Thereby, the candidate variants are only selected when contained within a large homozygous stretch. For example, Gilissen et al [2010] successfully used this strategy to reduce the number of candidate variants from around 140 to only 3 or 4 in two patients exhibiting the autosomal recessive Sensenbrenner syndrome (OMIM#218330). 1.4.2.2.3. Selection of candidate genes and recognition of pathogenic mutations In the traditional candidate strategy, computational prioritization is usually used to reduce the number of genes residing within the mapped homozygous regions [Masoudi-Nejad et al., 2012]. Several bioinformatics tools are available on line to prioritize candidate genes based on information relating to the genes’ functions or their expression profiles [Oti et al., 2011]. Examples of such prioritizing programs are GeneDistiller2, Suspects, and ToppGene Suite. Further prioritization of candidate genes can be gained from what is known or inferred about their pathophysiology in available genes databases such as the University of California Santa Cruz (UCSC) Genome Browser and the National Center for Biotechnology Information (NCBI) or from previous studies. In analyzing WES data, researchers usually prioritize the variants residing within the homozygous regions [Gilissen et al., 2012]. In this strategy variants will be prioritized on the basis of the predicted impact of the variant on protein function and structure in the following order: stop mutations, frame- 39 shifting mutations, and mutations in the canonical splice-sites [Ku et al., 2011; Gilissen et al., 2012]. These mutations are known to cause deleterious effects on the corresponding protein [Ku et al., 2011]. On the other hand, the impact of non-synonymous mutations on their proteins is less predictable and thus several computational programs have been developed for their prioritization [Dolled-Filhart et al., 2013]. Such programs include MutationTaster, Polymorphism Phenotyping v2 (Polyphen2), and Sorting Intolerant From Tolerant (SIFT). The main criterion currently applied on prioritizing nonsynonymous mutations is conservation of the substituted amino acid across species [Kumar et al., 2009; Adzhubei et al., 2013]. These programs presume that important amino acids will be conserved in the protein family, and so changes at highly conserved positions tend to be predicted as deleterious. These programs also consider the type of amino acid change. For example, if the changed amino acid is a hydrophobic substituted with another hydrophobic residue it is usually predicted to be tolerated. However, substituting a hydrophobic amino acid with another charged or polar residue is predicted to affect protein function and therefore is deleterious [DolledFilhart et al., 2013]. As a final step in this process, even candidate variants should be sequenced by traditional Sanger sequencing in all family members to validate their segregation with the pathogenic phenotype [Gilissen et al., 2012]. In addition, definite proof of pathogenicity requires novelty verification in ethnically matched healthy controls and/or functional experiments. 40 1.4.3. Conclusion In this chapter, I have given a broad overview of autosomal recessive disorders and the methods by which disease-causing genes and mutations may be identified. It is striking that, for the majority of these severe disorders the underlying molecular causes remain unknown. Undoubtedly, current and future advances in sequencing technologies and bioinformatics are likely to accelerate the identification of disease-causing genes in monogenic disorders. Evidently, the challenge of disease gene identification is shifting from the identification to the interpretation phase and although numerous genomic and exonic variants are found in each patient only one or two may explain the monogenic disease. 41 1.5. Potential contributions and limitations of the study The research described in this dissertation has primarily focused on the mapping and identification of novel disease genes and causal mutations of rare autosomal recessive disorders among mainly consanguineous families residing in the UAE. 1.5.1. Limitations Autosomal recessive disorders are individually rare, making it difficult to collect significant numbers of unrelated patients or families with similar phenotype to further verify the pathogenicity of some diseasecausing genes. The UAE population is ethnically heterogeneous encompassing several isolated subpopulations and most studied families were not originally Emirati making it difficult to validate rarity or prevalence of some candidate variants in their original populations. Limited functional data can be generated for some of the identified genes and mutations due mainly to sampling difficulties for the following reasons: 1) families cooperation and localization, 2) severity of the syndrome (many affected children die with no sufficient or limited samples to study), 3) inaccessibility of relevant tissues to study gene expression. Unavailability of animal models to study the identified genes and mutations in vivo. This is mainly because of limited resources, timing 42 and limited expertise at CMHS. Knock out models are expensive and beyond the resources that we have at our disposal. 1.5.2. Potential contributions The importance of my study is that I have elucidated the genetic, molecular and in some cases the cellular bases of several rare autosomal recessive disorders segregating in highly-inbred consanguineous families. The findings from this dissertation are anticipated to: Facilitate genetic counseling, carrier testing, prenatal and even preimplantation genetic diagnosis for at-risk family members. Allow more accurate diagnosis for other patients exhibiting one of the studied phenotypes. Shed some light on some pathophysiological processes thereby enhancing understanding of normal physiology and disease mechanisms, facilitating the development of therapeutic targets for future possibly curative treatments for some diseases. 43 CHAPTER 2: GENERAL METHODS 44 2.1. Families 2.1.1. Consent and ethics approval All the investigations included in this dissertation have been approved by the ethics committee at Al-Ain Medical Human Research Ethics Committee according to the national regulations (protocol number 10/09). All individuals participated in this study or their guardians provided written informed consent before being enrolled in the genetic study. 2.1.2. Families recruitment and patients assessment For the UAE-based families, a detailed clinical assessment including a full medical history and systemic examination was performed for each family by Prof. Lihadh Al-Gazali either at Tawam Hospital or at Al-Ain Hospital. Blood samples were taken from most affected individuals, both parents and any available unaffected siblings. For families based outside the UAE, clinical data were acquired by liaising with the referring clinicians. For such internationally acquired cases, the referring clinician sent consent forms, full clinical report, family’s pedigree, history and pictures as well as DNA samples for genetic testing. 2.2. Methods 2.2.1. DNA, RNA & protein extraction 2.2.1.1. DNA extraction Whole blood samples were collected in ethylenediamine tetra-acetic acid (EDTA) tubes. Genomic DNA was extracted using the Flexigene DNA extraction kit (Qiagen, USA) following the manufacturer’s instructions. Briefly, 45 the blood sample was mixed thoroughly with red blood cells (RBCs)-lysis buffer followed by centrifugation to recuperate the leukocytes. Then, the white blood cells (WBCs) were lysed using the denaturation buffer and treated with Proteinase K (Sigma Aldrich, USA) to digest the proteins. The genomic DNA was precipitated using isopropanol (Sigma Aldrich, USA) followed by a washing step using 70% of ethanol (Sigma Aldrich, USA). Finally, the pellet was allowed to air dry for a few minutes and at the end the DNA was suspended in DNase-free water (Sigma Aldrich, USA). The DNA yields and qualities were determined, as measured by absorbance at 260 nm, using a Nanodrop Spectrophotometer 1000 (ND-1000; Thermo Fisher Scientific, USA). High quality DNA has an A260/A280 ratio of 1.7 to 1.9. A ratio of > 1.7 indicates that the DNA is free of protein contamination. 2.2.1.2. RNA extraction Total RNA was extracted from blood using Qiazol reagent (Qiagen, USA) following the manufacturer’s instructions. Blood was mixed thoroughly with red blood cells (RBCs)-lysis buffer (Qiagen, USA) followed by centrifugation to recover the leukocytes. Then, 1x106 white blood cells (WBCs) were mixed with 1 ml Qiazol reagent. 200 µl chloroform (Sigma Aldrich, USA) was added and mixed vigorously then centrifuged for 10 minutes at ~13,000 xg. The aqueous layer was then taken and mixed with 600 µl isopropanol (Sigma Aldrich, USA) and centrifuged to precipitate the RNA. Then the RNA pellet was washed with 70% ethanol (Sigma Aldrich, 46 USA), air dried and then dissolved in DNase- and RNase- free water (Sigma Aldrich, USA). 2.2.1.3. Protein extraction Total proteins were extracted from adherent cells (skin fibroblasts, HeLa, Hek293 and COS7). The cell culture medium was removed and cells were washed once with a phosphate buffered saline (PBS; Gibco Life Technologies, USA). Cells were detached using 1x trypsin solution (Gibco Life Technologies, USA) by incubation at 37oC for 5 minutes or until they were visibly detached. Two milliliters of culture media with fetal bovine serum (FBS; Gibco Life Technologies, USA) was added to stop the protease reaction then cells were collected and centrifuged. The supernatant was decanted and cells were lysed by incubating in 150 µl radio immunoprecipitation assay buffer (RIPA; Thermo Scientific Pierce, USA) containing 1x Protease inhibitor (Sigma Aldrich, USA) for 15 minutes on ice. The cell lysates were collected and transferred to a micro-centrifuge tube. The samples were then centrifuged at maximum speed ~13,000 xg to pellet the cell debris. The supernatant was transferred to a new tube and quantified using bicinchoninic acid (BCA) protein assay kit (Thermo Scientific Pierce, USA). After mixing reagents A and B of the kit, the samples were added and incubated for 30 minutes then were read against protein standards at 562 nm. 2.2.2. Polymerase chain reaction (PCR) amplification 2.2.2.1. Standard PCR 47 PCR amplification of a DNA fragment of interest was performed on a 2720 thermal cycler (Applied BioSystems, USA). A total volume of 20 µl of PCR reactions were prepared containing 1x PCR buffer (Qiagen, USA), 0.2 mM dNTPs (Promega, USA), 5 µM of each forward and reverse primers (Metabion, Germany), 100 ng of template DNA and 0.5 units of Taq DNA polymerase (Qiagen, USA). The standard reactions were subjected to an initial denaturation period of 5 minutes at 95 oC followed by 40 cycles of 95 oC for 30 seconds, 56 oC-60 oC for 45 seconds and 72 °C for 45 seconds and final extension of 72 °C for 10 minutes. 2.2.2.2. Reverse transcription The cDNA was prepared by using GoScript reverse transcription system (Promega, USA) according to the manufacturer’s instructions. A total volume of 50 µl of reactions were prepared containing 1x buffer, 0.2 mM dNTPs, 2.5 µl of random primers, 1 µg of template RNA and 0.5 units of reverse transcriptase and RNase inhibitor. The standard reverse transcription reaction was composed of an initial annealing period of 5 minutes at 25 oC followed by 1 hour extension at 40 oC then the reverse transcriptase was inactivated at 70 oC for 15 minutes. 2.2.2.3. Real-time PCR The expression levels of mRNA were analyzed using target specific TaqMan gene expression assays (Hs01089850_m1 for LINS and Hs01097626_m1 for COL11A1; Applied Biosystems, USA) using the 7500 Real Time PCR system (Applied Biosystems, USA). Human HPRT1 48 (hypoxanthine phosphoribosyltransferase1; Hs99999909_m1; Applied Biosystems, USA) was used as an endogenous control. All reactions were run in duplicates and repeated in two different amplification experiments to ensure reproducibility. Target mRNA was amplified and quantified in a total volume of 20 µl containing: 10 µl of 2X TaqMan gene expression master mix (Applied Biosystems, USA), 1 µl of TaqMan assay, 2 µl of the cDNA samples and 7 µl of nuclease-free water. The standard amplification conditions are as follows: 10 minutes activation at 95 oC, followed by 40 cycles of amplification at 95 oC for 15 seconds and 60 oC for 1 minute. The threshold cycle (CT) and relative expression or quantification (RQ) values were calculated using the 7500 analysis SDS software v2 (Applied Biosystems, USA). 2.2.2.4. Site directed mutagenesis The mutations were introduced in the ready-made JAM3-pCMV6 plasmid tagged C-terminally Myc-Flag-tagged commercial construct (RC216073; OriGene Technologies, USA, see Appendix-A) using the QuickChange site-directed mutagenesis kit (Stratagene, USA). The primers used to introduce p.E116K mutation in the cDNA of JAM3 in pCMV6 plasmid are the forward: 5’ CCCTTTATCGCTGTAAGGTCGTTGCTCG 3’ and the reverse: 5’ CGAGCAACGACCTTACAGCGATAAAGGG 3’. While, the primers used to introduce the p.C219Y mutation CTGGGCAGTACTACTACATTGCTTCCAATG 3’ were the forward: 5’ and the reverse: 5’ CATTGGAAGCAATGTAGTAGTACTGCCCAG 3’. PCR amplification was performed on a 2720 thermal cycler (Applied BioSystems, USA). A total 49 volume of 20 µl of PCR reactions were prepared containing 1x PCR buffers (Qiagen, USA), 0.6 mM dNTPs, 5 µM of each forward and reverse primers (Metabion, Germany), 50 ng of template DNA and 1.25 units of PfuTurbo DNA polymerase (Stratagene, USA). The reactions were subjected to an initial denaturation period of 5 minutes at 95oC followed by 16-18 cycles of 95oC for 30 seconds, 60oC for 1 minute and 72°C for 18 minutes and a final extension of 72°C for 20 minutes. The parental DNA templates were digested using the DpnI (Promega, USA) endonuclease treatment for 3 hours at 37°C. 2.2.3. Sequencing 2.2.3.1. Sanger sequencing 2.2.3.1.1. PCR products sequencing The PCR products were cleaned up using 1 unit of Exonuclease I Shrimp Alkaline Phosphatase solution (USB ExoSAP-IT; Affymetrix, USA) at 37 ˚C for 30 minutes followed by 80 ˚C for 15 minutes in thermocycle machine. Sanger cycle sequencing was performed using the BigDye Terminator kit v3.1 (Applied Biosystems, USA) in a reaction containing 1.5 µl of PCR products, 0.5 µM of either forward or reverse primers, 2 µl of 5x BigDye Terminator sequencing buffer and 2 µl of BigDye terminator Taq mix (Applied Biosystems, USA). Cycle sequencing reactions were performed as follows: 96 °C for 1 minute, followed by 25 cycles of 96 °C for 10 seconds, 50 °C for 5 seconds, and final extension of 60 °C for 4 minutes. Fluorescent PCR products were purified by adding 1.25 µl of 125 mM EDTA (Promega, USA) and 25 µl of 95% ethanol (Promega, USA) to precipitate the DNA. After 30 50 minutes incubation in -20°C the PCR plates were centrifuged for 20 minutes at 4°C at 4000 rpm. The plates were flipped on tissue papers and centrifuged briefly to decant the supernatants. 50 µl of 70% ethanol was added to each well and previous steps were repeated to wash the pellets. The plates were left to dry for 15 minutes in the air. The DNA pellets were then dissolved and denatured in 15 µl of HiDi-Formamide reagent (Applied Biosystems, USA). The purified products were then separated and electrophoresed in the POP7filled 16 capillaries of the 3130xl Genetic Analyzer system (Applied Biosystems, USA). The results were analyzed using Sequencing Analysis v5.3 software (Applied Biosystems, USA). 2.2.3.1.2. Plasmid sequencing Plasmid DNA sequencing was performed using the Sanger dideoxy method on the 3130xl Genetic Analyzer (Applied Biosystems, USA). A total volume of 4µl of purified plasmids, 5 µM of either forward or reverse primers, 2µl of 5x BigDye Terminator sequencing buffer and 2 µl of BigDye terminator cycle sequencing mix (Applied Biosystems, USA). The reactions were subjected to an initial denaturation period of 5 minutes at 95oC followed by 35 cycles of elongation at 95 oC for 30 seconds and annealing at 60 oC for 4 minutes. PCR products were cleaned up using the above described ethanol purification method and then were run on the 3130xl Genetic Analyzer (Applied Biosystems, USA). The results were analyzed using Sequencing Analysis v5.3 software (Applied Biosystems, USA). 2.2.3.1.3. Whole-exome sequencing 51 Whole-exome sequencing (WES) selectively sequences all the coding regions (exons and intron/exon boundaries) of all the genome. Library construction, whole-exome capturing, whole-exome sequencing, variant calling and annotation were performed by two commercial companies (Macrogen, South Korea; Oxford Gene Technology; UK). The library construction was performed using SureSelect human all exon Kit (Agilent Technologies, USA) and sequencing was performed by Illumina HiSeq 2000 (Illumina, USA) according to the manufacturer`s protocol. Paired end (2×100 bases) DNA sequence reads that passed the quality control were mapped to the human reference genome build hg19 using the Burrows-Wheeler Aligner (BWA) and Sequence Alignment/Map (SAM) tools. 2.2.4. Genotyping 2.2.4.1. Genome wide SNP genotyping Genome-wide homozygosity mapping using the Genome-Wide Human SNP Arrays 250K and 6.0 (Affymetrix, USA) was performed on the DNA samples from the recruited family members. SNP genotypes were obtained from several commercial companies (ATLAS Biolabs; Germany; Expression Analysis; UK; and Aros Applied Biotechnology, Denmark) and from academic institutions (Geoff Woods’ laboratory, Cambridge Institute for Medical Research; UK). Briefly, total genomic DNA (250 ng) was digested with NspI and SpyI restriction enzymes and linked to adaptors. Generic primers that recognize the adaptor sequence were used to amplify adaptor-ligated DNA fragments. Standard PCR conditions preferentially amplify fragments in the 52 200–1100 bp size range. The amplified DNA was then fragmented with the Affymetrix fragmentation reagent following the manufacturer’s protocol. Genotype calling was performed by the Genotyping Console software (Affymetrix, USA) using the default settings. 2.2.4.2. Microsatellite genotyping Fine mapping to confirm and reduce the homozygous regions identified in section 3.1 was performed using fluorescently-labeled microsatellite markers or STRs designed and analyzed in Professor Geoff Woods’ laboratory (Cambridge Institute for Medical Research; UK). STRs used for genotyping the family in section 3.6 were selected according to their locations and frequencies using the Marshfield genetic map. STRs amplification was performed using GoTaq Flexi DNA Polymerase kit (Promega, USA) in a 2720 thermocycler (Applied Biosystems, USA). PCR mix of 20 µl was prepared containing: 2.5 µl 5x buffer, 1µl MgCl2, 0.1 µl dNTPs, 0.25µl DNA, 0.0625µl GoTaq DNA polymerase, 0.4 µl forward STR primer, 0.1 µl reverse STR primer, 0.4µl M13 universal fluorescently labeled forward primer and 15.2µl nuclease free water. The amplification conditions were as follows: hold at 94 °C for 5 minutes, then 30 cycles of 94 °C for 30 seconds, 57 °C for 45 seconds, 72 °C for 45 seconds, followed by 8 cycles of 94 °C for 30 seconds, 53 °C for 45 seconds, 72 °C for 45 seconds, with final extension hold at 72 °C for 10 minutes. Labeled PCR product aliquots were denatured in HiDiFormamide (Applied Biosystems, USA) and electrophoresed using the 3130xl Genetic Analyzer system (Applied Biosystems, USA). PCR products sizes 53 were determined by reference to GeneScan-500 LIZ size standard (Promega, USA) and GeneMapper software (Applied Biosystems, USA). 2.2.5. Electrophoresis 2.2.5.1. Agarose gel electrophoresis PCR products (~5 µl) were separated according to size on 2% agarose gel (Promega, USA) in 1x TBE buffer (Tris/Borate/EDTA). Ethidium bromide (EtBr) usually is added to the gel to a final concentration of approximately 0.20.5 μg/mL. EtBr binds to the DNA and allows the DNA visualization under ultraviolet (UV) light. Where needed, specific PCR products bands were sliced from the gel with a clean, sharp scalpel and purified using QIAquick gel extraction Kit (Qiagen, Germany) according to the manufacturer’s protocol. Briefly, 3 volumes of buffer QX1 was added to 1 volume gel and incubated at 50 °C for 10 minutes to dissolve the gel. Then, 1 gel volume of isopropanol was added to the sample, mixed, and centrifuged in a spin column. The collected DNA was washed and dissolved in nuclease free water. 2.2.5.2. Western blot analysis Total protein extracted from cells (~100 μg) was mixed with a 1x reducing sample loading buffer [2 ml of Glycerol, 6 ml of 20% sodium dodecyl sulfate (SDS), 1.8 ml of 2-mercaptoethanol, 5 ml of 1 M Tris-HCL 6.8, 0.1% bromophenol blue and 3.8ml ddH2O] (Sigma Aldrich, USA). The mix was denatured at 100 oC for 5 minutes and then electrophoresed in 8% SDSpolyacrylamide gel electrophoresis (PAGE). The gel was then blotted to a nitrocellulose membrane (Thermo Scientific Pierce, USA) using a transfer 54 apparatus according to the manufacturer’s protocols (Bio-Rad, USA). After incubation with 5% nonfat milk in Tris-buffered saline with Tween 20 [TBST; 10mM Tris, pH 8.0, 150mM NaCl, 0.5% Tween 20] (Sigma Aldrich, USA) for 60 minutes, the membrane was then washed once with TBST and incubated with 1 in 500 dilution of rabbit polyclonal Anti-COL11A1 antibody (ab64883;Abcam, USA) at 4°C for 12 hours. Membranes were washed three times for 10 minutes and incubated with a 1:30,000 dilution of horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Santa Cruz Biotechnology, USA) for 2 hours at room temperature. Blots were washed with TBST three times and developed with enhanced chemiluminescence (ECL Plus; Thermo Scientific Pierce, USA) and visualized by the Typhoon imaging system (GE Healthcare, USA). 2.2.6. Cellular studies 2.2.6.1. Culture of human cell line Human cell lines (HeLa; HEK293; COS7) were cultured in Dulbecco’s modified Eagle’s medium (DMEM ; Invitrogen, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; Invitrogen, USA) , 2mM L-glutamine and 100U/ml penicillin/streptomycin (Invitrogen, USA) at 37 °C with 5% CO2. When the cells were confluent they were detached by trypsin treatment as described in protein extraction (see 2.2.1.3) and then plated in 24 well plates for confocal microcopy or 6 well plates for western blotting analysis. 55 2.2.6.2. Transient transfection of cultured cells Human cell lines were grown for 24 hours on sterile cover slips (Ibidi, Martinsried, Germany) in 24 well plates (Nalge Nunc International, USA) in the culture media described above. Upon reaching 60-70% confluency, cells were transfected using 1.7 µl of the non-liposomal transfection reagent FuGENE HD (Promega, USA) and incubated for 5 minutes at room temperature with 26 µl of Optimem (Invitrogen, USA) and 1 μg of the purified cDNA plasmids. Co-transfection with 0.2 μg of hRas-GFP (Harvey rat sarcoma viral oncogene homolog - green fluorescent protein) plasmid was performed to serve as a transfection indicator and a plasma membrane (PM) marker. Twenty-four hours after transfection, the cells were fixed and stained for confocal microscopy with specific antibodies. 2.2.6.3. Confocal fluorescence microscopy For immunofluorescence, cells cultured on cover slips were washed with PBS and fixed by methanol at -20o C for 4 minutes. Before blocking, coverslips were washed in PBS three times. Then the coverslips were incubated in blocking solution of 1% Bovine serum albumin (BSA; Sigma Aldrich, USA) in PBS for 30 minutes at room temperature. After blocking, cells were incubated for 1 hour at room temperature with the relevant monoclonal or polyclonal primary antibodies. Mouse anti-flag (Sigma Aldrich, USA) and rabbit anti-calnexin (Santa Cruz Biotechnology, USA) were used at 1:1,000 and 1:100 dilutions, respectively. Then the cover slips were washed with PBS and re-incubated in the dark with the appropriate rohdamine/fluorescein 56 labeled secondary antibodies for 45 minutes at room temperature. Secondary antibodies (fluorescence-labeled “Alexa-Fluor-568” [anti-mouse] and “AlexaFluor-488” [anti-rabbit]; Santa Cruz Biotechnology, USA) were used. Finally, the cover slips were washed several times with PBS and mounted in mounting medium (ICN Biomedicals, USA). Data were acquired using a Nikon confocal microscope (Nikon Instruments, Japan). The images were presented as single sections in the z-plane, using Adobe Photoshop (Adobe, USA). Control experiments without the primary antibodies or with non-transfected cells were carried out and revealed very low-level background staining. 2.2.6.4. Fibroblast culturing Skin biopsies from patients and control samples were sliced into smaller pieces and cultured in 6 well plates in DMEM supplemented with 20% FBS, 2 mM L-glutamine and 100 U/ml Penicillin/Streptomycin at 37 oC with 5% CO2. After reaching confluency, cells were trypsinzed using 1x of trypsin solution and transferred to T25 (25 cm2) flasks in the same media to be further analyzed and maintained. 2.2.7. Bioinformatics analysis 2.2.7.1. Website addresses for internet resources A number of websites were utilized for various aspects of experimental design and data analysis (Table 2-1). 2.2.7.2. Primer design Primers for DNA and cDNA PCR amplification were designed using Primer3 or ExonPrimer (Table 2-1). Primers for site directed mutagenesis 57 were designed using PrimerX (Table 2-1). TaqMan assays were selected from the TaqMan gene expression assays listed in the company’s website (Applied Biosystems, USA). A list of all the primers used in this dissertation are illustrated in Appendix-B. 2.2.7.3. Homozygosity mapping analysis Generated SNPs derived from the family members DNA were loaded into the software package HomozygosityMapper or into the software package dChip and subjected to computational homozygosity mapping analysis. Copy number variations (CNVs) analysis was carried out using the affymetrix genotyping console software 4.1.4 (Affymetrix, USA). 2.2.7.4. Prioritization of genes and variants Candidate genes within homozygous regions were prioritized according to their expression profiles, interactions, functions, similarity and other criteria, using several prediction programs such as GeneDstiller2, Suspects, ToppGene Suite. In addition, those genes were ranked based on the knowledge gained from previous studies in PubMed, UCSC, OMIM and NCBI. 2.2.7.5. Analysis of Sanger sequences and exome sequencing data Exome variant filtering was performed against NHLBI exome variant database, Ensembl, 1000 Genome project, and dbSNP. ClustalW2 and Blast were used for aligning sequences generated from Sanger sequencing with the corresponding RefSeq sequences from the UCSC and NCBI. Online available software tools such as Polyphen2, SIFT, and mutation Taster were 58 used to predict the possible pathogenic effect of mutations, while to predict the effects of intronic mutations on splicing efficiency, human splicing finder v2.4.1, and GeneSplicer software were used. 59 Table 2-1. Web-based resources. UCSC NCBI Ensembl Primer3 PrimerX ClustalW2 dbSNP Affymetrix Blast ExonPrimer Online Mendelian Inheritance in Man (OMIM) PubMed Knockout Mouse Project PolyPhen-2 SIFT MutationTaster GeneDistiller 2 1000 Genome project NHLBI exome sequencing Project (ESP) Suspects ToppGene Suite ExPASy HomozygosityMapper Affymetrix TaqMan Gene Expression Assays GeneSplicer Human Splicing Finder - Version 2.4.1 dChip software HGMD Mutalyzer 2.0.beta-27 Marshfield human genetic map http://genome.ucsc.edu/ http://www.ncbi.nlm.nih.gov./ www.ensembl.org http://frodo.wi.mit.edu/primer3/ http://www.bioinformatics.org/primerx/ http://www.ebi.ac.uk/Tools/clustalw2/index.html http://www.ncbi.nlm.nih.gov./projects/SNP/ http://www.affymetrix.com/estore/ http://blast.ncbi.nlm.nih.gov/Blast.cgi http://ihg2.helmholtzmuenchen.de/ihg/ExonPrimer.html http://www.ncbi.nlm.nih.gov./sites/entrez?db=OMI M&itool=toolbar http://www.ncbi.nlm.nih.gov./pubmed/ http://www.knockoutmouse.org/data.shtml. http://genetics.bwh.harvard.edu/pph2/ http://sift.jcvi.org/ http://www.mutationtaster.org/ http://www.genedistiller.org/ http://www.1000genomes.org/ http://evs.gs.washington.edu/EVS/ http://www.genetics.med.ed.ac.uk/suspects/ http://toppgene.cchmc.org/ http://www.expasy.org/ www.homozygositymapper.org/ http://www.affymetrix.com/estore/ http://www.invitrogen.com/site/us/en/home/Product s-and-Services/Applications/PCR/real-timepcr/real-time-pcr-assays/taqman-geneexpression.html http://www.cbcb.umd.edu/software/GeneSplicer/ge ne_spl.shtml http://www.umd.be/HSF/ http://www.dchip.org http://www.hgmd.cf.ac.uk/ac/hahaha.php https://mutalyzer.nl/ http://research.marshfieldclinic.org/genetics/Geneti cResearch/compMaps.asp 60 CHAPTER 3: RESULTS AND DISSCUSSION 61 SECTION 1: Is autosomal recessive Silver– Russell syndrome a separate entity or is it part of the Three-M syndrome spectrum? 62 3.1.1. Background 3.1.1.1. Three M syndrome 3.1.1.1.1. Clinical features Three M syndrome (3-M; OMIM# 273750, 612921 and 614205) is a rare autosomal recessive disorder first described by Miller et al. [1975]. The disorder is characterized by low birth weight, proportionate dwarfism, dysmorphic facial features, and radiological abnormalities. The typical facial features include a relatively large head, frontal bossing, triangular face with a pointed chin, upturned nose with a long philtrum, and hypoplastic midface [Winter et al.,1984]. Radiological features include slender long bones and ribs, foreshortened vertebral bodies, and small pelvis [van der Wal et al., 2001]. 3.1.1.1.2. Genetic heterogeneity The 3-M syndrome is a genetically heterogeneous. Studying a series of 29 families with 3-M syndrome, Huber et al [2005] mapped the disease locus and the disease-causing mutations to chromosome 6p21.1 and in CULLIN7 (CUL7; OMIM*609577). Later on, Hanson et al [2009] described the mapping of a second locus for 3-M syndrome at 2q35-q36.1 and the subsequent identification of mutations in the OBSL1 gene (OMIM*610991) that encodes obscurin-like protein 1 (OBSL1), a putative cytoskeletal adaptor protein. In 2011, the same group used homozygosity mapping in five unrelated patients from consanguineous Asian families and identified another 3-M syndrome locus 19q13.2-q13.32 [Hanson et al., 2011a]. The region was 63 of 7.9 Mb and contained 301 protein-coding genes. Therefore the authors used exome sequencing to identify the causative gene and identified different homozygous mutations in the coiled-coil domain containing protein 8 (CCDC8; OMIM*614145) in all five patients. As a result, the authors proposed that CUL7, OBSL1, and CCDC8 are members of the same pathway that control mammalian growth. 3-M patients without a mutation in any of the aforementioned genes and who are not linked to any of the mapped regions have also been identified, indicating the involvement of additional genes in the pathway and causing this growth disorder [Hanson et al., 2009; Clayton et al., 2012]. 3.1.1.2. Silver–Russell syndrome 3.1.1.2.1. Clinical features Silver–Russell syndrome (SRS; OMIM#180860) is a clinically heterogeneous disorder characterized by severe intrauterine and postnatal growth retardation, relative macrocephaly and a characteristic small, triangular face [Hitchins et al., 2001]. Additional features include body or limb asymmetry, clinodactyly, and severe feeding difficulties in infancy [Eggermann, 2010; Wakeling et al., 2010]. No specific radiological abnormalities have been described in this syndrome [Galli-Tsinopoulou et al., 2008; Rossignol et al., 2008]. Although Bruce et al [2009] reported abnormally high lumbar vertebrae in hypomethylated SRS patients; it remains possible that some of the patients in their series had 3-M rather than SRS. 64 3.1.1.2.2. Genetic heterogeneity SRS is a genetically heterogeneous disorder, with different suggested modes of inheritance [Hitchins et al., 2001]. Autosomal recessive inheritance of SRS in several families with more than one affected child was suggested on the basis of parental consanguinity, affected sibling of both sexes and normal appearing parents [Callaghan,1970; Robichaux et al., 1981; Teebi, 1992; Gouda et al., 1996; Ounap et al., 2004]. Autosomal and X-linked dominant inheritance patterns have also been suggested in several SRS cases [Partington, 1986; Al-Fifi et al., 1996] . Maternal uniparental disomy (mUPD) of chromosome 7 accounts for 10% of SRS cases, and up to 50% of cases have methylation defects in imprinted regions on chromosome 11p15 [Abu-Amero et al., 2008]. The remaining 40% of SRS cases have an unknown genetic etiology [Abu-Amero et al., 2008]. 3.1.2. The purpose of the study Since the phenotypes of 3-M syndrome and SRS are overlapping, the conclusive diagnosis of either syndrome can be challenging. However, lack of radiological changes in SRS has usually been used to differentiate the two syndromes [van der Wal et al., 2001]. In this study, four consanguineous families from the UAE and Jordan were recruited. Three out of the four families were originally diagnosed with autosomal recessive SRS while the fourth family was showing clear radiological findings and therefore was diagnosed as a 3-M case with an unknown molecular cause. In order to further investigate these disorders in these families, molecular genetic 65 investigations were undertaken using homozygosity mapping strategy (based on parents’ consanguinity) and candidate gene sequencing approach. 3.1.3. Results 3.1.2.1. Clinical assessment of probands and families 3.1.2.1.1. Family A The parents are first cousins Emirati nationals of Omani origin (Figure 3.1-1a). The father was short with short fingers (no exact measurements were available) and the mother was of normal height. Six children out of 12 were affected but only three affected children were available for clinical evaluation. Three out of the six affected individuals are married and have apparently normal children. Patient 1 (III-10) was the product of a pregnancy complicated by oligohydramnios. He was noted to be small and short with short limbs but no birth measurements were available. He was evaluated in the Genetics Clinic at 3 months of age. His weight was 3,000 g (-2 SD), length 48 cm (-6 SD), and head circumference was 43.5 cm (+2 SD). He had feeding problems and was failing to thrive. His head was disproportionately big for his body, but his brain CT scan was normal. He had several dysmorphic features including prominent forehead, a triangular face, large eyes, a depressed nasal bridge, a short neck, and a pectus excavatum. His thorax was short. There was bilateral clinodactyly. His skeletal survey and chromosome analysis were normal. He was evaluated again at 5 years of age. His weight was 14 kg (-2 SD), height 86 cm (-5 SD), and head circumference 54 cm (+2 SD). He had 66 normal developmental milestones and had normal intelligence (Table 3.1-1). His mother complained of excessive sweating. Echocardiography and repeat skeletal survey were normal apart from delayed bone age (Figure 3.1-1c). He was evaluated by endocrinologist and his growth hormone analysis was normal. Patient 2 (III-11) is the brother of Patient 1. He was the product of a pregnancy complicated by oligohydramnios, with a normal delivery. His birth weight was 2,000 g ( - SD), length 41 cm (-5 SD), and head circumference 34 cm (<50th centile). He had feeding problems and failure to thrive with dysmorphic features similar to patient 1 (Table 3.1-1). Patient 3 (III-12) is the sister of Patient 1 and was the product of a pregnancy complicated by oligohydramnios, with a normal delivery. Her birth weight was 2,000 g (- SD), length 43 cm ( -4 SD), and head circumference 35 cm (50th centile). Dysmorphic features were similar to those of her brothers (Table 3.1-1, Figure 3.1-1c). Given the described phenotype above, the affected members of this family were initially diagnosed as autosomal recessive SRS cases. 3.1.2.1.2. Family B The parents are first cousins Emirati nationals of Omani origin (Figure 3.1-2a). The mother was short (149 cm) and the father was apparently of average height. Patient 1 (III-3), a female, was the product of a pregnancy complicated by oligohydramnios with a normal delivery at 36 weeks gestation. Intrauterine 67 Figure 3.1-1. Clinical features of some affected members from family A. a) Pedigree of family A showing consanguinity. b) X-ray of the lower limb of patient 1 (5 years; III-10) from family A showing normal bones. c) Patient 3 (3 years; III-12) from family A showing triangular face with large head. a I II 2 1 II 1 2 3 4 5 6 7 b c 68 8 9 10 11 12 Table 3.1-1. Clinical and radiological features of the studied patients compared to SRS and 3-M syndrome. Families Patients Family A III10 III11 Family B Family C Family D SRS 3M III12 III3 III4 III5 IV6 IV7 IV9 IV4 IV9 IV3 IV10 + + + + + + - - + ? ? ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + +/- + + + + + + + + + + + + + + - - - - - - - - - - - - - + - - - - - - - - - - + - General Clinical Features Intrauterine growth + restriction (IUGR) Postnatal + short stature Normal + intelligence Feeding + problems Excessive + sweating Cafe-au-laite spots Craniofacial Features Relative + macrocephaly High & prominent + forehead Triangular + face Long face Depressed + nasal bridge Thin upper lip + Posteriorly + rotated ears Short neck + Skeletal Features Deformed + sternum Clinodactyly + Body/facial asymmetry Prominent heels Radiological Features Slender tubular bones Tall vertebral bodies - + + + - - + + + ? + + + + + + + + + + + + + + + + + + + + + + + + + + + + - - + + + - - - - - - - - - + + - - + + + + + + + + + + + + + + + + + + + + - - - - - - - + - + + + + + - - - - - - - + - + + + + + - - - - + + + + - + + + - - + - - - - - - -/+ + + + + + + + + + + + + + + -/+ - - - - - - - - - - - - + - - - - - - - - - - - - - - + - - - - - - + - - + NA NA - + - - - - - - - - - + NA NA -/+ + NA=Not available 69 Figure 3.1-2. Clinical features of some affected members from family B. a) Pedigree of family B showing consanguinity b) and c) Photographs of head and hand of patient 1 (2 years; III-3) from family B showing triangular face, frontal bossing and clinodactyly.d) Lateral X-ray of the vertebrae of patient 1 (2 years; III-3) showing normal vertebrae. a I II 2 1 III 1 2 3 4 d b c 70 5 growth restriction (IUGR) was diagnosed prenatally. Her birth weight was 1,570 g (-2 SD), length 42 cm (-4 SD), and head circumference 34 cm ( +1 SD). At birth she was noted to have short limbs, a narrow and short thorax with a distended abdomen with a palpable liver and spleen. She has a triangular face with a prominent forehead, large eyes, a depressed nasal bridge, a thin upper lip, a short neck, and a bilateral clinodactyly (Figure 3.12b, 2c; Table 3.1-1). Her skeletal survey (Figure 3.1-2d), abdominal ultrasound, and chromosome analysis were normal. She had feeding difficulties and failure to thrive and was tube fed during infancy. Bone age at the age of 1.4 years was delayed (at the wrist it was 3 months and at the hands it was 1 year). At the age of 2 years her weight was 6.7 kg (-3 SD), and height was 70.2 cm ( -5 SD). Otherwise, she had normal developmental milestones. Endocrine evaluation revealed normal results including growth hormone. However, she was started on growth hormone therapy at the age of 3 years but the response was poor and therefore it was stopped. Patients 2 (III-4) and 3 (III-5), are brothers of Patient 1. Both were the products of pregnancies complicated by oligohydramnios and normal deliveries. Both had IUGR and dysmorphic features with feeding difficulties similar to their sister (Table 3.1-1). The diagnoses of the affected members of this family were initially assigned as autosomal recessive SRS. 71 3.1.2.1.3. Family C A Jordanian family with three affected children and consanguineous first-cousin parents was evaluated clinically in Jordan and UAE (Figure 3.13a). Patient 1 (IV-6; Table 3.1-1), a girl of 11 years, the eldest of four siblings was the product of a full-term caesarean for breech presentation. Her birth weight was 2,400 g (5th centile), birth length was 47 cm (-2 SD), and head circumference was 37 cm (+2 SD). The main problem of the family is the symmetrical short stature of patient 1 and her two brothers (Patients 2, IV-7 and 3, IV-9; Table 3.1-1). At 11 years, her weight was 28 kg (>10 th centile), height was 123 cm (-3 SD), and head circumference was 59 cm (+2 SD). She also had a similarly affected cousin (Patient 4, IV-4; Table 3.1-1), the son of her paternal aunt (Figure 3.1-3a), whose parents are also consanguineous. Her dysmorphic features are summarized in Table 3.1-1. In addition, she had partial syndactyly of the 2nd and 3rd toes, and hyper-extensible joints (Table 3.1-1). Her skeletal survey at the age of 12 years showed delayed bone age. Her abdominal ultrasound showed mild hepatosplenomegaly. Her brain MRI showed mild bifrontal brain atrophy and no abnormalities were detected on her pituitary MRI. Her karyotype was normal. Both affected brothers had similar features (Table 3.1-1). The younger brother had in addition, micrognathia and bluish sclerae. His voice was also unusual, but similar to his mother's. Skeletal surveys on both were reported normal (at 10 and 5 years, respectively) (Figure 3.1-3b). Endocrine investigation including growth hormone stimulation test, thyroid function tests, and 72 Figure 3.1-3. Clinical data of some affected members from family C. a) Pedigree of family C showing consanguinity. b) X-ray of the lower limbs of patient 3 (12 years; IV-9) showing normal bones. a I II III 1 2 3 4 5 IV 1 2 3 4 5 b 73 6 7 8 9 4 6-9 adrenocorticotropic hormone (ACTH) were normal. The diagnosis of the affected members of this family was initially assigned as autosomal recessive SRS. Following the identification of a mutation in OBSL1 in this family, the X-ray films were re-evaluated by the radiologist. It was concluded that the long bones on Patient 2 (IV-7) were slender. There were no vertebral abnormalities. 3.1.2.1.4. Family D The parents are Emirati second cousins of Baluchi origin (Figure 3.14a). They have a total of 11 children, 3 of them are affected. The parents and the other children are all of normal height. Patient 1 (IV-9) is a 12-year-old child who was the product of a normal pregnancy and delivery. No birth measurements were available. The parents noted short stature in the first year of her life. She was evaluated by different pediatricians and several investigations were done but no diagnosis was provided. She was re-evaluated at the age of 12 years. Her weight was 17 kg (-2 SD), height 115 cm (-5 SD), and head circumference 51.5 cm (50th centile). Her dysmorphic features are summarized in Table 3.1-1 and shown in Figure 3.1-4b. Her skeletal survey showed slender long bones and foreshortened vertebrae (Figure 3.1-4c; Table 3.1-1). Patients 2 (IV-3) and 3 (IV-10), are the 19-year-old sister and 7-year-old brother of Patient 1. Both were the products of normal pregnancies and delivery. Both have proportionate short statures and similar facial appearances to Patient 1. No radiological examinations were carried out (Table 3.1-1). 74 Figure 3.1-4. Clinical features of some affected members from family D. a) Pedigree of family D showing consanguinity. b) Patient 1 (IV9) and her brother (IV-10) from family D. note triangular face and frontal bossing. c) Lateral x-ray of the vertebrae of patient 1 (IV-9) family D at 12 years of age showing foreshortened vertebrae. a I II III 1 2 IV 1 2 3 4 5 6 7 8 c b 75 9 10 11 3.1.2.2.1. Family A Homozygosity mapping of family A, with the Affymetrix Gene-Chip Human Mapping 250K array, in affected (III-10, III-11, III-12), healthy children (III-4, III-6) and their mother (II-2) identified a unique homozygous region on chromosome 2q33.3-q36.3. Further polymorphic microsatellite marker analyses reduced the critical region to an 11 Mb (211-222 Mb) interval encompassing around 108 genes in the UCSC Genome Browser database (Figure 3.1-5; Table 3.1-2). A list of candidate genes in the region of homozygosity was generated using the GeneDistiller software. This showed that 84 genes of the 108 total were producing proteins, including OBSL1, which had recently been identified as a second cause for 3-M syndrome cases. Combining the similarities in the phenotype of 3-M and SRS with recent molecular findings and linkage results, led to sequence OBSL1 gene being identified as the sole target. A homozygous non-synonymous mutation was found in exon 2 of OBSL1 in all the screened affected members of family A, c.1119G>C (p.W373C) (Figure 3.1-6A). Screening the whole family for the detected mutation revealed the carrier status of the healthy members as well as the mother in which the same mutation was heterozygous. Screening for more than 100 normal controls showed no change in that specific location among the population. Most of the mutations reported so far in the OBSL1 gene were truncating mutations located within the first 8 exons of the gene affecting all known isoforms, resulting in 76 complete loss of OBSL1 function 77 2 Figure 3.1-5. Homozygosity mapping results in family A. A single homozygous region was identified on chromosome 2 for the disease in the family. The mapped region found between rs12474633 and rs1834129 and contains 108 genes including the OBSL1. 78 218,454,000 218,494,000 AC010886AC20 AC009469TC16 219,273,000 219,294,000 222,528,800 222,628,850 222,739,400 AC012510TG20TC27 AC012510AC20** AC009231GT18** AC009302TG20 AC009302GT21 170 218 163 167 215 171 456 155 214 159 169 215 171 426 184 294 208 229 155 214 159 169 215 171 426 184 294 - 229 289 264 155 214 159 169 215 171 426 184 294 - 229 289 264 III-4 (unaffected) 207 207 *The markers were designed by Dr. Geoff Wood’s lab. **Markers flanking OBSL1 gene (chr2: 220426438-220436268) 219,153,000 AC073838AC28 AC021016TC27AT 196 AC021016TC15 219,047,000 302 AC097483AGAA 204 229 289 264 218,427,000 287 264 218,184,719 GATA4G12 AC010886CA21 location 211,245,000 * AC008172AC15 MARKER II-2 (mother) 201 207 170 218 163 167 215 171 456 196 302 204 229 287 264 155 214 159 169 215 171 426 184 294 208 229 289 264 III-6 (unaffected) 201 207 170 218 163 167 215 171 456 196 302 204 229 287 264 170 218 163 167 215 171 456 196 302 204 229 287 264 III-10 (affected) 201 201 170 218 163 167 215 171 456 196 302 204 229 287 264 170 218 163 167 215 171 456 196 302 204 229 287 264 III-11 (affected) - Table 3.1-2. STR genotyping results for the homozygous region on chromosome 2 in family A. 170 218 163 167 215 171 456 196 302 204 229 287 264 170 218 163 167 215 171 456 196 302 204 229 287 264 III-12 (affected) 201 201 Figure 3.1-6. The p.W373C mutation found in family A. A) DNA sequencing revealed novel homozygous missense mutation (c.1119G>C) in OBSL1 in all the affected children. The mutation was found to be heterozygous in the carriers and absent in normal controls. B) Schematic diagram of OBSL1 protein showing the location of the two missense mutations detected so far. W373C is located in the immunoglobulin-like 3 (IgL 3) domain, while F697G found by Huber et al [2010] resided between fibronectin III (FN3) and IgL5 domains. A) Sequencing chromatogram of a normal individual carries the wild type sequence of OBSL1 Sequencing chromatogram of a carrier individual carries a heterozygous c.1119G>C Sequencing chromatogram of an affected individual carries a homozygous c.1119G>C B) 79 [Hanson et al., 2009]. The loss of OBSL1 leads to downregulation of CUL7 and results in a primordial growth disorder 3-M syndrome. One other missense mutation (p.F697G) was detected by Huber et al [2010] in OBSL1 gene in one Algerian family with 3-M syndrome. The reported missense mutation and the one described here are located in the first exons of the OBSL1 gene and therefore affecting all the validated isoforms (NM_015311.2, NM_001173431.1, and NM_001173408.1). OBSL1 protein is composed of 14 immunglobulin like (IgL) folds and one fibronectin III (FN3) domain (Figure 3.1-6B). The p.W373C resided in the IgL4 while the p.F697G is located upstream of IgL5 (Figure 3.1-6B). Bioinformatics tools such as PolyPhen-2 and MutationTaster showed that the substitution of the 373 tryptophan (W) by a Cysteine (C) has a destructive effect on the Obscurin-like protein 1. Substitutions with C were demonstrated to be pathological in the fibroblast growth factor receptors because they produce intermolecular disulfides which alter their function [Wilkie et al., 1995]. Furthermore, multiple alignments between species confirmed the importance of the substituted amino acid as a reflection to its persistent existence in all of them (Table 3.1-3). 3.1.2.2.2. Family B SNP genome wide genotyping using the Affymetrix 250K array detected 2 regions of homozygosity in family B (Figure 3.1-7, Table 3.1-3). The first region was the same one on chromosome 2 found in family A and contained the OBSL1 gene. The other region was unique for family B spanning 43.9 Mb of chromosome 6 bounded by SNPs rs1806719 and rs6454404. The 80 candidate regions were further inspected in all the family members by the use of STR markers (Table 3.1-4 and 3.1-5). The STR markers in the first region on chromosome 2 were not shared by all the affected (III-5) and one unaffected (III-1) was homozygous as well. However, the STR markers in the second region on chromosome 6 were homozygous in all the affected, but the mother (II-2) and one unaffected (III-1) were also homozygous. Hence, the genes in both regions were prioritized for sequencing. Within the second region, CUL7 was a candidate gene because of its known involvement in 3-M syndrome as well as OBSL1 in the first one. Both genes were sequenced in the whole family detecting a homozygous nonsense mutation in the CUL7 gene in all the affected members (lll3, lll4, lll5) and a heterozygous carrier status in both parents (ll1, ll2) (Figure 3.1-8). The nonsense mutation occurred in isoform 2 of CUL7 (c.203G>A; p.W68X) (NM_014780.4) but affected all the validated isoforms (NM_001168370.1, NM014780.4) and resulted in a truncated protein that most probably would not pass the nonsense-mediated decay (NMD) surveillance. 3.1.2.2.3. Family C Genotyping of the whole genome for the 12 members of family C (Figure 3.1-9) has linked their phenotype to the same region on chromosome 2 with the other 2 families (A, B) analyzed in the same study (Table 3.1-6). OBSL1 was targeted and sequenced revealing another novel mutation in its first exon c.681_682delinsTT (Figure 3.1-10). This indel resulted in 2 changes within the translated protein p.[H227H; Q228X], of which the first is a 81 82 Myotis brandtii] Xenopus tropicalis Anolis carolinensis Pan troglodytes Homosapiens Macaca mulatta Canis lupus Bos taurus Rattus norvegicus Mus musculus Gallus gallus Danio rerio Organism QDVEGREHGIAVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGTVRRL LKDIAVREGQDAVLECSVPDGSRTSWYLEDQRLHPDKRHHMEEQGPIRRL VDVEVLESQDAILECQVPVATIPTVWYLEDKRLHPSPKYLIEEQGLLRRL QDVEGREHGIAVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGTVRRL QDVEGREHGIAVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGTVRRL QDVEGREHGIAVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGTVRRL QDVEGREHGIAVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGTVRRL QDVEGREHGIAVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGTVRRL QDVEGREHGIVVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGTVRRL QDVEGREHGIVVLECKVPNSRIPTAWFREDQRLLPCRKYEQIEEGAVRRL EDVEVAEREDAVLECQVPLGSIPTAWFLEDRELQPSHKYVMEECGVVRRL QDSEFRERDVAVLECEVPEESISTAWYLEDQRLQHGNKYNMEQKGTWRRL OBSL1 protein sequence Table 3.1-3. Conservation of the 373-tryptophan (W) in the OBSL1 protein across species. 83 Figure 3.1-7. Homozygosity mapping results in family B. Wide Genome genotyping of family B identified a region of homozygosity on chromosome 6 between rs1806719 and rs6454404 containing the CUL7 gene. 84 219,153,000 219,273,000 219,294,000 222,528,800 222,628,850 222,739,400 AC073838AC28 AC012510TG20TC27 AC012510AC20** AC009231GT18** AC009302TG20 AC009302GT21 160 226 159 167 217 171 426 184 283 208 229 287 268 197 168 228 155 167 215 169 426 188 279 216 237 287 268 201 II1 (Father) 160 226 159 167 217 171 426 184 283 216 229 291 268 201 170 214 165 165 219 152 456 196 287 202 229 291 260 207 II2 (Mother) *The markers were designed by Dr. Geoff Wood’s lab. ** Markers flanking OBSL1 gene (chr2: 220426438-220436268) - 218,494,000 AC009469TC16 AC021016TC27AT 218,454,000 AC010886AC20 - 218,427,000 AC010886CA21 219,047,000 218,184,719 GATA4G12 AC021016TC15 211,245,000 AC008172AC15 AC097483AGAA Coordinates MARKER* 160 226 159 167 217 171 426 184 283 208 229 287 268 197 160 226 159 167 217 171 426 184 283 216 229 291 268 201 III1 (unaffected) CHROMOSOME 2 160 226 159 167 217 171 426 184 283 208 229 287 268 197 170 214 165 165 219 152 426 196 287 202 229 291 260 207 III2 (unaffected) 160 226 - 167 217 171 426 184 283 208 229 287 268 197 160 226 - 167 217 171 426 184 283 216 229 291 268 201 III3 (affected) 160 226 159 167 217 171 426 184 283 208 229 287 268 201 160 226 159 167 217 171 426 184 283 216 229 291 268 201 III4 (affected) 168 228 155 167 215 169 426 188 279 216 237 287 268 201 160 226 159 167 217 171 426 184 283 216 229 291 268 201 III5 (unaffected) Table 3.1-4. STR genotyping results for the homozygous region on chromosome 2 in family B. 85 43,192,350 43,346,000 54,000,150 70,589,500 84,463,800 87,032,500 AL355385TG24** AL133375GT22 AL139389AC23 AL359539TA24 AL109915CA21 AL390125TC19 222 214 170 208? 247 231 224 216 184 209 251 231 II1 (Father) 222 216 184 209 251 231 228 216 184 209 251 231 II2 (Mother) *The markers were designed by Dr. Geoff Wood’s lab. ** The nearest marker to CUL7 gene (chr6: 43005355-43021683) Coordinates MARKER* 222 214 170 208? 247 231 224 216 184 209 251 231 III1 (unaffected) CHROMOSOME 6 222 216 184 209 251 231 222 216 184 209 251 231 III2 (unaffected) 222 216 184 209 251 231 222 216 184 209 251 231 III3 (affected) 222 216 184 209 251 231 228 216 184 209 251 231 III4 (affected) 222 216 184 209 251 231 222 216 184 209 251 231 III5 (affected) Table 3.1-5. STR genotyping results for the homozygous region on chromosome 6 in family B. Figure 3.1-8. Mutation analysis results in family B. A nonsense mutation c.203G>A was detected in CUL7 gene in all the affected children of family B. The same mutation was found to be heterozygous in parents and some unaffected children. Sequencing chromatogram of a normal individual carries the wild type sequence of CUL7 Sequencing chromatogram of a Carrier individual carries a heterozygous c.203G>A Sequencing chromatogram of an affected individual carries a homozygous c.203G>A 86 87 Figure 3.1-9. Homozygousity mapping results in family C. A homozygous region was identified in family C on chromosome 2 between rs12474633 and rs1834129 containing the OBSL1 gene. 88 222,628,850 222,739,400 AC009302TG20 AC009302GT21 160 226 161 - 217 171 426 184 287 208 229 160 226 161 - 215 173 426 190 306 210 227 287 268 207 *The markers were designed by Dr. Geoff Wood’s lab. ** Markers flanking OBSL1 gene (chr2: 220426438-220436268) 222,528,800 AC009231GT18** - AC021016TC27AT 219,294,000 219,047,000 AC021016TC15 219,273,000 - AC097483AGAA AC012510AC20** 218,494,000 AC009469TC16 AC012510TG20TC27 218,454,000 AC010886AC20 219,153,000 218,427,000 AC073838AC28 268 218,184,719 GATA4G12 AC010886CA21 287 207 211,245,000 AC008172AC15 III3 (father) Coordinates MARKER* 160 226 161 161 217 171 426 184 287 208 229 287 268 201 170 214 161 173 227 171 426 184 279 212 229 287 264 207 III4 (mother) 160 226 161 161 217 171 426 184 287 208 229 287 268 201 160 226 161 165 217 171 426 184 287 208 229 287 268 207 IV6 (affected) 160 226 161 165 217 171 426 184 287 208 229 287 268 - 160 226 161 161 217 171 426 184 287 208 229 287 268 - IV7 (affected) 160 226 161 161 217 171 426 184 287 208 229 287 268 207 160 226 161 167 215 173 426 190 306 210 227 287 268 207 IV8 (unaffected) 160 226 161 161 217 171 426 184 - 208 229 287 268 207 160 226 161 165 217 171 426 184 - 208 229 287 268 207 IV9 (affected) 160 214 161 165 217 171 426 184 279 208 229 287 264 201 170 226 161 173 227 171 426 184 287 212 229 287 268 207 III1 (father) 170 226 159 165 215 171 426 184 287 208 227 287 268 207 170 226 161 167 217 173 426 190 306 210 229 287 268 207 III2 (mother) 170 214 161 165 217 171 426 184 279 208 229 287 264 201 170 226 161 173 227 171 426 184 287 212 229 287 268 207 IV2 (unaffected) 160 226 159 165 215 171 426 184 287 208 227 287 - 207 170 226 161 167 217 173 426 190 306 210 229 287 - 207 IV3 (unaffected) Table 3.1-6. STR genotyping results for the homozygous region on chromosome 2 in family C. 160 226 161 165 217 171 426 184 287 208 229 287 268 207 170 226 161 165 217 171 426 184 287 208 229 287 268 207 IV4 (affected) 170 214 159 165 217 171 426 184 279 208 229 287 264 201 170 226 161 173 227 171 426 184 287 212 229 287 268 207 IV5 (unaffected) Figure 3.1-10. Mutation analysis results in family C. Three nucleotides were found to be mutated in OBSL1 gene in family C c.[681_682delinsTT; 690delC]. The first one is silent (p. H227H), the second one is nonsense (p.Q228X) mutation while the third is a frame shift deletion. The same mutations were found to be heterozygous in parents and some unaffected children. The mutations were not detected in normal affected sibs and in 100 normal healthy controls. Sequencing chromatogram of a normal individual carries the wild type sequence of OBSL1 Sequencing chromatogram of a Carrier individual carries a heterozygous form of the detected mutations Sequencing chromatogram of an affected individual carries a homozygous c.[681_682delinsTT;690del C] 89 silent one while the other is a nonsense mutation that is predicted to cause premature and truncation of OBSL1 in those patients. The mutation affects all the validated isoforms (NM_015311.2, NM_001173431.1, NM_001173408.1) and thus is expected to be abolished by the NMD mechanism. In addition, a non-reported SNP was identified shortly after the nonsense mutation c.690delC which causes a frame shift and premature termination of the transcript (Figure 3.1-10).The three changes were found in all affected members (IV-4, IV-6, IV-7, IV-9) in a homozygous form and in all the unaffected (IV-1, IV-2, IV-3, IV-5, IV-8) as well as both parent pair (III-1, III-2, III-3, III-4) in a heterozygous form. 3.1.2.2.4. Family D Direct DNA Sequencing of CUL7 and OBSL1 genes in family D led to the identification of a homozygous deletion in exon 3 of CUL7. The deletion of 6 bases occurred in isoform 2 of CUL7 (c.649_654delAGCCGC) (NM_001173431.1), but affected all the validated isoforms (NM_015311.2, NM_001173431.1, NM_001173408.1) (Figure 3.1-11). This mutation resulted in an in-frame deletion of two highly conserved serine (S) and arginine (R) between species, p.217_218delSR in isoform 2 of CUL7 protein NP_055595.2 (Table 3.1-7). The mutational screening of the deletion that was performed on the genomic DNA from all the affected members (IV-3, IV-9, IV10) and one unaffected (IV-2) sib revealed the presence of the homozygous deletion in all the affected but not in the unaffected who has a homozygous normal wild type sequence of CUL7. Both parents (III-1, III-2) were found to 90 Figure 3.1-11. Mutation analysis results in family D. Sequencing of CUL7 gene in family D revealed a novel deletion of 6 nucleotides in exon 3 c.649_654delAGCCGC. All the affected individuals have the homozygous deletion but the parents were found to have the heterozygous form only of the deletion. Around 100 normal controls were screened for the same deletion but it was not found. Sequencing chromatogram of a normal individual carries the wild type sequence of CUL7 Sequencing chromatogram of a carrier individual carries a heterozygous form of the detected mutation Sequencing chromatogram of an affected individual carries a homozygous c.649_6654delAGCCGC 91 92 Monodelphis domestica (CUL9) Brachydanio rerio Takifugu rubripes SRSTLLELFAETTSSEEHCMAFEGIHLPQIPGKLLFSLVKRYLCVTSLLD NRYTLLELFAETTSSEEHGISFEGIHLPQIPGKLLFSLVGVFKETGALDL NRFTLLELFAETTSSEEHGISFEGIHLPQIPGKLLFSLVKRYLCVTSLMD SRCALLALFAQATLSEHPMSFEGIQLPQVPGRLLFSLVKRYLHVTFLLDQ Bos taurus SRCALLALFAQATLSEHPMSFEGIQLPQVPGRVLFSLVKRYLHVTSLLDQ Homo sapiens SRCALLALFAQATLSEHPMSFEGIQLPQVPGRLLFSLVKRYLHVTSLLDQ SRCALLALFAQATLSEHPMSFEGIQLPQVPGRVLFSLVKRYLHVTSLLDQ Macaca mulatta Canis lupus SRCALLALFAQATLTEHPMSFEGVQLPQVPGRLLFSLVKRYLCVTFLLDR Rattus norvegicus SRCALLALFAQATLSEHPMSFEGIQLPQVPGRVLFSLVKRYLHVTSLLDQ SRCALLALFAQATLTEHPMSFEGVQLPQVPGRLLFSLVKRYLHVTFLLDR Mus musculus Pan troglodytes CUL7 protein sequence Organism Table 3.1-7. Conservation of the 217-serine (S) and 218-arginine (R) in CUL7 protein across species. be carriers for the mutation as expected in autosomal recessive inheritance. Furthermore, the deletion was not identified in a panel of 100 ethnically matched control samples, suggesting that the sequence variant is not a common polymorphism. 3.1.4. Discussion In this study four consanguineous families with a total of 13 children affected by a growth retardation syndrome were investigated and found to have mutations in two of the three genes known to cause 3-M syndrome (Summary in Table 3.1-8). Three of these families from different origins were initially diagnosed by different clinical geneticists as autosomal recessive SRS (Families A, B, and C). As a result of the findings in this study, the diagnoses of the studied patients were re-evaluated and reassigned as autosomal recessive 3-M syndrome. 3.1.4.1. The differential diagnosis of Three-M syndrome The 3-M syndrome is a form of autosomal recessive low birth weight with dwarfism [Fuhrmann et al., 1972; Miller et al., 1975]. It is characterized by pre- and postnatal growth retardation associated with facial dysmorphic features and normal intelligence [Hennekam et al., 1987]. The diagnosis is usually based on this pattern of abnormalities and the associated radiological changes [van der Wal et al., 2001]. These include slender long bones with narrow diaphysis and foreshortened vertebrae. The main features of this syndrome are nonspecific if taken separately but the combination of growth retardation with the facial features and the radiological changes distinguish 93 94 deletion in exon 3 of CUL7 (NM_014780.4)* c.649_654delAGCCGC p.217_218delSR This indel resulted in 2 changes within the translated protein p.[H227H; Q228X] , of which the first is a silent one while the second is a nonsense mutation that is predicted to cause prematuration and truncation of OBSL1 in those patients. The nonsense mutation affects all the validated isoforms (NM_015311.2, NM_001173431.1, NM_001173408.1) and thus expected to be abolished by the NMD mechanism Multiple alignments between species confirmed that this mutation resulted in an in-frame deletion of two highly conserved serine and arginine between species "Chromosomal positions are based on UCSC Human Mar. 2006 (NCBI36/hg18) assembly. *This isoform was used for the mutation’s nomenclature. NA D nonsense mutation was found in CUL7 (NM_014780.4)* c.203G>A p.W68X Chr2: 211,512,854222,712,896 Chr6: 41,833,86685,740,223 Chr2: 211,512,854222,712,896 Non-synonymous mutation was found in exon 2 of OBSL1. c.1119G>C p.W373C Chr2: 211,512,854222,712,896 1. indel in the first exon of OBSL1 c.681_682delinsTT p.[H227H; Q228X] 2. A non-reported SNP was identified immediately after the nonsense mutation c.690delC which cause frame shift and premature termination of the transcript The mutation resides in the first 8 exons of the OBSL1 gene and therefore affecting all the validated isoforms (NM_015311.2, NM_001173431.1, and NM_001173408.1). Furthermore, bioinformatics tools showed that the p.W373C mutation has a destructive effect on OBSL 1 protein and multiple alignments between species confirmed the importance of the substituted amino acid The mutation affected all the validated isoforms (NM_001168370.1, NM_014780.4) and resulted in a truncated protein that most probably wouldn’t pass the nonsensemediated decay (NMD) surveillance. Likelihood of Pathogenicity Mutations Found Autozygosity Region/s" C B A Family Table 3.1-8. Summary of genotyping and mutational screening results for all the recruited families. this syndrome from other growth retardation syndromes [Feldmann et al., 1989; Clayton et al., 2012]. More than 94 families with genetically confirmed 3-M syndrome have been reported in the literature [Hanson et al., 2011b]. The majority of 3-M cases were found to carry mutations in CUL7 gene [Hanson et al., 2011b]. Two other disorders with similar features to 3-M were also found to be caused by mutations in CUL7 gene raising questions about their identity. The first one is the sad-appearing face syndrome which was described by Le Merrer et al [1991]. Children with this syndrome have primordial dwarfism and a round, sad-appearing rather than triangular face. In addition, radiological examination of the skeleton was normal in these children. The second disorder is Yakut short stature syndrome which was identified by Maksimova et al [2007] in a new population isolate in Asia. Affected children had pre- and postnatal growth retardation, a triangular face with hypoplastic midface, frontal bossing, a depressed nasal bridge, a short wide thorax, and short limbs with brachydactyly. Only a few patients showed the radiological features of slender bones and tall vertebral bodies. A founder mutation (p.Q1553X) in the CUL7 gene was identified in these children. The diagnosis of SRS in three out of four of the families in this study was based on the presence of pre- and postnatal severe growth retardation together with typical dysmorphic facial features, clinodactyly, and feeding difficulties [Price et al., 1999]. However, since SRS is a phenotypically heterogeneous condition and may be misdiagnosed [Hanson et al., 2011b; Clayton et al., 2012], the scoring system that was devised by Bartholdi et al 95 [2009] was used for the diagnosis of SRS in this study. All the children scored between 10–12 points suggesting that this diagnosis is likely. In addition, they all lacked the radiological changes seen in 3-M syndrome. Although SRS is usually sporadic and is listed in OMIM as autosomal dominant, there are several reports in the literature of families with apparent autosomal recessive inheritance. Teebi [1992] reported a consanguineous Arab family with six children (5 males and 1 female) with features suggestive of SRS and summarized the data of other similar families in the literature [Silver and Gruskay, 1957; Gray and Evans, 1959; Callaghan, 1970; Robichaux et al., 1981; Saal et al., 1985; Partington, 1986]. Another family with two sisters having features suggestive of autosomal recessive SRS was described by Ounap et al [2004]. However, the siblings in this family were found to have hypomethylation of H19 and IGF2 genes on chromosome 11p15. One other family with two affected children was found to have the same epigenetic mutation in 11p15 [Bartholdi et al., 2009]. Methylation analysis of the father revealed a normal result, most likely reflecting germ cell mosaicism in the father [Bartholdi et al., 2009]. It is possible that the apparent recessive inheritance in many SRS families is explained on this basis. It is also possible that some of those families have 3-M rather than SRS and therefore the identity of the autosomal recessive SRS is questionable. Since the radiological changes in 3-M are not a constant finding and are age dependent, the diagnosis of 3-M should be considered in children with primordial dwarfism with dysmorphic facial features suggestive of either SRS 96 or 3-M, with or without the X-ray changes seen in 3-M. In addition, the value of the radiological findings described in the 3-M syndrome is doubtful. The slenderness of the long bones is not specific to this syndrome as it is seen also in other forms of primary dwarfism [Le Merrer et al., 1991]. Also, vertebral findings of foreshortening of the vertebral body is only recognized in older children and adults [van der Wal et al., 2001] and therefore do not allow early recognition. 3.1.4.2. The Three-M syndrome pathogenesis To date, three cytoplasmic proteins have been implicated in the pathogenesis of the 3-M syndrome. Approximately 67% of mutations described in 3-M syndrome patients are found in CUL7, with 28% identified in OBSL1 and 5% identified in CCDC8 [Hanson et al., 2011b; Clayton et al., 2012]. Most the mutations reported so far in all the three proteins were shown to be loss of function mutations [Hanson et al., 2011b]. CUL7 mutations were found throughout the whole gene, with the majority of the reported mutations creating PTC and are therefore likely to lead to a complete loss of the CUL7 protein like the mutation I have identified in family B (Table 3.1-8) [Huber et al., 2009]. Splicing and non-synonymous mutations were also identified in CUL7 and predicted to lead to the generation of a nonfunctional protein product like the mutation I have identified in family D (Table 3.1-8) [Huber et al., 2009]. Most of the mutations identified in OBSL1 so far including the two mutations identified in this study were found to cluster at the N-terminal region of the protein, thus affecting all the known isoforms for this protein [Hanson et 97 al., 2009; Huber et al., 2010]. The majority of the mutations were presumptive null mutations creating PTC similar to the one found in family C (Table 3.1-8) [Hanson et al., 2011b]. Non-synonymous mutations in OBSL1 were also detected in 3-M patients resembling the one detected in family A (Table 3.1-8) [Hanson et al., 2011b]. CULLIN7 or CUL7 is an essential protein of the ubiquitin-proteasome pathway acting as a structural protein in the formation of a SKP1-CUL7FBXW8-ROC1 (SCF) E3 ubiquitin ligase multi-protein complex [Hanson et al., 2011b]. The ubiquitin-proteasome pathway is a major protein degradation pathway in which proteins are targeted for degradation. This process involves attachment of a small regulatory protein called ubiquitin to a specific target protein by the E3 ubiquitin ligase complex. This binding most commonly is for marking the target protein for degradation by the proteasome, but sometimes it may alter the cellular location or the activity of the target protein [for review see Willems et al., 2004]. To date, only two proteolytic targets of the CUL7 SCF complex have been identified namely cyclin D1 and insulin receptor substrate 1 (IRS-1) [Okabe et al., 2006; Xu et al., 2008]. Cyclin D1 is a wellknown regulator of cell cycle progression but the importance of its degradation mediated by the CUL7 SCF complex is still undetermined [Alao, 2007; Lin et al., 2008]. On the other hand, IRS-1 is an adaptor protein that acts in both the insulin and insulin-like growth factor 1 (IGF-I) signaling pathways stimulating cell growth and proliferation as well as inhibiting programmed cell death [Dearth et al., 2007]. Targeted disruption of Cul7 in 98 mice resulted in a reduction in cell proliferation and induction of senescence [Arai et al., 2003]. Those models exhibited an IUGR phenotype resembling the 3-M patients apparently due to the disruption of the CUL7 SCF complex. In addition, several knockout studies have shown that the disruption of the IGF-I pathway results in severe growth restriction in mouse models and induces cellular senescence [Xu et al., 2008]. Therefore, it was postulated that the primary mechanism responsible for the growth restriction seen in 3-M patients is due to the alteration of the IGF-1 pathway caused by IRS-1 unsuccessful ubiquitination and degradation. It has been shown through co-immunoprecipitation assays that CUL7 and OBSL1 physically interact [Hason et al., 2009]. In addition, silencing the expression of OBSL1 in mammalian cell lines led to a decrease in CUL7 expression [Hason et al., 2009]. From these findings it was concluded that these two proteins act in the same pathway to control cell proliferation and human growth. However, it is currently unclear as to how OBSL1 interacts with the IGF-I pathway. Hanson et al [2011b] suggested that OBSL1 as a cytoskeletal adaptor protein may function as a structural protein linking CUL7 to IRS-1. On the other hand, the function of CCDC8 is still unknown. Recently, it has been shown to interact with p53 and acts as a key co-factor in p53-mediated apoptosis [Dai et al., 2011]. Another study has demonstrated that CCDC8 interacts with OBSL1 but not with CUL7. Taken all together, it was concluded that that all three proteins exist in a common pathway [Hanson et al., 2011a]. 99 Several families with 3-M syndrome are not linked yet to any of the three genes indicating further genetic heterogeneity [Huber et al., 2009]. Therefore, investigating more 3-M or 3-M like cases will most likely lead to the identification of more genes causing 3-M syndrome revealing other members in the 3-M pathway and expanding the knowledge of how this pathway controls cell growth. 3.1.5. Conclusion IUGR is a nonspecific finding that can also be a significant feature of many recognized genetic syndromes including SRS, 3-M syndrome, Dubowitz syndrome, and Mulibrey nanism. Differentiation of 3-M syndrome from autosomal recessive SRS has been difficult because of the phenotypic variability of the latter. Limb length asymmetry is seen in over half of those with autosomal recessive SRS, but not in individuals with 3-M syndrome. Characteristic radiologic findings of 3-M syndrome are not present in SRS. In this study, I have identified the genes and the mutations underlyingseveral unrelated IUGR patients who were initially diagnosed as autosomal recessive SRS cases. Interestingly, the identified genes were known to cause 3-M syndrome. Therefore, the results of the molecular investigation in this study allowed accurate diagnosis of the exhibited phenotype in four families with multiple affected members and the subsequent genetic counseling. Identification of these homozygous mutations in CUL7 and OBSL1 genes (inherited in an autosomal recessive manner) has facilitated the possibility of future prenatal diagnosis for these families. In 100 addition, this study has raised a question regarding the identity of the autosomal recessive SRS. Molecular investigation of more apparently autosomal recessive SRS cases will hopefully provide the answer. 101 SECTION 2: Unraveling the genetic defects and the pathophysiologic mechanism underlying fibrochondrogenesis in patients from the UAE 102 3.2.1. Background Fibrochondrogenesis (FBCG; OMIM#228520 and # 614524) is a severe short-limbed skeletal dysplasia which was first described by Lazzaroni-Fossati et al in 1978 as a rare autosomal recessive neonatal lethal rhizomelic chondrodysplasia [Lazzaroni-Fossati et al., 1978]. Since then, several cases have been described in the literature originating from different ethnic backgrounds which include Italian and Japanese [Eteson et al., 1984], Spanish [Martínez-Frías et al., 1996], German [Sauer et al., 2000], French [Randrianaivo et al., 2002], Turkish [Leeners et al., 2004], and Indian [Kulkarni et al., 2005; Kundaragi et al., 2012]. In the UAE, FBCG was reported in 2003 as a relatively common recessive type of skeletal dysplasia among the 38,048 births during a 5-year study period [Al-Gazali et al., 2003]. All reported cases show distinctive abnormalities such as a flat face with prominent protuberant eyes, a flat nose with anteverted nares, a small mouth, a cleft palate with severe micrognathia and low set ears associated with severe shortening of the limbs. Typical radiological features include short long bones with very broad and slightly irregular metaphyses, small, inferiorly broad iliac bones with the lower margin having a caudally directed bump-like configuration boarded by spurs, short ribs which are cupped anteriorly and pear-shaped vertebrae with sagittal clefting [Al-Gazali et al., 1997; Al-Gazali et al., 1999]. Histological examination of different long bone sections showed severe abnormalities of chondrocytes and cartilaginous matrix in growth 103 plates and epiphyseal cartilages. Spaces of cystic degeneration of the matrix cartilage were also observed [Kulkarni et al., 2005]. 3.2.2. The purpose of the study Since the genetic defects causing FBCG were not yet known, three families from UAE exhibiting autosomal recessive FBCG were recruited. The families were investigated using homozygosity mapping and candidate gene sequencing approaches to elucidate the defective gene(s) underlying this severe phenotype. 3.2.3. Results 3.2.3.1. Clinical phenotyping of UAE patients expanded the phenotype especially in survivors 3.2.3.1.1. Family A (FA) The parents in this family are distantly related Emirati nationals of Omani origin and the affected child was their first (Figure 3.2-1). The parents appear to display normal height, vision and hearing. The parents reported no history of deafness or vision abnormalities in the family. The affected child was the product of a normal pregnancy with a normal delivery by vacuum extraction. At birth, she was noted to have typical clinical and radiological features of FBCG (Table 3.2-1). At the time of examination, she was 3.5 years old and had global developmental delay, short limbs with contractures and pectus carinatum with dorsal kyphosis. She had severe bilateral sensory– neural hearing loss and severe myopia. She was operated on for a bilateral cleft palate and a bilateral cataract. Detailed eye examination did not show any vitreous changes. 104 Figure 3.2-1. Pedigree of family FA. The proband is the first child (II-1) of distantly related parent. Distant relatives I 2 1 II 2 1 105 Table 3.2-1. The clinical and radiological features of the eight FBCG patients from three families from the UAE. Families FA Patients II1 Alive at 3.5 years Survival Origin FB FC III1 III2 III4 III5 Few hours Few days 1 day 3 days Omani III7 Alive at 6 years III8 II2 2 days Few hours Yeme ni Omani Clinical Features Wide anterior fontanelle Protuberant eyes + + Flat midface Small flat nose Small mouth Cleft palate Short neck Short limbs Joint contractures Myopia Severe SNHL Progressive kyphosis Cataract + + + + + + + + + + + Wide anterior fontanelle & sutures Short wide cupped ribs Platyspondyly Pear shaped vertebrae on lateral projection Small & broad iliac bones Small sciatic notch Caudally directed bump like configuration with medial & lateral spurs Short dumb-bell long bones Irregular metaphysis Ectopic extra calcification + + + + + + + + + + + + + + + + + + + + NA NA NA NA NA NA NA NA NA Radiological Features + + + + + + + + + + + + + NA NA NA - + + + + + + + + + + + + + + + + NA NA NA - + + + + + NA NA NA - + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + - + + - - + + - NA=Not available 106 3.2.3.1.2. Family B (FB) The parents are Emirati first cousins of Omani origin (Figure 3.2-2). Both parents are of normal height and hearing. The mother has normal vision but the father developed a cataract in his right eye at the age of 38 years. Both parents have no complaints regarding their joints. They had a total of eight children, six of whom were affected. Details of two of the affected children have been previously published [Al-Gazali et al., 1997; 2003]. These affected children had the typical features of FBCG as detailed in table 3.2-1 and five of the six affected children died in the neonatal period (Figure 3.23a). However, one child (FB-III-7) was still alive at the age of 6 years and this child was part of a twin pregnancy (Figure 3.2-3b and c). The other twin was an unaffected female who developed severe myopia and squint in infancy but had no other relevant complaints. The affected child (FB-III-7) was on respiratory support for one month after birth followed by tracheostomy for another few months. She was able to breathe by herself by 2 years of age. This surviving affected child is now 6 years of age but has developmental delay, short limbs with contractures of the large joints, severe pectus carinatum with narrow chest and dorsal kyphosis (Figure 3.2-3b and c). She walks with difficulty and has no speech. She has severe myopia and bilateral profound sensory–neural hearing loss requiring hearing aids. She was operated on for a cleft palate and a bilateral cataract. Detailed ophthalmological examination revealed myopia, estropia, aphakia and myopic degeneration of the retina, but there were no vitreous changes. 107 Figure 3.2-2. Pedigree of family FB. Six children out of eight of a consanguineous couple were affected with FBCG. All the affected children died except one (III-7). I II 1 2 III 1 2 3 4 108 5 6 7 8 Figure 3.2-3. Clinical features of selected affected members from family FB. a) The general appearance of patient III-1 in family FB. Note the typical facial appearance with short limbs and narrow chest. b) & c) Images of patient FB-III-7 at 6 years of age showing severe skeletal abnormalities including severe kyphosis, pectus carinatum and joint contractures. Note also the flat face with prominent large eyes reminiscent of Marshal syndrome. a) b) c) 109 3.2.3.1.3. Family C (FC) The parents are distantly related of Yemeni origin and this child was their first (Figure 3.2-4a). The parents appeared of normal height, had normal hearing and vision. The child was the product of a pregnancy complicated by polyhydramnios (Table 3.2-1). The delivery was normal. At birth the child was found to have the typical clinical and radiological features of FBCG (Figure 3.2-4b). He died few days after birth due to respiratory insufficiency. 3.2.3.2. Gene mapping and mutation analysis on UAE families with FBCG revealed homozygous two mutations in COL11A1 gene The genomes of seven affected (FB-III-1, FB-III-2, FB-III-5, FB-III-7, FB-III-8, FA-II-1, FC-II-2) one unaffected (FB-III-6) and two parents (FB-II-1, FB-II-2) were genotyped using SNP 6.0 array. Homozygosity mapping analysis on the generated genotyping data identified a block of homozygous SNPs covering around 4.5 Mb shared between all the seven affected individuals from the three families (Figure 3.2-5). The shared block between the affected children FB-III-1, FB-III-2, FB-III-5, FB-III-7, FB-III-8, was localized between rs12072388 (Chr1:99,920,791) and rs11185389 (chr1:104,549,369). FA-II-1 was homozygous only between rs12137590 (chr1: 101771970- 101772470) and rs550437 (chr1: 104319230–104319730), which narrowed the region further. The two parents (FB-II-1, FB-II-2) and the unaffected (FB-III-6) used for genotyping were heterozygous except for 364 SNPs covering the entire COL11A1 gene were homozygous in the father of the FB family (FB-II-1; Figure 3.2-5). The wider block contained around 42 genes. DNA sequencing of 20 candidate genes (including COL11A1 gene) 110 Figure 3.2-4. Clinical features of the affected child from family FC. a) Pedigree of family FC. b) Baby gram of FC –II-2 showing short dumb Bell appearance of long bones, short cupped ribs and typical appearance of the ilium. b) a) Distant relatives I 1 2 1 2 II 111 Figure 3.2-5. Homozygosity mapping of the UAE families with FBCG. Genome wide SNP genotyping of seven patients with FBCG (FB-III-5, FBIII-7, FB-III-8, FB-III-1, FB-III-2, FA-II-1 and FC-II-2) from three unrelated families mapped the disease to chr1p21.1-p21.2. A wide block (included in a red dotted square) was found to be shared between FB-III-5, FB-III-7, FB-III-8, FB-III-1, FB-III-2, and FC-II-2. FA-II-1 was only homozygous in part of the wide block (included in a blue dotted square). The mother (FBII-2) and an unaffected sib (FB-III-6) were heterozygous in this region while the father (FB-II-1) showed partial homzygosity having 364 homozygous SNP within the mapped region (indicated with a red arrow). The homozygous SNPs in patients are designated by green lines, while the homozygous SNPs in the unaffected individuals are indicated by red lines. 112 located within the homozygous block in family FB revealed no mutation within the exons and their splice junctions (listed in table 3.2-2 and Appendix B). However, a homozygous nonsense mutation c.4084C>T (p.R1362X) (NM_001854.3) was detected in family FA in exon 54 of COL11A1 (Figure 3.2-6) that is predicted to cause pre-maturation and truncation of procollagen chains in this patient and thus expected to be abolished by the non-mediated decay (NMD) mechanism. Clinical similarity of the phenotype between the recruited families highlighted COL11A1 as the candidate disease-causing gene. Therefore, to investigate COL11A1 further in family FB, RNA was extracted from cultured fibroblasts from the only surviving patient in this family (FB-III-7). Direct Sequencing of COL11A1 cDNA revealed a homozygous inclusion of a 50 bp pseudo-exon between exon 48 and exon 49 (Figure 3.2-7). Re-sequencing of intron 48 of COL11A1 revealed a homozygous genomic mutation c.3708+437T>G in all the affected individuals of family FB (Figure 3.2-8). Apparently this transversion created a strong donor splice site leading to the subsequent selection of a weaker ‘opportunistic’ acceptor site, as described previously in the literature (Figure 3.2-9) [Buratti et al., 2006]. The interesting finding is that the retention of this pseudo-exon has led to premature insertion of a termination codon in the mature mRNA and most probably resulted in its rapid degradation by NMD. Unfortunately, sequencing the 67 exons of COL11A1 gene in the affected member of the third family FC (FC-II-2) did not reveal any suspected change 113 Table 3.2-2. List of the twenty genes sequenced in this study. Gene RefSeq number Coordinates Exon count PALMD NM_017734 100111430 8 AGL NM_000642 100315639 34 SLC35A3 NM_001271685 100435991 8 HIAT1 NM_033055 100503788 12 SASS6 NM_194292 100549101 17 LRRC39 NM_001256385 100614003 10 DBT NM_001918 100652477 11 RTCA NM_001130841 100731713 12 CDC14A NM_003672 100818022 16 GPR88 NM_022049 101003727 2 VCAM1 NM_001078 101185195 9 EXTL2 NM_001261441 101337927 5 SLC30A7 NM_001144884 101361631 11 DPH5 NM_001077395 101455179 18 S1PR1 NM_001400 101702304 2 COL11A1 NM_080629 103342022 67 RNPC3 NM_017619 104068577 14 AMY2B NM_020978 104097265 12 AMY2A NM_000699 104159998 10 AMY1A NM_001008221 104198140 11 114 Figure 3.2-6. Mutation analysis results in family FA. A novel nonsense mutation was found c.4084C>T in COL11A1 in the affected child (FA-II1) in a homozygous form. The same mutation was found to be heterozygous in parents. The mutation was not detected in normal healthy controls. Sequencing chromatogram of a normal individual carries the wild type sequence of COL11A1 Sequencing chromatogram of a carrier parent carries a heterozygous c.4084C>T Sequencing chromatogram of the affected carries a homozygous c.4084C>T 115 116 b) a) Figure 3.2-7. A novel insertion of a 53 nucleotide between exons 48 and 49 of COL11A1 cDNA in family FB. a) Sequencing chromatogram showing the region between exons 48 and 49 of COL11A1 cDNA sequence of a normal control with the exons borders are underlined. b) Sequencing chromatogram showing the homozygous insertion of a 50 nucleotide (underlined) in FB-III-7 COL11A1 cDNA. Figure 3.2-8. Mutation analysis results for intron 48 of COL11A1 in family FB. A novel intronic c.3708+437T>G mutation was found in all the affected members of family FB in a homozygous form. The same mutation was heterozygous in parents and the unaffected sib (FB-III-6). The mutation was not found in 100 normal controls. Sequencing chromatogram of a normal individual carries the wild type sequence of COL11A1 Sequencing chromatogram of a carrier individual carries a heterozygous c.3708+437T> G Sequencing chromatogram of an affected carries a homozygous c.3708+437T> G 117 118 New E47 E48 E49 E49 E50 E51 E52 E53 E54 E55 E56 c.4084C>T Control gtattttcagACTATATAATGTTACAGCAATATGTGCGTGATAGGTGCACCCTTTATCATgtaaaatgtttt Patient gtattttcagACTATATAATGTTACAGCAATATGTGCGTGATAGGTGCACCCTTTATCAGgtaaaatgtttt E48 E46 c.3708+437T>G E57 Figure 3.2-9. Schematic representation of the genomic region encompassing exons E46 to E57 of the COL11A1 gene indicating the locations of the two detected mutations in the UAE families FA and FB. The two mutations detected in this study, c.4084C>T in FA and c.3708+437T>G in family FB, are located in the sequences encoding the triple helical domain of COL11A1 protein. The drawing demonstrates the effect of the c.3708+437T>G mutation in introducing a pseudo -exon (labeled as “New”) between exons 48 and 49. The drawing also shows the genomic sequence of the patient with c.3708+437T>G mutation aligned to the sequence of the normal control illustrating the T to G change in red. The change from AT to AG is predicted to create a favorable consensus sequence strengthening a cryptic donor splice site within the intron. including absence of the intronic mutation found in family B. No cDNA for any member in this family was available to confirm whether a splicing change in COL11A1 is the cause of the corresponding phenotype. Therefore, I was unable to confirm or exclude the involvement of this gene in their phenotype. However, genome wide-homozygosity mapping showed a large stretch of homozygosity on chromosome 1 with overall homozygosity of 7.8% suggesting a possible closer parental relationship than that indicated in the family history. In addition, the locus for the COL11A2 gene found to be responsible for some fibrochondrogenesis cases [Tompson et al., 2012] is heterozygous in this patient. However, I can not exclude the possibility that the phenotype is caused by compound heterozygous mutations in COL11A2. 3.2.3.3. Functional studies on the c.3708+437T>G mutation confirm the predicted loss of function mechanism underlying FBCG The expression level of COL11A1 mRNA in the patient’s (FA-III7) fibroblasts was investigated by real time (RT)-PCR using a specific Taq-Man probe for exons 14 and 15 of human COL11A1 transcript NM_001854.3 (Hs01097626_m1). This pre-designed probe can also detect all the other known isoforms of human COL11A1 mRNA. No detectable expression was observed for COL11A1 in the fibroblasts from this patient compared to normal healthy control fibroblasts (Figure 3.2-10a; Appendices B1 and B2). Absence of mRNA for this gene could be explained by NMD degradation due to the premature termination. Collagen 11A1 protein expression has been also evaluated by western blotting. Total protein was extracted from the patient’s (FA-III7) fibroblasts, 119 Figure 3.2-10. Expression analysis of the homozygous c.3708+437T>G mutation in COL11A1 gene. a) COL11A1 mRNA level in the patient fibroblasts was not detectable by TaqMan RT-PCR compared to its expression in the fibroblasts of a healthy control. b) 100 micrograms of total protein were extracted from the fibroblasts of the patient as well as a normal control and loaded into an SDS-PAGE and then analyzed by western blotting. A COLL11A1 band at the expected size (~181 KDa) was detectable in the control and totally absent from the patient. a) Relative Quantification (RQ) 1.2 1 0.8 0.6 0.4 0.2 0 Healthy Control Patient 1 0.002336927 COL11A1 mRNA b) B actin 120 quantified and resolved by SDS-PAGE followed by western blotting using rabbit polyclonal anti-collagen 11A1 raised against human epitope (ab64883). This antibody binds to an internal highly conserved sequence of COL11A1 protein (NP_001845.3; amino acids 601 to 630) which is similar in all the known isoforms. A protein band with expected size for human COL11A1 (~181 KDa) was detected in the sample from the healthy control which was completely absent from the patient’s sample (Figure 3.2-10b). 3.2.4. Discussion Human chondrodysplasias are genetically and clinically heterogeneous groups of cartilage disorders where the severity of the phenotypes, in part, correlated with the type of the causative mutation [Matsui, 2010]. In this study, I have discovered the novel association of a severe form of chondrodysplasia “the FBCG” in affected families from UAE with COL11A1 gene. This finding was independently confirmed by Tompson et al [2010] who described two patients with FCBG carrying compound heterozygous mutations in COL11A1. I have also reported the first homozygous loss-of-function COL11A1 mutations described in humans. 3.2.4.1. Clinical heterogeneity of type XI collagenopathies The COL11A1 gene (OMIM*120280) on chromosome 1p21 encodes the α1 chain of type XI collagen. Collagen XI is a minor structural protein (3– 10%) expressed in the hyaline cartilage, the vitreous of the eye, the nucleus pulposus of the intervertebral disc and the inner ear where it forms thin fibrils together with collagens II and IX [Cremer et al., 1998]. In fetal cartilage, 121 collagen XI consists of α1(XI)α2(XI)α1(II)-polypeptide chains encoded by COL11A1, COL11A2 and COL2A1 genes, respectively [Kadler et al., 2008]. The only exception is in the ocular vitreous where α2(XI) is replaced by the α1 chain of collagen V, which shares structural homology with collagen XI and their α chains are interchangeable in certain tissues and cell lines [Fernandes et al., 2007; Fichard et al., 1995]. Electron microscopy studies have shown that the triple helical or the C-terminal domains of collagen XI and V are located in the core of their fibrils with the N-terminal domains (NTD) at the fibrils’ surface [Fichard et al., 1995; Holmes and Kadler et al., 2006; Fallahi et al., 2005]. Variable locations of their functional domains suggest two roles for both collagens [Wenstrup et al., 2004], where they act as nucleators for fibril assembly [Blaschke et al., 2000] as well as controlling fibril diameters through the NTDs by sterically hindering their thickening [Kadler et al., 2008; Blaschke et al., 2000]. Mutations in the genes encoding collagen XI cause various chondrodysplasias. Autosomal dominant Stickler syndrome type II (STL2; OMIM#604841) and Marshall syndrome (OMIM#154780) are caused by heterozygous mutations in the COL11A1 gene [Fernandes et al., 2007]. Compound heterozygous and homozygous mutations in COL11A1 have been reported with FBCG1 (OMIM#228520) [Tompson et al., 2010] and a simple Stickler phenotype [Alzahrani et al., 2012]. Heterozygous and homozygous mutations in COL11A2 were detected in a number of chondrodysplasias including otospondylomegaepiphyseal dysplasia (OSMED; OMIM #215150 & 122 #277610), STL3 Heterozygous (OMIM#184840) mutations condrodysplasias such in as and COL2A1 are achondrogenesis FBCG2 (OMIM#614524). associated with (OMIM#200610), several Kniest dysplasia (OMIM#156550), and STL1 (OMIM#108300). 3.2.4.2. Genetic heterogeneity of FBCG FBCG has been shown to be genetically heterogeneous resulting from mutations in two genes out of three encoding the α chains of type XI collagen, COL11A1 and COL11A2 [Tompson et al., 2010; 2012]. Moreover, all the reported cases of FBCG so far show a recessive mode of inheritance for the disease [Lazzaroni-Fossati et al., 1978; Eteson et al., 1984; Martínez-Frías et al., 1996; Al-Gazali et al., 1997; 1999; Sauer et al., 2000; Randrianaivo et al., 2002; Leeners et al., 2004; Kulkarni et al., 2005; Kundaragi et al., 2012] except one case reported by Tompson et al [2012]. The authors have suggested an autosomal dominant inheritance for the disorder after identifying a de novo heterozygous 9 bp deletion in exon 40 of COL11A2 in one patient with FBCG. However, in that study no linkage analysis or sequencing of the patient’s cDNA was performed to exclude any other splicing mutation(s) which might be found in COL11A2 or in COL11A1. 3.2.4.3. Phenotypic spectrum of COL11A1 mutations Mutations in COL11A1 gene have variable phenotypes ranging from the absence of any symptoms or mild phenotypes to early lethality. Numerous heterozygous mutations in COL11A1 have been previously reported in patients with autosomal dominant Stickler and Marshall syndromes [Griffith et 123 al., 1998; Richards et al., 2006; 2010; Vijzelaar et al., 2013]. Initially, these two dominant conditions were regarded as distinct entities but more recently they have been considered allelic [Majava et al., 2007]. The mutations reported to cause these two conditions include heterozygous nonsynonymous substitutions of glycine residues and skipping or in-frame deletion of one or more exons (Table 3.2-3) [Richards et al., 2006; 2010; Griffith et al., 1998; Majava et al., 2007; Annunen et al., 1999; Martin et al., 1999; Poulson et al., 2004]. Four mutations in COL11A1 gene have been recently found in two unrelated American FBCG patients [Tompson et al., 2010]. The reported mutations were in the compound heterozygous states and they introduce a PTC in one allele and a glycine substitution in the other. The father had an early-onset hearing loss but the mother was normal (Table 3.2-3). One homozygous 1 bp deletion has been detected in exon 6 of COL11A1 gene creating a PTC in the N-terminal propeptide of the protein product [Alzahrani et al., 2012]. The patient carrying this mutation exhibited a mild phenotype resembling Stickler while the carrier parents were normal except for a mild myopia in the father (Table 3.2-3). In this study, two homozygous mutations in COL11A1 have been identified in two unrelated families. Both mutations created a PTC in the highly conserved triple helical domain of the protein product. All the affected patients in this study exhibited a severe FBCG phenotype which was lethal in most of them. While the carriers were almost normal showing early myopia in one case and adulthood cataracts in the other (Table 3.2-3). 124 125 p.G1042R Mother of family 1 Father of family 1 Mother of family 2 Father of family 2 Non-synonymous mutation Splice site mutations results in in-frame deletion of exon/s Prominent forehead, protruding eyes with marked midface hypoplasia, short nose , and long philtrum Mild phenotype with Less pronounced mid face hypoplasia -ve -ve flat face, small chin, and cleft palate -ve -ve -ve -ve -ve -ve -ve -ve -ve Orofacial Features -ve myopia, cataract, and rarely retinal detachment Mild myopia -ve 'beaded' or type 2 vitreous, , moderate to severe myopia in early childhood -ve -ve Severe myopia since infancy Myopia at 10 years Wore glasses at 6 years Mild myopia -ve -ve Cataract of right eye at 38 years Ocular Features -ve early-onset hearing loss congenital hearing loss Mild hearing loss and history of hearing loss in the family -ve -ve Mild to severe hearing loss Hearing loss at age 7 years -ve -ve -ve -ve -ve -ve -ve Auditory Features -ve *The heterozygous status was assumed but not actually confirmed by DNA sequencing Glycine substitutions Gross Deletions Stickler and Marshall overlap Splice site mutations results in in-frame deletion of exon/s Splice site mutations results in in-frame deletion of exon/s Gross Deletions Gross Deletions Frame shift led to PTC Frame shift led to PTC Non-synonymous mutation Non-synonymous mutation Nonsense mutation p.N398PfsX19 p.N398PfsX19 Glycine substitutions p.G1315X p.G796R Marshal Father Mother Stickler type II c.3708+437T>G c.3708+437T>G FB-II2 FB-III6 p.A596fsX8* Nonsense mutation Nonsense mutation Splicing mutation led to insertion of an exon and PTC = = p.R1362X p.R1362X c.3708+437T>G Non-synonymous mutation Frame shift led to PTC Implication Frame shift led to PTC Mutation 1-nt deletion Cases Cho/+ mice FA-I1 FA-I2 FB-II1 short stature more short stature and abnormalities in cranial ossification, -ve -ve Early onset osteoarthrosis Average stature and joint pain Mild short stature for her family Average stature Average stature -ve -ve Other Features Early onset Osteoarthritis -ve -ve -ve Majava et al., 2007 Griffith et al., 1998; Annunen et al., 1999 Alzahrani et al., 2012 Alzahrani et al., 2012 Richards et al., 2006; 2010; Griffith et al., 1998; Majava et al., 2007; Poulson et al., 2004 Tompson et al., 2010 Tompson et al., 2010 Tompson et al., 2010 Tompson et al., 2010 This study This study This study This study This study Reference Li et al., 1995 Table 3.2-3. Comparison of the phenotypes associated with disease causing heterozygotes genotypes in COL11A1 gene. Similarly, Col11a1 homozygous null mutation in mice ‘Cho mouse’ causes a lethal chondrodysplasia with clinical features strikingly similar to FBCG in humans including the patients in this study [Li et al., 1995; Fernandes et al., 2007]. The homozygous strains show abnormalities in the cartilages of limbs, ribs, mandible and trachea [Li et al., 1995]. Nonetheless, heterozygous mice Cho/+ survive normally and do not exhibit any observable symptoms except osteoarthritis (Table 3.2-3) [Rodriguez et al., 2004]. 3.2.4.4. Pathophysiological mutations mechanisms underlying COL11A1 The Stickler and Marshall syndromes causing mutations are probably acting via a dominant negative mechanism resulting in the dominant mode of inheritance of these chondrodysplasias [Richards et al., 2006; 2010]. Several models have been proposed to explain the deleterious effect of such mutations including the prevention of triple helix formation due to the incorporation of the defective chains and the subsequent accumulation of the unfolded procollagen molecules in the endoplasmic reticulum (ER) of growth plate chondrocytes [Prockop et al., 1989]. This accumulation is likely to lead to the rapid degradation of the whole complex [Rodriguez et al., 2004; Prockop et al., 1989] and/or the induction of the ER stress signaling pathway promoting chondrocyte apoptosis [Wilson et al., 2005; Nundlall et al., 2010]. Moreover, it has been suggested that the abnormal α1(XI) chain may fold into a triple helix which has kinks leading to abnormalities in fibril formation [Prockop et al., 1989; Gaiser et al., 2002]. Interestingly, in the FBCG cases described by Tompson et al [2010], the heterozygous carriers (parents) of the 126 glycine substitutions did not show the dominant Stickler or Marshall phenotypes. This may reflect the variable effects of the glycine substitutions and their dependence on the positions of these substitutions within the chains. In addition, variable phenotypes have been reported for different substitutions [Kuivaniemi et al., 1991]. For the COL11A1 homozygous mutations, the Cho mouse is considered to be the most appropriate animal model for the phenotype of a homozygous null mutation of the Col11a1 gene [Li et al., 1995; Fernandes et al., 2007]. All the symptoms of Cho/Cho mouse described [Li et al., 1995] are in concordance with the FBCG phenotype seen in the studied patients. In Cho/Cho model, a complete knockout of the type XI collagen has been assumed because of the lack of α1(XI) chains. This assumption was supported by the prominent presence of abnormally thick collagen fibrils in the cartilage of Cho/Cho mice [Li et al., 1995]. On the other hand, cartilage from Cho/+ mice contained a mix of normal thin and three time thicker fibrils [Xu et al., 2003]. Recently, Fernandes et al [2007] studied Cho mouse at the protein level and identified a mix of α1(V) and α1(XI) chains in the Cho/+ cartilage, whereas α1(V) and α2(XI) chains were observed in the Cho/Cho cartilage. Therefore, they concluded that alternative assemblies of type XI collagen chains were generated in Cho/Cho cartilage instead of a complete knockout of the type XI molecule. Similarly, no mRNA or protein expression has been observed in this study for the α1(XI) chain in one of the investigated patients. Therefore, the two homozygous null mutations in COL11A1 gene 127 detected in this study are considered to be the first to be observed on the effects of complete loss-of-function of this gene in humans. Although another homozygous null mutation in COL11A1 has been reported with a milder phenotype, yet the reported mutation has occurred in exon 6 of this gene which is missing or alternatively spliced in some of its known isoforms [Iyama et al., 2001; Chen et al., 2001]. By contrast, the mutations reported in this study and in the Cho mouse are located in highly conserved regions of COL11A1 found in all the known isoforms. Furthermore, additional isoforms seem to exist for COL11A1 showing tissue-specific preferential expression (http://www.uniprot.org/uniprot/P12107). Nevertheless, Yoshioka et al [1995] has demonstrated experimentally that Col11a1 has a broader spectrum of expression and concluded that the alpha 1(XI) chain may participate in the formation of stage- and tissue-specific trimers with distinct functional properties. Taken all together this may explain the phenotypic spectrum of COL11A1 null homozygous mutations. 3.2.5. Conclusion In this study, I have localized and identified the gene underlying FBCG in 2 out of 3 consanguineous families from the UAE. I have also revealed novel recessive mutations in COL11A1 and the phenotypes associated with them in affected and carrier individuals. As outlined in Chapter 1, identification of causative genes and mutations underlying heterogenous recessive disorders such as FBCG can be helpful in refining the diagnosis and the subsequent clinical management of the patients. In addition, identification of 128 these homozygous COL11A1 mutations (inherited in an autosomal recessive manner) has facilitated carrier screening and the subsequent appropriate genetic counseling as well as the possibility of future prenatal diagnosis or preimplantation genetic diagnosis for these families. The identification of a human model of COL11A1 inactivation has also implicated a novel disease pathway which can be explored in the future to elucidate the molecular basis of other unresolved chondrodysplasias. 129 SECTION 3: Delineation of the clinical, molecular and cellular aspects of several novel JAM3 mutations underlying the autosomal recessive hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts 130 3.3.1. Background Junctional adhesion molecule 3 (JAM3; OMIM*613730) is a member of the JAM subfamily of proteins, which include F11R (JAM1, JAM-A), JAM2 (JAM-B), JAM3 (JAM-C), and IGSF5 (JAM4) [Ebnet, 2008; Weber et al., 2007]. Other related members of this subfamily of proteins include the endothelial cell selective adhesion molecule and coxasackie virus and adenovirus receptor (CXADR) [Weber et al., 2007]. JAMs and related proteins are localized at intercellular contacts and participate in the assembly and maintenance of tight junctions (TJs) and the control of cellular permeability [Ebnet, 2008]. TJs are morphologically distinct subcellular structures that are located closer to the apical side (compared with other junctional complexes) and are highly regulated areas of close contact between the plasma membranes of adjacent epithelial and endothelial cells [Balda and Matter, 2008]. TJs are important for the formation of polarized epithelial and endothelial barriers by forming intermembrane diffusion barriers and by controlling diffusion along the paracellular pathway [Cereijido et al., 2008]. It has also been found that TJs are important for intracellular signaling [Matter and Balda, 2007; Steed et al., 2009; Terry et al., 2010]. JAMs have been found to regulate adhesion between leukocytes and endothelial cells and the paracellular transmigration of leukocytes across the endothelium [Bradfield et al., 2007; Weber et al., 2007]. Moreover, JAMs play a role in cell polarization during spermatogenesis [Gliki et al., 2004]. In addition to JAMs, TJs contain other proteins including claudins, occludins, and tricellulins [Ebnet et al., 131 2004; Ebnet, 2008]. JAMs are single-span transmembrane proteins, whereas claudins, occludins, and tricellulins are tetra span transmembrane proteins [Balda and Matter, 2008; Ebnet, 2008]. The tetra span proteins form the paracellular permeability barrier and therefore determine the selectivity and extent of paracellular diffusion. JAMs on the contrary, are postulated to mediate homotypic cell–cell adhesion [Bazzoni et al., 2000; Weber et al., 2007]. Several inherited conditions have been shown to be caused by mutations in genes encoding members of TJs protein complexes [O’Driscoll et al., 2010]. Hypomagnesaemia has been shown to be caused by mutations in genes encoding claudins 16 and 19 [Konrad et al., 2006; Simon et al., 1999] and familial hypercholanemia to be caused by mutations in the TJP2 and BAAT genes [Carlton et al., 2003]. Similarly, mutations in claudin 14 and tricellulin have been found to cause hereditary deafness [Riazuddin et al., 2006; Wilcox et al., 2001] and mutations in NHS to cause Nance-Horan syndrome [Burdon et al., 2003]. The variability in the clinical presentations reflects the variable functions of TJs in various tissues and organs. 3.3.2. The purpose of the study In a collaborative study, we mapped a rare recessive disorder segregating in an extended consanguineous family from the UAE to chromosome 11q25 using a homozygosity mapping approach [Mochida et al., 2010]. The disorder is characterized by hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts (HDBSCC; OMIM#613730). Sequencing of candidate genes residing within the 132 homozygous region revealed a homozygous mutation in the JAM3 gene [Mochida et al., 2010]. In this study, additional families from different ethnic backgrounds exhibiting similar phenotypes were recruited. Subsequently, direct sequencing of JAM3 gene was carried out in all the affected children to confirm the association of this gene with the aforementioned phenotype. Additionally, this study was carried out to identify more mutations in this gene that may expand the phenotype or provide insights into the pathophysiological mechanism underlying this severe phenotype. 3.3.3. Results 3.3.3.1. Clinical assessment of the studied families Three families from different ethnic backgrounds (one Turkish, one Afghani, and one Moroccan) were recruited to participate in this study (Figure 3.3-1). Consanguinity is evident in two of the families and the clinical features of all the affected children are listed in Table 3.3-1. 3.3.3.1.1. Family 1 The parents of the affected child in this family are second cousins of Turkish origin. The parents are normal. Their first child (II-1) was a preterm baby girl who had intracranial bleeding and died in the neonatal period. One of the mother’s sisters died with a similar phenotype but no clinical data were available. The second child (II-2) of the couple was a female who was the product of a normal pregnancy and term delivery. Her birth weight was 2,480 gm (<10th centile). She was admitted to the neonatal intensive care unit on day 5 due to tonic seizures, which were resistant to treatment. 133 134 Figure 3.3-1. Pedigrees of the three families in this study. Three families with different ethnic origins were recruited for this study. Family 1 is Turkish, family 2 is Afghani, whereas family 3 is Moroccan. Filled symbols denote affected individuals with the syndrome of hemorrhagic destruction of the brain, subependymal calcification, and congenital cataracts (HDBSCC). Sequenced individuals are indicated with an asterisk. 135 bilateral cataracts NA bilateral cataracts Seizures Ophthalmology Posterior fossa abnormalities Renal abnormalities Hepatomegaly Brain MRI Intraparanchymalhemorrhage Evidence of brain destruction Subependymal basal ganglia & white matter calcification NA 35cm Birth head circumference + + + + + + + - + + + 34cm died after birth died 9 days Survival 2 + 1 + Patients Ethnic origin Consanguinity Families - + + + NA NA bilateral cataracts, microphthalmia + + + + + bilateral cataracts, pale optic disc NA + + 3 4 UAE (Baluchi) + + alive 5 alive 7 years years with severe with mental & severe physical mental & handicap physical handicap 33cm NA Mochida et al [2010] + + + + + NA bilateral cataracts NA NA died 2 month + 5 6 + + + + + NA bilateral cataracts + + alive 11 years with severe mental & physical handicap NA Table 3.3-1. Clinical features of hemorrhagic brain destruction-JAM3 type. - + + + - bilateral cataracts + 35cm Died at 2 months Family1 1 Turkish + - + + + + - bilateral cataracts - NA 2 - + + + + - bilateral cataracts 35cm EEG bilateral epilepliform activity Died at 5 days Afghani - Family 2 Died at 14 days - 1 Present study - + + + - bilateral cataracts status epilepticus on EGG 34.5cm Died at 39 days Family 3 1 Moroccan + Physical examination revealed a head circumference of 30 cm, sloping forehead prominent, bulging anterior fontanelle, bitemporal grooving, and bilateral nuclear cataracts (Figure 3.3-2A-1). Cranial ultrasound showed a prominent expansive bleeding. Brain computerized tomography showed calcification of the subependymal region and multifocal intraparenchymal hemorrhage (Figure 3.3-2A-2). Serological tests were negative for toxoplasmosis, rubella, and cytomegalovirus. Cerebrospinal fluid PCR for cytomegalovirus and toxoplasma were also negative. Platelets levels were normal. The infant developed post hemorrhagic hydrocephalus and died at 2 months of age. 3.3.3.1.2. Family 2 The parents are non-consanguineous and healthy of Afghani origin. Patient 1 (II-1) was the first child of the family born at term by emergency caesarean section. His birth weight was 2,480 gm (<10th centile). He was noted to have bilateral cataracts and had feeding problems from birth. He was discharged home on day 4. At 2 weeks of age, he presented with vomiting, irritability, and a high pitched cry. A brain MRI at the age of 15 days showed Grade IV intraventricular hemorrhage, severe ventricular dilatation, and white matter abnormalities in both cerebral hemispheres and multiple subependymal cysts (Figure 3.3-2B). Renal ultrasound showed horseshoe kidneys with echogenic cortex and altered corticomedullary differentiation. He had progressive neurological deterioration and died at 2 weeks of age. Investigations were all normal and included blood karyotype, CGH array, and 136 Figure 3.3-2. Clinical features of some affected members in this study. A-1: Facial picture of the affected individual in family 1 shows bilateral nuclear cataracts and hypertelorism. A-2: Head CT image of the patient in family 1 reveals multifocal intraparenchymal hemorrhages (arrows) and subependymal calcification (arrowheads). B: T2-weighted axial brain MRI of patient 1 in family 2 reveals multifocal intraparenchymal hemorrhages of varying ages (arrows) as well as intraventricular hemorrhage (asterisks). C-1: Head CT of the patient in family 3 shows multifocal intraparenchymal hemorrhages (arrows) and subependymal calcification (arrowheads). Diffusely hypodensity of brain parenchyma is also noted, suggesting severe edema. C-2: T2- weighted coronal MRI image of the same patient shows intraventricular hemorrhage with associated enlargement of the ventricles. 137 a coagulation study including platelets. Patient 2 (II-3) was the third child of the family, with the family’s second child being a healthy male. Pregnancy with patient 2 was normal and delivery was by elective caesarean section at term. His birth weight was 3,315 gm (25th centile), length was 52 cm (50–90th centile), and head circumference was 35 cm (50th centile). At birth, he was noted to have bilateral cataracts. His tone and reflexes were normal and there was no clinical evidence of seizures. Cranial ultrasound showed periventricular echogenicity but there was no bleeding. However, he deteriorated on day 5 and his hemoglobin dropped from 180 g/l to 96 d/l, and he required ventilation. His EEG on the third day of life showed bilateral epileptiform activity. A cranial ultrasound revealed extensive bilateral intracerebral hemorrhage. He died on the fifth day of life. Abdominal ultrasound showed pelvicalyceal dilatation of the right kidney. Full metabolic work up was normal. 3.3.3.1.3. Family 3 The parents are first cousins of Moroccan origin who had a total of five pregnancies. The family history is significant in that the mother’s brother (I-3) died on the seventh day of life without any known diagnosis. This couple’s first child was a male born at 36 weeks gestation who had cerebral hemorrhage, hydrocephalus, and died at 10 days after birth. They then had two healthy children. The fourth pregnancy ended with a stillborn child who had hydrocephalus and the cerebral tissues showed necrotic degeneration. The index case (II-5) is 28 day old female, the product of a normal pregnancy. 138 Fetal tachycardia was detected at 37 weeks gestation and therefore the baby was delivered by caesarean section. Her birth weight was 2,520 gm (10–25th centile), length 46.5 cm (25th centile), and head circumference 34.5 cm (50th centile). Bilateral cataracts were detected at birth both central and nuclear. On the second day of life, she presented with irritability, crying, and a bulging anterior fontanelle. Her brain ultrasound showed dilated lateral ventricles, intraventricular hemorrhage in the right ventricle, and frontal periventricular cysts. On the eighth day of life, she was referred to another hospital for neurosurgical evaluation. A head CT revealed multifocal intraparenchymal hemorrhages, subependymal calcification, and diffusely hypodense brain parenchyma (Figure 3.3-2C-1). A MRI of the brain revealed intraventricular and intraparenchymal hemorrhage, edema, and cystic changes of the brain parenchyma, dilated ventricles, and thin corpus callosum with hemorrhagic damage (Figure 3.3-2C-2). There was normal circulation in the circle of Willis arteries. There were no seizures clinically. Metabolic screening was normal. Platelets levels were normal. She had severe anemia. Her brain CT scan demonstrated bilateral subependymal calcification in the lateral ventricles. On the eighteenth day of life, her head circumference was 41 cm (4 standard deviations above the mean) and she was discharged for palliative care at home. She died at home the age of 39 days. 3.3.3.2. JAM3 mutational analysis DNA sequencing revealed three novel homozygous non-synonymous mutations in JAM3 gene in the three studied families (Table 3.3-2; Figure 3.3- 139 3). In family 1, a non-synonymous mutation (c.656G>A) in exon 6 of JAM3 gene was detected, which led to an amino acid substitution at position 219 (p.C219Y). In family 2, a single nucleotide substitution (c.346G>A) in exon 4 of JAM3 was identified leading to a non-synonymous mutation at position 116 (p.E116K). The third mutation (c.2T>G) was detected in family 3 changing the initiation codon (methionine) of JAM3 leading to amino acid substitution to arginine (p.M1R). The three mutations were not reported in either the dbSNP or the 1000 genome project. All the amino acids affected by these mutations were highly conserved across species (Table 3.3-2) and were predicted to be disease causing by Polyphen2, SIFT, and Mutation taster. Furthermore, the detected mutations segregated with disease status in the three families; the parents were heterozygous, consistent with carrier status, and the available unaffected siblings were homozygous for the wild-type (WT) or heterozygous for the mutation. All the detected mutations were submitted to the LOVD database (www.lovd.nl/JAM3). 3.3.3.3. The p.C219Y mutation resulted in JAM3 trafficking defect To understand the pathophysiological significance of the two detected non-synonymous mutations (p.E116K and p.C219Y), confocal immunofluorescence microscopy was employed to establish their subcellular localization relative to the wild type (WT)-JAM3 protein. HeLa cells, known to lack endogenous expression of JAM3 [Betanzos et al., 2009], were either transfected with individual JAM3 cDNA constructs (WT, p.E116K or p.C219Y; see methods 2.2.2.4 and Appendix A) or co-transfected with GFP-hRas as 140 marker for the plasma membrane. The cells were then stained with anti FLAG antibodies to visualize the JAM3 protein. The cells transfected with JAM3 only constructs were co-stained with anti-calnexin polyclonal antibodies to visualize the endoplasmic reticulum (ER) network. As shown in panels A–F of figure 3.3-4, the WT JAM3 is predominantly localized to the plasma membrane and co-localized largely with the GFP-hRas. Similarly, the p.E116K mutant is predominantly localized to the plasma membrane as shown in panels G–L of figure 3.3-4. In contrast, the p.C219Y mutant colocalized predominantly with the ER marker (calnexin) and not with GFPhRas suggesting its retention within the ER (Figure 3.3-4, panels M–R). This retention was observed also in different cell lines (COS7; Figure 3.3-5). To further investigate the extent of the trafficking defect caused by the p.C219Y mutation, HeLa cells were transfected individually with the WTJAM3 and the p.C219Y constructs. Then these cells were stained by an anticalnexin antibody and counted using Image J software (Figure 3.3-6; 3.3-7). Several confluent fields of fluorescently labeled non-transfected cells (in green) were selected and counted (figure 3.3-6). In these fields, the number of transfected cells stained with anti-FLAG antibodies (red color) was also counted (Figure 3.3-6; Appendix-C). The percentage of transfected cells showing plasma membrane localization versus ER localization of JAM3 protein were calculated and assessed in both the WT and the p.C219Y transfection experiments (Figure 3.3-7). Around 96% of the cells transfected with the p.C219Y mutant construct showed ER localization pattern while the 141 142 c.656G>A c.346G>A 6 4 p.E116K p.C219Y Ig-like V-type Nonsynonymo us Homozyg ous C-terminus Ig-like C2-type Splice site Domain Homozyg ous Homozyg ous p.V205Lfs (*p.V250Lfs X26) c.612+1G>T* c.747+1G>T 5 Mutation type Nonsynonymo us Genotype Protein change ¥ ¥ cDNA change E Conservation FTAVHKDDSGQYYCIASNDAGSARCEE FTAVHKDDSGQYYYIASNDAGSARCEE FHAVHKGDTGRYSCIATNDAGFAKCEE FNAVHKDDSGQYYCIASNDAGAARCEG FSAVHKEDSGQYYCIASNDAGAARCEG FSAVHKEDSGQYYCIASNDAGSARCEE FSAVHKEDSGQYYCIASNDAGSARCEE FTAVHKDDSGQYYCIASNDAGSARCEE FTAVHKDDSGQYYCIASNDAGSARCEE FSAVRKEDAGEYYCRAKNEAGISECGP FRSVKKEDAGEYYCQARNEAGWSKCIR TRRDSALYRCEVVARNDRKEIDEIVIE TRRDSALYRCKVVARNDRKEIDEIVIE TRMDTATYRCEVAAPSDTKTIDEINIQ TRSDSAIYRCEVVALNDRKEVDEITIE TRSDSAIYRCEVVALNDRKEVDELTIE TRTDSALYRCEVVARNDRKEIDEIVIE TRKDSALYRCEVVARNDRKEIDEIVIE TRRDSALYRCEVVARNDRKEIDEIVIE TRRDSALYRCEVVARNDRKEIDEIVIE TRSDSADYRCEVTAPNDQKSFDEILIS SRSDTAQYRCEVAAIDDQKPFDEILIS G.gallus M.musculus R.norvegicus C.lupus B.taurus H.sapiens P.troglodytes D.rerio D.rerio Normal Mutated G.gallus M.musculus R.norvegicus C.lupus B.taurus H.sapiens P.troglodytes D.rerio D.rerio CAGGCACTTTGGTAAGATCTCTTC CAGGCACTCTGGTAAGATCTCTT GGCACTCTGGTAAGATCTCTT CAGGCACTCTGGTAAGA CAGGCACTTTGGTAAGATCTCTTC CAGGCACTTTGTTAAGATCTCTTC © Normal Mutated Human Chimp Cat Mouse Normal Mutated Table 3.3-2. Summary of all the mutations detected in JAM3. protein-protein interactions defects Misfolding of the protein leads to its retention in the endoplasmic reticulum 19bp insertion leads to a frame shift and created premature termination codon Likelihood of Pathogenicity This study This study Mochida et al 2010 Ref. 143 c.2T>G p.M1R Nonsynonymo us N-terminus -----------MSCAISYSLLFVFPGC MALSRRLRLRLYARLPDFFLLLLFRGC MALSRRLRLRLCARLPDFFLLLLFRGC FESVE---------------MAVLQGC MALRRRPSLVL--------LLLLVRGC MALRRPPRLRLCARLPDFFLLLLFRGC MALRRPPRLRLCARLPDFFLLLLFRGC TEHFTDSKMALTPLACVLLLLSMQCYI G.gallus M.musculus R.norvegicus C.lupus B.taurus H.sapiens P.troglodytes D.rerio Translation aberrations leading to loss or abnormal expression This study Mutation nomenclature is based on NM_032801.4 and NP_116190.3 starting from A of the ATG as 1. *The nomenclature used by Mochida et al [2010] based on NM_032801.3. © Conservation alignments for all the non-synonymous mutations are based on homoloGene results for JAM3 (ID: 83700), but the splice site mutation alignments were adopted from MutationTaster results. ¥ 1 Homozyg ous MALRRPPRLRLCARLPDFFLLLLFRGC RALRRPPRLRLCARLPDFFLLLLFRGC Normal Mutated 144 Figure 3.3-3. The identified mutations in JAM3. A) Schematic presentation of JAM3 protein was constructed using MyDomains software (http://prosite.expasy.org/mydomains). The figure shows JAM3 domains including Ig-like type V and C2, Transmembrane (TM) and PDZ motif. All the reported mutations in JAM3 are indicated by arrows. Red arrows for the missense mutations detected in this dissertation and the grey arrow is designated for the splice site mutation reported by Mochida et al. [2010]. B) Genomic DNA analysis of JAM3 in the studied families: 1) in family 3 a novel homozygous non-synonymous mutation c.2T>G in the affected child but the parents were heterozygous carriers compared to a normal control, 2) two affected individuals in family 2 found to have a homozygous missense mutation c.346G>A. The parents and one unaffected sib were carriers for the mutation, 3) sequencing chromatograms displaying the substitution c.656G>A detected in family 1. The affected individual carried the mutation in a homozygous form, while the parents were heterozygous carriers. 145 Figure 3.3-4. The p.C219Y mutation disrupts normal intracellular JAM3 protein localization. HeLa cells transiently transfected with Cterminal FLAG-tagged JAM3-pCMV5 vector constructs wild type (WT), A– F; p.E116K, G–L; and p.C219Y, M–R and stained with anti-FLAG monoclonal antibodies (red). WT vector localizes to the plasma membrane (PM) with the hRas-GFP-tagged marker (A–C) and not with the endoplasmic reticulum (ER) marker calnexin (green; D–F). JAM3– p.E116K–FLAG construct localization was similar to the WT with hRasGFP-tagged marker (G–I) and the calnexin (J–L). The p.C219Y construct do not localize at the PM like hRas-GFP-tagged marker (M–O) and appears to be accumulated or clustered within the ER with calnexin (P–R). 146 Figure 3.3-5. Subcellular localization of FLAG-tagged JAM3-WT and JAM3-C219Y in COS7 cells. WT JAM3 co-localizes with GFP-hRas at the plasma membrane (PM; panel a-c) while C219Y mutated protein is retained in the endoplasmic reticulum (ER) compared to the GFP-hRas protein at the PM (panel d-f). 147 Figure 3.3-6. Subcellular localization in HeLa cells of FLAG-tagged wild-type (WT) and C219Y mutant of JAM3 with different cellular confluency. Most of the mutant protein fails to reach the plasma membrane (PM), demonstrating a perinuclear distribution with calnexin, consistent with retention in the endoplasmic reticulum (ER) regardless of the number of the surrounding cells. Representative Images were taken at 40X magnification from four independent transient transfection experiments. 148 Figure 3.3-7. Comparison between subcellular localization of wild type (WT) and C219Y mutant of JAM3. C219Y mutated proteins were predominantly retained in the endoplasmic reticulum (ER), with 96% colocalization with ER-calnexin and the level of cell surface or plasma membrane (PM) localization of the C219Y-JAM3 proteins was significantly reduced compared to that of WT-JAM3 as shown in the bar chart. Between 2000 to 4000 cells were counted in multiple fields at a 40X magnification using image J software; values are mean ± SD. 120 % of transfected cells 100 80 60 40 20 0 WT C219Y PM 98.4496124 3.921568627 149 ER 1.550387597 96.07843137 WT was found at the cell surface of approximately 98% of the transfected cells (Figure 3.3-7). 3.3.4. Discussion In this chapter I have investigated three families with multiple affected children exhibiting autosomal recessive HDBSCC. I identified three different, novel, homozygous non-synonymous mutations in JAM3 gene underlying this severe phenotype in all the studied patients. 3.3.4.1. The impact of JAM3 mutations on the protein product Arrate et al [2001] and Santoso et al [2002] cloned and characterized JAM3 as an approximately 43 KDa transmembrane glycoprotein. This type I integral membrane glycoprotein consists of two immunoglobulin (Ig)-like domains with intrachain disulfide bonds at the extracellular region. By sequence analysis, Arrate et al [2001] concluded that the N-terminal domain is a V-type, whereas the membrane proximal domain is a C2-type. Also they predicted that the intrachain disulfide bonds that stabilize each Ig domain are between C53–C115 and C160–C219. Ig-like domains are implicated in a range of vital functions, including cell–cell recognition, cell-surface receptors, muscle structure, and the immune system [Teichmann and Chothia, 2000]. The C- terminal region of the protein is composed of a short transmembrane domain followed by an intracellular domain containing a PDZ binding motif (Figure 3.3-3A). The p.C219Y mutation detected in family 1 alters one of the four highly conserved cysteine residues of JAM3 disrupting one out of two disulfide 150 bonds known to stabilize the Ig-folds of the protein [Arrate et al., 2001; Santoso et al., 2002]. Several functional studies demonstrated that cysteinesubstitutions disrupt the structure of cell surface proteins causing their misfolding, instability, and ER retention [Fukuda et al., 2011; Rudarakanchana et al., 2002]. Consistent with this, comparison of the subcellular localization of flag-tagged C219Y mutants transiently expressed in HeLa (Figure. 3.3-4 and 3.3-6) and COS7 (Figure 3.3-5) clearly showed that most of the mutated protein failed to reach the cell membrane. ER retention is a well-established disease causing mechanism in many human syndromes caused by loss-offunction mutations [Aridor, 2007; Ali et al., 2010; 2011; Chen et al., 2005]. The p.E116K mutation found in family 2 did not seem to affect the subcellular localization or trafficking of the protein (Figure. 3.3-4). However, its position in the Ig-like V-type domain suggests an interfering role of this mutation with the dimerization or with the interaction(s) of this protein with one or more of its interacting partners (Figure 3.3-3A). JAM3 controls TJ maintenance by engaging in homophilic and heterophilic interactions with neighboring JAM molecules [Ebnet, 2008]. Arrate et al [2001] and Santoso et al [2002] predicted the first ATG by homology alignments between JAM3, JAM1, JAM2, and mouse Jam3. Arrate et al [2001] found using SignalP V1.1 the signal sequence of JAM3, which should be cleaved off after glycine 30 from the putative ATG. The mutated methionine in family 3 is predicted to be the first methionine of the JAM3 protein and such mutations almost invariably interfere with the translation of 151 the protein (Figure 3.3-3). However, there are two in-frames ATG’s upstream of this start codon and the improbable use of one of them or for the next inframe ATG would encode a non-functional or unstable protein. 3.3.4.2. Clinical consequences of JAM3 deficiency Mouse Jam3 is expressed at the apical junctions of endothelial cells, smooth muscle cells, fibroblasts, Schwann cells, spermatids, and hematopoietic stem cells [Mochia et al., 2010; Scheiermann et al., 2007]. More recently, Jam3 expression was detected in the TJs of neural stem cells in the embryonic ventricular zone and the adult ependymal cell layer of mouse brain [Stelzer et al., 2012]. More than half of mice with homozygous mutation in Jam3 exhibit postnatal lethality. Jam3 deficient mice exhibit growth retardation, granulocyte muscle homeostasis weakness, defects, abnormal spermatogenesis, electrophysiological defects, and hypersensitivity to mechanical stimuli [Colom et al., 2012; Scheiermann et al., 2007]. Furthermore, mouse Jam3 apical expression was found on the embryonic retinal neuroepithelia, and its deficiency caused nuclear congenital cataracts [Daniele et al., 2007]. The brains of mice lacking Jam3 are abnormal in some strains [Wyss et al., 2012] but relatively unaffected in others [Stelzer et al., 2012]. In humans, Mochida et al [2010] demonstrated that a homozygous mutation in JAM3 is the cause of a severe autosomal recessive HDBSCC syndrome segregating in a consanguineous family. The affected members in this family were born with congenital cataracts and developed severe brain 152 abnormalities from progressive hemorrhagic destruction of the brain tissue, including the cerebral white matter and basal ganglia. Three out of six affected individuals died early in infancy. Brain imaging in all the affected individuals showed calcification in the subependymal region of the brain multifocal intraparenchymal hemorrhage with associated liquefaction, and massive cystic degeneration resulting in large ventricles [Al-Gazali et al., 1999; Mochida et al., 2010]. The phenotype of the children in this study is very similar to the phenotype of the affected children in this previously reported family. All the affected children in this study had bilateral cataracts and develop progressive neurological impairment, associated with seizures in some individuals. In addition, neuroimaging in all patients showed intraparanchymal hemorrhage with evidence of brain destruction and subependymal, basal ganglia, and white matter calcification. These changes are very similar to those found in the original family. Some of the other features described in the original family, such as renal anomalies and liver enlargement, were more variable in this patient group. All the affected children in this report died. Therefore, of the total 10 cases reported so far the disorder was lethal in 7 cases. Death occurred in the first few weeks of life. Those who survived (three in total) are severely retarded with severe spasticity and seizures. Other conditions that could be considered in the differential diagnosis include the syndrome of band-like intracranial calcification, simplified cerebral gyri, and polymicrogyria [Abdel-Salam et al., 2008; O’Driscoll et al., 2010]. 153 This syndrome is caused by mutations in another TJ gene, occludin (OCLN; OMIM *602876). However, patients with this syndrome do not have intracranial hemorrhage or congenital cataract [Mochida et al., 2010]. Furthermore, the patients here did not show any evidence of developmental malformation of the cerebral cortex and polymicrogyria. Similarly mutations in collagen type IV alpha (COL4A1; OMIM*120130) can cause overlapping clinical presentation, which include antenatal intracranial hemorrhage [de Vries et al., 2009], brain small vessel disease with hemorrhage or vascular leukoencephalopathy, porencephaly [Gould et al., 2006; Sibon et al., 2007], and hereditary angiopathy with nephropathy, aneurysms, and muscle cramps [Plaisier et al., 2007]. All these disorders are inherited as autosomal dominant and the clinical presentations are different from the cases in this study. Proliferative vasculopathy and hydranencephaly–hydrocephaly syndrome (Fowler syndrome) is an autosomal recessive perinatal lethal disorder characterized by hydrocephalus associated with progressive destruction of the central nervous system (CNS) tissue as a result of an unusual and characteristic proliferative vasculopathy [Fowler et al., 1973]. The hallmark of the syndrome is the microvascular proliferation, which is associated with extensive necrosis and calcification of the CNS tissue. There are no visceral malformations [Lalonde et al., 2010]. It is caused by mutations in the FLVCR2 gene (OMIM *610865). Clinically affected fetuses present with fetal akinesia deformation sequence with muscular neurogenic atrophy [Fowler et al., 1973; 154 Meyer et al., 2010], which is different from the clinical presentation of the cases in this study. 3.3.5. Conclusion In this study, I identified several non-synonymous mutations scattered throughout the coding sequence of the JAM3 gene affecting different domains of the protein product. However, the affected children shared similar lethal phenotype indicating homogeneity of this disorder at the clinical and molecular level. This is likely due to the total loss of the protein function as a result of those mutations. Therefore, this study confirms the importance of JAM3 as a component of the junctional complexes and its deficiency leading to a distinctive and catastrophic neonatal presentation of cataracts and hemorrhagic destruction of the brain. Furthermore, elucidation of the genetic basis of this disorder is of clinical benefit to patients and families. Identification of pathogenic mutations can confirm the clinical and biochemical diagnosis and can also contribute to accurate genetic counseling, prenatal diagnosis and preimplantation genetic diagnosis. Molecular characterization of this syndrome may also provide a basis for the future development of effective therapeutic strategies. 155 SECTION 4: LINS and TTC23 are candidate genes for autosomal recessive intellectual disability 156 3.4.1. Background Intellectual disability (ID) is a health condition characterized by low intelligence and associated limitations in adaptive behavior. ID is a highly heterogeneous condition and one of the most important socio-economic health care problems worldwide [Ropers, 2008]. Molecular karyotyping is the first diagnostic test for congenital ID as most severe cases occur due to chromosomal abnormalities [Ropers, 2010]. High resolution comparative genomic hybridization (CGH) was developed to detect pathogenetically relevant deletions and duplications too small to be detectable by conventional karyotyping [Miller et al., 2010]. Sequencing, on the other hand, has become the method of choice to diagnose causes of ID that cannot be explained by routine karyotyping or CGH [Topper et al., 2011]. During the past decade, hundreds of defective genes have been identified to be the underlying causes of ID [van Bokhoven et al., 2011]. Different modes of Mendelian inheritance have been reported to cause ID. The vast majority of cases were inherited as autosomal recessive traits in consanguineous populations [Ropers, 2010]. 3.4.2. The purpose of the study Several autosomal recessive ID genes have been identified from investigating consanguineous families using the concept of homozygosity mapping and candidate gene sequencing approaches [Cantagrel et al., 2010; Garshasbi et al., 2011], and more recently using both homozygosity mapping and exome sequencing [Ali et al., 2012a, 2012b; Martinez et al., 2012; Schuurs-Hoeijmmakers et al., 2012]. Therefore, this study was carried out to 157 discover a potentially novel gene causing non-specific ID of autosomal recessive mode of inheritance in two children from a consanguineous family from the UAE. The molecular defect in this family was investigated using homozygosity mapping and whole-exome sequencing (WES). 3.4.3. Results 3.4.3.1. Clinical assessment of patients The parents of the two affected children are Emirati first cousins once removed of Yemeni origin (Figure 3.4-1A). They have 2 children; both of them are affected by intellectual disability. In the family history the father’s brother had a child who died at 6 months of age of unknown cause and a 14 year old child with intellectual disability of unknown etiology. No further information was available on this child and we were unable to evaluate her because she lives in Yemen. The first child of this family is a boy and currently aged 9 years (II-1; Figure 3.4-1A). The pregnancy was complicated by gestational diabetes and mild hypertension, delivery was induced but otherwise was normal. His birth weight was 3000 gm but no other measurements were available. The neonatal period was complicated by poor feeding requiring admission to the Special Baby Care Unit (SCABU) for several days. At the age of 9 months he was not responding to the mother and was noted to have head nodding and repetitive rotatory hand movements. The hand movements disappeared but the head nodding has continued till now. He crawled at the age of 16 months and walked at the age 19 months but he still has no speech. He was 158 extremely hyperactive with aggressive destructive behavior for which he required medications to calm him down. There was no history of seizures. Examination at the age of 8 years revealed a weight of 18 kg (<3rd centile) and height of 105 cm (<3rd centile), and head circumference of 51 cm (<50th centile). He had a slightly flat mid-face with a depressed nasal bridge (Figure 3.4-1B-1) otherwise no other dysmorphic features were noted. He was continuously nodding his head from side to side. His neurological examination was normal. EEG and skeletal survey examinations were reported to be normal. His brain MRI showed right frontal lobe vascular malformation with cortical and subcortical distribution. No associated cortical abnormalities were observed. There was no hemorrhage or gliosis and MRI Spectrometry was normal. Blood and urine amino acid and organic acid screening, thyroid function tests, mucopolysaccharides screening, transferrin isoelectric focusing, very long chain fatty acids and phytanic acids, Fragile X mutation, MECP2 gene analysis were all normal. CGH microarray analysis was normal. The second child is a 3 year old female (II-2; Figure 3.4-1A; 3.4-1B-2). She is the product of normal pregnancy and delivery. Her birth weight was 2950 g. The mother noted head nodding in the first few months of life. She was hypotonic and had head lag at the age of 7 months. All her developmental milestones were delayed. She walked at 20 months of age and she has no speech till now. Examination at 7 months revealed mild flattening of the midface. No other dysmorphic features were noted. Neurological examination revealed hypotonia with head lag, side to side head nodding, otherwise there 159 Figure 3.4-1. Clinical Data of the studied family. A) Pedigree showing the mode of inheritance for an autosomal recessive intellectual disability phenotype in a consanguineous family from United Arab Emirates. The studied members are indicated by numbers and asterisks. B) Appearance of the two affected sibs, 1) II-1 at age 8 years and 2) II-2 at age 3 year. Note the flattening of the mid face. 160 were no other abnormalities. EEG showed bilateral centro-temporal discharge without generalization. Her brain MRI was normal. Creatine phosphokinase (CPK), uric acid, lactate, urine and blood amino acids and organic acids were normal. Transferrin-isoelectric focusing and very long chain fatty acids and phytanic acids were normal. CGH microarray showed interstitial deletion of 4 oligonucleotide probes at 7p22.1 spanning approximately 197 kb. However, testing the parents showed that the mother has these changes and the other affected child did not have them, indicating that this deletion is not related to the phenotype. At 3 years her weight was 13 kg (3rd centile), height 92 cm (3rd centile) and head circumference 48 cm (3rd centile) (Figure 3.4-1B-2). 3.4.3.2. Genome-wide linkage analysis revealed four homozygous regions The results of the genome-wide SNP genotyping and linkage analysis for the studied pedigree are illustrated in Table 3.4-1 and Figure 3.4-2. The genotyping data showed four blocks of homozygosity shared between the two affected members of the studied family (Figure 3.4-2A). One block mapped to chromosome 8 between rs7388114 and rs4738955 flanking a 2.5 Mb genetic interval (8q12.1-q12.3) (Figure 3.4-2B). The second block of homozygosity mapped to chromosome 10 between rs293303 and rs10994485 flanking a 9.2 Mb genetic interval (10q21.1-q21.2) (Figure 3.4-2C). Blocks of homozygosity were observed on chromosome 13, which together comprised 19.9 Mb genetic intervals (13q21.2-q33.3) (Figure 3.4-2D, Table 3.4-1). The last block of homozygosity detected was found on chromosome 15 spanning 3.8 Mb 161 162 60,741,289 53,237,721 61,358,370 100,588,543 107,638,292 97,559,785 8 10 15 Total 13 Start Chromosome 63,260,182 62,487,332 74,891,328 105,390,934 109,171,572 101,322,542 End rs7388114 rs293303 rs3119852 rs7994515 rs11616550 rs1588752 SNP start rs4738955 rs10994485 rs9543569 rs773354 rs11618877 rs11637451 SNP end Genetic interval q12.1-q12.3 q21.1-q21.2 q21.2-q22.1 q32.3-q33.2 q33.3 q26.2-q26.3 2,518,893 9,249,611 13,532,958 4,802,391 1,533,280 3,762,757 35,399,890 Length (kb) Table 3.4-1. Intervals of shared homozygosity between the two affected individuals of the studied family. 24 163 89 17 33 Genes 163 A Figure 3.4-2. Genome-wide homozygosity in the studied family. Genotyping all the family members using Affymetrix SNP 6.0 array revealed multiple regions of shared homozygosity between the two affected sibs. A) HomozygosityMapper view of the Genome wide homozygosity analysis results (Max homozygosity score: 2000, 868063 markers). B) Zoom in view into the homozygous region detected on chromosome 8 (q12.1-q12.3), C) Zoom in view into the homozygous region detected on chromosome 10 (q21.1-q21.2), D) Zoom in view into the homozygous region detected on chromosome 13 (q21.2-q22.1), (q32.3-q33.2) and (q33.3).E) Zoom in view into the homozygous region detected on chromosome 15 (q26.2-q26.3). B 164 C 165 D 166 E 167 between rs1588752 and rs11637451 (Figure 3.4-2E). The parents were heterozygous at all the homozygous segments. 3.4.3.3. Whole-exome sequencing identified two splicing mutations in LINS and TTC23 genes The co-segregating homozygous segments together encompass around 163 genes (Table 3.4-1). In order to reveal the molecular basis of the ID in the studied family, WES was carried out on the two affected children. A minimum of 79.70% of the on-target regions were covered to a depth of at least 20x. Around 45,800 variations from the reference genome were identified (Table 3.4-2). Among these 3,500 novel variants were recognized and approximately 700 variations indicative of serious consequences in coding sequences were found. Across the variations, 160 variants were found to be homozygous, of which only two were shared between the two affected children. Both variants were within the same homozygous region on chromosome 15q26. Both were splicing mutations affecting a splice donor in LINS (NM_001040616.2: c.1219_1222+1delAAAGG) and a splice acceptor in TTC23 (NM_001040655.1:c.456-1G>T) (Figure 3.4-3, Figure 3.4-4). Both variants were confirmed to be homozygous by Sanger sequencing in the two affected children, heterozygous in parents and not found in 200 healthy controls with matching ethnic origin (Figure 3.4-5). 3.4.3.4. The two splicing mutations were confirmed to be deleterious at the mRNA level of both genes To investigate the consequences of the molecular defect caused by the detected splicing mutation, reverse transcription-PCR was performed 168 169 ¥ including those in dbSNP release 132. filtering out those contained in dbSNP release 132. * Number of Variations Variations Overlapping Genes Variations Overlapping Transcripts Variations Overlapping Regulatory Regions Variations Overlapping Protein Domains Intergenic Variations Variations With Predicted Serious Consequences Variations With Other Predicted Consequences Homozygous Variations With Predicted Serious Consequences All Variants* II1 II2 45,912 45,795 45,565 45,476 45,565 45,476 9,314 9,223 30,375 30,524 347 319 10,050 9,940 43,154 43,039 8122 8096 Table 3.4-2. Summary metrics of all and novel variants identified by the exome sequencing. Novel Variants¥ II1 II2 3,658 3,424 3,596 3,365 3,596 3,365 876 804 2,425 2,261 62 59 793 680 3,418 3,211 165 160 170 Figure 3.4-3. Integrative Genomics Viewer (IGV) visualization of homozygous mutation c.1219_1222+1delAAAGG in LINS gene from exome data. All reads show 5 bp deletion, sequence of wild type gene and exon annotation at bottom. The adjacent homozygous substitution G>A (C>T on reverse strand) is a common variant rs12719734G>A. 171 Figure 3.4-4. Integrative Genomics Viewer (IGV) visualization of the second novel mutation detected in this study by whole exome sequencing. A homozygous splicing mutation c.456-1G>T has been identified in the two affected sibs on Chr15:99758919C>A in TTC23 gene. Read depth= 218. Figure 3.4-5. Sanger sequencing verification of the c.1219_1222+1delAAAGG mutation in LINS gene. DNA sequencing chromatograms confirmed the segregation of the AAAGG (inside the brown square) deletion detected by exome data with the assessed phenotype. The deletion was found to be homozygous in the patients (II1 and II2) and heterozygous in parents (I1 and I2). The deletion was not found in 100 normal controls. The rs12719734G>A (designated with a red star) was found in all the screened individuals. Control Parent Patient 172 using total RNA isolated from a normal control, parents and patients’ leukocytes as templates (Figure 3.4-6). The control sample (Con.; Figure 3.46) showed multiple bands at around 1000 bp indicating the presence of multiple transcripts for this gene in leukocytes. On the other hand, the two patients showed similar multiple bands patterns, albeit at lower sizes of around 400 bp (Figure 3.4-6-II-1 and –II-2). The parents showed both the upper and the lower multiple bands which is consistent with being heterozygous carriers for the predictable splicing aberration (Figure 3.4-6-I-1 and –I-2). To further characterize the spliced products, I gel-purified all the PCR products and sequenced them using Sanger sequencing. The analysis demonstrated that in the normal control the upper band (1014 bp) represented the NM_001040616.2 cDNA fragment spanning from exon 3 to exon 6 (Figure 3.4-7). In addition, the higher band was accompanied by at least two bands recognized to be alternatively spliced transcripts that lacked some parts of exon 6 (Figure 3.4-8). The exon-intron 5 splice defect mutation present in the patients’ gene caused the skipping of exon 5 resulting in a smaller sized band (423 bp) noted in the parents and patients but not in the normal control (Figure 3.4-7). This was also accompanied by bands of lower sizes representing multiple transcripts for the mutated allele (Figure 3.4-8). As indicated above, these additional splice variants that lack parts of exon 6 are also present in the control DNA and therefore not related to the pathogenic phenotype. 173 On the other hand, sequening of the TTC23 cDNA in all the family members revealed a deletion of 7 nucleotides at the beginning of exon 7 (Figure 3.4-9). The deletion was predicted to cause a frameshift in the nascent transcript introducing a premature termination codon and subsequently could be eliminated by the cellular nonsense mediated decay. 3.4.3.5. To The impact of exon 5 skipping in LINS on the mRNA expression investigate the effect of the identified mutation c.1219_1222+1delAAAGG on the expression of LINS mRNA, total RNA was extracted from the leukocytes of all the family members and healthy controls. Subsequently, the expression of LINS mRNA was quantified by real timePCR using specific Taq-man probe spanning exons 3 and 4 of LINS (NM_001040616.2; Hs01089850_m1). The levels of LINS mRNA were unexpectedly more than 2-fold higher in patients compared to the normal controls (Figure 3.4-10, Appendices E1 and E2). LINS mRNA expression in parents was around 1-fold higher than a normal control and around 1-fold less than the patients’ as expected in heterozygous carriers (Figure 3.4-10, Appendices E1 and E2). Katoh [2002] characterized human LINS (NP_001035706.1) and mouse Lins by their similarity with Drosophila lines. The two proteins shared a homologous domain with Drosophila lines with the human protein consisting of 757 amino acids (aa). Translating NM_001040616.2 lacking exon 5 by Expasy translate tool predicted a truncated protein lacking 197 amino acid (p.Glu211_Lys407del). Most of these deleted amino acids are evolutionarily 174 175 Figure 3.4-6. Exon skipping analysis by reverse transcription-PCR. Agarose gel of RT-PCR reaction products from LINS cDNA amplification in a control (con), parents (I-1, I-2) and patients (II-1, II-2) compared to a DNA 100 bp ladder (M). The gel showed a ~1014 bp band of the wild type LINS transcript encompassing exon 5 in a normal control (con) accompanied with multiple isoforms of varying length (~1000 bp). In patients’ (II-1, II-2) lanes, only smaller bands were seen (~400 bp) suggesting a homozygous deletion of around 600bp. The parents (I-1, I-2) have both upper and lower bands suggesting that they carry the 600 bp deletion in a heterozygous state. 176 Figure 3.4-7. Schematic diagram of the splicing defect seen in patients based on Sanger sequencing data of the cDNA. The upper most bands of the higher and lower bands seen in RT-PCR gel were purified and sequenced. This higher band noticed in control and parents was found to include exons 3, 4, 5 and 6. On the other hand, Exon 5 (E5) was found to be missing in the lower-size band seen in both patients and parents. These results suggested that the genomic deletion at the end of E5 abolished a canonical splicing site masking the exon from the splicing machinery which considered it to be part of intron 4 and cut it out of the nascent mRNA. 177 Figure 3.4-8. Proposed alternative splicing variants of LINS. Analyzing the accompanying upper and lower bands amplified by RT-PCR suggested the presence of at least 3 LINS transcripts alternatively spliced in exon 6 with the putative multiple splice junctions are shown. All the lower bands lack exon 5 while the upper bands include it compared to the RefSeq NM_001040616.2. Figure 3.4-9. Sequencing resuts of the TTC23 cDNA. Sequencing chromatograms showing the homozygous deletion of 7 nucleotides ATTTAAG (inside the brown square) in the beginning of exon 7 of TTC23 mRNA in the patients. The deletion was heterozygous in the parents and absent in a normal control. Control Patient Parent 178 Figure 3.4-10. Expression analysis of the homozygous c.1219_1222+1delAAAGG mutation in LINS gene. Relative quantification (RQ) of LINS mRNA level was evaluated through Real time PCR. The expression of LINS mRNA in the patients’ leukocytes was found to be around 2-fold higher than its expression in healthy controls. The level of LINS mRNA in parents was also abnormal. LINS mRNA levels were quantified in two healthy controls, the two patients as well as the parents in duplicates and in two different experiments. RQ values were calculated by the SDS software. The normalized RQ values represented in the bar graph are the mean values ± SD. 3.5 Relative Expression of LINS mRNA 3 2.5 2 1.5 1 0.5 0 RQ values Healthy Controls 0.938155 179 Patients Parents 3.060203791 2.197383523 180 the patients. The representative alignments were adopted from NCBI HomoloGene (http://www.ncbi.nlm.nih.gov/homologene) and are showing that this part of LINS protein is highly conserved signifying its importance. Figure 3.4-11. Conservation across species of the amino acids that are predicted to be deleted from LINS protein in conserved across species (Figure 3.4-11), suggesting an important role for this domain in the protein structure and/or function. Part of the deletion (30 aa) lies within the Drosophila lines homologous domain found by Katoh [2002]. The deletion also included Lys407 which is found experimentally to be a potential regulator of the protein ubiquitination and the subsequent regulation of its proteasome-mediated degradation [Kim et al., 2011]. 3.4.4. Discussion As mentioned in the introduction of this chapter, the clinical features of inherited ID are not conclusive and may associate with different genetic defects. Therefore, several studies were carried out to map the locus of a nonspecific autosomal recessive ID [for example, Abou Jamra et al., 2011] and to elucidate the genes underlying ID [for example, Najmabadi et al., 2011]. In this study, I studied two siblings, a male and a female with early onset ID. I mapped the disease locus and identified a novel five nucleotide homozygous deletion in LINS gene and a splicing mutation in the TTC23 gene in both patients. The mutation in LINS affects a donor splice site leading to exon skipping and a large deletion in the expressed transcripts. On the other hand, the mutation in TTC23 affects an acceptor splice site leading to a frameshift and a premature termination of the corresponding transcript. However, LINS has been favored over TTC23 as indicated in the next section because it has been recently reported as a disease-causing gene for an autosomal recessive ID phenotype in an Iranian family [Najmabadi et al., 181 2011]. However, the possible involvement of TTC23 or both LINS and TTC23 in the phenotype can not be categorically excluded. In humans, LINS was described in 2002 by Katoh as a protein containing Drosophila lines homologous domain [Katoh, 2002]. The author detected LINS 2.8 kb-transcript (NM_001040616.2) in human fetal brain and kidney. However, since then not many experiments have been performed to characterize human LINS further. However, the association between LINS and autosomal recessive ID was inferred from two lines of evidence in the literature. The first clue was deduced from a homozygosity mapping study that was performed on 64 Syrian consanguineous families with autosomal recessive ID [Abou Jamra et al., 2011]. This study has revealed 11 novel ID loci including the locus on chromosome 15q23-26 in one of the studied families. The mapped family was a big family with 8 children in total of whom 5 were exhibiting moderate ID, delayed speech and epilepsy. The other evidence of association between LINS and ID was noticed in another collaborative study that was carried out on 136 consanguineous Iranian families with autosomal recessive ID [Najmabadi et al., 2011]. The authors combined homozygosity mapping and targeted exome sequencing of the mapped regions to unravel the molecular basis of ID in these families. This study has revealed new mutations in 23 genes previously implicated in autosomal recessive ID, and potentially disease causing variants in 50 novel candidate genes including LINS (OMIM*610350). The authors identified a homozygous deletion of four 182 nucleotides in LINS exon 5 (NM_001040616.2:c.985_988delCATG). This deletion was predicted to cause a frame shift producing a truncated protein (p.His329*). The mutation was found in four affected children of consanguineous parents exhibiting microcephaly and early onset ID. The patients under investigation here had no microcephaly but showed ID and head nodding as the only clinical features. In addition the deletion identified in this study in LINS is also predicted to produce a truncated protein that may be functionally abnormal or structurally unstable. This might explain the lack of a negative feedback control over LINS gene expression as seen in patients and to a lesser extent in their parents (Figure 3.4-10). Moreover, both mutations affected all the three known isoforms of LINS, while other reported frame shift mutations p.D511Ffs*10, p.S594Ffs*6, and p.Q717* reported in the exome variant database affected only one isoform (Appendix-F) In addition, TTC23 is a cervical cancer proto-oncogene 8 protein that was found to be associated with cervical cancer and cervicitis (GeneCards database). Consistently, TTC23 and similar proteins were predicted and demonstrated experimentally to be cell cycle regulators [Warnatz et al., 2011; Izumiyama et al., 2012]. In addition, TTC23 is not expressed in fetal brain (NextBio database.com). Yet, the TTC23 can not be excluded as a candidate for the ID phenotype in this family or any other less obvious cosegregating phenotype. 3.4.4.1. Lines/LINS is important for segment polarity in Drosophila and plays a putative dual role in WNT canonical pathway 183 LINS (formerly known as WINS1 or LINS1) is the human homologue of the Drosophila segment polarity gene lin [Katoh, 2002]. Lines is the protein product of lin which was originally identified in Drosophila melanogaster in the 1980s [Nüsslein-Volhard and Wieschaus, 1980; Nüsslein-Volhard et al., 1984]. Drosophila studies revealed that lines is an essential protein for patterning and morphogenesis of Drosophila dorsal epidermis [Bokor and DiNardo, 1996; Hatini et al., 2000; Hatini et al., 2005], hindgut [Iwaki et al., 2001, Green et al., 2002; Johansen et al., 2003] and muscles [Volk and VijayRaghavan, 1994]. Lines was also found to play an important role in the development of Drosophila wings [Nusinow et al., 2008; Benítez et al., 2009], and testis [DiNardo et al., 2011]. Lines is believed to be a transcriptional regulator, playing a dual role as both an activator and repressor of downstream target genes listed in Table 3.4-3 [Hatini et al., 2000, Iwaki et al., 2001]. Hatini et al [2000] demonstrated that lines is essential for late wg signaling activity in the developing dorsal epidermis, acting downstream of arm but upstream of wg target genes (Figure 3.4-12). The author showed that with wg signaling, lines accumulates in the nucleus to modulate transcription of wg and ve (veinless) that are known targets of wg signaling. The author also proved that there is an interaction between lines and Drosophila hedgehog (hh) which exports lines from the nucleus to the cytoplasm antagonizing wg signaling. During Drosophila embryogenesis, lines was found to be implicated in dorsal muscle patterning by regulating groovin expression [Volk and VijayRaghavan, 1994]. In the developing hindgut, Iwaki 184 et al [2001] demonstrated that lines promotes the expression of genes of the large intestine (otp, dpp, en, and dri), and represses the expression of genes of small intestine (hh, upd, and ser). Castelli-Gair [1998] proposed lines to be a transcriptional cofactor for Abdominal-B for the activation or repression of its downstream target genes. These genes include cut that represses a neural cell fate, spalt that affects the development of the fly's gut, and ems which is necessary for proper head formation and is also involved in brain morphogenesis (http://flybase.org/) [Castelli-Gair, 1998]. It was shown that lines is part of a molecular regulatory pathway composed of drm, an inhibitor of lines by exporting it to the cytoplasm, and bowl a downstream target of lines in the nucleus [Hatini et al, 2005]. Interestingly it was observed that, hh promotes drm expression, while wg represses drm expression regulating the drm/lines/bowl pathway which consequently regulates the patterning and cell rearrangement in the Drosophila embryonic epidermis, foregut, hindgut, gonads and imaginal disc [Iwaki et al., 2001; Hatini et al., 2005; Nusinow et al., 2008; DiNardo et al., 2011]. In the developing wing, Benítez et al [2009] noticed that bowl protein represses Wg pathway and activates Notch (N) and Hh pathways. Therefore, they concluded that lines is essential for normal functioning of Wg/WNT, Hedgehog (Hh) and Notch (N) pathways during embryogenesis in Drosophila. In the Drosophila testis, lin mutant cells were not differentiating into cyst stem cells (CySC) and expressed niche cell fate markers hh and cactus [DiNardo et al., 2011]. The observation suggested that 185 186 Dorsal Epidermis rho (ve) rhomboid Hindgut Hindgut Hindgut/Posterior spiracles Hindgut Hindgut otp dpp en retn (dri) hh orthopedia decapentaplegic engrailed retained hedgehog wingless Tissue/ System Dorsal Epidermis Gene symbol wg Gene name Down Up Up Up Up Down Up/Dow n Up SHH - EN1 EN2 SMAD3 OTP PARL and Human homologue WNT1 This gene encodes a protein that is crucial in Both genes encode homeodomain-containing transcription factors that have been implicated in the control of mid-hindbrain pattern formation during embryogenesis. En1 deficient mice lack most of the cerebellum and midbrain, whereas En2 mutants survive with cerebellar defects. This protein functions as a transcriptional modulator thought to play a role in the in neural stem cells where it is essential to activate TGFβ-responsive genes activating the neural developmental program. This gene encodes a mitochondrial integral membrane protein that plays an important regulatory role in mitochondrial-mediated apoptosis. Parl knockout mice undergo progressive multi-tissue atrophy, including atrophy in the thalamus and striatum, mediated by increased apoptosis. This gene encodes a homeodomain-containing transcription factor that is implicated in the development of the brain, specifically hypothalamus, in vertebrates. Otp knockout mice displayed progressive impairment of crucial neuroendocrine developmental events. Putative roles in the development of the central nervous system in vertebrates WNT1 protein involved in the proliferation and differentiation of neural progenitors. Wnt1 deficient mice embryos have showed severe abnormalities in the development of the midbrain and cerebellum. Table 3.4-3. The reported downstream target genes of lines in Drosophila. [Iwaki et al., [Iwaki et al., 2001] 2001; [Iwaki et al., 2001; Hanks et al. 1995; Orvis et al., 2012] [Iwaki et al., 2001; Estarás et al., 2012] [Iwaki et al., 2001; Acampora et al., 1999; Del Giacco et al., 2008; García-Moreno et al., 2010] [Hatini et al., 2000; Cipolat et al., 2006; Jones et al., 2008] [Hatini et al., 2000; Thomas and Capecchi, 1990; Marei et al., 2012] References 187 Dorsal epidermis/ muscle Testis bowl ct salm ems sr cact Cut Spalt major empty spiracles Stripe or Groovin Cactus Dorsal epidermis/ foregut/ hindgut/ gonads/ imaginal disc Posterior spiracles Posterior spiracles Posterior spiracles Hindgut Brother of odd with entrails limited Serrate Hindgut os (upd) ser outstretched Down Up Up Up Up Up Down Down NFKBIA - EMX2 - - - This gene encodes a member of the nuclear factorκB (NF-κB) inhibitor family that is involved in inflammatory responses. NF-κB pathway plays a significant role in neurite outgrowth, activitydependent plasticity, and cognitive function. NFKBIA is often deleted in glioblastomas. The encoded protein is expressed in the dorsal telencephalon during development and is involved in regional patterning of the neocortex into defined functional areas. Emx2 deficient mice displayed defects in archipallium structures that are believed to play essential roles in learning, memory and behavior. [DiNardo et al., 2011; Bredel et al., 2011; Roussos et al., 2013] [Volk and VijayRaghavan, 1994 [Castelli-Gair, 1998; Yoshida et al.,1997; Zembrzycki et al., 2007] [Castelli-Gair, 1998] [Castelli-Gair, 1998] [Hatini et al., 2005] [Iwaki et al., 2001; Sander et al., 2003; Stump et al., 2002; Mead and Yutzey, 2012] JAG2 The encoded protein is one of several ligands that activate Notch and related receptors. It was found in most neuron subtypes. Notch signaling plays a pivotal role in the regulation of vertebrate neurogenesis and brain development. [Iwaki et al., 2001] Komada, 2012] - patterning and cell-fate specification, particularly in the central nervous system. SHH plays different roles depending on its concentration, area, and timing of exposure. 188 Figure 3.4-12. Lines/LINS plays a putative dual role in WNT canonical pathway. In WNT canonical pathway, the absence of a signal leads to the hyper-phosphorylation of arm/CTNNB1 leading to its ubiqitination and degradation by the proteasome in the cytoplasm. Binding of wg/WNT1 ligand to an Fz and arr/LRP receptor complex leads to stabilization of hypo-phosphorylated arm/CTNNB1, translocating it to the nucleus. In the nucleus, arm/CTNNB1 competes with and displaces gro/TLE interacting with pan/TCF proteins to activate transcription. In Drosophila, lines/LINS was found to act as a modulator of wg/WNT canonical pathway acting in parallel with or downstream of arm/CTNNB1 in response to wg/WNT1 signaling to enhance or represses the transcription of target genes. Frizzled (Fz), arrow (arr), LDL receptor-related protein (LRP), armallido (Arm), β-catenin (CTNNB1), groucho (gro), transducin-like enhancer of split (TLE), pangolin (pan), T-cellfactor (TCF). lines represses niche fate and promotes CySC fate antagonizing Bowl and N pathways which promotes niche cell fate. 3.4.4.2. LINS in the cognitive pathways Numerous studies have revealed that correct corticogenesis is an outcome of the interplay between multiple signaling pathways including Wg/WNT, Hedgehog (Hh) and Notch (N) pathways [Lei et al., 2006; Tang et al., 2010; Ulloa and Martí, 2010; Dave et al., 2011; Roussel and Hatten, 2011; Marei et al., 2012; Wilson and Stoeckli, 2012]. This crosstalk provides mitogenic signals, positional information, migratory cues and differentiation signals [Dave et al., 2011]. In addition, the coordinated interaction between these critical pathways is a prerequisite for the precise regulation of symmetric/asymmetric division during neurogenesis in the developing vertebrate central nervous system (CNS). Many of these pathways were first identified in genetic studies in Drosophila [Roussel and Hatten, 2011]. Mammalian orthologs were subsequently identified and genes within the pathways have been cloned and studied. However, the exact outcomes of these interactions are not fully understood. In addition, not all the interactive players or factors that affect the number and type of divisions that a neocortical progenitor cell undergoes are known In Drosophila, lines (the homologue of LINS) has been recognized to be a tissue- and a stage-specific modulator of wingless signaling [Hatini et al., 2000]. Lines was found to be activated by Drosophila wingless (wg) [Hatini et al., 2000]. Wingless-type MMTV integration site family-1 (WNT1) is the 189 human homologue of the Drosophila wg and its discovery led to the subsequent elucidation of the WNT pathway [Saito-Diaz et al., 2012]. The activation of the canonical wingless/WNTsignaling pathway occurs through the binding of wg/WNT ligand to the seven-pass transmembrane Frizzled (Fz) receptor and its co-receptor, the arrow (arr)/low-density lipoprotein receptor related protein (LRP) (Figure 3.4-11) [MacDonald and He, 2012]. This binding stabilizes the cytosolic co-activator armadillo (arm)/β-catenin1 (CTNNB1) and its translocation to the nucleus [Saito-Diaz et al., 2012]. Thus, leading to competitive displacement of groucho (gro)/transducin-like enhancer of split (TLE) from the transcription factors pangolin (pan)/T cell-specific transcription factor (TCF) initiating the transcription of the pathway target genes. WNT1 is secreted from a signaling center located at the boundary between prospective mid and hindbrain (mid-hindbrain boundary) and mediate development of these two brain regions [Wittmann et al., 2009]. Disturbed WNT pathways due to inherited mutations in positive and negative regulators of signaling have been reported to cause autosomal recessive ID [Ekici et al., 2010; Buchman et al., 2011]. Therefore, my finding that a mutation in another regulator of the WNT signaling pathway is responsible for a form of recessive ID further illustrates the importance of this pathway in human cognition and/or brain development. 3.4.5. Conclusions Inherited ID conditions are a group of genetically heterogeneous disorders that lead to variable degrees of cognition deficits. It has been shown 190 that inherited ID can be caused by mutations in over 100 different genes and there is evidence for the presence of as yet unidentified genes in a significant proportion of patients. Therefore, I aimed in this study to identify the defective gene underlying an autosomal recessive ID in two sibs of an Emirati family. Using a combined approach involving homozygosity mapping and whole-exome sequencing I have identified two possible disease-causing genes (LINS and TTC23). The detected mutations in both genes were demonstrated to be damaging to the mRNA. However, fly studies and human mutations suggested LINS as the most likely causative. However, more ID cases are needed to confirm pathogenicity. The direct benefit of these findings is to facilitate the prenatal and preimplanting diagnosis for this consanguineous couple to have a healthy child. In addition, this study shed light on new genes in the cognition pathway and further highlights the importance of WNT pathway in cognitive development. Future identification of LINS mutations may expand the phenotypic spectrum, provide further insight into genotype-phenotype correlations and facilitate molecular investigation of the consequences of LINS dysfunction. Understanding LINS-related disease mechanisms may then facilitate the development of novel therapeutic strategies. 191 SECTION 5: Characterizing the genetic basis of a clinically heterogenous autosomal recessive congenital muscular dystrophy in a highly inbred Arab family 192 3.5.1. Background Congenital muscular dystrophy (CMD) is a clinically and genetically heterogeneous group of inherited muscle disorders [for review see Bertini et al., 2011]. CMDs are present at birth. Affected infants typically have severe hypotonia, weakness, feeding difficulty, and respiratory insufficiency. Joint contractures can be present at birth or develop as muscles weaken. Spinal rigidity and later on spinal deformity such as scoliosis are common manifestations in the affected children. Muscle weakness is progressive. Infants may die early due to respiratory or feeding problems. The classification for the CMD subtypes is mainly based on the gene in which disease-causing mutations occur and the protein encoded by that gene [Sparks et al., 2001; Bertini et al., 2011]. The main subtypes of CMD are: 1) Laminin-α2–deficient CMD (MDC1A), 2) Collagen VI-deficient CMD, 3) Dystroglycanopathies, 3) SEPN1-related CMD (previously known as rigid spine syndrome, RSMD1) and 4) LMNA-related CMD (L-CMD). Several less common CMD subtypes have been reported in a limited number of individuals with unidentified molecular cause. Most of the CMD subtypes are recessively inherited yet dominant inheritance is also established in some subtypes. A phenotypic classification for the CMD subtypes can be also used with cognitive impairment ranging from severe mental retardation to mild developmental delay, structural brain and/or eye anomalies, and seizures are found almost exclusively in the dystroglycanopathies [Sparks et al., 2001]. While, laminin alpha-2-deficient CMDs are characterized by white matter 193 abnormalities without major cognitive involvement [Sparks et al., 2001]. However, the phenotypes of the CMD subtypes overlap significantly even though the genetic defect may differ. Also mutations in one of the so far identified CMD genes can be associated with a spectrum of clinical phenotypes. 3.5.2. The purpose of the study Accurate diagnosis of CMD subtypes has proven difficult because of their clinical and genetic heterogeneity [Valencia et al., 2013]. Also, many CMD patients do not have a mutation in one of the known genes, meaning that many more genes could be involved [Valencia et al., 2013]. Therefore, this study was initially performed to identify an anticipated novel gene underlying an autosomal recessive CMD in a consanguineous family from the UAE. 3.5.3. Results 3.5.3.1. Clinical assessments of patients The parents of the affected children in this family are first cousins from Sudan. They have a total of six children; two of them are affected (Figure 3.51). The parents are normal. In the family history the mother’s sister who married her cousin had ten children; six of them died early in infancy of unknown reasons. No further information was available on these children and we were unable to evaluate them because they live in Sudan. The index case (II4) is a girl. She is the product of a normal pregnancy with normal term delivery. A large head was diagnosed prenatally. Her birth 194 weight was 3000 g (25th percentile). At birth a large head was confirmed with a head circumference of 36cm (75th percentile). She was also noted to have a nystagmus and bilateral cataract which was operated on. Developmental delay and hypotonia were noted in the first year of her life. In addition, there were generalized seizures which were controlled with anticonvulsants. Evaluation at the Genetic Clinic at the age of 5 years revealed a head circumference of 52.7 cm (90th percentile), hypotonia with contractures of the knee, ankle, elbow, wrist and interphalangeal joints and severe psychomotor retardation. The sister of the index case (II-3) is also the product of a normal pregnancy and delivery. Her birth weight was 3000 g (25th percentile). She was noted to have a left sided cataract which was operated on in the first year of life. At two weeks the child developed left sided seizures which responded to anticonvulsants. Evaluation at the age of 10 years revealed a severely globally retarded girl, wheelchair bound with contractures of most large and small joints. Her creatine phosphokinase (CPK) was elevated. The magnetic resonance imaging (MRI) of the brain at the age of 13 years showed several structural brain abnormalities. The corpus callosum was hypoplastic and there was diffuse gliosis within the fronto-temporal lobes. There was a dilatation of all ventricles with a decrease in the amount of white matter. The posterior cranial fossa was shown to be enlarged with cystic formation widely communicating with the fourth ventricle. The cerebellum and vermis appeared hypoplastic. 195 196 II I 1 1 2 3 4 5 2 6 4 Figure 3.5-1. Pedigree of the studied family. The Pedigree is showing the mode of inheritance for an autosomal recessive congenital muscular dystrophy phenotype in an Arab consanguineous family. 3.5.3.2. Molecular genetic investigations 3.5.3.2.1. Homozygosity mapping analysis revealed homozygous regions across the genome multiple The results of the genome-wide SNP genotyping and linkage analysis for the studied pedigree are shown in figure 3.5-2, figure 3.5-3 and table 3.51. The genotyping data showed nine blocks of homozygosity shared between the two affected members of the studied family (Figure 3.5-2). The biggest homozygous block was observed on chromosome 1 between rs12066191and rs17091690 flanking a 30 Mb genetic interval (1p34.2-p31.1) (Figure 3.5-3). Two blocks of homozygosity were noticed on chromosome 2 with one of them located between rs9808571 and rs12993911 flanking a 7 Mb genetic interval (p15-p13.3) (Table 3.5-1). The other stretch of homozygosity on chromosome 2 was less than 1 Mb (2p25.1). Three blocks of homozygosity were seen on chromosome 7, with the biggest one of them (~7 Mb) bordered by rs6466076 and rs821783 (7q22.3-q31.1; Table 3.5-1). Another homozygous region (~1.6 Mb) was observed on chromosome 19 between rs11084444 and rs16988328. The last block of homozygosity detected was found on chromosome 21 spanning 3.9 Mb between rs9636576 and rs8127726 (Table 3.5-1). The parents and all the unaffected sibs were heterozygous at all the homozygous segments. 3.5.3.2.2. Whole-exome sequencing revealed a novel nonsense mutation in POMGNT1 gene The co-segregating homozygous regions together encompass around 469 genes (Table 3.5-1). In order to reveal the molecular basis of the phenotype in the studied family, WES was carried out on the two affected 197 children. A minimum of 79.70% of the on-target regions were covered to a depth of at least 20x. Around 49,000 variations from the reference genome were identified (Table 3.5-2). Filtering out all the common SNPs reduced the number to 4,365 novel variants generated from each exome. Of these novel variants, 900 were found in coding sequences with serious consequences. Approximately 700 variants were homozygous, of which only one stop gained mutation was identified in POMGNT1 gene (NM_001243766.1: c.1462C>T; p.Arg488*) (Figure 3.5-4). Mutations in this gene are known to cause CMDdystroglycanopathies. The mutation was novel, and predicted to cause the complete loss of the protein product by Mutation Taster. Moreover, screening all the family members showed that the mutation segregated with the phenotype (Figure 3.5-5). In carrier screening, the parents and all the unaffected sibs were carriers except one individual (II5). 3.5.4. Discussion In this chapter, I have characterized the genetic basis of vague clinical features of an autosomal recessive CMD with brain and eye abnormalities in two affected children in a Sudanese extended family. I mapped the disease to specific chromosomal 1 loci (1p34.2-p31.1) using homozygosity mapping approach. In addition, I found that the phenotype is associated with a homozygous loss-of-function mutation in the POMGNT1 gene by investigating the exomes of the two affected children. Combining this genetic finding with the clinical features of the studied patients, the broad diagnosis of CMD was refined into a specific phenotype of POMGNT1-related CMD- 198 199 Chromosome Start 1 42,165,131 62,666,178 2 11,540,489 16115413 7 105478527 156762248 12 115939302 19 56817035 21 28563550 Total End 72,195,715 70,011,952 11,761,309 16412625 112469778 158054285 119530488 58491733 32514524 SNP start rs12066191 rs9808571 rs4669727 rs10277151 rs6466076 rs12919 rs11834961 rs11084444 rs9636576 SNP end Genetic interval Length (kb) Genes rs17091690 p34.2-p31.1 30,030,584 219 rs12993911 p15-p13.3 7,345,774 51 rs2304401 p25.1 220,820 rs10253784 p21.2 297,212 rs821783 q22.3-q31.1 6,991,251 51 rs13222149 q36.3 1,292,037 rs11064657 q24.21-q24.23 3,591,186 23 rs16988328 q13.43 1,674,698 73 rs8127726 q21.3-q22.11 3,950,974 52 55,394,536 469 Table 3.5-1. Intervals of shared homozygosity between the two affected individuals of the studied family. 200 Figure 3.5-2. HomozygosityMapper view of the Genome wide homozygosity analysis results. Genotyping all the family members using Affymetrix SNP 6.0 array revealed multiple regions of shared homozygosity between the two affected sibs. 201 Figure 3.5-3. Zoom in view into the homozygous region detected on chromosome 1. The block of homozygosity on chromosome 1 was spanning 30 Mb genetic intervals (p34.2-p31.1) and flanked by rs12066191and rs17091690 encompassing 219 genes. 202 ¥ including those in dbSNP release 132. filtering out those contained in dbSNP release 132. * Number of Variations Variations Overlapping Genes Variations Overlapping Transcripts Variations Overlapping Regulatory Regions Variations Overlapping Protein Domains Intergenic Variations Variations With Predicted Serious Consequences Variations With Other Predicted Consequences Homozygous Variations With Predicted Serious Consequences All Variants* II3 II4 48,920 49,351 48,544 49,002 48,544 49,002 9,911 9,964 32,553 32,858 376 349 10,604 10,689 45,989 46,400 10,157 10,245 Table 3.5-1. Summary metrics of all and novel variants identified by the exome sequencing. Novel Variants¥ II3 II4 4,365 4,365 4,295 4,278 4,295 4,278 1,096 1,058 2,908 2,886 70 78 933 911 4,080 4,069 784 777 203 Figure 3.5-4. Integrative Genomics Viewer (IGV) visualization of homozygous mutation c.1460 in POMGnT1 gene from exome data. All reads show the homozygous substitution of G>A (C>T on reverse strand) and the changed amino acid arginine (R) at the bottom. Figure 3.5-5. Mutation analysis results in the studied family. Novel missense mutation was found c.1462C>T in POMGnT1 in all the affected children in a homozygous form. The same mutation was found to be heterozygous in parents and some unaffected children. The mutation was not detected in normal affected sibs or in normal healthy Arab controls. Sequencing chromatogram of a normal individual carries the wild type sequence of POMGnT1 Sequencing chromatogram of a carrier individual carries a heterozygous c.1462C>Y Sequencing chromatogram of an affected individual carries a homozygous c.1462C>T 204 dystroglycanopathies with brain and eye anomalies. 3.5.4.1. Genetic heterogeneity of CMD-dystroglycanopathy with brain and eye anomalies Dystroglycanopathies with brain and eye anomalies is a group of genetically heterogeneous autosomal recessive disorders that can be caused by mutations in several genes involved in the alpha-dystroglycan (α-DG) glycosylation pathway. Including the POMT2 gene (OMIM*607439), POMGNT1 gene (OMIM*606822), FKTN gene (OMIM*607440), FKRP gene (OMIM*606596), LARGE gene (OMIM*603590), ISPD gene (OMIM*614631), GTDC2 gene (OMIM*614828), TMEM5 gene (OMIM*605862), B3GALNT2 gene (OMIM*610194), SGK196 gene (OMIM*615247) and B3GNT1 gene (OMIM*605517). The unifying feature in all these disorders is deficient posttranslational glycosylation of α-DG on the sarcolemma of skeletal muscle fibers. α-DG is a heavily glycosylated, peripheral membrane protein that directly binds, probably through its sugar chains, to the alpha-2 chain of laminin-2 (laminin-α2; an extracellular matrix protein) and to betadystroglycan (ß-DG). ß-DG is a transmembrane protein that binds to dystrophin protein which in turn binds to the intracellular actin filaments [Ibraghimov-Beskrovnaya et al., 1992]. The dystroglycan complex works as a transmembrane linkage between the cytoskeleton of the muscle fiber and the surrounding extracellular matrix [Ibraghimov-Beskrovnaya et al., 1992]. DG hypoglycosylation was found to directly abolish the binding activity of the protein with its several extracellular matrix ligands such as laminin-α2 (OMIM*156225), neurexin (OMIM*600565), and agrin (OMIM*103320) 205 [Michele et al.; 2002]. Neurexin and agrin proteins are known to be involved in synaptic signaling of nerves and muscles neurons [Gingras et al., 2002; Reissner et al., 2008]. 3.5.4.2. Clinical heterogeneity of CMD-dystroglycanopathy with brain and eye anomalies To date in OMIM there are at least 12 congenital muscular dystrophies-dystroglycanopathies (MDDG) with brain and eye anomalies classified according to the mutated dystroglycanopathy gene mentioned above. Namely, MDDGA1 (OMIM#236670), MDDGA2 (OMIM#613150), MDDGA3 (OMIM#253280), MDDGA4 (OMIM#253800), MDDGA5 (OMIM#613153); MDDGA6 (OMIM#613154), MDDGA7 (OMIM#614643), MDDGA8 (OMIM#614830), (OMIM#615181), MDDGA10 MDDGA12 (OMIM#615041), (OMIM#615249), and MDDGA11 MDDGA13 (OMIM#615287). These disorders share the common clinical feature of muscular dystrophy with variable neurologic and ophthalmic phenotypes. The genetic basis of at least 40% of MDDG cases still is unidentified [Godfrey et al., 2007; Mercuri et al., 2009; Messina et al., 2010; Devisme et al., 2012; Cirak et al., 2013]. Screening known and candidate genes for causative mutations in congenital MDDG would most probably miss the causative genes for the unsolved dystroglycanopathy cases [Cirak et al., 2013]. 3.5.4.3. Phenotypic spectrum of POMGNT1 mutations POMGNT1 gene encodes a glycosyltransferase involved in the glycosylation of α-DG and is responsible for the transfer of N- acetylglucosamine (GlcNAc) to O-mannose glycoproteins [Clement et al., 206 2008b]. Mutations in this gene disrupt its function and cause a wide spectrum of clinical severity. POMGNT1 mutations were first identified in patients with MDDGA3 (formerly known as Walker-Warburg syndrome or muscle-eye-brain disease), a congenital muscular dystrophy with structural eye and brain defects and severe mental retardation [Yoshida et al., 2001]. POMGNT1 Mutations are also associated with a less severe CMD-dystroglycanopathy with mental retardation (MDDGB3; OMIM#613151) [Clement et al., 2008a]. Clement et al [2008b] have broadened the spectrum of POMGNT1 mutations to include relatively mild adult onset limb-girdle muscular dystrophy with no mental retardation (MDDGC3; OMIM#613157). Due to the wide spectrum of POMGNT1 mutations, Taniguchi et al [2003] recommended searching for POMGNT1 mutations in any CMD patients worldwide. Having said that, Hehr et al [2007] studied the clinical phenotypes of nine MEB patients and noticed a broad phenotypic variability between patients irrespective to the type or location of the underlying POMGNT1 mutation. The authors concluded that the type and position of the POMGNT1 mutations are not of predictive value for the clinical severity. Several therapeutic approaches are now under study such as viral-mediated gene therapy which has proved to be successful [Reed, 2009]. For example, overexpression of LARGE gene was found to improve glycosylation of α-DG required for extracellular matrix (ECM) binding even in disorders where other genes in the pathway are mutated including POMGNT1 [Yu et al., 2013]. This treatment rescued the muscular dystrophic phenotype in a Pomgnt1-knockout mouse model [Yu et al., 2013]. 207 3.5.5. Conclusions In this study I have identified a homozygous loss-of-function mutation in POMGNT1 gene in two affected children of a consanguineous Arab family residing in the UAE. This genetic finding in addition to the involvement of brain and eye anomalies in the phenotype, has refined the initial broad diagnosis of CMD in these patients into a specific disease called MDDGA3. Understanding the molecular basis of this dystroglycanopathies had led to a more precise diagnosis and genetic counseling in the family. Moreover, therapeutic strategies are being developed and tested in the preclinical models and it is hoped that these observations will pave the way to therapeutic interventions in humans [Reed, 2009]. 208 SECTION 6: A novel mutation in PRG4 gene underlying camptodactyly-arthropathy-coxa vara-pericarditis syndrome with the possible expansion of the phenotype to include congenital cataract 209 3.6.1. Background Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome (OMIM#208250) is characterized by congenital flexion contractures of the fingers, non-inflammatory swelling of the joints, deformity of the hips, and pericarditis. Camptodactyly may be seen in many other genetic syndromes, including oculo-dental-digital, orofacial-digital, cerebrohepato- renal, Catel Manzke, and Pena-Shokeir I syndromes [Choi et al., 2004]. Camptodactyly in CACP is usually present at birth and mostly bilateral and progressive [Offiah et al., 2005]. The arthropathy (swollen joint) in CACP usually appears in the first year of life and mainly involves the large joints such as elbows, hips, knees, and ankles [Choi et al., 2004]. This arthropathy is non-inflammatory because it lacks signs of inflammation, such as heat, and is not responsive to anti-inflammatory drug therapy [Offiah et al., 2005]. Synovial hyperplasia is also prominent in these patients, without any evidence of inflammatory cell infiltration or vasculitis. Tenosynovectomy of the hands and synovectomy of the knee joints have been used in some patients to maintain mobility of affected joints [Ochi et al., 1983; Martin et al., 1985]. Progressive coxa vara was seen in half of the CACP cases reported, whereby the angle between the ball and the shaft of the femur is reduced, leading to severe impairment of hip mobility [Choi et al., 2004]. Approximately 30% of CACP cases develop either mild self-limiting or life-threatening non-inflammatory pericarditis [Choi et al., 2004]. 210 3.6.2. The purpose of the study This study was performed to investigate the molecular cause underlying a severe CACP syndrome inherited in an autosomal recessive manner in an extended consanguineous family from the UAE. An initial partial Sanger sequencing of the PRG4 gene failed to reveal any pathogenic mutation. Therefore, the genetic investigation in this family was carried out using whole-exome sequencing and STR genotyping approach. 3.6.3. Results 3.6.3.1. Clinical assessments A highly inbred family with four affected children in two branches exhibiting severe autosomal recessive CACP syndrome was recruited in this study (Figure 3.6-1). All affected children in this family presented at birth with camptodactyly of either some fingers or all fingers. Two of them had trigger fingers. Joint swelling started in the first few months of life starting with the knee joints and progressing to involve the elbows, ankles, wrists and hips. Some of the children experienced mild pain but there were no signs of inflammation. Coxa vara was present in all of them. However, there was no history of pleuritis, pericarditis or spinal abnormalities. Camptodactyly was operated on successfully in all of them. One of the children was found to have a bilateral congenital cataract which was noticed at birth. This was operated on successfully. 211 Figure 3.6-1. Pedigree of the studied family. The camptodactylyarthropathy-coxa vara-pericarditis syndrome presented in four affected children in two branches of this consanguineous family. I 3 2 1 4 II 1 2 3 4 212 5 6 7 8 3.6.3.2. Molecular genetic investigation In order to reveal the molecular basis of the phenotype in the studied family, whole-exome sequencing (WES) was carried out on three affected children II-5, II-7, and II-8. In total 68660 variants were generated of which 29675 were homozygous. Those variants were substantially reduced to 1350 novel variants of which approximately 110 were predicted to alter the protein product. To identify the causative gene and variant, shared homozygous regions were investigated using the exome data. Two homozygous regions were noticed to be shared between the three affected children (Table 3.6-1). The first block of homozygosity was noticed on chromosome 1 spanning around 26 Mb, while the second one was found on chromosome 12 spanning 5 Mb. Specific STR markers were used to confirm and narrow down the homozygous regions in all the available family members (Table 3.6-1). STR genotyping refined the homozygous region on chromosome 1 around 14 Mb flanked by the markers AFMA116YC5 (chr1:182,873,495) and the marker AFMC023WE9 (chr1:196,650,533). On the other hand, the two STR markers flanking the second homozygous block were found to be heterozygous in two out of the three affected children (Table 3.6-1). Within the assigned homozygous region on chromosome 1, only a homozygous frame shift mutation in PRG4 gene was found in all the investigated affected individuals. The detected mutation is an insertion of a cytosine mono-nucleotide in a stretch of five cytosines at the beginning of exon 7 of PRG4 gene NM_005807.3:c.1320dupC (Figure 3.6-2). The 213 214 Location I1 140/140 216/218 263/265 168/170 244/248 22,351,420 24,093,565 AFMC027ZD1 AFMA119XE5 276/285 251/255 Chr12: 21,727,315-26,848,663=5Mb 207,759,412 AFM059YH4 196,650,533 AFMC023WE9 201,121,159 189,344,581 AFMA057VB5 AFMB002YA5 182,873,495 AFMA116YC5 Chr1:182,800,727-209,788,213=26Mb Marker 276/278 255/257 136/140 214/218 263/263 166/168 242/244 I2 276/278 255/257 136/140 214/216 263/265 166/168 242/244 II3 276/285 251/255 140/140 216/218 263/265 168/170 244/248 II4 276/276 255/255 140/140 216/218 263/263 168/168 244/244 II5 278/285 257/257 140/140 218/218 261/263 166/168 242/244 I3 276/285 241/255 140/142 218/218 261/263 168/172 244/246 I4 278/285 241/257 140/142 218/218 263/263 168/172 244/246 II6 276/278 255/257 140/142 218/218 261/263 168/168 244/244 II7 II8 276/278 255/257 140/142 218/218 263/263 168/168 244/244 Table 3.6-1. STR genotyping results for the two homozygous regions identified in the studied family. Figure 3.6-2. Representative DNA sequencing chromatograms displaying the c.1320dupC mutation of PRG4. The first chromatogram shows the 5 cytosine homopolymers in the reference sequence of a normal control, while the second one shows the duplication of the c.1320 cytosine (indicated by a star) carried by the two alleles of the affected. The last chromatogram shows the mutation in one allele carried by the parents and the unaffected carriers. Sequencing chromatogram of a normal individual carries the wild type sequence of PRG4 gene Sequencing chromatogram of a Carrier individual carries a heterozygous form of the detected mutations Sequencing chromatogram of an affected individual carries a homozygous c.1320dupC 215 duplicated nucleotide caused a frame shift in the coding DNA sequence of the gene and created a PTC in its protein product p.Lys441Glnfs*198. The mutation was verified by Sanger sequenced in all family members. All the unaffected children and parents were found to be carriers for the mutation. This mutation is located at the beginning of the mucin-like domain of the protein product (Figure 3.6-3). 3.6.4. Discussion In this study I investigated a consanguineous family in the UAE with four affected children in two branches exhibiting severe autosomal recessive CACP syndrome. Interestingly, one of the affected children has a bilateral congenital cataract in addition to the typical CACP phenotype. This feature has not been reported previously in this syndrome. I identified a novel mutation in PRG4 gene in this family by whole-exome sequencing and STR genotyping. CACP is a rare genetic disorder inherited in an autosomal recessive manner [Alazami et al., 2006; Basit et al., 2011]. In 1998, the disorder was linked to chromosome 1q25-31 using homozygosity mapping [Bahabri et al., 1998]. In 1999, Marcelino et al identified the causative gene [Marcelino et al., 1999]. All the mutations reported so far in all the studied CACP cases are nonsense or frame shift deletions and insertions that premature termination of the lubricin protein which is encoded by the PRG4 gene (OMIM*604283; Figure 3.6-3) [Marcelino et al., 1999; Alazami et al., 2006; Basit et al., 2011]. These mutations are predicted to cause the degradation of the mutated 216 217 K441 Figure 3.6-3. Schematic presentation of PRG4 protein domains showing the position of all the reported mutations. All the detected mutations thus far in PRG4 are either nonsense or deletions and insertions that created premature termination codons (PTCs). Our insertion (represented in red) also caused a frame shift creating PTC shortly after. mRNA by the nonsense mediated decay mechanism of the cell. It has been documented that complete absence of the truncated protein product is the molecular mechanism underlying the multiple phenotypes of CACP [Rhee et al., 2005; Alazami et al., 2006]. Although the mutation in this family has not been reported previously it is similar in many ways to the previously reported ones as the fate of truncated protein is predicted to be completely lost [Rhee et al., 2005; Alazami et al., 2006; Basit et al., 2011]. However, the detected mutation in this study is situated in a high repetitive part of the lubricin protein, the large mucin-like domain that contains 41 repeats of the degenerate KEPAPT motif [Alazami et al., 2006]. For this reason, this region (p.350-855, NP_005798.2) was usually missed or avoided when performing mutational screening by Sanger sequencing technology [Alazami et al., 2006; Marcelino et al., 1999]. Therefore, I was unable to recognize the disease causing mutation using Sanger sequencing although the diagnosis was clear. As a result,I decided to use the more accurate sequencing technology WES. Because of the short read sequence nature of the WES and the sophisticated analysis methods that follows, this technology is more suitable to obtain highquality variant calls [Shiqemizu et al., 2013]. Homozygous Prg4 null mice developed with age abnormal morphologic changes such as irregular endochondral growth plates and altered cartilage surface [Rhee et al., 2005]. Also those mice exhibited camptodactyly, progressive synovial hyperplasia which eventually led to failure of joint function [Rhee et al., 2005]. The biological roles of PRG4 are 218 the focus of intensive studies partly due to its possible usage as a biotherapeutic target in arthritic diseases [Bao et al., 2010]. Rhee et al [2005] demonstrated that lubricin, the protein product of PRG4, is an essential boundary lubricant in the mouse knee joints. The authors also observed that decreased lubrication in the ankle joints of the mutated animals due to the absence of lubricin causes abnormal changes within the tendon and tendon sheath. The authors suggested that this is the leading cause of the camptodactyly seen in CACP patients. Novince et al [2011] confirmed experimentally that PRG4 contributes to endochondral bone formation, trabecular bone formation and skeletal homeostasis. Congenital camptodyctyly and non-inflammatory arthropathy are characteristic manifestations of CACP seen in all reported cases. Other manifestations including pericarditis, pleuritis and coxa vara are variable and many patients do not develop them throughout the course of the disorder. On the other hand, congenital cataracts have never been reported to be associated with PRG4 mutations before. In the studied family here, one of the affected was born with a cataract without any previous history of such phenotype in the family or in any of the others affected. The process leading to cataracts actually originates with events occurring in the lens epithelium which performs important metabolic functions for the entire lens [Andley, 2008]. PRG4 mRNA was detected in the human lens epithelial cells as well as in the cornea, the conjunctiva, the meibomian gland secretions, and the iris of the eye [Morrison 219 et al., 2012] (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GPL96). In addition, Morrison et al [2012] displayed that PRG4 is an effective ocular surface boundary lubricant, alone or in combination with hyaluronan. Schmidt et al [2013] has proved experimentally that PRG4 is transcribed, translated, and expressed by ocular surface epithelia. The authors have demonstrated that PRG4 presence significantly reduces friction between the cornea and conjunctiva. Therefore, the authors have concluded that PRG4 deficiency may play a role in promoting corneal damage. 3.6.5. Conclusion CACP is a clinically heterogenous congenital disorder caused by mutations in PRG4 gene encoding a chondroitin sulfate proteoglycan that acts as a lubricant for the cartilage surface. In this chapter, I have investigated a family from the UAE with typical features of CACP in whom one of the children had in addition, a bilateral congenital cataract. In this study, I have identified a novel null mutation in PRG4 gene confirming the genetic homogeneity of CACP thus far. The clinical and molecular findings in this chapter have expanded the spectrum of PRG4 deficiency to include ocular abnormalities. Furthermore, elucidating the genetic defect in this family confirmed the diagnosis and facilitated carrier screening. 220 SECTION 7: Mapping of an autosomal recessive progeroid syndrome with neonatal presentation and long survival to a single locus on chromosome 19p13.3-13.2 221 3.7.1. Background Progeroid syndromes are a group of disorders characterized by an “old man” appearance either at birth or later in life. Hutchinson-Gilford progeria syndrome, mandibuloacral dysplasia, and cockayne syndrome are examples of progeroid syndromes, which do not manifest at birth but later in life [Pollex and Hegele, 2004; Garg et al., 2005]. In several other syndromes, the progeroid phenotype is usually seen at birth. Examples of the latter group include Wiedemann-Rautenstrauch syndrome (WRS; OMIM#264090), PettyLaxova-Wiedemann syndrome (PLWS; OMIM#612289), Fontaine-Farriaux syndrome [Castori et al., 2009], Hallerman-Strief syndrome (OMIM#234100), and De Barsy syndrome (OMIM#219150). Other conditions with neonatal presentation include cutis laxa type 1A (OMIM#219100), cutis laxa type IIB (OMIM#612940), geroderma osteodysplastica (OMIM#231070), and some forms of Marfan syndrome [Graul-Neumann et al., 2010; Goldblatt et al., 2011]. WRS is a very rare autosomal recessive progeroid syndrome. The syndrome is characterized by an aged appearance at birth with relative macrocephaly, craniofacial disproportion, reduced subcutaneous fat, thin skin, and neonatal teeth [Rautenstrauch et al., 1994; Courtens et al., 1997; Stoll et al., 1998; Pivnick et al., 2000]. The syndrome was first described in two patients by Rautenstrauch and Snigula in 1977, followed by a report of two more cases by Wiedemann in 1979 [Rautenstrauch and Snigula 1977; Wiedemann, 1979]. Since then, more reports have been published with about 222 30 cases being reported so far from different ethnic backgrounds [Devos et al., 1981; Bitoun et al. 1995; Hou and Wang, 1995; Arboleda et al., 1997, 2011; Pivnick et al., 2000; Korniszewski et al., 2001; Hoppen et al., 2004; Hou, 2009; Tunc et al., 2009; Pandey et al., 2011; Puente et al., 2011]. The molecular basis of the WRS is still largely unknown. 3.7.2. The purpose of the study Progeroid syndromes of infancy and childhood are often associated with a wide spectrum of etiologies (including structural abnormalities and inborn errors of metabolism) and identified genetic defects account for a number of cases. In many children the underlying cause is not elucidated, and it is likely that a significant proportion of these cases have a currently undetermined genetic basis. In order to elucidate the genetic defect underlying one of these disorders, I performed homozygosity mapping and whole-exome sequencing on three affected children from a consanguineous Arab family residing in the UAE who presented with a syndrome that overlaps to a large extent with the autosomal recessive progeroid syndrome WRS. 3.7.3. Results 3.7.3.1. Clinical assessments of patients. The parents of the affected children are first cousins of Palestinian origin. They have a total of six children; three of them are affected (Figure 3.7-1; Table 3.7-1). The parents appeared normal, and there was no history of similar conditions in the family. Patient 1 (II-1), a 27-year-old man, was evaluated in the Genetic clinic 223 Figure 3.7-1. Pedigree of the family affected with autosomal recessive progeroid syndrome. The pedigree of the studied family showing the affected members (filled symbols) and non-affected members (open symbols). The double line in middle generation (the parents) indicates consanguinity. 224 225 IUGR=Intrauterine growth restriction GR=Growth retardation Features Birth Weight Length Aged appearance at birth Relative macrocephaly Wide fontanelle & Sutures Sparse scalp hair Prominent scalp veins Small facial bones Flat malar region Large eyes Downslanting palpebral fissures Large beaked nose Short philtrum Micrognathia Low set prominent ears Neonatal teeth Altered dentition Large hands & feet Lack of subcutaneous fat Fat accumulation in buttocks Contractures at elbow & knee joints Scoliosis/kyphosis Hypoplasia of distal phalanges Coronal synostosis Genital anomalies CNS anomalies Endocrine Abnormalities Psychomotor retardation Chronic lung disease Age at report Weight at report Height at report II-1 2.3kg 51cm + + + + + + + + + + + + + + + + + + + + + 27y 32.7kg 143cm II-5 2.4kg 47cm + + + + + + + + + + + + + + + + + + 12 y 20kg 120cm II-6 1.8 kg (34week) 45cm + + + + + + + + + + + + + + + + + Repeated infections 7y 12kg 105cm WRS IUGR IUGR + + + + + + + + +/+ + +/+/+ + + + +/+/+ in survivors +/+/+/+ Birth-17y Failure to thrive GR Table 3.7-1. Clinical features of the three affected sibs in this report compared to Wiedemann-Rautenstrauch syndrome (WRS). because of his appearance. He was the product of a normal pregnancy and normal term delivery. His birth weight was 2300 g (<10 centile) and his length was 51 cm (>10 centile) (Table 3.7-1). At birth, he was noted to have an aged appearance, lacking subcutaneous fat on his face and body, and his skin was wrinkled. In addition, he had several dysmorphic features including a relatively large head with wide fontanelle and sutures. He had visible superficial veins, sparse scalp hair, eyebrows, and eye lashes with lower eyelid entropion. His face was triangular with a large nose and small mandible. There were two upper neonatal teeth. He was kept in a special care baby unit for 3 weeks. No further information about his neonatal period was available. During his childhood, he was failing to thrive and he developed repeated chest infections. He had normal motor milestones, but he had delayed speech and his intelligence was low. He had few primary teeth, which fell out at 7 years and were not replaced by permanent teeth except two premolar teeth, which fell out later in childhood. Evaluation at 20 years old revealed an old looking man with a generalized reduction of subcutaneous fat and dysmorphic features. He had areas of sparse scalp hair, large eyes, and a big beaked nose with low insertion of the columella, short philtrum, flat malar region, and micrognathia (Figure 3.7-2-1). The mouth appeared large, and there were no teeth (Figure 3.7-2-2). His nails were normal. There were contractures at the knee and elbow joints and scoliosis. There was no fat accumulation in the buttocks. He had low voice similar to that of old people. 226 Figure 3.7-2. Representative images of patient 1. 1) and 2) Patient 1 at 20 years of age: note the large eyes, large beaked nose, short phitrum, flat malar region, and small mandible (1), note lack of teeth (2). 3) and 4) Patient 1 at 27 years of age: front view of the patient, note lack of subcutaneous fat, contractures at the elbow, and PEG tube (3), lateral view of the patient showing the posture and contractures at the knee joints (4). 227 He had suffered from severe constipation since infancy. He attained puberty at 13 years of age and he had normal secondary sexual characteristics and normal genitalia. His vision and hearing were normal. He continued to suffer from repeated chest infections, which were attributed to aspiration as he had some degree of functional dysphagia. At 27 years of age, he was on continuous oxygen therapy by mask and used a PEG tube for feeding (Figure 3.7-2-3, 2-4). His weight was 32.7 kg (-3 SD), and his height was 143 cm (-3 SD). His deterioration was because of his chronic lung disease and his inability to swallow. Skeletal survey showed generalized osteopenia. There was no other neurological or cardiovascular deterioration. Fasting blood lipids were normal. Endocrine functions including thyroid and adrenal functions were normal. His karyotype was normal (46XY). His CGH array has shown a male profile with 21 copy number variants (CNVs) of no clinical significance. Patient 2 (II-5) is the sister of patient 1 (Figure 3.7-3A-1). She was the product of a normal pregnancy and a normal term delivery. Her birth weight was 2400 g (<10 centile), and her length was 47 cm (<10 centile). She was noted to have the same phenotype as her brother (Table 3.7-1). During infancy and childhood, she failed to thrive. She had few primary teeth, which fell out and were not replaced by permanent teeth. She had normal motor milestones, but her speech was delayed and her IQ was low. She was evaluated by us at the age of 12 years. Her weight was 20 kg (-2 SD) and height was 120 cm (-3 SD). Her eyes were large with down-slanting palpebral fissures (Figure 3.7-3A-2). She had a large nose with a broad nasal tip, short 228 philtrum, and a flat malar region. Her mouth was large with no teeth, and she had micrognathia. Her scalp hair was sparse with areas of alopecia (Figure 3.7-3A-3). The ears were prominent and low set. She is 12 years old now and has signs of puberty including breast development (Tanner stage 2), sparse axillary, and pubic hair. Patient 3 (II-6) is the brother of patients 1 and 2 (Figure 3.7-3B-1). He was the product of a normal pregnancy and a normal delivery at 34 weeks gestation. His birth weight was 1800 g (10 centile), and his length was 45 cm (>10 centile). He was noted to have similar features to his siblings (Table 3.71). He is now aged 7 years and has delayed speech (Figure 3.7-3B-2, 3B-3). 3.7.3.2. Molecular genetic investigation 3.7.3.2.1. Sequencing of LMNA and ZMPSTE24 genes showed negative results The molecular cause of Hutchinson-Gilford progeria has been shown to be mutations in the genes encoding nuclear lamin A (LMNA) [De SandreGiovannoli et al., 2003; Eriksson et al., 2003]. On the other hand, it has been shown that mandibuloacral dysplasia is caused by mutations in ZMPSTE24 gene, which encodes for a metalloprotease involved in lamin A maturation [Agarwal et al., 2003; Navarro et al., 2004]. Sequencing of LMNA and ZMPSTE24 genes by the Sanger conventional method did not reveal any pathogenic mutation. 3.7.3.2.2. Genome-wide homozygosity mapping linked the condition to 19p13.3p13.2 The results of the genome-wide genotyping and linkage analysis for the studied pedigree are shown in figure 3.7-4 and Appendix-G. The 229 Figure 3.7-3. Representative images of patient 2 and 3. A-1) Patient 2: in the first year of life. A-2) patient 2 at 12 years of age, note the large eyes, large nose with short phitrum, flat malar region, and small mandible. A-3) Lateral view of patient 2, note sparse hair, large and low set ears, and very small mandible. B-1) Frontal view of patient 3 at 3 years of age. B-2) Patient 2 at 7 years of age and B-3) side view of patient 2 at the same age. 230 231 Figure 3.7-4. Genome-wide genotyping results. (A) Genome-wide SNP Genotyping of all the family members mapped the disease locus to chromosome 19p13.3-p13.2. (B) Visualizing the only block of homozygosity shared between the three affected sibs (II1, II5, and II6). The shared homozygous interval appears in red and blue colors. This diagram was constructed using dChip software. (C) List of all the RefSeq genes located within the mapped region showing the potential candidates in bold. genotyping data showed two shared blocks of homozygosity between the three affected members of the studied family. One block mapped to chromosome 5 between rs4522972 and rs11957206, flanking a 3.5-Mb genetic interval (5p13.2; Appendix-G). However, one of the unaffected children (II4) was homozygous at that locus too, and therefore it has been excluded. The second block of homozygosity observed to be shared between the three affected sibs was found at a single locus spanning 6.9 centimorgan (cM) on chromosome 19 (Figure 3.7-4 and Appendix-G). All the non-affected sibs were heterozygous at that locus. The co-segregating segment is flanked by rs2240743 and rs479448, demarcating a genetic interval of 2.3 Mb on chromosome 19p13.3-p13.2 (Figure 3.7-4). CNVs analysis detected simultaneously by the Affymetrix SNP 6.0 array failed to identify any significant shared gains or losses. 3.7.3.2.3. Whole-exome sequencing convincing causal mutation analysis failed to reveal a To reveal the molecular basis of this syndrome and to identify the causative mutation, the whole coding regions and splice sites of the three affected individuals were sequenced by next generation sequencing WES. All the detected variants shared between the three affected members were filtered against dbSNP, 1000Genome project, NHLBI exome sequencing project, and in-house exome variants databases. The sequencing data confirmed the linkage but I could not identify any unreported homozygous disease-causing mutation shared between the three affected individuals within or outside the linked region. 232 3.7.3.2.4. Computational prioritizing of the genes within the mapped region Expression studies of progeroid syndromes demonstrated that among the most important mechanisms proposed to be involved in induction of aging are aberrations in lipid synthesis and cellular metabolism [Zouboulis and Makrantonaki, 2011]. In addition to alterations in DNA repair, mitochondrial function, cell cycle control, apoptosis, extracellular matrix expression, proteolysis, and hormonal levels [Zouboulis and Makrantonaki, 2011]. The potential disease loci 19p13.3-p13.2 detected in our study contains around 70 RefSeq genes (Figure 3.7-4). Prioritizing these genes using GeneDistiller2 pointed out several candidates that are not associated with known diseases and may play a role in one or two of the aforementioned pathways. Among these genes are VAV1, XAB2, SAFB, KHSRP, MLLT1, LONP1, TNFSF14, TNFSF9, ACER1, CLPP, ARHGEF18, CRB3, FCER2, and TUBB4A. However, extensive exome sequencing showed no pathogenic mutation in the coding regions of any of the nominated genes. Unfortunately, RNA samples from the affected patients were not available to investigate the presence of expression and splicing aberrations in any of the candidate genes. 3.7.4. Discussion A consanguineous family of Palestinian origin living in the UAE with three affected children exhibiting a syndrome that overlaps with autosomal recessive WRS to a large extent was recruited in this study. To identify the disease locus I performed whole-genome genotyping SNP-based and 233 homozygosity mapping on all the family members and mapped the disease to a novel locus 19p13.3-p13.2. In addition, I investigated the exomes of the three patients to identify a shared potentially disease-causing mutation but my attempts were unsuccessful. The features in the three siblings in this study are very similar to those seen in WRS patients. They all had neonatal teeth and aged appearance since birth. Their scalp hair was sparse, as well as the hair of their eyebrows and eyelashes. They had also large beaked noses. All of them had developmental delay, and all survived the first 6 years of life (27, 12, and 7 years). The average survival in WRS, however, is 7 months. Only two cases in the literature had survived beyond the age of 10 years, and clinical details are available at the ages of 16 and 17 years [Rautenstrauch et al., 1994; Arboleda and Arboleda, 2005]. Both of these surviving patients had altered dentition. Oral radiography on the patient reported by Arboleda and Arboleda [2005] at 9 years of age showed only four maxillary teeth, two premolars at each side. There was no dentition in the mandible and only scant alveolar tissue with very small nuclear teeth [Arboleda and Arboleda, 2005]. Similarly, the patients in this study had altered dentition. Two of them had only two premolar teeth, which fell out during childhood and one had no permanent teething at all. It was not possible to do oral radiography to assess if there are permanent teeth, which have not erupted. Both surviving patients in the literature had delay in their secondary sexual characteristics and onset of puberty. Whereas the 27-year-old patient in this report developed puberty at 234 the appropriate age (13 years). In addition, his 12-year-old sister had some secondary sexual characteristics but had not yet had started her menarche. Scoliosis, which was reported in the two patients with WRS who survived, was also present in our patient. Neurological deterioration was documented in the case reported by Rautestrauch et al [1994] but not in the case reported by Arboleda and Arboleda [2005]. The patients here had no neurological or cardiovascular deterioration. The main deterioration noted in our patient was the repeated chest infections caused by aspirations because of the inability to swallow, which has progressed gradually necessitating gastrostomy for feeding and continuous oxygen therapy. It is possible that the siblings in the family studied here have a new syndrome, which overlaps with WRS. Identification of the molecular basis of this syndrome will reveal if it is part of the WRS spectrum or is a new entity. The segregation of the syndrome in the family described here was in favor of an autosomal recessive mode of inheritance. Mutations in LAMNA and ZMPSTE24 genes have been excluded. The three affected sibs shared two homozygous regions on chromosomes 19 and 5. Only the region on chromosome 19 was found to be heterozygous in the three non-affected sibs. The 2.3 Mb linked region reported here (chr19p13.3-p13.2) is gene rich encompassing around 70 genes. Therefore, I decided to investigate the whole coding regions of the genome in the three affected sibs by whole-exome sequencing, which failed to reveal any potentially causal variants. This could be because of a deficit in the sequence coverage, which may have resulted in 235 missing the causative mutation [Bloch-Zupan et al., 2011]. Alternatively, the causative mutation may be located outside the coding exons within the introns or the regulatory elements of the genes located in that locus. Structural variations cannot be excluded, although CGH array and CNVs on SNP 6.0 array seemed to be normal. In addition the causative mutation could have been reported in the dbSNP which may have led into being missed in the filtering steps. Future work will attempt to identify a causative mutation in candidate genes. This in turn may help in solving at least one of the pieces of the complex puzzle of the molecular mechanisms underlying human aging. 3.7.5. Conclusions In this chapter, I have described a Palestinian family with three affected individuals exhibiting progeroid syndrome characterized by intrauterine growth retardation, a progeroid appearance, failure to thrive, a short stature, and hypotonia. The progeroid features were evident at birth. All the affected members of this family have survived beyond the neonatal period and one of them is currently a 27-year-old adult. As parental consanguinity suggested an autosomal recessive mode of inheritance, I performed homozygosity mapping using SNP-based arrays followed by next generation whole-exome sequencing to identify the disease causing gene. I was able to identify a single block of homozygosity shared between all the affected members of the studied family spanning 2.3 Mb on chromosome 19p13.3-p13.2. However, Sanger sequencing of known genes and whole exome sequencing of the three affected sibs did not reveal a 236 shared causal mutation. These findings are anticipated to open the way for the identification of the molecular causes underlying this syndrome. 237 CHAPTER 4: GENERAL CONCLUSIONS & FUTURE PERSPECTIVES 238 4.1. General conclusions The prevalence of monogenic autosomal recessive disorders in the population of UAE is relatively high. This is mainly due to the high rates of consanguineous marriages among the Emirati people as well as many of the subpopulations residing in the country as indicated in chapter 1. Many autosomal recessive disorders have catastrophic effects on affected infants as illustrated in sections 3.2 and 3.3 of the results; while the rest can cause severe physical and intellectual disabilities as seen in sections 3.1, 3.4, 3.5, 3.6 and 3.7. Therefore, mapping and identifying causative genes and mutations responsible for autosomal recessive disorders have been the primary goals of my PhD dissertation. Understanding the inheritance mode of a given rare genetic disease and utilizing a suitable mapping strategy are crucial for the identification of the causative mutation(s). Autosomal recessive inheritance of the studied phenotypes in the recruited families has been facilitated by the consanguinity of the apparently healthy parents (predominantly first cousins). Moreover, most of the families in this study were highly inbred with multiple affected offspring with female and male siblings or cousins being equally affected. Homozygosity mapping has been shown since its discovery by Lander and Botstein [1987] to be a very powerful tool for the identification of genetic defects underlying recessive disorders in consanguineous families. It has been used to map almost all the studied disorders in my dissertation which narrowed down the search from genome wide to a limited number of genetic 239 intervals as seen in sections 3.4, and 3.5. In this dissertation, I have successfully mapped four autosomal recessive disorders to a single chromosomal locus as seen in sections 3.1, 3.2, 3.6 and 3.7. Scientists have applied successfully the candidate gene direct sequencing approach to search for causative mutations within homozygous regions for several decades which I also achieved in sections 3.1, 3.2, and 3.3. Advanced sequencing technologies such as next-generation sequencing platforms have pushed the boundaries and made it possible to carry out direct sequencing of the whole-genome or whole-exome. DNA sequencing using those technologies generates large volumes of data in a relatively short period of time. However, there are many genetic variants across the genome to sift through, which increases the difficulty in finding the real diseasecausing variant. Since the monogenic disorders studied in my dissertation have a clear mode of autosomal recessive inheritance and the corresponding phenotypes are mostly caused by mutations that disrupt protein functions, I have combined homozygosity mapping with whole-exome sequencing to identify the causative mutations. This two-step approach proved to be successful in finding the causative mutations in sections 3.4, 3.5 and 3.6 but failed to pinpoint the genetic defect in section 3.7. This might be due to the low quality of the exome sequencing performed at a particular service provider rather than flaws in the used approach. Therefore, further investigations are still ongoing in this family using higher quality sequencing data. 240 Finding the gene and the genetic variation for a disease is just the start. Providing evidence to confirm that the genetic variant is causal requires further work. Therefore, correct segregation of all the detected variants with the corresponding disease phenotypes was confirmed in all the studied families. All the non-synonymous variants identified were validated for novelty in at least 200 chromosomes of healthy controls and were absent as normal variants in public databases. Several freely available bioinformatics tools were also employed to predict the impact of all the detected variants on the corresponding proteins functions and/or structures. Functional studies at the RNA or the protein level were performed when applicable as seen in sections 3.2, 3.3, and 3.4. In conclusion, seven different rare autosomal recessive disorders have been investigated in this dissertation. The main achievements for each section of the project are as follows: I have successfully mapped and found novel causative mutations for an autosomal recessive intrauterine growth restriction (IUGR) in four consanguineous families with multiple affected children. Three of the four families were initially diagnosed as autosomal recessive SilverRussell syndrome (SRS) cases and after the genetic findings in section 3.1 the patients were re-categorized as Three-M syndrome cases. All the detected mutations were found in CUL7 and OBSL1 genes that are known to cause the Three-M syndrome. These findings put into question the existence of autosomal recessive SRS. 241 I have successfully mapped and found novel causative mutations for a severe autosomal recessive disorder called fibrochondrogenesis (FBCG) in two families out of three in section 3.2. The detected mutations disrupted the COL11A1 gene that was not associated with FBCG before. I have identified a non-sense mutation in one family and a genomic splicing defect in the other family, which I showed experimentally to be destructive to the nascent mRNA sequence. In addition, I have elucidated the loss-of-function effect of this mutation by proving lack of expression of the mutated transcript and protein. I have successfully identified several novel causative mutations for an autosomal recessive catastrophic hemorrhagic destruction of the brain in three families in section 3.3. In this section, three non-synonymous mutations were identified in the JAM3 gene that was mapped and characterized previously in a collaborative study. In this dissertation, I proved the relative homogeneity of the clinical presentations of JAM3 mutations with the detected mutations disrupting different domains of the JAM3 protein. Previous knowledge from the literature of JAM3 (structure and function) and prediction programs were employed to predict the effect of the three mutations. Cellular studies and confocal fluorescence microscopy were used to detect any cellular trafficking defects caused by the disease-causing mutations and this has been demonstrated for one of them. 242 I have successfully mapped and identified 2 candidate splicing mutations for an autosomal recessive intellectual disability (ID) in the two children of a consanguineous couple. The splicing mutations detected in section 3.4 were identified in two unstudied genes namely LINS and TTC23. Both mutations were demonstrated experimentaly to be damaging. The TTC23 mutation caused a frameshift introducing a premature termination codon shortly after, which is most likely to lead to its degradtion by the NMD. The LINS mutation caused skipping of an exon and the expression level of the transcription in patients was higher than that of normal controls and parents suggesting a loss of negative feedback control. This control might be originally brought about by LINS protein which is predicted to be misregulated and thereby nonfunctional in the patients’ brains. I have successfully mapped and found a novel causative mutation for a very heterogeneous autosomal recessive congenital muscular dystrophy (CMD) in a consanguineous family with multiple affected children. I detected a loss-of-function mutation in one of the CMD- dystroglcanopathies-causing genes POMGNT1. The genetic finding from this study combined with the clinical involvement of the eye and brain anomalies in patients have refined the diagnosis in this family to be a specific disease called dystroglycanopathies type A3 (MDDGA3). 243 muscular dystrophies- I have successfully mapped camptodactyly-arthropathy-coxa a severe autosomal vara-pericarditis recessive syndrome in a consanguineous family from the UAE with multiple affected children. I detected a novel null mutation in PRG4 gene which confirmed the initial diagnosis. Moreover, in this study the carrier status was determined for all the family members from the two branches. I have successfully mapped an autosomal recessive progeroid syndrome with neonatal presentation in a consanguineous family with multiple affected children. In this section a rare most interesting phenotype was described and mapped to a novel single locus on chromosome 19. The mapping results were published attracting more collaborative research to further study the molecular causes of this phenotype. 4.2. 4.2.1. Future perspectives Identifying the genetic recessive disorders bases of additional autosomal The power to detect the genetic bases of ARDs will increase with the advances in massively parallel sequencing technologies at lower costs. It is noticed that these technologies are accelerating the discovery of novel genes and rare variants using chips that have greater genetic coverage, sensitivity and specificity. Nevertheless, several bioinformatics challenges remain to be addressed and improved. The storage and backup of the huge data generated from high throughput sequencing is still challenging to most laboratories. In addition, many analysis methods require deep methodological 244 knowledge and a powerful computational infrastructure. To overcome these challenges, an automated pipeline for analyzing the generated data should be developed locally. However, choosing appropriate analysis tools, applying sufficient filtering steps, utilizing suitable parameters, and then implementing them all in an automated pipeline is a challenge which requires advanced bioinformatics skills. 4.2.2. Establishing pathogenicity of mutations the identified genes and Proving causality of recessive mutations in candidate genes is essential for proper counseling of families. Establishing pathogenicity of mutations can be achieved using functional assays and/or animal models. Yet, detection of mutations in unrelated families with similar or overlapping phenotypes is the most definitive way to establish pathogenicity of novel disease-causing genes. This will be useful for many of those genes in which a defect has only been found in a single family. 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Exon Forward Primers Reverse Primers OBSL1 gene 1 CAGTCTGGGCTCTTGTCCTC GGCAGTAAAGCACCTTGAGC 2 and 3 GTCAGGGCTGGATGAGAACT CTTGTGGCCCTTGAACATCT 4 TCCCGTACCACTACGCTTCT TCTTCCCCGTCGTATACCTG 5 GTGGTGTTCCACGGTTCTG TGTTGCATGTGGGTTGAAAT 6 TGCTTGGGAATCCAAGAAT TGAGAGCCTGTCTCCAAAAA 7 CTCTGGTGCCACATGATCTC AGGCTTCCTTAGCCAAGACC 8 GCAGAAAGTCTGCTGTGCTT AGGGGCTTGTTTCCTTGAAT 9A TCTGGAATCCAAAGCATTCTG CGAATGGATGAATGAACGAA 9B AGCTCTGCTCCACAGCTCAT TAGACTGGCCCTCAACAACC 10 and 11 TGGTCAGAGTAACTGATTGAAAAA AGGCTCAGGGCTTTGAGGA 12 GTGAGTACAGCCTGGGCATT AGCCCCTGCTTTTAACCACT 13 TGGGGTGCTAGGCTAGGATT CAGCAGCTCTGTCTCTGGTG 14 GCAGGGACCGTGACTTTG GCAAAATCCACATGGACTTCTAA 15 ACACTTCTCCCCCTTCCTGT CCACACAGGGCCTATGAGTC 16 TAACCCATTGGCCCTGTG TTACAGTGACAAGACCTACCTCCT 17 and 18 AGAGGCTCCTGGCACGAC CTAGAGGTCCGGGACTAGGG 19 AGCCTCGTCCCTGTTCTTTC TTGCACCTCAGACCCAAAGT 20 CAAGAACGCTGCCTCTCTCT GATCTGAGCCAAGCAGTTCC. 21 TATCCCAAATCCCAGCATTC CCAAACTGTGTGGCCTCTG CUL7 gene 1 CCCCCATGAAGAGTTACGTG AGGCCCCATAAGCTAGAACC 2 GTGCAGCATGCAACTCCTG TTCCTTTGCATAAAAAGCAACT 3 GATCGTGTTTATGTGCGTGTG GGAGAAGAGCACCCTTCCTG 4 AGGGCATTCAGCTACCACAG ACCCAGATTTTCCCAGCTCT 5 and 6 GGAGAAGGCATCCTGTGTGT ACCCTCCCACCTCTGAAAAT 7 GAGGAGGGGTCTGGAATTTT GCTTCTCCGTTTGTTGCTTC 8 and 9 AGCGAGACTCCGTCTCAAAG GCGTTTTGATTTGTGAACCA 10 and 11 AACCACCTGGCTCTTGTGTC TAGGTCTTGGGGTTGTGGTC 12 and 13 CAACCCATCAACATCCCTTT TCCAGTCCCCTCACTGTTTC 14 and 15 TCTTGAGCCCCACAGAAAGT CAGCCCACTGGAGAAATCTT 16 CTCCAGTGGGCTGGGAAT ATGGTGTCCCCTGACACTTG 17, 18, and 19 CCTCAAGCGATCCTCTCATC CTCCACTTCCTGGGTTCAAA 20, 21, and 22 TGCGCCTGGTCTTATTTTCT CACACCCGGCTAATTTCTGT 23 and 24 ATCCTGAGGCTTGCCATAGA CCGTCTCTTCTCCAAGTTCTG 25 and 26 TCTCTGAAGGCGGTCTCTGT CTCTGAGAAGGGGCTGAGTG PALMD gene 283 1 CCCCTAGAGCTAGCAAGCAG ACTCGTCCATGAAGGCTGAC 2 CACTTCGCTCTCCTTTGCTT TGGCCTCCCCTATGGTTATT 3 GCAAGGCACTGACAGAAAAT TGAAAGCGTGCCTTAAAAA 4, 5 and 6 CGGGCATTAGGTATCTTCCA CGGTGACTCACATAACTCACAGA 7.1 CATCTCACATACAAGAACATGAAGG CACTGATCGGGGATGAGG 7.2 GGACTGGGTATTGGTGTAAATGA AGGTGGTCAGAGAAGCCTGA 8 TCAATGCTCACACTGTTGGA CAAGCTCTTATTAGAAATGCTTTGG 2 AGL gene GAATTTGGGAATGGGGAGAT TGGGAATTAGTACGCAGAGAAA 2b TCAGCATTTGATGACGGGTA GAATGGGCAGGGGATATTTT 3 CCATGGTAGCTCCCAAGTTT GCTCTGCAATGGACCATAAAA 4 CCAACATTCTTGGATATTTTGTG 5 AAACTTGCTTAAAGTCTCTTGTGG 6 and 7 TGCTGAAGCGAATGATAGG TAAGACATGCCTGCTTCCC GATCACTCTCCTGTATTTATAAGCAA C TGCCCTTTAGATGTTTCTGC 8 GAGGAAAACGGGTTCCACTA TGGCTGATGATGTTTTCCAA 9 and 10 CAAACCTTTATGGCTAGTATGATTTTC TGCAGTCCTCCAGAAACATT 11 GATATGCTGTCATGTTAACTCCC TTTGTTGGCTCCATGTTTTC 12 TCCCTTATGATGCGACTTGG TGCACTCCATATTATCAGAAACG 13 TCTCAATATCTTTGCAAAATCTAATC AAATGGTTTTGTTAGTGAGTCTTCAG 14 and 15 TGAGCCATTTCTCCAGTTAAG ACCTGTGTTTGAAGGCAATG 16 and 17 TCAGCGTATGATGCTCTTCC TCATACCTGGCCAAGTTACC 18, 19 and 20 TTCCCTAGAGCTAAGCTATTATTTTG TGCATGTAGGCATGTGTGTATC 21 TGGTTTGGGAGTAATGTCGC 22 and 23 TTCTTGAGATGCTTTTGGGC 24 AATGGGTGAAATGAAAGCAG GAAATTGGCACACAAAGAGG GGCAAATTTCATTCATAGTAATAGTG G CACACATAAATGCCTGGTGC 25 TGACAAGATAATGGAATCTCATTTG TGACTTCAAAGATTTTCACCCC 26 GGGAGTCACACAGATTGTTAGC TTCAGGCAAAGACCAATCAC 27 TTGTATGCTCGTCTGTATTATTTGG TTTCAAACTGGCTGACAAAAG 28 AATGCAAGCAATGTGGCAG CAACCCACGAGCACTTACAG 29 TGTGAGGATGATAGACAATGGC CAAAGTTGCGACAAATATGGTTC 30 TTGGGTGGTGCTACATCTTC TGTTACAGGTTAAAAGTGATACGC 31 GGCTTTCCTAACTTCTACGGC TACAGCCTTGATCTCCTGGG 32 and 33 GATTAAAGTCCTGTGAACTCAGTATG AACAACCCAAAATAAGAGAAAGTG 34 TTCCAAAGATGGAATGTCAAAG TGAACTCAAACACATTTCATTGG SLC35A3 gene 1 TAGGGACAGAGCTGCCAAGT CACCAAGTAAGAAACTGCACCA 2 GCTGCTGTAGCACCCAAAGT ACTAGGCCATTCGACAATGC 3 AACTTTGAGATTTTGGCTGTCA 4 GCATGCTGCCATAGTCCTTA CCTGATTTGATCATTACACATTGG TCACATAAAGGATGCCCTCA 5 CTCTCCTTTGCAGTCCCAAA AAAGACAAGAGCATTGCCAGA 284 6 CTGGGCAACAGAGCAAGACT CAAAGTTCATTAAAGACCCCTGT 7 GAGGGCTATTGAACAAAACCTG TTTTCAGGAGAAGACTTTCTTTCA 8.1 CTTAGCCAGATACCTGGAATGTA TTTGCAAAAATTGGTATCTGACA 8.2 TTGCTGCAGAAATGTCCTATG TGGCAATCTGGAATGTCTCC 1 HIAT1 gene GCGAGGATTTAGCTAGCTGGT GAGCAGCGGAGGACGATT 2 TTTCGTTGTTGCCTTGTCTG AAATTCAAACACCTCCACGC 3 CCAATGTAGTGCCCTGTGAC GGTTTATTATTATGCATGATTGGC 4 GGAGCCCCACAGATGATTC GCCAGAAGTCTAGATGCCTCC 5 GCTGAGCTGCTATTGAAGGG AAAACTTACATAGCCTCATACCGC 6 and 7 GCAACAGAAGGCCTGACATA AGGCGTGAGCCACTATACCA 8 CCCACCATGGATAAGTAGGC CATGAAACTTCTAACTTCATCCAGAC 9 TCAGCAGAGGGACTTTTCATC AAAATTCAGCCTGTCATTTAGC 10 TTGTGAATTAAGTTGCTGTCTTTG GCCACATATTCTAAGTGAGAGAAAAG 11 TCAGAGCTCATGTTTGCATAGG AGCCAATTTTCACAAGGTGG 12 TTTGCCAGTTGTTATTCCCAG TGGAAAACTGTCTGGTTCTAAGTTC 1 SASS6 gene TAACCGCATCTACCCTCAGC CTCACACCCTTCCTCCAGTC 2 TTACTCACTAGTGGAACAAGAAGAAA ACTCCAGCCTGGTGACAGAG 3 GAGCGGAATGTCTACCCCTA TCAGCAAAAGCTTCAAACTTCA 4 AGGTTGCAGTGAGCCAAGAT CGGATGACTTTAGTGCCTTTG 5 TGGCACTCAGTGAATTTTTGTT GTCCAGATCCATCCCGACTA 6 TGGGGATCTTGTTAAAATGATTTAG AATGAAGACTCGAGGGAACA 7 GGCCGTTTTGGGTCTAAGAT GAACAAAACCAAAAACCCATT 8 CCTTTTCAGAATGGGGATCT CAGCTGTGCCAGTAATCTGC 9 and 10 GAATTATTTTCCCTTGATATGATTGAA TCCTCCTTCTCAGCCAAGAG 10, 11 and 12 GCCACGAGAAAGAAAAGCAC CGGGAGGATAACTTGAACCA 13 GGTGAACCTATATTGCTCTCCTG 15 GAAGGATGAGGTGGGAGGAT TCAGCCTCTGTTCAGAGCAA AGTGCATATATTTCCTGTACACATAG A TGTTGGAGGAGGAAACAGAAA 16 and 17 CCATTTGCTTAAGAAACCAGTG GCCATGTTCACAAACCAAAA 1 LRRC39 gene TGCAGATCCATGGTTTACTGG ATGAACACCCCGCAAAAA 2 GCAATGGGTAGAGCCATGAT AGAATCGCTTGAACCCAAGA 3 TGAGACCCCCAAGAAAATTG CGCATAGACAAATTTAATTGAGTCAG 4 TGAAACTGGAAGGCAGAGGT GTTTAGGCAGGGTTGAAGCA 5 ATCCGGAAGTCACACAAAGC TCCACAAACTTGAATCCCCTA 6 CAGGAAAATGGCGTGAACC TTTCTCCAGTTAAAACCTGCATT 7 CCCTGGAGGTCATTTAGCTTT CCCAAACCCTTTTACTTACTTGG 8 GAGGGGGAGAATAGCCTGAG GGATTCTTTCTTGAATAGACCAGTG 9 TGCAACTAGACTAACAGTTGCTTTT CCTTCCCCCACTTGAAAGAC 14 GCAGAGTTGTGTGGCATGAT 285 10 TCCAGATCTGTAGGGAAAAATG GGTCGAATCCAGTATTTGCAG DBT gene 1 TCCTAGGGTTTAGCTCAGGC TTCCACTCCAGACTGGTCG 2 TCAGAAGGAATTTTGGGTAAGG AAAGGGGTGCCAGTGTTAAG 3 GGCAGTGCCAGTAAGTCTGTG CACCACTATATCATCAACACAAGG 4 TTCTTGCAGATACATTGAAGAGAC 6 TTACCACATGCATATGTGAACAG TTCTATTTTGTGTTGAACTTGCTACC AAATGGTAGCTCTTACTGTTATTAGG C GCACTACAGTCTGGACAACAAG 7 GATGCAGTCAGTGTTCCAGC GTGGCAGGTGCCTGTATTC 8 TGGCCTGAAGGTAACATTGG TCTGCCATACAGCTATGTATTTCC 9 and 10 CAGGTAATCCAGCCACCTTG CTGTCGTGGGGTCTGGG 11 ATGGGAAGAGTAGGAATCAGC TAAAATGTGACAGCCCCAGG 1 and 2 RTCA gene CCAGGATCCTCGAGAAAACA CTGGATCGCCCATACCTG 3 TCAGCGGTCTCAGTCTTTTG CCCTCCGTATCTCAGAATGTG 4 AACTGGTCTGCAAGGAAGTG AGTGAGCAGTGATTGCACCA 5 AAATACTGTTTTTCTTTAAAATTCAGG AGCCTTGGCAACAGCACT 6 AAAAATGGAACCAGCCTACA CCCATGCTCCTGCTATTCAT 7 TCACATATCTCAAGGCTACCACA GCCATATTGCCACAAAGGAC 8 CATAAGCATATTTAAAAATTCAGTGC 5 AGGAGTGACATATCCACCAGG 10 TTGTGTATCCCAAACACTCAGAA GGACAACAGAGCGAGACTCC TGAAAGTGTAATTGACTCTAAAAGAG A GTCAATTGCGCAAAACACTG 11 TGTCATCTTCTGAGTATGTCTGGA AGCTCAGTGCCTGTCACACA 12 TCATCAATCATTTGATCTTTGTGTC CAGCTATCACACATATCCACCAA 1 CDC14A gene CTGCTGCGGAGAAAGGAG AAGAAAGAGAAGCCGGAAGC 2 GAGGGATGAGGTTTCTGCAA CCAGGCACCTGAGTCTTAAA 3 GAGGGCATTAGGACGAAACC TTCCAGTGACTTCATTCAGCTC 4 AACCCTCAAATGCCATAGAAA CAGATCAATTCCCAAACTGCT 5 ATGGCCTCTTCATGGATCTG TCTTGCTAGCTAGGGGGATAAA 6 GCAATTTCAGAAGTGCTTCAGTT TCATCCTTCAAGAAGTGAAAAGC 7 AGACAGTCTTAATGCAATATAACCAG CCAAGGCTGAAAGGACAAAA 8 TTGAGAATCAGGTGCATGTTTT TGGGGGTCAGGTAAAGTGAA 9 TTCTCTCACACAACACACTTTCC TTGTGTCCCAGTTGTCTGAAA 10 CCTTCATTTTGCCCTTTTCA TGCATTAGCTGTCAAATCAACA 11 TTTTGGCTTTTTGAAAGTGAC TTGCTATATAGACGGATGGCTCT 12 TGACAATGCTGATTCACAGTACC GAGATGGCAGCAGTTGCTTT 13 GATAGCAAGCCTGGTTCTGG AAATCCTTTTCATGGTGAGCA 14 TTAGAACAGTGCCTGGCAGA TCCCTCCACAACCTTGTCTC 15 AGCCATCCTGACATCATTCAC GCTCAGAAGGCTTCCTTGG 9 TGGTAAAGGATACTACTGGTCTGA 286 16 CCTAGCTAATGCTTTGTGTCTCCT AAATTTTTGGTAGCAAACAGCAG 2.1 GPR88 gene CTGCTGAATCCAATGCAAAG CCAGGTAGCAGTGCAGCAG 2.2 CTCGTGCTGCTGCTCCC GACTTTAGGATTCCTCTAAAACACAG 1 VCAM1 gene TTCTGCAATCAGCATTGTCC CTCAATTGCCTTTGGAAAAA 2 GCATCAAGGTTGGAACTGAG TTTGGATTCCTGCTCTCTCTC 3 TTTGCTTCCAATTCTTGTGC AAGCTCAAGCCTTCTCAGTCC 4 TTACCCTTAAGTGGGAGGTTAAG AAGCAGAGCCATGGAAACTC 5 TCATTCTTAAAAGATGTGGGCTC AAGGATTGTGGGTGCTGTG 6 GTCAAGCCATGGCACACAG ACTGCCTAGCTGGTCCCTTC 7 ACCATGACGTGTCCATGTTC CATGAGAAAAGACAATTTCAGGAG 8 GAACATGAAAGAAGCATAAGCC CAACTTCCTGGCTAAACTTTATAGG 9 CAAGTTTGTGGAAGCCAACA CAAAGGTCAGCTGTCAGCAA 1 EXTL2 gene TCTTTCGCTTCCAGGTCATC ACGCTAACAAAGGCAAATACA 2 AAGGTGCCTGCTTCATCTTC GGGCTGGGAGTAGGAAGAAA 3 TAACAAGCAGGGCAAGAAGG TAACCTCTTCGCCAGGAAAC 4 TCCCTTCAGGCAGTTATACAATC CTTTCAGGATGGCTGAACTAAG 5 TTGTGATGCCATTTGACCTG GGATAAGACCAACTTCTTGGC 1 and 2 SLC30A7 gene GTAAGCGAATTCCCGGGTG CGTGACAAGAGAGGTTGCAC 3 CTTGTGCAGGAGGTACGAGG GAAAGTGGTTACCCTTGGGG 4 TCTGTTTTGTGGCATTCTGC CCAGATTGTGCCTCTACATTTC 5 CCACATAAGAACAAGTTTTGGACC AGAAGGCACTGAGATGGAGG 6 GCTTTCATTACCTTCTGGTACTCC AAAAGCGAACATCCACTGC 7 TGCCACTAAATTTACCTCCACTC GACATGCCAAGTGCACCC 8 CAACCCCTGTTGATTTGAAAC 9 TTGAAAATGTGCTGGGTTTAG 10 11 AATACTTGTCATGGCACCTGG TGGAGTATGGTTTGTACATTATTCTC A TCACCTGGATATCTGGATAGGG GCTATGAGCCGAGACAGTGC AACTCTATGTTGAATGAATATCTCTG C GGACTATAGGCGCATGTCAC 2 DPH5 gene CTTCTGCGGAAAGGTGGTAG GCTCCCTCCTCCCATAACAT 3 TGGACCCAGCCTTACCTACA AAAATTTATAAGGATTGCTGTAGTGC 4 CCCCAAATACAGAGTTTAATTGC ACTAGCTTTTCTTCACTAATCTCAAAG 5 TGCAGCATCACAATGCTCTT 8 and 9 CCAAGGCTGCTTTAGGAGTG GAATTGATAATTGCTAGAAGAAAGAA A AACACTCCTTCATACACACAGACAT 10 CCAAAGAAACCTTGGCTGTC ATTTATTGAATGAAAATTTGAGTACCT 10b 6 and 7 287 CACACAATTGTTACATATTCATCCAA GAAAAGACCAAAAGTCCAGGAG AAGTCTGAGAACCACAGATTTAGG TGGAGATGGTGGAAAAGGAC 11 and 12 TGCCTTATGCCTAATGAGTGG GCCTAGTAGTGCCTTGCACA 13 ACCCAAGGCTGAGAAAAGTG CACCCAGGTTCACCATCAC 14 and 15 GAGCATTCCCTCAGGCTAGA CTAGGGCAGGGCATAAGAGG 16 TTCCCTTGTCTTCTTTTCCAA TCTCAGTGTTGAATCCACAAAA 17 CCAAATTGCCACTTACTTTGAA CCTCACTTTCTGGAATGGTGT 18 TTCTGAAAGGGTAAAGTTTGGAA AGAGCCAGTAGGGGAAATGC S1PR1 gene 1 TTGTTTAAGGCTGCGGTTTC ATGGCGAGGAGACTGAACAC 2 GCCACCACCTACAAGCTCA TTCGTATTCTCCCCCTTCCT COL11A1 gene 1 TCAGCCTGCTTGTCAGTTTC CGGGGAGGAAGGGTAAAGT 2 GTTAGGGAAGCATGGCAAGT TGGTGACCACAAGGTATTTGG 3 AAACCTTAATGGAGGACCCCTA TCACCAGCCTCTAGAAAAACC 4 TGCTTGTGTCCTTTTGGGTA GGTCACCCTTTAGAGTTTTCAA 5 GCCGATTTGTAAAATAGGTGTTTC AACAGCAAGAAAACAAGTACCAAA 6a and 7 6b GGAAATGTGCTTTGTCACTCC GGAAATGTGCTTTGTCACTCC GCAAAACCTGAATTGGCATT GCTCCCAACTGAGGCTTAAA 8 and 9 TCCCATCACACGCTATCAAA TCATTGGTAAAACACGAACATACA 12 and 13 TAAGGTGGAAAGGGCAAATG AATGGTAGCATCTTCCGTATGT 14 TTTGCAGTTCTTCAGTAACCTGAC ATGCACAATCCCTGGAAGAA 15 TCAATGCAAGTGCTGGAAAA AAACCCTCATACATCAATGGAA 16, 17 and 18 TGAAACCATCTCTCTTGATGCT TTTTTCTGATTTTGCAAATTGTT 19 and 20 CCTGCCAAATGTCCTGTTTT TTTCGCATGGCAATTATCTG 21 and 22 TGGACATCACTTTCAGTGTGTTA TTTCCCTCATGAGATTCTGGA 23 and 24 TGTTTGAGGGTGGGTAGAGG TTTCCATGTCGACTCCAGATT 25 TTACATGGCATTTCCTCCAA TGCAATATGGAAGTGATATGATGA 26, 27 and 28 AGCCATACACACTGATGCAAA TTCAAGAAATAAAATGTCTGTTGAAT 29 AGTTGGAAAAGGGAGGAGGA TCTCCACAAAAATTAAAATCCTTAAA 30 GATCAAGGGCAGTTTGAAGG TTTCTGGAGGCAAATTTTAATG 31 32, 33, 34 and 35 CTGCAAATCTTCCTTTTGAGTG GGCGTCCACACACTCTATGA TGTGAAAATCACTCTGTTCCAAA GACATGCACATGTATTAGCAGACA 36 TGTCACTGTGATGAAGAAGCTG TTCAGAAACGATCTATGAACCTTA 37 GAGCAAAGTAAAATAGCTAATGGAAA AATTTTTCCATTTTTCTCAACCT 38 TGGCAAGTTTTGAATTTTAATGA TTGATGTACGAAAGCTGCATA 39,40 and 41 ACTTGGACTGGCCACACTCT CCCACACACTGTGGAATCAC 42 TGGATTCAACTGTTTCTCTTTGG CCAGGGTATTTACATGCCAGA 43 TCACAAATGAAGGTAGGGAACA TGTGTGTGAAACTTTGAAAAAGG 44 GGGAACCCAAGATTTTCCAT AACTGCAGAGGTAATGTAGTATTGG 45 and 46 TCCAAAGGAGTGCAGAAGTG TTTACATCCACCAGAAAACCTG 47 CATGTGGTCAACATTTGCAT CCTGCTCCAAATCAAAATCC 48 and 49 AACCCTGATGTGCTTCACTAAC GGCTTAGAAACACACATACTAGAAGC 288 50 and 51 CAGCATCAAGCCTGCATATT ATTTCTCTTCTCCCGCCAAA 52 and 53 TTGGCGGGAGAAGAGAAATA GAGCTATGTTTTTCAAAGGCTGA 54 TGGAAATGAAGAAAGAGGGTTG AGCACAGAGGAGTGGAAAGC 55 and 56 AAAATGGGTTTTGATTTTCGTT TCCCACAAAATTATCCACGTT 57 CATATGGTGGAACTTAAGGGTGA TCTGATCAAGTGGTTTATTTTCCA 58 59,60,61 and 62 GAAGCCTTAAAAATAAAGCAACAT TGGGAAGCTAAGGATTGAAGC CCAATGCTTAATAAAGCCCTCA TGGCAGAATGTGCTTTTTGT 63 GCTAATGAAAAGCTAAACCAACTC GACAAGGATTTTCCAAAGCAA 64 TACCCCAATATGCCCAAAAG TAGTGAGCAAGCAGGTGCAG 65 CAAATTTTTCTCTTTGTCTCATTTTG GAGACCACAGGGAATCCTCA 66 TGCTTCTGGCATCACAGTTC AACATGTCAAGATTAAGCAACAAA 67 CCTCCACTCAGAAGCCCAAG TTCAAAGCTTTTGCCATGTG RNPC3 gene 5 CTGCCAAGAAATGCAGTAGC CCGATTAAAACTCCACTTCGC AACACTTGAAACATCTTACCTTGTAA C CCCTTGCCACTTCACAATG GACAAACTCAACTCAAAATGTTTATT C CAAATTACATTTCCCAAACTAGC 6 7 CATGTAGAATTGACATTTTAGGAAGG TTCAGCTGAGGGTAAAGGAAG GCATTTCTTTCATATTCCGGC CAAATAACAAGGCTCGAACG 8 AAGATGAAAGCTAAGCAAGGC CAGAGAAGAGAGTAGTGGTCAACTG 9 ATTGACATCGCTGAACCTCG TCTCACCGTTTACCATCAATTTC 10 TCATGGCTAGTCACCCTTTAAC GAAACACTGTATGGAAAGGAACG 11 GGTTTGGGAAATTCACTTTAAGC TTGTTTCAGCCACTTAGCCC 12 GCGAACTTTCACCTTCCTTG CAACTACAAAACTGAGTAATTCCAAC 13 TTGTAGAATCAATTTTAGTGCAGAAC AGGTAAAGAAGCAAAGGCCC AATTGAACAACCTCATGCTGC 1 2 3 4 14 AAGAGTCTTCGAAGGGTTGC GGTTGCATAGCTTTCATCTCAAG TCAGCTGCCTGGGTATAAGC AATACAGAAAGGATTCAGTGAAGG GAGTAACAGGCACACTCCCC AMY2B gene 3 TTGGAGGGCTCTTGTTGAAG 4 CCCAGCAATATATCATTGTGTATG AGGCAACATTTTACTTCACAGG 5 TGTCTAGAAGGCATGTAGGTGTTTAG AAAGTGGGCAAATGTGTATTG 6 AAGCTCGTCGACTTTATTTCC 7 TTTAACCTCCTCTTCACATACAGC TTAATTGGCTCACCACCCA GGCATACTCACTTGTGATAGACAC 8 and 9 TCATATGTCTGAAAAGGCTTGC 10 and 11 GGAGTGCCTCTAAATGATAATGTG TGGCAAAAGAGAACCAGAGG 12 AAGGTTACTTTTGGTCCTAGAAAGC TCTTCTAAAGCATAACAACTTGCTG 1 AMY2A gene CCTTGAGTTGGAAGGGGTTC GAGGGCTCTTGGTGAAGAAA 2 ATCTGTGAAGCTTGGGCAAC TTCCGGGAAATACGAACAG 3 AGGTGTTTAGTTCACATTACTTTCC AGAAGTGGGCAAATGTGTATTG 289 TCTGCTTTAAACTTGTTTCTTTGG TGAAGGAGTAACAGCCATCATC 4 TCCCAATTAAAAATCTCATCGAC TGTCAAAGATACCTGTTGTCCTG 5 AGTTTGGGACCATCCTGGAC TTGCCTTGTGACAGACACTC 6 and 7 TGCTGAAACCTCTGAAAGGAC GTTTCCCTGCGGGCTAAG 8 and 9 TGCCAGAAGAAAACCAGAGG GCATATGATACTGCAAGGGTG 10 AAGGTTACTTTTGGTCCTAGAAAGC TTTCTGAGACATCCTTTTGCC 2 AMY1A gene GGGCTGTTACTTGCCTTGAG ACATCGGAGGGCTCTTGTT 3 TGGGCAACATTTTACTTCACAG CAGGAAATATGGATAGTTATAGCGG 4 TGGTAGTTTCCGGTTCTCTCAG TTTTCAATTGGTTGGGAGC 5 CTCTTAGGGACAGAGGTTAACAAG CATTCTGTGTACCTCCATAAAACC 6 GAGAATTCCTTGAGGCCTGG AGCGTTCCTAGGCATACTGTC 7 and 8 AATGTGCTGAAACCTCTGAAAG TTCCAATTAAACCTGAGGAAGG 9 and 10 TGGCAAAAGAGAACCAGAGG GAAGGAGTAACAGCCATCATCC 11 TGAAAAGGAAATTGGTAGGTTTTC TGTTGGTAGAATGCCAAGGA COL11A1 cDNA AACCATCAAATTTAGAAGAA ATCCGAGCCTGCTGAAGAAT ATGGAACAATGGAAAGTTACC AATCCAGTGGAACCCTTTGGA GGCCCACCTGGCCCAATGGG GGACCAGTCTCACCGGTTGG AACCAGGACTTGCTGGACTT CCATCAGCTCCATTGGGACC GTGCTGATGGGCCTCCTGGT AGGACCCGGTTGACCAGGAT GACAAGGGTGAAATTGGTGAG CAAGTCTCACCACCAGATGTG ATCCTGGTCAACCGGGTCCT GGACCAACTTCAAATCCGAAC TTGTGAGTTTGAGTTCACAG GGTAACTTTCCATTGTTCCAT TGACAATGATTGTTGATTGT CATGGCCATTTATGCTTGT ATGGAACAATGGAAAGTTACC ATCCGAGCCTGCTGAAGAAT ACAAGCATAAATGGCCATG AGGACCTTGAGGACCTCGTT AACGAGGTCCTCAAGGTCCT AATCCAGTGGAACCCTTTGGA GTATGGCAGGTGTAGATGGC GGTCCAGGGAATCCAACTGG TCCAAAGGGTTCCACTGGATT CTCACCAATTTCACCCTTGTC CCAACCGGTGAGACTGGTCC CCATCAGCTCCATTGGGACC GAGGTCTTCCTGGAGCTCAG AGGACCCGGTTGACCAGGAT GGTCCCAATGGAGCTGATGG CTGCTTGCATGCCTTCAGTAT ATCCTGGTCAACCGGGTCCT CAAGTCTCACCACCAGATGTG ATACTGAAGGCATGCAAGCAG JAM3 gene GGACCAACTTCAAATCCGAAC 1 TGGAAATATACCCTGCTTCACTC CATCCCAAATTAGCTCCCC 2 CTCTTTCCCTTGGCTCCTTC AAAACTGCACAGGGCTCACT 3 GGGTGTTTAAGGGAGGGGTA ACTGCACTTGTTGATGCTGG 4 AGTGTGTCTTGCACACCAGG TCAGGCAGTCTCAGCTCTCA 5 TGGCTTGTCAGTCTGCAGTT TCCCTCTTCGGTCTCAATCT 6 GGAGCTGTCAGGCAGGTATC TTGCAGTGAAAGTTGCAAGG 290 7 TTTCAGTGCGACTGCATTTC AAGGGGGAATGTTGTTTTCC 8 GTACTGGCCCTGATCACGTT CTGCAAAGCCAACACACAGT 9 GTACCCAGCAGGGAAAACAA TTTACCGGGTCCATCTTGAG 2 CAGCGCTGTTCGACTGG AGAGGGGTCCCTGCCTC 3 CACTGTCTAGGCACACAGACTC GGGGAGGGCCTAGGTAGAAG 4 CCCACCTCTCAGCTTCCTTC CCAAGTCTGTCATCACCCAG 5 CCCTGGGTCTTGGCCTC GGGTAAGGGTAGGGCTGC 6 GTGATGCCCAACTCAGGC GGCTTCCAGCCTGCATC 7 TTGTGATGTTCAGAGCTGGC TGGGATGAGTGAGAGGAAGC 8 GGAGATGGCCGAGATGC GTCTTGCCACTCTCTCCCTG 9 and 10 CCAAGAGCCTGGGTGAGC GCAAACTTGGGTTGGGG 11 GGACAGAGTAAGCAGCAGGC CCAGCGAGTAAAGTTCCAAAAC 12 CCTGAGTGGTCAGTCCCAG CTACCCCTCGATGACCAGG 13 CTCCCTTCTAGGGGCCAG GAGGTGAGGAGGACGCAG 1 ZMPSTE24 gene GCTCTGAAGGGACGAGTGTC GAAGCCAAGGCTACTCCAGG 2 TGGCAAGCTATAAACCATTCG TTCTGGGACTTGTAAGTGTGG 3 TGTCCTTTCTTTCTTTATACCATGC TTAGTGGAAAGCCTGCCAAG 4 AAGCATATTGCTTGGGATGTAG CCCCAAGACTTTCAGCTATG 5 TGCAAGACATTTACCCATTG LMNA gene 7 CTCTCCAAAGGACCCCAAAC TGGTCAGTCAATACTCCTGTGTC AAGGTAAACTCCTAGCCTTTAAATAA C TCCCTGAATTCAACACTCACTG 8 GAAGGGCTATTACTGGGTTAAAAG CTCTCATGCCTGCCATAGTTC 9 TGGATGCTACTGATCCCATAG AAGCTCTAGATTTGAAGCAGGC 10 CAGTCTCAGCTCATGGAACC GGAACATGCTGCCAGGAC LINS gene CATCATGTAGCGGTGGACAT TGTAGGCACAAAAAGCTGACA 6 CCTGGGAATACCAGAGCAAG LINS cDNA CGATTCTAAATTAATCTGCATGTTCC CATCCTCTGGTCAGTGTTAAG POMGNT1 gene AGTGGCCTACACCGGAAAAG 291 GGATAGCTCTTTCCCCAAGG Appendix C1- (Real Time-PCR COL11A1 Gene Expression Results). Block Type Chemistry Experiment Run End Time Instrument Type Passive Reference Reporter Quencher Ct Threshold Sample Name Target Name Cт 96alum TAQMAN 2013-06-26 09:53:47 AM GST sds7500 ROX FAM NFQ-MGB 0.2 Cт Mean Control HPRT1 24.40 24.48 Control HPRT1 24.42 24.48 Control HPRT1 24.55 24.48 Control HPRT1 24.56 24.48 Control COL11A1 20.78 20.83 Control COL11A1 20.91 20.83 Control COL11A1 20.54 20.83 Control COL11A1 21.08 20.83 Patient HPRT1 24.06 24.21 Patient HPRT1 24.11 24.21 Patient HPRT1 24.15 24.21 Patient HPRT1 24.52 24.21 Patient COL11A1 29.04 29.37 Patient Patient Patient COL11A1 COL11A1 COL11A1 29.38 29.42 29.66 29.37 29.37 29.37 292 Cт SD ΔCт Mean ΔCт SE ΔΔCт RQ 0.08 0.08 0.08 0.08 0.22 -3.65 0.12 0 0.22 -3.65 0.12 0 0.22 -3.65 0.12 0 0.22 -3.65 0.12 0.25 5.16 0.16 8.819986 0.25 5.16 0.16 8.819986 0.25 5.16 0.16 8.819986 0.25 5.16 0.16 8.819986 1 1 1 1 0.20 0.20 0.20 0.20 0.00221 0.00221 0.00221 0.00221 Appendix C2- (Real Time-PCR COL11A1 Gene Expression Results). Block Type Chemistry Experiment Run End Time Instrument Type Passive Reference Reporter Quencher Ct Threshold Sample Name Target Name Control HPRT1 Control HPRT1 Control HPRT1 Control HPRT1 Control COL11A1 Control COL11A1 Control COL11A1 Control COL11A1 Patient HPRT1 Patient HPRT1 Patient HPRT1 Patient HPRT1 Patient COL11A1 Patient COL11A1 Patient COL11A1 Patient COL11A1 96alum TAQMAN 2013-06-25 10:54:40 AM GST sds7500 ROX FAM NFQ-MGB 0.2 ΔCт Mean ΔCт SE 0.08 -3.81 0.08 0 20.96 0.08 -3.81 0.08 0 20.89 20.96 0.08 -3.81 0.08 0 21.03 20.96 0.08 -3.81 0.08 0 24.4 24.45 0.02 24.4 24.45 0.02 24.4 24.45 0.02 24.4 24.45 0.02 Cт Cт Mean Cт SD 24.73 24.78 0.12 24.67 24.78 0.12 24.76 24.78 0.12 24.95 24.78 0.12 20.88 20.96 20.99 ΔΔCт RQ 1 1 1 1 29.29 29.310 0.074 4.86 0.04 8.66644 0.0024611 29.31 29.310 0.074 4.86 0.04 8.66644 0.0024611 29.22 29.310 0.074 4.86 0.04 8.66644 0.0024611 29.40 29.310 0.074 4.86 0.04 8.66644 0.0024611 293 Appendix D- (JAM3-Cell Counting Results). WT counting, n=6 no. of cells/field no. of transfected cells PM ER %PM %ER 385 27 27 0 100 0 380 29 29 0 100 0 451 30 28 2 93.3 6.66 380 13 13 0 100 0 291 23 23 0 100 0 244 7 7 0 100 0 2131 129 127 2 98.45 1.55 2.72 2.72 % SD C219Y counting, n=12 no. of cells/field no. of transfected cells PM ER %PM %ER 465 8 0 8 0 100 372 6 0 6 0 100 410 8 2 6 25 75 227 7 0 7 0 100 455 3 0 3 0 100 453 4 0 4 0 100 156 5 0 5 0 100 209 2 0 2 0 100 226 2 0 2 0 100 368 2 0 2 0 100 312 2 0 2 0 100 315 2 0 2 0 100 3968 51 2 49 3.92 96.07 7.21 7.21 % SD 294 Appendix E1- (Real Time-PCR LINS Gene Expression Results). Block Type Chemistry Experiment Run End Time Instrument Type Passive Reference Reporter Quencher Ct Threshold Sample Name Control1 Target Name LINS Control1 LINS Parent1 96alum TAQMAN 2013-04-24 14:43:50 PM GST sds7500 ROX FAM NFQ-MGB 0.2 31.8161 Cт Mean 31.8475 31.8789 31.8475 2.263409 29.0390 RQ Cт 1 1 LINS ΔCт SE ΔΔCт 0.04444 ΔCт Mean 5.761385 0.04391 0 0.04444 5.761385 0.04391 0 29.0487 0.01360 4.582888 0.07329 -1.1785 Cт SD Parent1 LINS 2.263409 29.0583 29.0487 0.01360 4.582888 0.07329 -1.1785 Patient1 LINS 3.191751 28.3452 28.5400 0.27551 4.087037 0.19489 -1.6743 Patient1 LINS 3.191751 28.7349 28.5400 0.27551 4.087037 0.19489 -1.6743 Control1 HPRT1 26.0554 26.0861 0.04338 Control1 HPRT1 26.1168 26.0861 0.04338 Parent1 HPRT1 24.5384 24.4658 0.10275 Parent1 HPRT1 24.3931 24.4658 0.10275 Patient1 HPRT1 24.4474 24.4530 0.00797 Patient1 HPRT1 24.4587 24.4530 0.00797 295 Appendix E2- (Real Time-PCR LINS Gene Expression Results). Block Type Chemistry Experiment Run End Time Instrument Type Passive Reference Reporter Quencher Ct Threshold Sample Name Target Name Control1 LINS Control1 LINS Control1 LINS Control2 LINS Control2 LINS Control2 LINS Parent2 LINS Parent2 LINS Patient2 LINS Patient2 LINS Patient2 LINS Control1 HPRT1 Control1 HPRT1 Control2 HPRT1 Control2 HPRT1 Parent2 HPRT1 Parent2 HPRT1 Patient2 HPRT1 Patient2 HPRT1 96alum TAQMAN 2013-05-06 10:23:32 AM GST sds7500 ROX FAM NFQ-MGB 0.1 Cт 31.1906 Cт Mean 31.3139 Cт SD 0.12455 ΔCт Mean 4.498495 ΔCт SE 0.0996 ΔΔCт 0 31.4397 31.3139 0.12455 4.498495 0.0996 0 31.3115 31.3139 0.12455 4.498495 0.0996 0 30.8503 30.7672 0.07572 4.688981 0.3170 0.19048 0.87631 30.7021 30.7672 0.07572 4.688981 0.3170 0.19048 0.87631 30.7493 30.7672 0.07572 4.688981 0.3170 0.19048 0.87631 27.3032 27.3032 -1.0917 2.13135 27.2055 27.2055 -1.0917 2.13135 28.5758 28.4577 0.10242 2.948256 0.2532 -1.5502 2.92865 28.4033 28.4577 0.10242 2.948256 0.2532 -1.5502 2.92865 28.3939 28.4577 0.10242 2.948256 0.2532 -1.5502 2.92865 26.7464 26.8154 0.09757 26.8844 26.8154 0.09757 26.3923 26.0782 0.44410 25.7642 26.0782 0.44410 23.32828 23.19516 0.188264 23.06204 23.19516 0.188264 25.75568 25.50945 0.348221 25.26322 25.50945 0.348221 296 0.06910 0.06910 4.108123 4.108123 0.062 0.062 RQ 1 1 1 Appendix F- (The Alignment of the Three Known LINS Isoforms and Showing the Reported Homozygous Mutations) uc002bwi.3 uc010usa.2 uc002bwg.3 MKVFCEVLEELYKKVLLGATLENDSHDYIFYLNPAVSDQDCSTATSLEWANTCGIQGRHQ 60 MKVFCEVLEELYKK---------------------------------------------- 14 MKVFCEVLEELYKKVLLGATLENDSHDYIFYLNPAVSDQDCSTATSLEWANTCGIQGRHQ 60 ************** uc002bwi.3 uc010usa.2 uc002bwg.3 PISVGVAPIAVAPVCLKTNSQMSGSREVMLLQLTVIKVMTTRILSVKTEFHAKEQYRDVI 120 -----------------------------------------------------------PISVGVAPIAVAPVCLKTNSQMSGSREVMLLQLTVIKVMTTRILSVKTEFHAKEQYRDVI 120 uc002bwi.3 uc010usa.2 uc002bwg.3 KILLESAKVDSKLICMFQNSDKLLSHMAAQCLALLLYFQLREKITLSNSWIAFCQKNLSE 180 -------------ICMFQNSDKLLSHMAAQCLALLLYFQLREKITLSNSWIAFCQKNLSE 61 KILLESAKVDSKLICMFQNSDKLLSHMAAQCLALLLYFQLREKITLSNSWIAFCQKNLSE 180 *********************************************** uc002bwi.3 uc010usa.2 uc002bwg.3 YSESNKAIYCLWTLTAIIKEIFKDSCSQKTEILKQFLTHFDTIFEVFYNSLFSQHFENCR 240 YSESNKAIYCLWTLTAIIKEIFKDSCSQKTEILKQFLTHFDTIFEVFYNSLFSQHFENCR 121 YSESNKAIYCLWTLTAIIKEIFKDSCSQKTEILKQFLTHFDTIFEVFYNSLFSQHFENCR 240 ************************************************************ uc002bwi.3 uc010usa.2 uc002bwg.3 DTSKIVNILMCFLDLLELLIASRIHLKLHFTCQRILFLKPSCMLEVITWPIQAFVKRKVI 300 DTSKIVNILMCFLDLLELLIASRIHLKLHFTCQRILFLKPSCMLEVITWPIQAFVKRKVI 181 DTSKIVNILMCFLDLLELLIASRIHLKLHFTCQRILFLKPSCMLEVITWPIQAFVKRKVI 300 ************************************************************ uc002bwi.3 uc010usa.2 uc002bwg.3 IFLKKCLLCKVGEDLCRGSVPALMPPDHHVAVDMLALANAVLQAVNSGLLKTLSVYEKHS 360 IFLKKCLLCKVGEDLCRGSVPALMPPDHHVAVDMLALANAVLQAVNSGLLKTLSVYEKHS 241 IFLKKCLLCKVGEDLCRGSVPALMPPDHHVAVDMLALANAVLQAVNSGLLKTLSVYEKHS 360 ************************************************************ uc002bwi.3 uc010usa.2 uc002bwg.3 FFGGDEVQPECELITSPDHVILRAASLVIMKSLEIKFQNYSSASEVKGNSP--------- 411 FFGGDEVQPECELITSPDHVILRAASLVIMKSLEIKFQNYSSASEVKGNSP--------- 292 FFGGDEVQPECELITSPDHVILRAASLVIMKSLEIKFQNYSSASEMKVDLQRFMSELLTF 420 *********************************************:* : uc002bwi.3 uc010usa.2 uc002bwg.3 ----------------------------------------------------------------------------------------------------------------------LKPHLQPSLQLHNPCKWLSRVFIEQDDDMLEAAKASLGIYLTLTRGCEATESLTQGKEMW 480 uc002bwi.3 uc010usa.2 uc002bwg.3 ------NSFCMQCVIIYL-------STVIHNYQISGLV---------------------- 436 ------NSFCMQCVIIYL-------STVIHNYQISGLV---------------------- 317 DHHTHENGYNPHCIFLFFLKNIGFDSTVLLDFLISSETCFLEYFVRYLKLLQKDWDNFFT 540 *.: :*::::: ***: :: **. . uc002bwi.3 uc010usa.2 uc002bwg.3 ----------------------------------------------------------------------------------------------------------------------ICNNFDATESKYDISICGCVPSLVQDQSSNQTIPHRLTAPHSHRDVCARHSWASDAPSEP 600 uc002bwi.3 uc010usa.2 uc002bwg.3 ----------------------------------------------------------------------------------------------------------------------LKAVMSKGAHTMCASSLSSPRASQSLVDYDSSDDSDVESTEQCLANSKQTSLHQQATKEI 660 uc002bwi.3 uc010usa.2 uc002bwg.3 ----------------------------------------------------------------------------------------------------------------------QDAAGTSRDKKEFSLEPPSRPLVLKEFDTAFSFDCEVAPNDVVSEVGIFYRIVKCFQELQ 720 uc002bwi.3 uc010usa.2 uc002bwg.3 ------------------------------------------------------------------------DAICRLQKKNLFPYNPTALLKLLKYIEVISNKTMNTL 757 p.E211_K407del p.H329* p.D511Ffs*10 p.S594Ffs*6 p.Q717* 297 Appendix G- (Blocks of Shared Homozygosity between the Affected Individuals in the Progeoid Family) Chromoso me #Marke rs cM homozygo us Mb homozygo us 19 538 6.869973 2327817 5 1134 3.360131 3478008 15 60 2.5372 856473 18 199 1.963434 3189103 5 198 1.690854 880798 7 96 1.43476 397722 11 145 1.354594 241667 16 147 1.299843 406761 5 484 1.279213 1901109 2 239 1.217237 745831 15 80 0.635543 214538 16 258 1.14732 14728452 5 219 1.144042 486677 19 69 1.027368 279433 5 185 1.134247 438269 7 28 1.109348 1456646 14 114 1.08893 493953 8 454 1.109099 912749 1 83 1.08187 825468 16 131 1.071606 149463 16 98 1.003512 402941 3 81 0.998388 360452 2 151 1.001 487989 16 51 0.940892 676479 1 119 0.956286 325695 13 106 0.949888 183279 1 90 0.918146 337078 11 87 0.906477 384678 8 273 0.909864 710823 4 54 0.871355 134956 298 First flanking affy SNP_A4242649 SNP_A8294257 SNP_A4239006 SNP_A8340389 SNP_A8541326 SNP_A8660044 SNP_A8597963 SNP_A8711753 SNP_A1921210 SNP_A8464169 SNP_A4219989 SNP_A8591256 SNP_A2166411 SNP_A1899719 SNP_A8354405 SNP_A1816484 SNP_A8637820 SNP_A4266781 SNP_A8625319 SNP_A4239889 SNP_A8410619 SNP_A1893390 SNP_A2105394 SNP_A2060840 SNP_A8314872 SNP_A2251827 SNP_A1852783 SNP_A2045808 SNP_A1920451 SNP_A4264103 first flanking rs rs2240743 rs4522972 rs2305252 rs2775801 rs7711157 rs4716622 rs1487185 rs4784248 rs6869276 rs287307 rs1828774 rs9931752 rs17295893 rs8102561 rs4869582 rs37612 rs1269068 rs4345557 rs2990245 rs4786804 rs11642531 rs1350226 rs13387359 rs173236 rs331642 rs9586847 rs4971264 rs2510389 rs4629861 rs2284659 last flanking affy SNP_A8552994 SNP_A4294262 SNP_A2026514 SNP_A4280665 SNP_A1806657 SNP_A4282407 SNP_A8443151 SNP_A2129800 SNP_A4243654 SNP_A1962314 SNP_A8712802 SNP_A8453942 SNP_A8300458 SNP_A8405110 SNP_A8553759 SNP_A1990170 SNP_A8623013 SNP_A8489209 SNP_A1967458 SNP_A8356391 SNP_A8453945 SNP_A1928014 SNP_A8356124 SNP_A8405141 SNP_A2296960 SNP_A8346187 SNP_A2125755 SNP_A8703516 SNP_A8429469 SNP_A8690626 last flanking rs rs479448 rs2972935 rs1828775 rs4800370 rs1917753 rs10263848 rs7119267 rs17302707 rs6451656 rs3788961 rs1877911 rs1551300 rs32631 rs10403600 rs4869458 rs1167829 rs6573632 rs2952172 rs6427299 rs4786829 rs7197420 rs1522938 rs1568238 rs2641906 rs1777958 rs1549058 rs6697616 rs12806559 rs7825113 rs13112159 20 94 0.835394 263734 15 135 0.884564 542767 15 94 0.881762 244125 20 169 0.865832 462428 2 162 0.870471 668482 15 114 0.843549 260896 2 218 0.847482 816865 15 90 0.833313 184456 5 166 0.825269 742132 12 98 0.78654 147670 2 78 0.793548 422820 14 89 0.783678 416264 2 350 0.798067 1421880 17 31 0.743335 138451 18 126 0.788235 327142 14 19 0.782552 93768 16 140 0.763843 297202 7 70 0.764332 385798 4 103 0.7792 372630 18 38 0.714986 152816 5 165 0.761398 388099 22 24 0.744101 185073 21 61 0.728308 234022 15 68 0.753502 123124 13 100 0.747969 438391 12 122 0.729415 191442 2 90 0.707302 455912 10 84 0.71719 271789 10 66 0.711424 269604 15 55 0.707444 104024 18 144 0.729519 406840 16 130 0.708305 665065 1 40 0.691 78227 12 155 0.714274 796955 299 SNP_A2228677 SNP_A8302872 SNP_A8400845 SNP_A8570714 SNP_A8575630 SNP_A4273883 SNP_A1782810 SNP_A8344505 SNP_A2238942 SNP_A8562733 SNP_A8376108 SNP_A1928232 SNP_A8426004 SNP_A8507978 SNP_A8353174 SNP_A8312243 SNP_A1905019 SNP_A4301558 SNP_A2262947 SNP_A4200882 SNP_A8665235 SNP_A8482732 SNP_A8343534 SNP_A2114261 SNP_A2244339 SNP_A2137525 SNP_A8334169 SNP_A8421335 SNP_A8282169 SNP_A4289616 SNP_A2146836 SNP_A1856738 SNP_A1802652 SNP_A2083007 rs2865718 rs11639209 rs11637779 rs159088 rs17701375 rs936532 rs16847958 rs7166757 rs7734243 rs7308676 rs7587659 rs4901542 rs10204835 rs12453336 rs9304001 rs11629400 rs8055417 rs2236426 rs6842825 rs10502344 rs1362801 rs138877 rs2836912 rs17501060 rs9318105 rs4391872 rs4674301 rs1954211 rs1934944 rs1058696 rs12960235 rs13333771 rs947379 rs10506938 SNP_A8428647 SNP_A1907056 SNP_A4301578 SNP_A8365695 SNP_A8500556 SNP_A8645433 SNP_A4243961 SNP_A8289300 SNP_A2241969 SNP_A2267526 SNP_A1813575 SNP_A2141881 SNP_A2146202 SNP_A8361730 SNP_A8688101 SNP_A8612300 SNP_A1787393 SNP_A8415488 SNP_A8518164 SNP_A8393102 SNP_A2010842 SNP_A2117236 SNP_A8344015 SNP_A1828272 SNP_A2156861 SNP_A8303828 SNP_A8583570 SNP_A8393955 SNP_A8358186 SNP_A2004441 SNP_A1922875 SNP_A1885978 SNP_A8459089 SNP_A4284955 rs7270917 rs1498590 rs10518966 rs212600 rs7581360 rs4924389 rs16858115 rs8034873 rs2416258 rs1610177 rs4241112 rs8009660 rs6714124 rs9896300 rs1443614 rs2771324 rs8049029 rs887056 rs12331468 rs412604 rs2939335 rs137866 rs11088473 rs11858005 rs1333099 rs7978886 rs11695967 rs11253896 rs7071399 rs174274 rs587274 rs756718 rs2268147 rs2063568 2 151 0.711294 453046 8 101 0.672038 223511 16 118 0.706724 201207 5 74 0.71054 178820 3 94 0.706327 281706 7 62 0.703338 272134 17 82 0.700361 257307 8 97 0.697072 475299 13 159 0.698595 310055 20 35 0.610627 192775 2 77 0.681771 345629 2 49 0.685898 231078 14 68 0.647605 174679 3 98 0.666032 336930 18 96 0.665213 485311 15 93 0.656077 286336 8 129 0.663736 1424992 13 192 0.646459 341782 12 150 0.64195 428501 12 106 0.652405 1026078 5 133 0.643308 300666 16 79 0.641216 257468 20 68 0.646856 207737 18 27 0.567697 45341 12 34 0.59654 113347 1 150 0.631848 435514 19 72 0.595177 217721 7 137 0.62604 360170 3 33 0.55585 133228 5 155 0.630424 321046 21 33 0.627964 107706 2 119 0.617122 487010 21 132 0.622652 239251 16 32 0.613937 656960 300 SNP_A4202663 SNP_A1871827 SNP_A8677307 SNP_A8665201 SNP_A8619422 SNP_A8510961 SNP_A8488533 SNP_A4266940 SNP_A2306548 SNP_A2118501 SNP_A8694468 SNP_A4298368 SNP_A2240938 SNP_A8493455 SNP_A8589802 SNP_A8445054 SNP_A8697323 SNP_A8306221 SNP_A2060882 SNP_A4255153 SNP_A8663562 SNP_A8631323 SNP_A8566265 SNP_A1834941 SNP_A2175187 SNP_A4291941 SNP_A8695332 SNP_A8517212 SNP_A8302720 SNP_A8666560 SNP_A4215562 SNP_A8601198 SNP_A4270622 SNP_A1794911 rs1374706 rs7833668 rs1533822 rs930049 rs9828988 rs6969684 rs2286593 rs1384830 rs1998612 rs13039229 rs12463788 rs2251692 rs17091694 rs10804733 rs299238 rs7164548 rs13252978 rs2699342 rs7975189 rs7971845 rs224912 rs9937099 rs1821809 rs11151031 rs10773715 rs968304 rs2081894 rs11980816 rs9863733 rs2099041 rs2823718 rs155106 rs2898402 rs33957528 SNP_A8577227 SNP_A2288165 SNP_A2055674 SNP_A2144218 SNP_A4296026 SNP_A1796724 SNP_A8682473 SNP_A8283345 SNP_A8678340 SNP_A2223591 SNP_A8675760 SNP_A8482377 SNP_A2100205 SNP_A8635652 SNP_A1937145 SNP_A8604831 SNP_A8285471 SNP_A8411172 SNP_A4226886 SNP_A1861049 SNP_A1982329 SNP_A2222656 SNP_A4242325 SNP_A8563155 SNP_A2117433 SNP_A1898128 SNP_A8658540 SNP_A2190907 SNP_A2269156 SNP_A2127960 SNP_A8342629 SNP_A8468836 SNP_A4209148 SNP_A8511559 rs10490277 rs17220174 rs11074815 rs12186616 rs9832791 rs2893375 rs8074682 rs993106 rs9533573 rs5019252 rs1729080 rs10210963 rs14042 rs2331675 rs7233350 rs6598242 rs6985209 rs6491764 rs4913518 rs10219717 rs10040233 rs17772159 rs2870718 rs1893833 rs1675874 rs7542088 rs4802981 --rs9858714 rs6897517 rs440294 rs13405639 rs2837623 rs8045689 16 178 0.592246 326992 2 163 0.622669 801017 7 47 0.613196 175015 5 70 0.617952 193152 8 30 0.593169 578831 21 115 0.61343 465623 7 67 0.607217 185040 19 28 0.59319 186654 11 70 0.530497 415466 8 36 0.546771 130125 5 57 0.594322 108125 3 62 0.579503 150913 3 47 0.56195 140347 18 69 0.573347 399776 13 178 0.586322 346625 4 256 0.579508 733741 13 59 0.551896 191968 21 117 0.553616 299471 1 48 0.555842 311906 18 58 0.554748 230670 9 122 0.564931 290713 16 95 0.515708 94137 11 84 0.549639 203944 20 58 0.545111 232843 8 137 0.552171 488622 8 34 0.510436 257916 3 75 0.52682 320793 10 79 0.534242 309687 3 45 0.538907 418941 4 118 0.539872 201162 5 36 0.494215 101513 3 140 0.53708 769730 4 63 0.535694 272982 13 149 0.531531 660443 SNP_A8650559 SNP_A2035326 SNP_A8534863 SNP_A8713807 SNP_A8415724 SNP_A2017840 SNP_A8386966 SNP_A2040097 SNP_A4279373 SNP_A8587261 SNP_A8665085 SNP_A1844296 SNP_A2073526 SNP_A8625867 SNP_A1901882 SNP_A8662143 SNP_A8567696 SNP_A2015339 SNP_A8494641 SNP_A8541348 SNP_A2112983 SNP_A8360633 SNP_A2065676 SNP_A2282624 SNP_A8401245 SNP_A4304357 SNP_A8439239 SNP_A2001383 SNP_A8640809 SNP_A8320864 SNP_A2287706 SNP_A4216907 SNP_A2134181 SNP_A2054587 301 rs4614753 rs11896227 rs13225903 rs11742964 rs6983214 rs2244721 rs834433 rs12461453 rs11230633 rs4427135 rs1862581 rs4686945 rs12696555 rs4624285 rs2706410 rs10518378 rs1415411 rs2823059 rs1000997 rs616329 rs10868516 rs1102967 rs2103173 rs3827963 rs12678203 rs7836362 rs7616037 rs522951 rs4630960 rs16894263 rs35580 rs12485995 rs379519 rs1970270 SNP_A8474184 SNP_A8483255 SNP_A8691497 SNP_A8462702 SNP_A8360655 SNP_A8578379 SNP_A8337232 SNP_A8657796 SNP_A2133800 SNP_A1894187 SNP_A2232408 SNP_A8296797 SNP_A4240247 SNP_A8321335 SNP_A4261207 SNP_A2140218 SNP_A2053167 SNP_A8341833 SNP_A8415696 SNP_A8422546 SNP_A2051868 SNP_A8427362 SNP_A2249626 SNP_A8496777 SNP_A4222297 SNP_A8518046 SNP_A8478724 SNP_A1851303 SNP_A2312494 SNP_A2273392 SNP_A1872737 SNP_A1973688 SNP_A8418952 SNP_A2092010 rs12599583 rs11694706 rs13234269 rs3906345 rs7841468 rs2898175 rs3778813 rs7250294 rs11230775 rs7014406 rs2136193 rs7645379 rs11720187 rs1434527 rs4883908 rs6534279 rs9573281 rs2823262 rs11260711 rs13380899 rs927524 rs7206547 rs11602446 rs12626071 rs7842793 rs7844699 rs197849 rs11190643 rs16852464 rs600940 rs250350 rs9844218 rs17792161 rs1504432 5 115 0.526687 234596 5 122 0.516283 618999 4 49 0.51586 118106 10 25 0.517875 73660 5 82 0.513358 284695 8 46 0.511769 189610 15 62 0.510099 322420 5 110 0.506285 349460 20 72 0.515149 96721 16 152 0.503385 449585 18 136 0.485107 496290 19 38 0.456893 150237 11 155 0.496645 354259 11 101 0.489891 393418 2 62 0.490528 170480 11 34 0.494443 229173 14 70 0.457911 319580 5 37 0.463147 118137 15 39 0.477471 95359 1 86 0.489027 259071 8 73 0.43779 195432 12 20 0.399639 117994 22 6 0.299485 108609 4 82 0.30739 370683 14 43 0.241404 57155 14 17 0.222499 67035 14 22 0.161097 112431 9 2 0.000604 372 SNP_A2038519 SNP_A8505148 SNP_A4245693 SNP_A1949159 SNP_A2258350 SNP_A1930592 SNP_A2248316 SNP_A2136005 SNP_A8306556 SNP_A2004616 SNP_A8442961 SNP_A2310289 SNP_A8399281 SNP_A8292783 SNP_A1814793 SNP_A1789365 SNP_A8565893 SNP_A1920263 SNP_A8550064 SNP_A2137263 SNP_A8583481 SNP_A8383045 SNP_A8523422 SNP_A8345245 SNP_A2188880 SNP_A8457061 SNP_A1927639 SNP_A1960831 302 rs577395 rs4632762 rs10028944 rs7091695 rs6893638 rs17729816 rs1025199 rs12697229 rs6043342 rs8050676 rs680798 rs2574764 rs11603534 rs4939346 rs3769095 rs7108368 rs10873515 rs2569094 rs12595826 rs12038162 rs13282231 rs7959658 rs4822313 rs11936246 rs17533457 rs10139382 rs10147424 rs2211079 SNP_A8461210 SNP_A8549210 SNP_A2054181 SNP_A8699730 SNP_A8388863 SNP_A8645898 SNP_A1876769 SNP_A2255648 SNP_A8591660 SNP_A2270222 SNP_A8435863 SNP_A2153655 SNP_A1802721 SNP_A1789702 SNP_A8658230 SNP_A8371549 SNP_A8550766 SNP_A8603505 SNP_A4240727 SNP_A1803596 SNP_A4202799 SNP_A1785324 SNP_A1795337 SNP_A8639376 SNP_A2206884 SNP_A1800625 SNP_A8565893 SNP_A1997383 rs2047322 rs6873479 rs7694098 rs10998696 rs6882146 rs7843437 rs1426721 rs4513709 rs200745 rs17256727 rs878971 rs553611 rs34869754 rs11230463 rs11898345 rs10793063 rs10151090 rs3095905 rs1444860 rs1889543 rs1440744 rs12313899 rs451115 rs12331558 rs12432655 rs1957684 rs10873515 rs1556858 الخالصة إن األمراض الوراثية المتنحية لها تأثير مدمر على المرضى و عائالتهم كما ولها تأثير كبير على نظام الرعاية الصحية في البالد .لذا معرفة األسباب الجينية والجزيئية والخلوية لهذه األمراض يعتبر إجباري لما سيؤدي إليه من تحسين تشخيصهاوالسيطرة عليها والوقاية منها وربما عالجها. في هذه األطروحة أصف المظاهر السريرية و األسس الجينية لسبعة أمراض وراثية متنحية في عائالت مصابه .لقد تم التعرف على مناطق الجينات المسببة لأل مراض المدروسة بالقيام بعملية تنميط جيني على نطاق واسع يليها قراءة للجينات المرشحة أو لكل الجينات المنتجة للبروتينات معا لتحديد الطفرات .كما تم إستخدام بعض الوسائل المعلوماتية الحياتية والدراسات العملية إلثبات صحة النتائج. لقد تم إيجاد مناطق كل األمراض المدروسة كما تم التعرف على عدة طفرات مرضية جديدة متماثلة. الطفرات التي تم التعرف عليها في الجينين OBSL1و CUL7في 4عائالت أدت إلى تشخيصهم كحاالت مصابة بالمرض .Three-Mالطفرات الجديدة التي تم التعرف عليها في الجين COL11A1في عائلتين كانت أول صلة بين هذا الجين و مرض .fibrochondrogenesisالطفرات الجديدة التي تم الكشف عليها في الجين JAM3في 3عائالت تؤكد أهميته في حاجز الدم في الدماغ .الطفرة التي تم التعرف عليها في الجين LINSفي أخين مصابين كانت الدليل القاطع على دوره في التطور العقلي .كما تم الكشف عن طفرات جديدة في الجينين POMGnT1و PRG4في عائلة مصابة بضمور العضالت الوالدي و في عائلة مصابة بمتالزمة camptodactyly-arthropathy-coxa vara-pericarditisعلى التوالي .وأخيرا ،تم إيجاد منطقة واحدة على كروموسوم 91لمرض progeroidلحديثي الوالدة. إن التعرف على األسس الجينينة لهذه األمراض الوراثية المتنحية ذو فائدة مباشرة لألسر المصابة بها وذلك بتقديم تشخيص جزيئي دقيق مما يجعل هناك إمكانية لمنع حدوث حاالت جديدة .كما ساهمت بعض النتائج في هذه الدراسة في فهم اآللية المرضية الكامنه خلف هذه األمراض الوراثية المتنحية و قدمت رؤى جديدة في بعض نواحي التطور البشري. جامعة اإلمارات العربية المتحدة كلية الطب و العلوم الصحية التعرف على العوامل الوراثية المسببة لبعض ألمراض الوراثية المتنحية ناديا عوني عكاوي تقدم هذه األطروحة إلستيفاء جزء من المتطلبات المطلوبة لدرجة الدكتوراة في علم الوراثة بإشراف األساتذة بسام علي و لحاظ الغزالي 12/2013