In addition, 9 reference probes are included in the P116 probemix, detecting several autosomal locations.

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1 mix P116-B1 SGC Lot B1-0115, B As compared to version A1 (lot A1-0708), one extra probe each for SGCB, SGCD and FKRP have been included. Five reference probes and the control fragments have been replaced. Mutations in SGC cause limb-girdle muscular dystrophy (LGMD). Over time (usually after many years), patients with LGMD lose muscle bulk and strength. The distal muscles are affected late in LGMD, if affected at all. LGMD is typically an inherited disorder, though it may be inherited as a dominant, recessive or X- linked genetic defect. The muscle cells of patients with LGMD cannot properly form the proteins needed for normal muscle function. Defects of different proteins are involved in LGMD, each related to a specific type of muscular dystrophy. Autosomal recessive LGMD is a genetically heterogeneous disorder. Of the many genes that can result in this disorder, the following genes are present in the P116 SGC probemix: Gene Number of Number of Location LGMD type exons probes SGCA 10 exons kb 17q21 LGMD2D SGCB 6 exons kb 4q12 LGMD2E SGCD 9 exons kb 5q33 LGMD2F SGCG 8 exons kb 13q12 LGMD2C FKRP 4 exons kb 19q13 LGMD2I This includes a flanking probe located downstream of the SGCA gene. This includes a probe specific for the FKRP L276I mutation. This probe will only generate a signal when the mutation is present. In addition, 9 reference probes are included in the P116 probemix, detecting several autosomal locations. SD030 Sample DNA Please note that the FKRP L276I mutation-specific probe has only been tested on control plasmids and not on positive human DNA samples with the point mutation! This SD030 sample DNA is provided with each probemix vial and can be used in data binning in the fragment analysis and as a positive control for the mutation-specific probe (see next page). This probemix is designed to detect deletions/duplications of one or more sequences in the SGCA, SGCB, SGCD, SGCG and FKRP 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 test. probemixes 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 test probemixes and reagents includes a limited license to use these products for research purposes. The use of a 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). Related probemixes P268 DYSF: Contains probes for DYSF involved in LGMD2B. P176 CAPN3: Contains probes for CAPN3 involved in LGMD2A. P116 SGC probemix Page 1 of 7

2 P061 Lissencephaly: Contains probes for POMT1 and POMGNT1 involved in LGMD2K and LGMD2O. P436 ANO5: Contains probes for ANO5, involved in LGMD2L. P048 LMNA/MYOT: Contains probes for LMNA, MYOT and CAV3. 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 P116-B1 SGC probemix contains 48 s with amplification products between 130 and 492 nt. This includes one probe, specific for the FKRP L276I mutation which will only generate a signal when the mutation is present. 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. SD030 Sample DNA The SD030 Sample DNA provided with this probemix can be used as Binning DNA sample for binning of the FKRP L276I mutation-specific probe (FKRP probe L13479). Inclusion of one reaction with SD030 DNA in MLPA experiments is recommended as it can aid in data binning of the peak pattern using Coffalyser.Net software and as an artificial positive control for the specific point mutation. Please note that SD030 DNA consists of female DNA mixed with plasmid DNA that contains the target sequences detected by the above mentioned probe + the sequence of the 105 nt chromosome Y specific control fragment. The amount of plasmid used (relative to the genomic DNA) results in a relative probe signal for the 105 nt probe on this female DNA which is identical to the relative probe signal obtained on male DNA samples. As a result, the 100 and 105 nt control fragments indicate the presence of two copies chromosome X and one copy chromosome Y and one copy of the mutation-specific probe (heterozygous mutation). The product description of the SD030 Sample DNA can be found on This product is for research use only. 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, 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 at. Info/remarks/suggestions for improvement: info@mlpa.com. P116 SGC probemix Page 2 of 7

3 Table 1. MLPA P116-B1 SGC probemix Chromosomal position reference SGCA SGCB SGCD SGCG FKRP 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 101 X-fragment: Specific for the X chromosome 105 Y-fragment: Specific for the Y chromosome 130 Reference probe L q SGCA probe L SGCG probe L SGCA probe L Reference probe L q SGCB probe L SGCA probe L SGCG probe L SGCB probe L SGCB probe L ± SGCB probe L SGCD probe L SGCD probe L Reference probe L p ± FKRP probe L SGCG probe L SGCD probe L SGCD probe L Reference probe L q SGCA probe L ± FKRP probe L13479 L276I mutation 265 SGCG probe L SGCD probe L SGCD probe L Reference probe L p SGCA probe L SGCG probe L SGCD probe L SGCA probe L Reference probe L q SGCG probe L SGCB probe L SGCA probe L COL1A1 probe L07764 Down stream 373 SGCG probe L SGCD probe L SGCD probe L SGCA probe L Reference probe L p ± FKRP probe L ± FKRP probe L SGCA probe L SGCB probe L Reference probe L q SGCA probe L ± FKRP probe L SGCG probe L Reference probe L q11 Mutation-specific probe. This probe will only generate a signal when the FKRP L276I mutation is present. It has been tested on artificial test DNA but not on positive human samples! P116 SGC probemix Page 3 of 7

4 ± 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 to facilitate the 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: 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. Table 2. P116 probes arranged according to chromosomal location Table 2a. SGCA SGCA NM_ start codon (ex 1) L CTCTTCTGGACT-CCTCTCCTCGTG 1.4 kb L TGTGCACACCTT-GGACCATGAGAC 0.3 kb L TGCCACCCCAGA-AGATCGTGGGCT 0.3 kb L GGACAGCTTTGA-TACCACTCGGCA 0.5 kb L CTGCCCTCAACA-CCTGCCAGCCGC 0.8 kb L CTTGCTACGACA-CCTTGGCACCCC 1.1 kb L CTTCTTGGTGGA-TGCTCTGGTCAC 0.4 kb L AGAGACCTGGCT-ACCTCCGAGTGA 4.7 kb L CCATGTTCAATG-TGCACACAGGTG 0.4 kb L , reverse GAGAAGGGAGGA-TGAAGTCAGGGC 9.3 kb stop codon (ex 9) L07764 COL1A1 AAGACACAGGAA-ACAATGTATTGT Flanking probe. Included to facilitate the 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. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. SGCB MLPA probe SGCB NM_ start codon (ex 1) 196 ± L nt before exon 1, reverse GGCGCGTTGTAT-TGCACAGGGGCC 5.0 kb L ATACATTCCGAT-TGATGAAGATCG 3.8 kb L CGATTTAAGCAA-GTATCTGACATG 0.9 kb L TGTAGAAAACAA-CAAAACTTCTAT 0.9 kb L TGTATTCATTAT-GGGCAAAACCAT 4.0 kb L GGGACGCTCTTC-AAGGTGCAAGTA stop codon (ex 6) ± 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. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. P116 SGC probemix Page 4 of 7

5 Table 2c. SGCD SGCD NM_ start codon (ex 2) L CTGACTGGGGCA-GCTTCTGAGCGC 2.5 kb L nt after exon 2 AGGTGGAGATGG-TGAGTAATTCCC 15.0 kb L AGCACCATGCCT-GGCTCTGTGGGG kb L AAAGAAATCCAG-TCCCGACCAGTA 80.6 kb L TCTGCCAGAAAT-GTTACAGTGAAC 5.7 kb L , reverse CTACTACCACTT-CATTATTGTCTG 52.5 kb L CCTAAATCTATA-GAAACACCTAAT kb L AGAAGCTGGCAA-TATGGAAGCCAC 1.6 kb L CAGAAGGTCTTC-GAGATCTGCGTC stop codon (ex 9) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2d. SGCG SGCG NM_ start codon (ex 2) L TGGTAGAGCTCG-GGCCAGCTGTAG 22.8 kb L TCTACTTGTTTG-TTCTTCTTTTAC 30.9 kb L TTGGAAGGGGAA-TCAGAATTTTTA 16.0 kb L GTGACTGTAAAT-GCGCGCAACTCA 28.7 kb L AGATCAACTCCA-ACGACGGCAAGC 16.0 kb L TTTGAACATTCA-GTGGAGACACCC 25.2 kb L GAGTCTAAGCAT-GGATGCCCCAAG 3.7 kb L ACCCAAGCTGGT-GCAGGGGACGTG stop codon (ex 8) + 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 is a reference standard in the NCBI RefSeqGene project. Table 2e. FKRP FKRP NM_ start codon (ex 4) 471 ± L nt after exon 1 TCGTGCTGGATA-AAGTGCAGGATC 1.8 kb 222 ± L TGCCCTCCTGGA-ACTCCCCCAGCC 0.5 kb 409 ± L , reverse CTGGGTCTGAGT-TGCGATTTGGCC 7.7 kb 259 ± L L276I mutation; , reverse CCAGCTCACTAT-GCGGATGCCCAG 0.9 kb 418 ± L CCAGATTTATCA-AATGGTCATGCC stop codon (ex 4) Mutation-specific probe. This probe will only generate a signal when the FKRP L276I mutation is present. It has been tested on artificial test DNA but not on positive human samples! ± 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. The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Note: numbering used here may differ from literature! Complete probe sequences are available on request info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. P116 SGC probemix Page 5 of 7

6 mix P116-B1 SGC sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with mix P116-B1 SGC (lot B1-0115). Figure 2. Capillary electrophoresis pattern of SD030 sample DNA (approximately 50 ng) analysed with mix P116-B1 SGC (lot B1-0115). The location of the FKRP L276I mutation-specific probe at 259 nt is indicated. P116 SGC probemix Page 6 of 7

7 Implemented Changes compared to the previous product description versions. Version July 2015 (54) - Product description adapted to a new lot (lot number added, new pictures included). - Various textual changes. Version 07 (53) - Replaced peak area by peak height. - Updated link for Database of Genomic Variants. - SD information added on page 1 and 2. - SD electropherogram added. - Removed the terms old and new MLPA buffer. - Various minor textual changes. Version 06 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 05 (48) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Various minor textual changes. - Remark on RefSeqGene standard and transcript variant added below Table 2. - Warnings about 259 nt probe L13479 and 301 nt probe L02785 added to Table 1. - Small correction of chromosomal locations in Table 1 and 2. Version 04 (46) - s of the probes targeting the SGCA, SGCB and SGCG genes updated according to new version of the NM_reference sequence. - Warning added in Table 1, 184 nt probe L02961, 247 nt probe L00720 and in Table 2, 148 nt probe L 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. P116 SGC probemix Page 7 of 7