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

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1 SALSA MLPA probemix P186-C2 PAX3 MITF SOX10 Lot C2-0916: As compared to version C1 (lot C1-0212), one reference probe has been replaced and several probe lengths have been adjusted. Several types of Waardenburg Syndrome (WS) can be casued by mutations in the MITF gene (WS2A), the PAX3 gene (WS1 & WS3) or the SOX10 gene (WS2E & WS4C). MITF transactivates the gene for tyrosinase, a key enzyme for melanogenesis, and is critically involved in melanocyte differentiation. Absence of melanocytes affects pigmentation in the skin, hair, and eyes, and hearing function in the cochlea. Therefore, hypopigmentation and hearing loss in WS2A are likely to be the results of an anomaly of melanocyte differentiation caused by MITF mutations. PAX3 and SOX10 regulate MITF, and failure of this regulation due to mutations in these genes results in the auditory-pigmentary symptoms in at least some individuals with WS1/WS3 (PAX3) or WS2E/WS4C (SOX10). The PAX3 gene (8 exons) spans ~100 kb of genomic DNA and is located on chromosome 2q36.1, ~223 Mb from the p-telomere. The P186-C2 probemix contains two probes for each exon of the PAX3 gene except for exon 2 (no probe) and exon 1 (one probe). The MITF gene (9 exons) spans ~32 kb of genomic DNA and is located on chromosome 3p14.1, ~70 Mb from the p-telomere. The P186-C2 probemix contains two probes for exons 1 and 2 of the alternative transcript variant 1 (NM_ ) and one probe for exons 2 to 9 of the MITF gene main transcript variant 4 (NM_ ). The SOX10 gene (4 exons) spans 12.2 kb of genomic DNA and is located on 22q13,1, 38.4 Mb from the p-telomere. This P186-C2 probemix contains one probe for exons 1-3 and two probes for exon 4 of the SOX10 gene. In addition, ten 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 Related SALSA probemixes P153 EYA1: Contains probes for the EYA1 gene, involved in branchio-oto-renal syndrome. P163 GJB-WFS1: Contains probes for GJB2, GJB3, GJB6, WFS1 and the POU3F4 area. P191/P192 Alport: Contains probes for the COL4A5 gene, involved in Alport syndrome. P280 SLC26A4: Contains probes for the SLC26A4 gene, involved in Pendred syndrome. P169 Hirschsprung 1: Contains probes for RET, ZEB2, EDN3 and GDNF. SALSA probemix P186 PAX3 MITF SOX10 Page 1 of 6

2 P318 Hirschsprung 2: Contains probes for SOX10, PSPN, NRTN, EDNRB, GFRA1, GFRA2, GFRA3, PHOX2B. P354 KIT SNAI2: contains probes for KIT and SNAI2, involved in piebaldism. References of SALSA probemix P186 PAX3 MITF SOX10 Kanno, A et al. (2016) Frequency and specific characteristics of the incomplete partition type III anomaly in children. The Laryngoscope. Matsunaga, T et al. (2013) Genetic analysis of PAX3 for diagnosis of Waardenburg syndrome type I. Acta Otolaryngol. 133(4): Wildhardt, G et al. (2013) Spectrum of novel mutations found in Waardenburg syndrome types 1 and 2: implications for molecular genetic diagnostics. BMJ open 3(3): e Milunsky, J et al. (2007) The value of MLPA in Waardenburg Syndrome. Genet Test. 11: Data analysis The P186-C2 PAX3 MITF SOX10 probemix contains 41 MLPA probes with amplification products between 130 and 472 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 at. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA probemix P186 PAX3 MITF SOX10 Page 2 of 6

3 Table 1. SALSA MLPA P186-C2 PAX3 MITF SOX10 probemix Chromosomal position SALSA MLPA probe reference MITF PAX3 SOX 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 «SOX10 probe L11858 Exon MITF probe L Reference probe L q PAX3 probe L05421 Exon Reference probe L q PAX3 probe L05433 Exon PAX3 probe L05425 Exon Reference probe L q MITF probe L PAX3 probe L29550 Exon MITF probe L11435 Exon «SOX10 probe L16376 Exon PAX3 probe L21404 Exon Reference probe L q MITF probe L «SOX10 probe L16380 Exon PAX3 probe L21163 Exon Reference probe L q PAX3 probe L21168 Exon PAX3 probe L05430 Exon MITF probe L21164 Exon MITF probe L21165 Exon PAX3 probe L21166 Exon «SOX10 probe L11857 Exon Reference probe L q MITF probe L11437 Exon MITF probe L04849 Exon MITF probe L11434 Exon PAX3 probe L29511 Exon PAX3 probe L11442 Exon * Reference probe L q PAX3 probe L05427 Exon MITF probe L29510 Exon PAX3 probe L11441 Exon PAX3 probe L05428 Exon MITF probe L21167 Exon Reference probe L p «SOX10 probe L16387 Exon MITF probe L Reference probe L q22 * New in version C2 (from lot C onwards). Changed in version C2 (from lot C 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 can be due to incomplete sample DNA denaturation, e.g., due to the presence of salt in the sample DNA. Notes The PAX3 & MITF exon numbering has changed. From description version 11 onwards, we have adopted the NCBI exon numbering that is present in the NG_ sequences for these two genes. The 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 identity of the genes detected by the reference probes is available on request: info@mlpa.com. SALSA probemix P186 PAX3 MITF SOX10 Page 3 of 6

4 Table 2. P186 probes arranged according to chromosomal location Table 2a. PAX3 SALSA MLPA probe PAX3 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L21166 Exon CCGCACTCGCCT-TTCCGTTTCGCC 2.7 kb No probe Exon L05421 Exon 3 (3a) 300 nt before exon 3 TTCCAGGGATGA-GAAAGTATAACC 0.4 kb L21168 Exon 3 (3a) AATCCGAGACAA-ATTACTCAAGGA 1.3 kb L29511 Exon 4 (4a) ATCCTGAGAAGT-AAATTCGGGAAA 0.4 kb L29550 Exon 4 (4a) 258 nt after exon 4 CTGGGATTCTAG-AACTGTGAATTG 61.7 kb L05425 Exon TCTGAACCAGAT-TTACCACTAAAG 0.1 kb L21404 Exon TACCCTGACATT-TATACTAGGGAG 10.5 kb L05427 Exon nt before exon 6 CTGTTGTTGCAA-GATCATGGTGGG 0.2 kb L05428 Exon GCCGTGCAAGAT-GGAGGAAGCAAG 1.0 kb L11441 Exon CCTCAACCGCTT-CCTCCAAGCACT 0.1 kb L05430 Exon TACAGACAGCTT-TGTGCCTCCGTC 18.1 kb L11442 Exon 8 (8b) GCAGTCAGAGAC-TAGACCATATGA 0.1 kb L21163 Exon 8 (8b) ACAGGCTACAGT-ATGGACCCTGTC 0.6 kb L05433 Exon 8 (9b) 282 nt after exon 8 ( GTGCCTTTCATT-ATCTCAAGCCAG (NM_ )) stop codon (ex 8) Changed in version C2 (from lot C onwards). Small change in length, no change in sequence detected. The NM_ sequence represents transcript variant PAX3 and is a reference standard in the RefSeq gene project. Exon 9 is only present in the alternative transcript variant NM_ (PAX3D). Note: The PAX3 exon numbering has changed. From description version 11 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for this gene. The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. Table 2b. MITF SALSA MLPA probe L L L L21861 MITF Ligation site Exon NM_ start codon (ex 1) (1) (1) (5) (5) 197 kb before exon ) 197 kb before exon ) 57 kb before exon ) 57 kb before exon ) Partial sequence (24 nt adjacent to ligation site) CCAGCAGTGGAA-GGACGGGAAGCG CCGGATTTCGAA-GTCGGGGAGGAG TAAGCTCCTCCA-GTATGACATCAC GCGGCCCAGTTC-ATGCAACAGAGA Distance to next probe 0.1 kb kb 0.1 kb 58.7 kb No probe Exon L04849 Exon 2(7) CATGCCACCGGT-GCCGGGGAGCAG 1.2 kb L29510 Exon 3(9) AGTGCCCAGGCA-TGAACACACATT 2.1 kb L21165 Exon 4(10) TGGGCTTGATGG-ATCCTGCTTTGC 7.8 kb L11434 Exon 5(11) TCTTTATGGAAA-CCAAGGTCTGCC 2.8 kb L11435 Exon 6(12b) AATCACAACCTG-AGTAAGTTGGTT 4.6 kb L21164 Exon 7(13) GTCAAATGATCC-GTGAGTACAATC 2.8 kb SALSA probemix P186 PAX3 MITF SOX10 Page 4 of 6

5 L11437 Exon 8(14) AAAGTTGCAACG-AGAACAGCAACG 5.6 kb L21167 Exon 9(15) AACTGCAGCCAA-GACCTCCTTCAG stop codon (ex 9) Changed in version C2 (from lot C onwards). Small change in length, no change in sequence detected. The NM_ sequence represents transcript variant 4 and is a reference standard in the RefSeq gene project. Note: The MITF exon numbering has changed. From description version 11 onwards, we have adopted the NCBI exon numbering that is present in the LRG for this gene (LRG_776; NM_000248). The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Complete probe sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. Table 2c. SOX10 SALSA MLPA probe SOX10 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 2) 442 «17728-L16387 Exon CACTTCCTAAGG- ACGAGCCCCAGA 0.8 kb 310 «14383-L11857 Exon ACGATGACAAGT-TCCCCGTGTGCA 5.5 kb 136 «11174-L11858 Exon AGCGGCTCCGTA-TGCAGCACAAGA 4.0 kb 256 «11175-L16380 Exon AGCCACGAGGTA-ATGTCCAACATG 1.3 kb 226 «11176-L16376 Exon AATCAGAGACAA-TTCACAGAGCCT stop codon (ex 4) «The probes targeting SOX10 are 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 RefSeq gene project. The same SOX10 probes are present in P318 Hirschsprung-2. 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 probemix P186 PAX3 MITF SOX10 Page 5 of 6

6 SALSA MLPA probemix P186-C2 PAX3 MITF SOX10 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P186 PAX3 MITF SOX10 (lot C2-0916). Implemented Changes compared to the previous product description version(s). Version October 2016 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - Exon numbering of the PAX3 and MITF genes have been changed. - Various minor textual changes. Version 10 (54) 15 July Figure based on the use of old MLPA buffer (replaced in December 2012) removed. Version 09 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 08 (47) - Product description adapted to a new product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor textual changes. Version 07 (46) - The MITF exon numbering has been changed. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Ligation sites of the probes targeting the PAX3 and MITF genes updated according to new version of the NM_reference sequence. - Partial probe sequences extended to 24 nt. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. - Various minor textual changes. - Various minor layout changes. SALSA probemix P186 PAX3 MITF SOX10 Page 6 of 6