MRC-Holland MLPA. Description version 11; 7 September 2017

Transcription

1 SALSA MLPA mix P268-A2 DYSF Lot A2-1115, A Compared to previous version A1-0608, two reference s have been replaced and one reference has been added. In addition, the control fragments have been replaced (QDX2). Limb-girdle muscular dystrophies (LGMD) are a group of phenotypically and genotypically heterogeneous disorders, characterised by progressive weakness and atrophy of the muscles of the pelvic and shoulder girdle. Mutations of the Dysferlin gene (DYSF) are the cause of limb-girdle muscular dystrophy type 2B (LGMD2B). Patients with LGMD2B have symmetrical and selective involvement of proximal limb-girdle muscles. The disease shows wide intra- and interfamilial clinical variability. The age at onset ranges from 2 to 40 years, but the disease usually first appears in the second or third decade of life, with the development of proximal weakness in the lower limbs. Mutations in DYSF result in a cascade of events leading eventually to muscular dystrophy. The precise underlying mechanisms have yet to be elucidated. The DYSF gene comprises 55 exons, spans ~220 kb of genomic DNA and is located on chromosome 2p13.2, approximately 72 Mb from the p-telomere. Copy number changes of this genomic region are found in healthy individuals and can be found in the Database of Genomic Variants. This P268-A2 DYSF mix contains s for 40 different DYSF exons. In addition, 8 reference s located on other chromosomal locations are included. This SALSA mix is designed to detect deletions/duplications of one or more sequences in the DYSF gene in a DNA sample. Heterozygous deletions of recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that. Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some 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 test. SALSA mixes and reagents are sold by 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 test mixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA mix 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). Related SALSA mixes P061 Lissencephaly: Contains s for the POMT1 gene involved in LGMD2K. P116 SGCA: Contains s for the SGCA, SGCB, SGCD, SGCG and FKRP genes involved in LGMD2D, 2E, 2F, 2C, and 2I. P176 CAPN3: Contains s for the CAPN3 gene, involved in LGMD2A. P048 LMNA/MYOT/ZMPSTE24: Contains s for the LMNA, MYOT, ZMPSTE24 and CAV3 genes. References for SALSA P268 DYSF mix Walter, M.C. et al. (2013) Treatment of dysferlinopathy with deflazacort: a double-blind, placebocontrolled clinical trial. Orphanet J Rare Dis 8(1): 26. Maznaric, M. et al. (2011) Abnormal expression of dysferlin in skeletal muscle and monocytes supports primary dysferlinopathy in patients with one mutated allele. EJoN. 18(7): SALSA mix P268 DYSF Page 1 of 6

2 More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands Data analysis The P268-A2 DYSF mix contains 48 MLPA s with amplification products between 130 and 490 nt. In addition, it contains 9 control fragments generating an amplification product smaller than 120 nt: four DNA Quantity fragments (Q-fragments) at nt, three DNA Denaturation control fragments (Dfragments) at 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 mix can first be normalised intra-sample by dividing the peak height of each s amplification product by the total peak height of only the reference s in this mix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference s. 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, can be downloaded free of charge from our website Many copy number alterations in healthy individuals are described in the database of genomic variants: 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 s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This mix was developed at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA mix P268 DYSF Page 2 of 6

3 Table 1. SALSA MLPA P268-A2 DYSF mix Length Chromosomal position SALSA MLPA (nt) reference DYSF 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 100 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 * Reference L q DYSF L08865 Exon DYSF L08882 Exon DYSF L22495 Exon DYSF L08885 Exon * Reference L q DYSF L08890 Exon DYSF L22505 Exon DYSF L22287 Exon DYSF L08879 Exon DYSF L08898 Exon DYSF L08875 Exon DYSF L12802 Exon DYSF L08881 Exon DYSF L11237 Exon DYSF L22497 Exon DYSF L08867 Exon DYSF L08886 Exon DYSF L22498 Exon DYSF L08888 Exon DYSF L08872 Exon DYSF L22499 Exon DYSF L08878 Exon DYSF L08894 Exon DYSF L08880 Exon DYSF L22500 Exon DYSF L08897 Exon DYSF L13321 Exon DYSF L08884 Exon * ± Reference L q DYSF L22501 Exon DYSF L22502 Exon ± Reference L p DYSF L22504 Exon DYSF L08869 Exon DYSF L11241 Exon DYSF L08873 Exon DYSF L08896 Exon DYSF L12801 Exon DYSF L08899 Exon Reference L q DYSF L08889 Exon DYSF L08868 Exon DYSF L11242 Exon DYSF L08863 Exon Reference L p Reference L q Reference L q22 * New in version A2 (from lot A onwards). Changed in version A2 (from lot A onwards). Small change in length, no change in sequence detected. ± This is located within, or close to, a very strong CpG island. A low signal of this can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. The identity of the genes detected by the reference s is available on request: info@mlpa.com. SALSA mix P268 DYSF Page 3 of 6

4 Table 2. DYSF s arranged according to chromosomal location Length (nt) SALSA MLPA DYSF Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next Start codon (ex 1) L22502 Exon , reverse GCTTAGAGCAGT-TGCTCTTAAAGG 27.0 kb L22499 Exon 2 (3) , reverse GATGACTTTGGT-TCTCTTCTTCAC 22.4 kb No Exon L13321 Exon 4 (5) , reverse TGGGCTGCTTCT-TGGTGTCCAGCA 8.6 kb L08863 Exon 5 (6) , reverse GGGAGGGCTCCA-GAGGAGTAGGGG 1.9 kb L22500 Exon 6 (8) AGGAAGACACAG-AGGACCAGGGAC 2.0 kb L08865 Exon 7 (9) CCCACTCTTCAA-TGAGGTGGGAGA 1.3 kb No Exon L11237 Exon 9 (11) TCTCTCAGGACA-GATGCTCTCCTC 3.2 kb L08867 Exon 10 (12) TTGATTGCAGAT-GGACGTGGGCAC 6.1 kb No Exon L08868 Exon 12 (14) GGAGGACATTGA-AAGCAACCTGCT 2.1 kb L08869 Exon 13 (15) TGAAACAGATCT-TTGGCTTCGAGA 6.7 kb L22495 Exon 14 (16) GCAGCAAGATCT-TGGAGAAGACGG 4.2 kb No Exon L22498 Exon 16 (18) TACCACCTACCT-GAGTATGTCGAA 10.2 kb L08872 Exon 17 (19) , reverse AGTCTGAGGCTT-TCGAAGGCTTGA 1.7 kb L08873 Exon 18 (20) CAGTCCCAGAGA-GTTCACAGGCTT 2.0 kb No Exon L22287 Exon 20 (22) ACTACGGGAACA-AGTTCGACATGA 2.8 kb No Exon L08875 Exon 22 (24) 7 nt before ex 22, reverse GCTTCCTGTGGA-ATGGGCAGGCAA 5.8 kb L12801 Exon 23 (25) GTGACATCCATG-AGACACCCTCTG 2.4 kb L11242 Exon 24 (26) , reverse AGCTTCCCACAA-TTCTTGCCACAG 3.9 kb L08878 Exon 25 (27) GTTCAACCAGTT-TGCTGAGGGGAA 1.8 kb No Exon L08879 Exon 27 (29) GGAAGAGGTGTT-TGAGAACCAGAC 0.9 kb No Exon L08880 Exon 29 (31) GGAAGCACTGAA-AAGGGTGAGCCA 3.5 kb L08881 Exon 30 (32) TTTTGGCTGGAA-GTTCCACCTCGA 15.4 kb L08882 Exon 31 (33) CGTCTCCACCTT-GAGCTTCGGTGT 9.0 kb No Exon L22497 Exon 33 (35) TCTTCTACGAGA-TCGAGATCTTTG 2.1 kb L08884 Exon 34 (36) ACCGAGTCTGGA-ACGGATGCCACG 2.0 kb No Exon L08885 Exon 36 (38) GTGCAGGAGACA-TCAAGGATCCTG 8.5 kb L08886 Exon 37 (39) , reverse GGAACCATGTAG-ATGTTGGCCTCC 2.1 kb No Exon 38 No Exon L22501 Exon 40 (42) CTCATCGACATT-GATGACAAGGAG 30.6 kb L08888 Exon 41 (44) ATTGGTGGAGCA-AATTCTTTGCCT 12.2 kb L08889 Exon 42 (45) , reverse TCCAGCTGTGTG-TCATAGACCTGC 2.8 kb L08890 Exon 43 (46) ATTGTCCGAGCA-TTTGGCCTGCAG 1.6 kb L22504 Exon 44 (47) GAAGAAATCAGT-GAGTGACCAGGA 3.8 kb L12802 Exon 45 (48) TCGACCTGGAGA-ACAGGCTGCTGT 3.0 kb No Exon L22505 Exon 47 (50) 22 nt before ex 47 CTCCTGCAACTT-TTTTGTCTTCTC 1.8 kb No Exon L08894 Exon 49 (52) TATTATCTGGAA-TACCAGAGATGT 5.1 kb No Exon L11241 Exon 51 (54) GACTGAGAGCAA-AATCCCAGCACG 4.9 kb L08896 Exon 52 (55) , reverse TCTTGGCTGTCT-TGGCTGGCTTGG 1.9 kb L08897 Exon 53 (56) TGACCTTGGAGA-TTGTAGCAGAGA 1.6 kb SALSA mix P268 DYSF Page 4 of 6

5 Length (nt) SALSA MLPA DYSF Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next L08898 Exon 54 (57) TCCTGCTGCTGT-TCCTGGCCATCT 3.8 kb L08899 Exon 55 (58) TGCCATGAAGCT-GGTGAAGCCCTT Stop codon (ex 55) We have been informed by a customer that a variant of unknown significance, c.341g>a, influences the 319 nt exon 4 and a two nt deletion (c.5698_5699delag mutation) affected the 382 nt exon 51 signal in some samples. In case of apparent deletions, it is recommended to sequence the region targeted by these s. Note: The DYSF exon numbering has changed. From description version 10 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. This exon numbering used here may differ from literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. The NM_ sequence represents transcript variant 4 and is a reference standard in the NCBI RefSeqGene project. The exon numbering used here may differ from literature! Complete sequences are available on request: info@mlpa.com. SALSA MLPA mix P268-A2 DYSF sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA mix P268-A2 DYSF (lot A2-1115). SALSA mix P268 DYSF Page 5 of 6

6 Implemented Changes compared to the previous product description version(s). Version 11 7 September 2017 (55) - Warning added in Table 1, 359 nt L Version 10 (55) 05 February Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - DYSF exon numbering adjusted. - Manufacturer s address adjusted. - Updated link for Database of Genomic Variants. - Peak area replaced with peak height. Version 09 (48) - Electropherogram picture of old buffer (introduced Dec. 2012) removed. Version 08 (48) - Warning added in Table 1, 335 nt L Version 07 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 06 (48) - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes. Version 05 (48) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Small correction of chromosomal locations in Table 1. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes on page 1. - Various minor layout changes. - Ligation sites of the s targeting the DYSF gene updated according to new version of the NM_reference sequence. Version 04 (45) - Warning added in Table 2 for the exon 5, 22 and 54 s. - Exon numbering has been changed in tables 1 and 2. The numbering as used in the Genbank mrna reference sequences (NM_ files) has now been adopted. - Minor textual and layout changes, tables have been numbered, - Ligation sites in table 2 have been adjusted according to the new version of the NM_ reference sequence. - Data analysis section has been modified. SALSA mix P268 DYSF Page 6 of 6