MRC-Holland MLPA. Description version 08; 02 June 2017

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1 SALSA MLPA probemix P367-A2 BEST1-PRPH2 Lot A2-0616, A As compared to version A1 (lot A1-0809), two reference probes have been replaced and the control fragments have been adjusted (QDX2). Please note that this P367-A2 probemix contains the probes for BEST1 (=VMD2) and PRPH2 (=RDS) that have been removed from SALSA probemix P229 OPA1. Vitelliform macular dystrophy is a genetic eye disorder that can cause progressive vision loss. This disorder affects the retina, the specialized light-sensitive tissue that lines the back of the eye. The early-onset form (known as Best disease) usually appears in childhood; both the onset of symptoms and the severity of vision loss vary widely. The adult-onset form starts mid-adulthood, and tends to cause vision loss that worsens slowly over time. The two forms of vitelliform macular dystrophy each cause characteristic changes in the macula that can be detected during an eye examination. Defects in the gene BEST1 (=VMD2) on chromosome 11 are responsible for Best disease and for some cases of the adult-onset form of vitelliform macular dystrophy. Changes in the PRPH2 (=RDS) gene on chromosome 6 can also cause the adult-onset form of vitelliform macular dystrophy. The BEST1 gene (11 exons) spans ~15 kb of genomic DNA and is located on 11q12.3, ~62 Mb from the p- telomere. The P367-A2 probemix contains one probe for each exon of the BEST1 gene. The PRPH2 gene (3 exons) spans ~26 kb of genomic DNA and is located on 6p21.1, ~43 Mb from the p-telomere. The P367-A2 probemix contains two probes for PRPH2 exon 2 and one probe each for PRPH2 intron 1 and exons 1 and 3. In addition, 8 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 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 Acids 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 P367 BEST1-PRPH2 probemix Page 1 of 5

2 Related SALSA probemixes P219 PAX6: Contains probes for the PAX6, SOX2, WT1 genes, involved in multiple ocular malformations. P221 LCA1: Contains probes for the AIPL1, CRB1, CRX, RPE65 genes, associated with Leber Congenital Amaurosis. P222 LCA2: Contains probes for the GUCY2D, RDH12, RPGRIP1, CEP290 genes, associated with Leber Congenital Amaurosis. P229 OPA1: Contains probes for the OPA1 gene, associated with optic atrophy type 1. References Boulanger-Scemama E. et al., Next-generation sequencing applied to a large French cone and conerod dystrophy cohort: mutation spectrum and new genotype-phenotype correlation. Orphanet J Rare Dis. 10:85. Data analysis The P367-A2 BEST1-PRPH2 probemix contains 24 MLPA probes with amplification products between 130 and 274 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 J. Coffa & N. Laddach at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the probemix designers could be included as co-authors. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P367 BEST1-PRPH2 probemix Page 2 of 5

3 Table 1. SALSA MLPA P367-A2 BEST1-PRPH2 probemix Chromosomal position SALSA MLPA probe reference BEST1 PRPH 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 BEST1 probe L14703 Exon Ø PRPH2 probe L14441 Intron Reference probe L q BEST1 probe L15175 Exon * Reference probe L p BEST1 probe L06515 Exon PRPH2 probe L07114 Exon BEST1 probe L06516 Exon Reference probe L q BEST1 probe L06521 Exon BEST1 probe L14704 Exon PRPH2 probe L13648 Exon BEST1 probe L06514 Exon Reference probe L q BEST1 probe L06523 Exon PRPH2 probe L13649 Exon * Reference probe L q BEST1 probe L13711 Exon PRPH2 probe L13791 Exon BEST1 probe L06519 Exon Reference probe L q BEST1 probe L06522 Exon «Reference probe L p24 * New in version A2 (from lot A onwards). Ø Intron probe. Only included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of a single intron probe are unlikely to be related to the condition tested. «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. SALSA P367 BEST1-PRPH2 probemix Page 3 of 5

4 Table 2. P367 probes arranged according to chromosomal location Table 2a. BEST1 gene SALSA MLPA probe BEST1 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 2) L15175 Exon reverse GGTGGGTCCGAT-GATCCCACAGAA 1.5 kb L06514 Exon TGCTCTGCTACT-ACATCATCCGCT 3.2 kb L06515 Exon AACAGCTGATGT-TTGAGAAACTGA 0.6 kb L06516 Exon GACGCTGGTCGT-GACCCGCTGGTG 1.2 kb L14704 Exon ACCACACAACAT-GTTCTGGGTGCC 0.7 kb L13711 Exon nt after ex 6 TCTGGACTTTGA-AGTGCCAAGTTC 0.7 kb L06519 Exon CTTCTTCTATGT-TGGCTGGCTGAA 1.2 kb L14703 Exon reverse CAGTTGGTCTCA-AAATCATCATCA 0.4 kb L06521 Exon GCACCAGGACCT-GCCTCGGATGGA 2.4 kb L06522 Exon GACGAGGAGGAT-GCTCACGCTGGC 2.1 kb L06523 Exon reverse GCTGTCTTGGAT-TCAGATAGAACT stop codon (ex 11) The NM_ sequence represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. Table 2b. PRPH2 gene SALSA MLPA probe PRPH2 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L13648 Exon GCAACAACGGTT-TTCGGGACTGGT 3.3 kb 142 Ø L14441 Intron nt after ex 1 CTGCAGTGGGCT-ACGTGTTCTTCC 14.0 kb L07114 Exon TGGATGGGCGGT-ACCTGGTGGACG 0.2 kb L13791 Exon CATGGGTGTCGT-CACGCTCCTCAT 6.3 kb L13649 Exon GTGAAGCTCCCT-TCAGGCCCGCTG stop codon (ex 3) Ø Intron probe. Only included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations a single intron probe are unlikely to be related to the condition tested. 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 P367 BEST1-PRPH2 probemix Page 4 of 5

5 SALSA MLPA probemix P367-A2 BEST1-PRPH2 sample picture Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P367-A2 BEST1-PRPH2 (lot A2-0616). Implemented Changes compared to the previous product description versions. Version June 2017 (55) - Product description adapted to a new lot (lot number added, small changes in Table 1 and Table 2, new picture included). - New reference added. - Manufacturer s address adjusted. - Peak area replaced with peak height. Version 07 (49) - Figure(s) based on the use of old MLPA buffer (replaced in December 2012) removed. Version 06 (49) - 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). Version 05 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added Version 04 (48) - Remark on RefSeqGene standard and transcript variant added below Table 2. Version 03 (46) Version 02 (46) - Various minor textual changes on page 1. - Various minor layout changes. - Tables have been numbered. - Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section. Version 01 (43) - Not applicable, new document. SALSA P367 BEST1-PRPH2 probemix Page 5 of 5