Furthermore, 8 reference probes are included in this probemix, detecting several different autosomal chromosomal locations.

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1 SALSA MLPA mix P328-A2 EYS Lot A As compared to version A1, two reference s have been replaced and one length has been adjusted. Retinitis Pigmentosa-25 (RP25) is characterised by progressive peripheral vision loss and night vision difficulties (nyctalopia) that can lead to central vision loss. The gene EYS (Eyes shut homolog) at the Retinitis Pigmentosa, RP25, locus on chromosome 6q12 encodes the protein SPAM and is commonly mutated in autosomal recessive retinitis pigmentosa-25. The EYS gene (43 exons) spans ~2 Mb of genomic DNA and is located on chromosome 6q12, ~64-66 Mb from the p-telomere. The P328-A2 mix contains one for each exon of the gene except for exon 2, 7, 9, and 27. Exon 12 and 17 are covered by two s. Additionally, the mix includes a for intron 1, 7, 11 and two s for intron 27, as well as three wildtype s for three pathogenic mutations as described in Abd El-Aziz M.M. et al. (Nat Genet (11): ). Furthermore, 8 reference s are included in this mix, detecting several different autosomal chromosomal locations. This SALSA mix 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. Note that a mutation or polymorphism in the sequence detected by a can also cause a reduction in relative peak height, even when not located exactly on the ligation site! In addition, some 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 mixes 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 mixes and reagents includes a limited license to use these products for research purposes. The use of a SALSA mix 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 SALSA mixes P235 Retinitis pigmentosa: contains s for the autosomal genes IMPDH1, PRPF31, RHO and RP1. P366 CHM-RP2-RPGR: contains s for the CHM, RP2 and RPGR genes, which are involved in X-linked retinitis pigmentosa. 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 P328 EYS mix Page 1 of 7

2 References Pieras J.I. et al. (2011). Copy-Number Variations in EYS: A Significant Event in the Appearance of arrp. Invest Ophthalmol Vis Sci. 52: Coppieters F. et al. (2014). Identity-by-descent guided mutation analysis and exome sequencing in consanguineous families reveals unusual clinical and molecular findings in retinal dystrophy. Genet Med. 16: Perez-Carro, R. et al. (2016). Panel-based NGS Reveals Novel Pathogenic Mutations in Autosomal Recessive Retinitis Pigmentosa. Sci. Rep. 6: Data analysis The P328-A2 EYS mix contains 54 MLPA s with amplification products between 128 and 500 nt. This includes three s, specific for three pathogenic mutations, which will show a decreased signal when a 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. Data generated by this mix can first be normalised intra-sample by dividing the peak height of each s amplification product by the total peak height of only the reference s in this mix (block normalisation). Secondly, inter-sample normalisation can be achieved by dividing the intra-normalised ratio in a sample by the average intra-normalised ratio of all reference samples. Please note that this type of normalisation assumes no changes occurred in the genomic regions recognised by the reference s. 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 s are unlikely to be the cause of the condition tested for. Users should always verify the latest scientific literature when interpreting their findings. This mix was developed by N. Laddach at. In case the results obtained with this mix lead to a scientific publication, it would be very much appreciated if the mix designer could be included as co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P328 EYS mix Page 2 of 7

3 Table 1. SALSA MLPA P328-A2 EYS mix Chromosomal position SALSA MLPA reference EYS 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 128 Reference L q EYS L20974 Exon EYS L14831 Exon EYS L14832 Exon EYS L14833 Exon EYS L14834 Exon EYS L14835 Exon EYS L14836 Intron Reference L q EYS L16559 Intron EYS L14922 Exon 12 (WT for c.1971delt) 190 EYS L14839 Exon EYS L14840 Exon EYS L14841 Exon EYS L14842 Exon EYS L14843 Exon EYS L14923 Exon EYS L14844 Exon * Reference L p EYS L14845 Exon EYS L14846 Exon EYS L14847 Exon EYS L14848 Exon EYS L22534 Exon EYS L22535 Exon EYS L14850 Exon EYS L14851 Exon EYS L14924 Exon 17 (WT for c.2710_2726del17) 295 Reference L q EYS L20978 Exon EYS L14853 Exon EYS L29703 Exon EYS L14855 Exon Ж EYS SP0435-L20979 Exon EYS L20980 Exon EYS L20981 Exon EYS L20982 Exon Reference L q EYS L14860 Intron EYS L21925 Exon EYS L17499 Exon ± EYS L14926 Exon 28 (WT for c.5857g>t) 400 EYS L14862 Exon EYS L14927 Exon EYS L14864 Exon EYS L14865 Intron Ж EYS SP0434-L20084 Exon Reference L q EYS L17898 Exon EYS L17507 Exon 32 SALSA P328 EYS mix Page 3 of 7

4 Chromosomal position SALSA MLPA reference EYS 472 EYS L14870 Exon EYS L20086 Intron * Reference L q Reference L q22 * New in version A2 (from lot A onwards). Changed in version A2 (from lot A onwards). Small change in length, no change in sequence detected. Wild type sequence detected. The presence of the c.1971delt mutation will result in a decreased signal. Wild type sequence detected. The presence of the c.2710_2726del17 mutation will result in a decreased signal. ± The presence of the c.5857g>t mutation close to the ligation site may result in a decreased signal. Ж This consists of three parts and has two ligation sites. Note: Exon numbering used here may differ from literature! Please notify us of any mistakes. The identity of the genes detected by the reference s is available on request: info@mlpa.com. SALSA P328 EYS mix Page 4 of 7

5 Table 2. EYS s arranged according to chromosomal location SALSA MLPA Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next start codon (ex 4) L17898 Exon 1 71 nt after exon 1 TAGCTGAACACA-ACTACAAAGATC 66.7 kb L14865 Intron nt before exon 2 GGTTTCCTGCCA-AGTTGAAGCCCT kb No Exon L20981 Exon 3 40 nt after exon 3 AACCAGAACTTT-TCAGAATCAGGT 0.4 kb L14835 Exon ACTGACAAATCA-ATCGTCATTCTG 4.8 kb L22534 Exon 5 24 nt after exon 5 TATGTATACAAC-CATATACACAAG 85.3 kb L21925 Exon TTATACTTATGA-ATGCCCAAAAGG 3.2 kb No Exon L14836 Intron nt after exon 7 AGCAGATAGATA-TTAGTTAGGGTC 17.6 kb L20982 Exon TCTGAATGAAGA-ATGGTGTTTCAA 40.3 kb No Exon Ж SP , TAGGTTGCATCT-30nt spanning Exon 10 L20084 reverse oligo-gcatcaataacc 9.0 kb L14832 Exon GGTATCTATGTT-TTCTCAGATGGG 5.6 kb L20086 Intron kb after exon 11 (NM_ ; GCCAGTCTTGAA-ACCACTAGATCA 33.6 kb ) L14922 Exon 12 (WT for c.1971delt) AATGGAACAACT-AGTACACATTTA 0.2 kb L14927 Exon nt after exon 12 GGATTGCTTCTT-TGGGCAATTTAA kb L14834 Exon GCCTCCATTTAA-AGGTAATGAACA 60.0 kb L14831 Exon reverse AGGATGCAGTCA-TCAATGTCCTGT 51.8 kb L14843 Exon CAAGAACAATTC-CACCTGTACTGA 33.2 kb L20974 Exon AATGGAGGTCTT-TGTCATGAATCT 9.9 kb L14923 Exon nt before exon 17 TCTCAGGGGATT-TAGTGGAGTATT 0.3 kb L14924 Exon 17 (WT c.2710_2726del17) TGTGAAGATATG-GTCAACAATTTC 0.3 kb 333 Ж SP0435- TTTTCTGGATCT-36nt spanning Exon , L20979 oligo-ccttgcaaaaat 15.4 kb L14839 Exon CTGATGGATACA-ACTGCCTCTGTG 64.0 kb L14844 Exon GTGACTGCAAGA-GTGGGTTTTTTG 1.0 kb L20980 Exon GTTCATGTGATG-CAGATGGGACTA 8.1 kb L14842 Exon TTGTATGAATGA-AGGCTTCTGTCA kb L14862 Exon nt after exon 23 GACTACTAATCA-AGTTTTAAGCAA 8.5 kb L14870 Exon GCTTGAGTGCAT-TCCCAACTCATG 24.4 kb L20978 Exon CAGTTGATCAAG-GTAAGTAGAAGT 2.0 kb L22535 Exon AGCCAGCTGGTA-TGCACTAATGGG kb No Exon L16559 Intron nt after exon 27, 1294 nt before exon ATTTTAGCACAA-AGCACATGTGAA 0.9 kb L14860 Intron nt before exon 28 CTAGATTGTATT-AACCATTCTTTG 0.4 kb 392 ± L14926 Exon 28 (WT c.5857g>t) GTCCTGGTGAAG-CAAAATTTAAAA 47.5 kb L14847 Exon ATCTATCAATCA-TGTACTCGGAAA 80.5 kb L14855 Exon CAGTGAAAAACT-ATCACATTAACA 76.2 kb L14841 Exon ACAAGGGGTTGA-TACCATGTGGAC kb L17507 Exon GGGAATTCCTAT-TTAGAACTGCCC 15.6 kb L14864 Exon nt after exon 33 ACGGTAGTTCTT-TCGTGCTGAAAT 67.2 kb L14833 Exon 34 2 nt after exon 34 reverse TTTGCATACCTA-CCTGAGAGGCAT 14.5 kb L14845 Exon reverse CCTGAACCCATA-AACAGGACCTGC kb L14851 Exon TGCCTCTGCCCA-TATGGGAGGTCT 58.0 kb L29703 Exon 37 9 nt after exon 37 ATGGTAAGATTA-ACTGAACCCTCT 17.1 kb L14848 Exon nt after exon 38 GTAAAGAGTCAT-TCCTTCGTCACT 0.9 kb SALSA P328 EYS mix Page 5 of 7

6 SALSA MLPA Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next L17499 Exon TGGCTACAGTGA-ATACACTCCAGA 10.1 kb L14850 Exon ATAGAGAGTGGA-ACTAGTGTTTAG 15.4 kb L14840 Exon GAGACAGTTTCT-ACCTGTGATCCT 35.8 kb L14853 Exon nt before exon 42 AAGGTTTGATGT-ACTCACCTACAA 5.2 kb L14846 Exon AGGCTACCTGGA-TCTAGATGGGAT stop codon (ex 43) Changed in version A2 (from lot A onwards). Small change in length, no change in sequence detected. Wild type sequence detected. The presence of the c.1971delt mutation will result in a decreased signal. Wild type sequence detected. The presence of the c.2710_2726del17 mutation will result in a decreased signal. ± The presence of the c.5857g>t mutation close to the ligation site may result in a decreased signal. Ж This consists of three parts and has two ligation sites. This detects the coding sequence of NM_ , which represents transcript variant 2 of the EYS gene. 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! Complete sequences are available on request: info@mlpa.com. Please notify us of any mistakes: info@mlpa.com. SALSA P328 EYS mix Page 6 of 7

7 SALSA MLPA mix P328-A2 EYS sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA mix P328-A2 EYS (lot A2-0217). Implemented Changes compared to the previous product description versions. Version March 2017 (55) - Product description adapted to a product version (version number changed, lot number added, small changes in Table 1 and Table 2, new picture included). - New reference added on page 1. - Various minor textual and layout changes. Version December 2015 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - New references added on page 1. - Various minor textual changes on page 1. - Various minor layout changes. - Exon numbering of the EYS gene has been changed on page 3 and 4. Version 04 (48) - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) 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, small changes in Table 1 and Table 2, new picture included). - New references added on page 1. - Various textual changes on page 1. - Various minor layout changes. - Small changes of lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - Remark on database of genomic variants has been added on page 2. SALSA P328 EYS mix Page 7 of 7