* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Product description P048-C1-0315 LMNA-MYOT - MRC
Bisulfite sequencing wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
No-SCAR (Scarless Cas9 Assisted Recombineering) Genome Editing wikipedia , lookup
Human genome wikipedia , lookup
Metagenomics wikipedia , lookup
Neuronal ceroid lipofuscinosis wikipedia , lookup
Cell-free fetal DNA wikipedia , lookup
Gene therapy wikipedia , lookup
Saethre–Chotzen syndrome wikipedia , lookup
Gene expression programming wikipedia , lookup
Primary transcript wikipedia , lookup
Gene expression profiling wikipedia , lookup
Gene nomenclature wikipedia , lookup
Gene desert wikipedia , lookup
Genome (book) wikipedia , lookup
History of genetic engineering wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Point mutation wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Frameshift mutation wikipedia , lookup
Designer baby wikipedia , lookup
Genome evolution wikipedia , lookup
Helitron (biology) wikipedia , lookup
Alternative splicing wikipedia , lookup
Microevolution wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
MRC-Holland Description version 17; 01 June 2016 ® MLPA SALSA MLPA probemix P048-C1 LMNA/MYOT/ZMPSTE24 Lot C1-0315, C1-0112. Laminopathies have emerged as clinically heterogeneous genetic disorders due to mutations in lamins or lamin-associated proteins. Lamins are structural protein components of the nuclear lamina, a protein network underlying the inner nuclear membrane that determines nuclear shape and size. The lamins constitute a class of intermediate filaments. Three types of lamins, A, B, and C, have been described in mammalian cells. Laminopathies regroup at least eight distinct diseases, belonging to the groups of skeletal and/or cardiac muscular dystrophies, axonal neuropathies, premature ageing syndromes and familial lipodystrophies. These diseases, such as Emery-Dreifuss muscular dystrophy, Hutchinson-Gilford progeria syndrome and limb-girdle muscular dystrophy type 1B (LGMD1B), result from alterations in the LMNA gene, encoding type A-lamins. Pathophysiological mechanisms explaining how mutations in a unique gene could lead to such various phenotypes are still unknown, but probably involve alterations in cellular mechanical stress responses, in gene expression, and/or in post-translational maturation of lamin A. One gene that is involved in the posttranslational processing of Lamin A precursor is ZMPSTE24 (also known as FACE-1 in human). Loss of function of the ZMPSTE24 gene and accumulation of precursor lamin A has been correlated with restrictive dermopathy (RD). Mutations in the MYOT gene are associated with LGMD1A and myofibrillar myopathies. Mutations in the CAV3 gene are associated with LGMD1C. The LMNA gene (12 exons) spans ~25.4 kb of genomic DNA and is located on 1q22, ~156 Mb from the ptelomere. The ZMPSTE24 gene (10 exons) spans ~36.1 kb of genomic DNA and is located on 1p34.2, ~40.7 Mb from the p-telomere. The MYOT gene (10 exons) spans ~20 kb of genomic DNA and is located on chromosome 5q31.2, ~137.2 Mb from the p-telomere. The CAV3 gene (2 exons) spans ~13 kb of genomic DNA and is located on chromosome 3p25.3, ~8.8 Mb from the p-telomere. The P048-C1 probemix contains probes for all 12 exons of the LMNA gene. Two probes are present for exon 1. This probemix furthermore contains probes for all exons of the ZMPSTE24, MYOT and CAV3 genes. Finally, 9 reference probes are included in this probemix, detecting several different autosomal chromosomal locations. This SALSA® MLPA® probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned gene(s) in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Note that a mutation or polymorphism in the sequence detected by a probe can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some probe signals are more sensitive to sample purity and small changes in experimental conditions. Therefore, deletions and duplications detected by MLPA should always be confirmed by other methods. Not all deletions and duplications detected by MLPA will be pathogenic; users should always verify the latest scientific literature when interpreting their findings. We have no information on what percentage of defects in these genes is caused by deletions/duplications of complete exons. Finally, note that most defects in this gene are expected to be small (point) mutations which will not be detected by this SALSA® MLPA® test. SALSA® MLPA® probemixes and reagents are sold by MRC-Holland for research purposes and to demonstrate the possibilities of the MLPA technique. They are not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA® MLPA® test probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA® MLPA® probemix and reagents requires a thermocycler with heated lid and sequence type electrophoresis equipment. Different fluorescent PCR primers are available. The MLPA technique has been first described in Nucleic Acid Research 30, e57 (2002). SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix Page 1 of 6 MRC-Holland Description version 17; 01 June 2016 ® MLPA References Van Spaendonck-Zwarts K et al. (2013). Genetic analysis in 418 index patients with idiopathic dilated cardiomyopathy: overview of 10 years' experience. Eur J Heart Fail. 15:628–636. Weterings A et al. (2013). A novel lamin A/C mutation in a Dutch family with premature atherosclerosis. Atherosclerosis. 229:169-173. Norton N et al. (2011). Assessment of LMNA copy number variation in 58 probands with dilated cardiomyopathy. Clin Transl Sci. 4:351–352. Marsman R et al (2011). A complex double deletion in LMNA underlies progressive cardiac conduction disease, atrial arrhythmias, and sudden death. Circ Cardiovasc Genet. 4:280-287. Related SALSA® MLPA® probemixes P116 P176 P268 P436 P061 SGC: Contains probes for the SGCA, SGCB, SGCD, SGCG and FKRP genes, involved in LGMDs. CAPN3: Contains probes for the CAPN3 gene, involved in LGMD2A. DYSF: Contains probes for the DYSF gene, involved in LGMD2B. ANO5: Contains probes for the ANO5 gene, involved in LGMD2L. Lissencephaly: Contains probes for the POMT1 gene, involved in LGMD2K. More information Website : www.mlpa.com E-mail : [email protected] (information & technical questions); [email protected] (for orders) Mail : MRC-Holland bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Data analysis The P048-C1 probemix contains 44 MLPA probes with amplification products between 129 and 463 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at 64-70-76-82 nt, three DNA denaturation control fragments (Dfragments) at 88-92-96 nt, one X-fragment at 100 nt and one Y-fragment at 105 nt. More information on how to interpret observations on these control fragments can be found in the MLPA protocol. Data generated by this probemix can first be normalised intra-sample by dividing the peak height of each probe’s amplification product by the total peak height of only the reference probes in this probemix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised probe ratio in a sample by the average intra-normalised probe ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference probes. Data normalisation should be performed within one experiment. Only samples purified by the same method should be compared. Confirmation of most exons deletions and amplifications can be done by e.g. Southern blotting, long range PCR, qPCR, FISH. Note that Coffalyser, the MLPA analysis tool developed at MRC-Holland, can be downloaded free of charge from our website www.mlpa.com. Many copy number alterations in healthy individuals are described in the database of genomic variants: http://dgv.tcag.ca/dgv/app/home. For example, a duplication of a complete gene might not be pathogenic, while a partial duplication or a deletion may result in disease. For some genes, certain in-frame deletions may result in a very mild, or no disease. Copy number changes of reference probes are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This probemix was developed at MRC-Holland. Info/remarks/suggestions for improvement: [email protected]. SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix Page 2 of 6 MRC-Holland ® MLPA Description version 17; 01 June 2016 Table 1. SALSA MLPA P048-C1 LMNA/MYOT/ZMPSTE24 probemix Length (nt) 64-70-76-82 88-92-96 100 105 129 136 148 154 160 166 172 179 184 190 196 202 208 214 220 230 238 247 256 262 266 274 280 286 292 301 310 315 321 330 337 346 355 Ж 361 371 382 391 400 409 418 427 436 454 463 SALSA MLPA probe reference Chromosomal position LMNA ZMPSTE24 MYOT CAV3 Q-fragments: DNA quantity; only visible with less than 100 ng sample DNA D-fragments: Low signal of 88 or 96 nt fragment indicates incomplete denaturation X-fragment: Specific for the X chromosome Y-fragment: Specific for the Y chromosome Reference probe 11622-L12379 ZMPSTE24 probe 15689-L21730 ZMPSTE24 probe 15691-L17658 LMNA probe 11552-L12299 LMNA probe 11553-L12300 MYOT probe 11554-L12301 MYOT probe 11555-L12302 ZMPSTE24 probe 15687-L18494 MYOT probe 11556-L12303 MYOT probe 11557-L12304 Reference probe 10138-L10600 MYOT probe 11558-L13504 ZMPSTE24 probe 15684-L18495 MYOT probe 11559-L13505 LMNA probe 05939-L05368 Reference probe 01783-L01347 CAV3 probe 09681-L10061 LMNA probe 02478-L01971 Reference probe 10702-L11284 ZMPSTE24 probe 17350-L17657 Reference probe 08600-L21175 CAV3 probe 17349-L21793 LMNA probe 12287-L13799 LMNA probe 12027-L13939 MYOT probe 11560-L12307 LMNA probe 01916-L01460 ZMPSTE24 probe 16705-L19884 LMNA probe 15694-L20323 Reference probe 05802-L20124 LMNA probe 01918-L21732 LMNA probe 01919-L01463 ZMPSTE24 probe 15692-L17659 ZMPSTE24 probe 16895-SP0379L19286 LMNA probe 13216-L14549 ZMPSTE24 probe 17417-L18992 LMNA probe 01922-L01466 LMNA probe 15693-L17660 Reference probe 15010-L11786 MYOT probe 11561-L21731 MYOT probe 11562-L12309 MYOT probe 11563-L14166 Reference probe 09614-L09909 ZMPSTE24 probe 15695-L17662 Reference probe 10108-L10532 10q25 Exon 7 Exon 10 Exon 5 Exon 9 Exon 4 Exon 1 Exon 2 Exon 8 Exon 5 18q11 Exon 9 Exon 1 Exon 3 Exon 12 13q14 Exon 1 Exon 11 6p12 Exon 8 17p11 Exon 2 Exon 1 Exon 1 Exon 7 Exon 2 Exon 3 Exon 7 15q15 Exon 4 Exon 6 Exon 4 Exon 5 Exon 8 Exon 9 Exon 10a Exon 3 2p24 Exon 6 Exon 2 Exon 10 20p12 Exon 6 8q22 Ж This probe consists of three parts and has two ligation sites. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference probes is available on request: [email protected]. SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix Page 3 of 6 MRC-Holland ® MLPA Description version 17; 01 June 2016 Table 2. P048 probes arranged according to chromosomal location Table 2a. ZMPSTE24 gene Length SALSA MLPA ZMPSTE24 (nt) probe exon start codon 208 179 310 346 355 Ж 454 136 262 371 148 15684-L18495 15687-L18494 16705-L19884 15692-L17659 16895-SP0379L19286 15695-L17662 15689-L21730 17350-L17657 17417-L18992 15691-L17658 Exon Exon Exon Exon 1 2 3 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10 Ligation site NM_005857.3 212-214 (exon 1) Partial sequence (24 nt adjacent to ligation site) 187-188 375-376 523-524 653-654 16 nt before exon 5; 700-701 900-901 1011-1012 1215-1216 1356-1357 2878-2879 GGTGCACGCTGA-AGGAGCCGGCGG ACCACCGGAGTT-AGGACAGATCAT CTCTGGAGACTT-TCTGGACGGTTC ATAATACTTTTG-TGATAGAAGAAA TTCTTGTGGTAA-31 nt spanning oligo-ATGAAAGATGCA CACACCTCTGCC-TGAGGGAAAGCT CAGCAATGCTTA-TTTTTATGGCTT TGTACTAGGCCA-TGAACTGGGGCA TTATGATAGCCA-ACCCACTCTTAT AAAGTGGGATCA-ACTGTACGCCTT Distance to next probe 2.6 6.9 0.7 1.5 kb kb kb kb 2.0 kb 9.4 4.6 5.0 3.0 kb kb kb kb stop codon 1637-1639 (exon 10) Ж This probe consists of three parts and has two ligation sites. The NM_005857.3 sequence is the reference standard in the NCBI RefSeqGene project. Table 2b. LMNA gene Length SALSA MLPA (nt) probe 280 286 301 391 330 154 337 315 361 160 382 247 220 12287-L13799 12027-L13939 01916-L01460 15693-L17660 01918-L21732 11552-L12299 01919-L01463 15694-L20323 13216-L14549 11553-L12300 01922-L01466 02478-L01971 05939-L05368 LMNA exon start codon Ligation site NM_170707.2 250-252 (exon 1) Partial sequence (24 nt adjacent to ligation site) Exon 1 Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10a Exon 11 Exon 12 534-535 589-590 639-640 781-780 reverse 936-937 1067-1066 reverse 1335-1336 1515-1516 161 nt before exon 8 1831-1830 reverse 1893-1894 2094-2095 9 nt before exon 12 CTTGACTCAGTA-GCCAAGGAGCGC GTGAGGAGTTTA-AGGAGCTGAAAG CTGATAGCTGCT-CAGGCTCGGCTG CTTCTTGGCCTC-ACCTAGGGCTGC CTGGTGGAGATT-GACAATGGGAAG ACTGCCTGGCAT-TGTCCAGCTGGA TACCAGGAGCTT-CTGGACATCAAG CGCAAACTGGAG-TCCACTGAGAGC TGTGTCCACAGA-TCATGGCTATTA GATGAGAGCCGT-ACGCAGGCTGTT CGCTCAGTGACT-GTGGTTGAGGAC TCCTCTGGCTCT-TCTGCCTCCAGT CTCCTCTGTTTT-CTCTCTTAGAGC start codon 2242-2244 (exon 12) Distance to next probe 0.1 15.4 3.8 0.4 0.3 0.9 0.3 0.4 0.4 0.5 0.9 0.4 kb kb kb kb kb kb kb kb kb kb kb kb The NM_170707.2 sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2c. CAV3 gene Length SALSA MLPA (nt) probe 238 274 09681-L10061 17349-L21793 CAV3 exon start codon Ligation site NM_033337.2 78-80 (exon 1) Partial sequence (24 nt adjacent to ligation site) Exon 1 Exon 2 142-143 435-436 CTGCAAGGAGAT-TGACCTGGTGAA GCATCAGCCACA-TCTACTCACTCT stop codon 531-533 (exon 2) Distance to next probe 11.8 kb The NM_033337.2 sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix Page 4 of 6 MRC-Holland ® MLPA Description version 17; 01 June 2016 Table 2d. MYOT gene Length SALSA MLPA (nt) probe 172 418 214 166 190 409 292 184 202 427 11555-L12302 11562-L12309 11559-L13505 11554-L12301 11557-L12304 11561-L21731 11560-L12307 11556-L12303 11558-L13504 11563-L14166 MYOT exon start codon Ligation site NM_006790.2 310-312 (exon 2) Partial sequence (24 nt adjacent to ligation site) Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Exon 6 Exon 7 Exon 8 Exon 9 Exon 10 13 nt after exon 1 164-165 726-727 889-890 957-956 reverse 1063-1064 1163-1164 1431-1432 1611-1612 1686-1687 AAGTCCCTTCTT-TATTCCTACTAC TCAGGATCTCAA-CAAGGAAGAGCA ACTGCAAATGCT-AAGCCCATACCA TAGGACCACAGA-ATGCAGCTGCTG AGTCGCGCATGT-TCTGAGTTGTGT ACCCACCACGTT-TCATTCAAGTGC GTCATGGTATCT-AAATGGAAGAAC CAGATCTCGGCT-ATACCTCCACCA GTGACTACATGT-AACACAAGATTA GTTCGACCAACA-TTCAGCAAATAT stop codon 1804-1806 (exon 10) Distance to next probe 2.5 5.4 1.7 3.2 1.2 1.4 2.7 0.8 0.3 kb kb kb kb kb kb kb kb kb The NM_006790.2 sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering used here may differ from literature! Complete probe sequences are available on request: [email protected]. Please notify us of any mistakes: [email protected]. SALSA MLPA probemix P048-C1 LMNA/MYOT/ZMPSTE24 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P048-C1 LMNA/MYOT/ZMPSTE24 (lot C1-0315). SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix Page 5 of 6 MRC-Holland Description version 17; 01 June 2016 ® MLPA Implemented Changes – compared to the previous product description versions. Version 17 – 01 June 2016 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Warning on probe located in CpG island removed. - Various minor textual changes. - New references added on page 2. Version 16 – 12 August 2015 (54) - Various minor textual changes. - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed. - “Peak area” replaced with “peak height”. Version 15 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 14 (48) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor textual changes on page 1. Version 13 (48) - Warning about salt sensitivity of 337 nt probe added. - Various minor textual changes. Version 12 (46) - Product description adapted to a new version (lot number added, small changes in Table 1 and Table 2, new pictures included). Version 11 (46) - Ligation sites of the probes targeting the CAV3 gene updated according to new version of the NM_reference sequence. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Warning added in Table 1, 178 nt probe 08416-L08305 and 364 nt probe 01921-L01465. - Data analysis method has been modified. - Various minor textual changes on page 1. SALSA MLPA P048 LMNA/MYOT/ZMPSTE24 probemix Page 6 of 6