In addition, 12 reference probes are included in this probemix, detecting several different autosomal chromosomal locations.

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1 SALSA MLPA probemix P350-B1 CLCN1-KCNJ2 Lot B1-0711: As compared to the previous version A1 (lot A1-1109), two CLCN1 probes, one KCNJ2 probe and four reference probes have been replaced, one new KCNJ2 probe and two flanking probes for CLCN1 have been included and the 88 and 96 nt control fragments have been replaced (QDX2 fragments). Myotonia congenita is a hereditary chloride channel disorder characterised by delayed relaxation of skeletal muscle (myotonia). A subset of mutations in the CLCN1 gene, located on chromosome 7, can cause either recessive myotonia congenita (Becker's disease) or dominant myotonia congenita (Thomsen's disease). Thomsen s disease is less common and less severe than Becker s disease. Andersen-Tawil syndrome is an autosomal dominant multisystem channelopathy characterized by periodic paralysis, ventricular arrhythmias, and distinctive dysmorphic facial or skeletal features. Hypoplastic kidney and valvular heart disease have also been reported. Mutations in the KCNJ2 gene (which is located on chromosome 17 and encodes the inward-rectifying potassium channel protein, Kir2.1) have been reported to be responsible for this disorder. The CLCN1 gene (23 exons) spans ~35.9 kb of genomic DNA and is located on 7q34, 143 Mb from the p-telomere. The P350-B1 probemix contains one probe for each exon of the gene and two flanking probes, one in the CASP2 gene (upstream) and one in the FAM131B gene (downstream). The KCNJ2 gene (2 exons) spans ~10.5 kb of genomic DNA and is located on 17q24.3, 68 Mb from the p-telomere. The P350-B1 probemix contains two probes for exon 1 and three probes for exon 2 of the gene. Please note that the database of genomic variants mentions a small number of copy number changes in this genomic region in healthy individuals (see In addition, 12 reference probes are included in this probemix, detecting several different autosomal chromosomal 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 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 probemixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA 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). More information Website : info@mlpa.com (information & technical questions); order@mlpa.com (for orders) Mail : bv; Willem Schoutenstraat 1, 1057 DL Amsterdam, the Netherlands SALSA P350 CLCN1-KCNJ2 probemix Page 1 of 6

2 Data analysis The P350-B1 CLCN1-KCNJ2 probemix contains 42 MLPA probes with amplification products between 130 and 481 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 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 by K. Tuin 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 included as co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P350 CLCN1-KCNJ2 probemix Page 2 of 6

3 Table 1. SALSA MLPA P350-B1 CLCN1-KCNJ2 probemix Length Chromosomal position SALSA MLPA probe (nt) reference CLCN1 KCNJ 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 CLCN1 probe L15525 exon CLCN1 probe L15528 exon CLCN1 probe L15547 exon * Reference probe L q CLCN1 probe L20767 exon * ๑ KCNJ2 probe L21186 exon Reference probe L q ๑ KCNJ2 probe L15548 exon Reference probe L q * CASP2 probe L kb upstream 203 ± CLCN1 probe L20768 exon CLCN1 probe L15526 exon * Ж CLCN1 probe SP0473-L20778 exon * Reference probe L q CLCN1 probe L15533 exon KCNJ2 probe L15549 exon * Reference probe L q CLCN1 probe L15529 exon CLCN1 probe L15537 exon * Reference probe L q CLCN1 probe L15544 exon CLCN1 probe L15527 exon Reference probe L q * Ж CLCN1 probe SP0474-L21318 exon * FAM131B probe L kb downstream 337 CLCN1 probe L20766 exon CLCN1 probe L15534 exon CLCN1 probe L15540 exon Reference probe L q CLCN1 probe L15538 exon CLCN1 probe L15543 exon Reference probe L p CLCN1 probe L15541 exon ± CLCN1 probe L21284 exon KCNJ2 probe L15550 exon CLCN1 probe L15535 exon * KCNJ2 probe L20782 exon CLCN1 probe L15546 exon CLCN1 probe L15539 exon Reference probe L q Reference probe L p25 * New in version B1 (from lot 0711 onwards). Changed in version B1 (from lot 0711 onwards). Small change in length, no change in sequence detected. Ж This probe consists of three parts and has two ligation sites. 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. ๑ The significance of exon 1 deletions is not clear as this exon is non-coding. ± SNP rs (203 nt) and rs (419 nt) could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. SALSA P350 CLCN1-KCNJ2 probemix Page 3 of 6

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. 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. Table 2. P350 probes arranged according to chromosomal location Table 2a. CLCN1 Length SALSA MLPA CLCN1 Ligation site Partial sequence (24 nt Distance to (nt) probe exon NM_ adjacent to ligation site) next probe 196 * L20777 CASP2 gene ACTTATCAAGGA-TCGGGAAGGTTA 11.3 kb start codon (ex 1) L15525 exon reverse TCAAAGGGCATA-TACTGGTACTGG 3.5 kb L15526 exon CACAAAGAACAA-TTCTCAGACAGG 1.0 kb L15527 exon GGACTACGTCAG-TGCCAAAAGCCT 0.7 kb L15528 exon TCCCCAGGCTGT-TGGTGAGAACTT 0.2 kb L15529 exon GAAATGAAGACA-ATACTTCGTGGG 1.6 kb L20766 exon reverse GAACACAGACAT-GAATTTGCTGAG 1.2 kb L20767 exon 7 87 nt after exon 7 reverse AGTAACCTGGTA-ATACCAGCACTT 6.3 kb 419 ± L21284 exon AACTACTGGAGA-GGATTCTTTGCA 0.4 kb L15533 exon GTTCAGAACCAA-TTTCCGAATGGA 0.3 kb L15534 exon reverse TGGCGATGCAGA-TACACAAATACA 0.9 kb L15535 exon TTGTTACCTTTG-TCATTGCCTCAT 0.4 kb 220 * Ж SP0473- CTGGGCCAGTCA-28 nt spanning exon ; L20778 oligo-ttgtcatcatca 6.5 kb L15537 exon reverse CCTAGCACAAAC-ACAGGCATGAAG 0.2 kb L15538 exon GGTAGGAGAAAT-CATGGCCATGCT 2.4 kb L15539 exon reverse AATTCGAAGCAA-ATCACAGCTGTG 0.4 kb L15540 exon reverse GCAACTCCCCAT-ATGTGTAAGAAG 3.2 kb L15541 exon TCGGAGCTGCCT-TACGACGGGAAG 0.5 kb 317 * Ж SP ; 24 nt before GGGGCAGTAGTA-53 nt spanning exon 18 L21318 exon 18 reverse oligo-agcctgggagca 0.5 kb L15543 exon 19 5 nt before exon 19 reverse TTTGACCTGAGA-GGACAGGTGAGT 0.4 kb L15544 exon TGTCACCTGAAG-AGGTGAGTAAGG 3.5 kb 203 ± L20768 exon TGTCTGTTTTGA-TTCCTGCTGTAT 0.2 kb L15546 exon TCCACCTCGCTT-ACGTGACCAGCA 1.2 kb L15547 exon reverse GGGAGGCAGCAA-TCACATCCCCTG 5.1 kb stop codon (ex 23) 328 * L20779 FAM131B gene AGAGTATCCTGA-AGCTGGTTGGGA * New in version B1 (from lot B onwards). Changed in version B1 (from lot B onwards). Small change in length, no change in sequence detected. Ж This probe consists of three parts and has two ligation sites. 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 (203 nt) and rs (419 nt) could influence the probe signal. In case of apparent deletions, it is recommended to sequence the region targeted by this probe. 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. Note: Exon 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. SALSA P350 CLCN1-KCNJ2 probemix Page 4 of 6

5 Table 2b. KCNJ2 Length (nt) SALSA MLPA probe KCNJ2 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 2) 184 ๑ L15548 exon reverse AGTGGTTTGTAA-AAAGCGAGTGAG 0.1 kb 166 * ๑ L21186 exon GCTCCTGCGCCA-GCAACAGGTAAG 5.5 kb L15549 exon reverse TCTTTCTTCACA-AAGCGGCTCCTG 0.2 kb L15550 exon TGTCCGAGGTCA-ACAGCTTCACGG 0.6 kb 445 * L20782 exon GGGCCACCGCTA-TGAGCCTGTGCT stop codon (ex 2) * New in version B1 (from lot B onwards). ๑ The significance of exon 1 deletions is not clear as this exon is non-coding. The NM_ sequence 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: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P350 CLCN1-KCNJ2 probemix Page 5 of 6

6 SALSA MLPA probemix P350-B1 CLCN1-KCNJ2 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P350-B1 CLCN1-KCNJ2 (lot B1-0711). Implemented Changes compared to the previous product description versions. Version June 2015 (54) - Small textual change on page 1. - Electropherogram picture using the old MLPA buffer removed. Version 03 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 02 (48) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Warning added: ๑ The significance of exon 1 deletions is not clear as this exon is non-coding. - Various minor textual changes on page 1. - Warning added in Table 1, 203 nt probe L20768, 419 nt probe L21284, and 454 nt probe L Information about reference sequences added below Table 2. SALSA P350 CLCN1-KCNJ2 probemix Page 6 of 6