MRC-Holland MLPA. Description version 07; 23 Feb 2015

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1 SALSA MLPA probemix P326-A2 LARGE, FKTN, POMT2 Lot A Compared to previous lot (A1-0709), one reference probe has been replaced and the control fragments have been adjusted (QDX2). Walker-Warburg syndrome, a severe recessive congenital muscular dystrophy associated with defects in neuronal migration that produce complex brain and eye abnormalities, is associated with defects in among others the LARGE, FKTN and POMT2 genes on chromosome 22, 9 and 14, respectively. The LARGE gene (16 exons) spans ~647 kb of genomic DNA and is located at chromosome 22q12.3, ~34 Mb from the p-telomere. This probemix contains two probes for every exon, with the exception of exons 6, 7, 9, 13 and 14, which are only detected by one probe. Also, one probe located upstream of the LARGE gene is included. The FKTN gene (11 exons) spans ~83 kb of genomic DNA and is located at chromosome 9q31-q33, ~108 Mb from the p-telomere. This probemix contains probes for exon 1, 4, 6, 7, 8 and 11. The POMT2 gene (21 exons) spans ~46 kb of genomic DNA and is located at chromosome 14q24.3, ~78 Mb from the p-telomere. This probemix contains probes for exon 1, 3, 4, 6, 8, 12, 15, 18 and 21. In addition, nine reference probes are included detecting several autosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned genes 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 test. SALSA probemixes are sold by for research purposes and to demonstrate the possibilities of the MLPA technique. This probemix is not CE/FDA certified for use in diagnostic procedures. Purchase of the SALSA test probemixes includes a limited license to use these products for research purposes. The use of this SALSA probemix 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 probemixes P048 LMNA-MYOT: Contains probes for LMNA, MYOT and CAV3 genes, involved in limb-girdle muscular dystrophies (LGMD). P061 Lissencephaly: Contains 4 probes for POMT1 gene involved in LGMD2K. P116 SGC: Contains probes for SGCA, SGCB, SGCD, SGCG and FKRP genes involved in LGMD2D, 2E, 2F, 2C, and 2I. P176 CAPN3: Contains probes for the CAPN3 gene, involved in LGMD2A. P268 DYSF: Contains probes for the DYSF gene, involved in LGMD. More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 6, 1057 DN Amsterdam, the Netherlands References for SALSA MLPA probemix P326 Costa C. et al. (2013) A Portuguese case of Fukuyama congenital muscular dystrophy caused by a multiexonic duplication in the fukutin gene. Neuromuscul Disord. 23: SALSA probemix P326 LARGE Page 1 of 8

2 Data analysis The P326-A2 LARGE probemix contains 52 MLPA probes 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 (D-fragments) 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 probemix can first be intra-sample normalized 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, 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 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 by K. A. de Groot at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P326 LARGE Page 2 of 8

3 Table 1. SALSA MLPA P326-A2 LARGE probemix Length Chromosomal position SALSA MLPA probe (nt) reference LARGE FKTN POMT 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 probe L q FKTN probe L13414 Exon LARGE probe L13415 Exon POMT2 probe L13416 Exon LARGE probe L13417 Exon ± LARGE probe L14168 Exon FKTN probe L13419 Exon POMT2 probe L13420 Exon LARGE probe L13421 Exon FKTN probe L13422 Exon POMT2 probe L13423 Exon Reference probe L q LARGE probe L13425 Exon FKTN probe L14684 Exon POMT2 probe L14685 Exon LARGE probe L13428 Exon Reference probe L q LARGE probe L13430 Exon FKTN probe L13431 Exon LARGE probe L13432 Exon LARGE probe L13433 Exon ± LARGE probe L14686 Exon POMT2 probe L13435 Exon LARGE probe L13436 Exon LARGE probe L13437 Exon POMT2 probe L13438 Exon LARGE probe L13439 Exon FKTN probe L13440 Exon * Reference probe L q LARGE probe L13442 Exon POMT2 probe L13443 Exon LARGE probe L13444 Exon POMT2 probe L13445 Exon LARGE probe L13446 Exon Reference probe L q LARGE probe L13448 Exon LARGE probe L13449 Exon POMT2 probe L13450 Exon LARGE probe L13451 Exon LARGE probe L13452 Exon Reference probe L q LARGE probe L13454 Exon EP300 probe L13223 Upstream 418 Reference probe L q LARGE probe L13457 Exon LARGE probe L13458 Exon LARGE probe L13459 Exon Reference probe L q LARGE probe L13461 Exon LARGE probe L13462 Exon LARGE probe L13463 Exon Reference probe L q22 SALSA probemix P326 LARGE Page 3 of 8

4 * New in version A2 (from lot A onwards). ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Flanking probe. Included only to facilitate determination of the extent of a deletion / duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. SNP (rs ) could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. 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: info@mlpa.com. SALSA probemix P326 LARGE Page 4 of 8

5 Table 2. P326 probes arranged according to chromosomal location Table 2a. LARGE Length (nt) SALSA MLPA probe L13223 LARGE Exon EP300 Exon 14 Ligation site NM_ NM_ ; Partial sequence (24 nt adjacent to ligation site) ACCACCAACAAC-ACAACTTCCCCA Distance to next probe kb start codon (ex 3) 256 ± L14686 Exon reverse CCGCGCTCCAAG-CCGATTCGTCCC 0.6 kb 160 ± L14168 Exon nt after exon 1 reverse TCCTCCTGGCTC-GCTCCCTGCCAG 63.0 kb L13415 Exon GCACCTGTCGTG-AGCCAGGTAAGA 0.1 kb L13452 Exon 2 71 nt after exon 2 TTGCCGTTTTTC-AGCAGGGCGATC 95.1 kb L13462 Exon 3 37 nt before exon 3 ATCTCCAGTGCT-AAGAACTCAGGC 0.2 kb L13421 Exon ATCACCTGGATT-TACCTGTTTTCT kb L13437 Exon 4 82 nt before exon 4 GCCTCGCCATGT-AGTAAGGGATGT 0.4 kb L13442 Exon GGTCGTGGAGAA-ATGCGAGGTAAA 23.8 kb L13446 Exon nt before exon 5 TATTTGAGCCAA-TCACCATCTCTC 0.3 kb L13433 Exon 5 2 nt after exon 5 TGTTCCATAGGT-AAGAACAACTTC 21.8 kb L13430 Exon TACAATGCAGAC-GAGCTCAAGGTA 39.6 kb L13444 Exon 7 18 nt after exon 7 reverse CGGGGAAAAAAC-GAATGGGCTACC kb L13463 Exon 8 66 nt before exon 8 reverse CAGTGACTATTG-AAGACACAGGGT 0.2 kb L13428 Exon 8 9 nt after exon 8 CAGGTAAATCCT-CAGGGTGGTATG 48.0 kb L13461 Exon 9 23 nt after exon 9 AGGAATAGCTGC-ACCTTCGAACCT 2.2 kb L13458 Exon GCTCCCCTGCTT-CTGGAATGTGCA 0.1 kb L13439 Exon GTGTCTGATCTA-AAGGTAGGGTCA 44.1 kb L13457 Exon CCTGCCAGGTCA-TTCACTGGAACT 0.1 kb L13425 Exon CAGTGAGGCTGA-TGTCAACAGTGA 21.4 kb L13417 Exon AGCAGCTGTCTG-AGCTGGACGAGG 0.1 kb L13448 Exon CCTGTACTTCCT-GCACTACGAGTA 11.9 kb L13451 Exon 13 2 nt after exon 13 AGTACCTCAGGT-AAGGACCTGCAG 20.9 kb L13454 Exon GTCATCCAGCTC-GATCTTGCCAAC 6.1 kb L13436 Exon reverse TGCGTGGCCTTT-CGTCCAGACGTG 0.2 kb L13432 Exon nt after exon 15 ATCCACCTGGTA-TGGTCGACGGGG 2.5 kb L13449 Exon TCTCAAAACCCT-CAAGGAAGAGTT 1.1 kb L13459 Exon CAGGAAGAAAGT-GATTGGGTTCCT stop codon (ex 16) Flanking probe. Included only to facilitate determination of the extent of a deletion / duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. ± Those probes are located within, or close to, a very strong CpG island. A low signal of those probes can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. SNP (rs ) could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. 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: info@mlpa.com. SALSA probemix P326 LARGE Page 5 of 8

6 Table 2b. FKTN Length SALSA MLPA FKTN Ligation site Partial sequence (24 nt Distance to (nt) probe Exon NM_ adjacent to ligation site) next probe start codon (ex 3) L13414 Exon 1 55 nt after exon 1 GCCTTCAGACAG-GTACCGCTGGCG 38.4 kb L13422 Exon AGGAAGCCGAAT-TGGATTTGATAG 7.9 kb L13431 Exon AGCTTTTGACAG-GTAAGTTCAGAG 3.3 kb L14684 Exon AGTTACAGCAAG-TTACTGTTGATG 7.5 kb L13440 Exon AGTGCAAAGGAA-TTACTGCAACTA 23.1 kb L13419 Exon TCACATCTGGAG-ACATCAGCCTTT stop codon (ex 11) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2c. POMT2 Length SALSA MLPA POMT2 Ligation site Partial sequence (24 nt Distance to (nt) probe Exon NM_ adjacent to ligation site) next probe start codon (ex 1) L13416 Exon nt after exon 1 GCTACAGTGCAT-CACCTGGCTTGC 13.8 kb L13423 Exon TCTTGCTGGCTA-CCTGAGTGGATA 1.7 kb L13438 Exon CCTACCTCACTG-TACTGGATCTGT 3.2 kb L14685 Exon nt before exon 6 CTGGTCACTTGT-GAAGAAGGAGCC 2.8 kb L13420 Exon TGCACAATGCTT-CCATCCCTGAAC 12.1 kb L13443 Exon nt after exon 12 TCTGGGAAAAGC-AGCCTATGAAGT 2.8 kb L13445 Exon ATCCCACATGGT-CATGATCCGGGT 4.1 kb L13450 Exon nt after exon 18 reverse GGTTGTTACCTT-TCAAAAGCAAAG 4.6 kb L13435 Exon CACTTGGCAGAA-ACGTGATGTGTC stop codon (ex 21) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Note: Exon numbering might be different as compared to literature! Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA probemix P326 LARGE Page 6 of 8

7 SALSA MLPA probemix P326-A2 LARGE sample picture Figure. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P326-A2 LARGE (lot A2-0513). Implemented Changes compared to the previous product description versions Version 07 (54)-23 Feb New sample picture included in product description. Version 06 (51) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1, new picture included). Version 05 (48) - Warning added in Table 1, 297 nt probe L Version 04 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 03 (48) - Various minor textual changes. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Small correction of chromosomal locations in Table 1 and 2. - Ligation sites of the probes targeting the LARGE, FKTN and POMT2 genes updated according to new version of the NM_reference sequence. - Remark on RefSeqGene standard and transcript variant added below Table 2. Version 02 (46) - Warning added in Table 1, 481 nt probe L Exon numbering of the FKTN gene has been changed on page 3 and 6. - Ligation sites of the probes targeting the POMT2 gene updated according to new version of the SALSA probemix P326 LARGE Page 7 of 8

8 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. - Partial probe sequences extended to 24 nt. - Data analysis method has been modified. - Various minor textual changes on page 1. - Various minor layout changes. SALSA probemix P326 LARGE Page 8 of 8