Download characterizing the genetic bases of autosomal recessive disorders

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Winter 12-2013
CHARACTERIZING THE GENETIC BASES
OF AUTOSOMAL RECESSIVE DISORDERS
Nadia Awni Akawi
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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. The way to find additional
families with mutations in candidate genes is by sharing genetic and
phenotypic data. Another way of sharing could be achieved via establishing a
mutation database for rare disorders with a forum where researchers can
easily communicate and present their candidate genes and mutations.
Establishing such a database requires advanced bioinformatics skills, a
strong computational infrastructure and willingness to share information
among clinicians and scientists.
245
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APPENDICES
281
Appendix A - (JAM3 cDNA Cloning Site and Associated Tags).
282
Appendix B - (All the Primers Used in This Dissertation).
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
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SNP_A2312494
SNP_A2273392
SNP_A1872737
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‫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