The SCN5A protein is found primarily in cardiac muscle and mediates the voltage-dependent sodium ion permeability of excitable membranes.

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1 SALSA MLPA probemix P108-B3 SCN5A Lot B As compared to the previous lot (B2-0312), two reference probes have been replaced and two probe lengths have been adjusted. The Brugada syndrome-1 and long QT syndrome type 3 are two hereditary cardiac diseases associated with mutations in the SCN5A gene. Both syndromes are ion channel diseases of the heart manifest on surface electrocardiogram by ST-segment elevation in the right precordial leads and prolonged QT interval, respectively, with predilection for polymorphic ventricular tachycardia and sudden death, which may be the first manifestation of the disease. Apart from these syndromes, mutations in the SCN5A gene are associated with several other cardiomyopathies such as familial atrial fibrillation, dilated cardiomyopathy, Romano-Ward syndrome and sick sinus syndrome 1. The SCN5A protein is found primarily in cardiac muscle and mediates the voltage-dependent sodium ion permeability of excitable membranes. The SCN5A gene (29 exons) spans 102 kb of genomic DNA and is located on chromosome 3p22.2. The P108 probemix contains one probe for each exon and one probe upstream of the first exon. For exon 29, two probes are present. In addition, 9 reference probes are included in this probemix, detecting different autosomal chromosomal locations. This SALSA probemix is designed to detect deletions/duplications of one or more sequences in the aforementioned 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 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 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 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 Related SALSA probemixes P114 LQT: Contains probes for KCNQ1 and KCNH2, which are involved in congenital long QT syndrome. References for SALSA MLPA probemix P108 SCN5A Garcia-Molina et al. (2013). A study of the SCN5A gene in a cohort of the 76 patients with Brugada syndrome. Clin Genetics. 83(6): Eastaugh et al. (2011). Brugada syndrome caused by a large deletion in SCN5A only detected by multiplex ligation-dependent probe amplification. J Cardiovasc Electrophysiol. 22: Macarie et al. (2009). The electrocardiographic abnormalities in highly trained athletes compared to the genetic study related to causes of unexpected sudden cardiac death. J Med Life. 2: SALSA P108 SCN5A probemix Page 1 of 6

2 Data analysis The P108-B3 SCN5A probemix contains 40 MLPA probes with amplification products between 142 and 453 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 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. SALSA P108 SCN5A probemix Page 2 of 6

3 Table 1. SALSA MLPA P108-B3 SCN5A probemix Length Chromosomal position SALSA MLPA probe (nt) reference SCN5A 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 142 Reference probe L q SCN5A probe L02930 Exon * Reference probe L p SCN5A probe L09329 Exon SCN5A probe L17222 Exon Reference probe L q SCN5A probe L02924 Exon SCN5A probe L02932 Exon SCN5A probe L07545 Exon SCN5A probe L02925 Exon SCN5A probe L17787 Exon 19a 214 SCN5A probe L09330 Exon Reference probe L q SCN5A probe L17217 Exon SCN5A probe L29551 Exon SCN5A probe L09326 Exon SCN5A probe L02927 Exon SCN5A probe L02935 Exon Reference probe L p SCN5A probe L02928 Exon SCN5A probe L02936 Exon SCN5A probe L17790 Exon SCN5A probe L17789 Exon SCN5A probe L17791 Exon SCN5A probe L17792 Exon * Reference probe L q ± SCN5A probe L02940 Exon SCN5A probe L02941 Exon SCN5A probe L07543 Exon Reference probe L q SCN5A probe L17213 Exon SCN5A probe L07538 Exon SCN5A probe L17793 Exon Reference probe L p SCN5A probe L17223 Exon SCN5A probe L17225 Exon SCN5A probe L07547 Exon SCN5A probe L17220 Exon ± SCN5A probe L17214 Upstream 453 Reference probe L q25 * New from lot B onwards. Changed from lot B onwards. Small change in length, no change in sequence detected. ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe (false positive deletion) can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. SALSA P108 SCN5A probemix Page 3 of 6

4 Table 2. SCN5A probes arranged according to chromosomal location Length (nt) SALSA MLPA probe SCN5A exon Ligation site NM_ start codon (exon 2) Partial sequence (24 nt adjacent to ligation site) Distance to next probe 445 ± L17214 Upstream 1746 nt before exon 1 GAAACGTTGACA-CTGCCTGCACTT 2.1 kb 337 ± L02940 Exon nt after exon 1 CGTCTCTAAACA-CCGTGCGCCCCC 16.0 kb L09329 Exon GCTTCCGCAGGT-TCACACGGGAGT 2.9 kb L02924 Exon CCAACGCCTTGT-ATGTCCTCAGTC 7.9 kb L02925 Exon CCAACTGCGTGT-TCATGGCCCAGC 1.6 kb L17790 Exon reverse GCCATGATAATC-ACACTAAAGTCC 6.9 kb L17793 Exon in NM_ ACTATTTCAGTT-ATCCCAGGTAAG 0.1 kb L17217 Exon reverse CAAATTCAGTTG-TGTATCTGTAAC 4.0 kb L02927 Exon CCTCAGCGTCTT-TGCCCTCATCGG 1.7 kb L07538 Exon CTCTGATGTGTT-ACTGTGTGGGAA 1.4 kb L02928 Exon CCGACCACGGCT-ACACCAGCTTCG 0.7 kb L17225 Exon CAAGCCACCATC-GCTGAGACCGAG 1.3 kb L17791 Exon GATGGTCCCAGA-GCAATGGTAATC 0.9 kb L17223 Exon CATGGCAAAAAG-AACAGCACTGTG 4.9 kb L17220 Exon GTCAGCGTCCTC-ACCAGCGCACTG 1.1 kb L09326 Exon ATGGACCCGTTT-ACTGACCTCACC 10.4 kb L02930 Exon CCTGTCCCGCAT-GAGCAACTTGTC 1.7 kb L07543 Exon TCTTGCTTGTTA-TGGTCATTGGCA 4.6 kb L17222 Exon reverse GTGTTTCCTTGC-GGGTGGGAGGCA 1.7 kb L17787 Exon 19a GTGCATCTCAGG-CCGACTGGCGGC 2.7 kb L02932 Exon CAACACCGCTGA-GCTCCTGGAGCA 1.4 kb L07545 Exon TGGTTCGAGACA-TTCATCATCTTC 8.8 kb L09330 Exon AGATGTTCACAT-ATGTCTTCGTGC 4.0 kb L29551 Exon ACTGCGGACGCT-GCGTGCACTCCG 2.2 kb L02935 Exon ACAAGAGCCAGT-GTGAGTCCTTGA 2.9 kb L17213 Exon nt before exon 25 GAAGCTCAAGCG-AGGTACAGAATT 0.8 kb L02941 Exon reverse AAGATGATGAAA-ATGACAAAATAG 0.8 kb L07547 Exon 27 5 nt before exon 27 GTGCCTTCTCTT-TGCACTTAGGGG 1.3 kb L17792 Exon reverse GCAGATCAGAAA-CATGATGGTGAC 3.0 kb L02936 Exon CATCATCCAGAA-GTACTTCTTCTC 0.8 kb L17789 Exon reverse GAGATCTTGGAT-GGGTTGGCTGCC stop codon (exon 29) Changed from lot B onwards. Small change in length, no change in sequence detected. ± This probe is located within, or close to, a very strong CpG island. A low signal of this probe (false positive deletion) can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. # Exon 6 is only present in transcript variant 3, which is represented by the reference sequence NM_ The ligation sites of the probes are indicated in the NM_ sequence, which is a reference standard in the NCBI RefSeqGene project, representing transcript variant 1. This sequence is the longer transcript, and encodes the longer isoform a. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P108 SCN5A probemix Page 4 of 6

5 SALSA MLPA probemix P108-B3 SCN5A sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P108-B3 SCN5A (lot B3-0916). SALSA P108 SCN5A probemix Page 5 of 6

6 Implemented Changes compared to the previous product description version(s). Version October 2016 (55) - Product description adapted to a new product version (version number changed, lot number added, changes in Table 1 and Table 2, new picture included). - Reference added at page 1. - Several minor textual changes throughout the document. Version July 2015 (54) - Figure based on the use of old MLPA buffer (replaced in December 2012) removed. - Various minor textual changes throughout the document. Version 11 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 10 (48) - Various minor textual and layout changes. Version 09 (46) - Remark on RefSeqGene standard added below Table 2. - 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 and layout changes. Version 08 (45) - Product description adapted to a new product version (version number changed, lot number added, 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. - Ligation sites updated according to new version of the NM_reference sequence. - SCN5A exon numbering changed! - Warning for false positive results due to incomplete denaturation added for the exon 1 probe. - Various minor textual changes on page 1. Version 07 (45) - Warning added about the 154 nt reference probe (03404-L02795). - Various minor textual changes on page 1 and 2. Version 06 (44) - Various minor textual changes on page 1. Minor changes in the data analysis section on page 2. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. - Various minor layout changes in Table 1 and 2. Tables have been numbered. SALSA P108 SCN5A probemix Page 6 of 6