MRC-Holland MLPA. Description version 14; 21 January 2015

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1 SALSA MLPA probemix P229-B2 OPA1 Lot B As compared to version B1-0809, two reference probes and the 88 and 96 nt control fragments have been replaced (QDX2). The OPA1 gene product is a nuclear-encoded mitochondrial protein with similarity to dynamin-related GTPases. It is a component of the mitochondrial network. Mutations in this gene have been associated with optic atrophy type 1, which is a dominantly inherited optic neuropathy resulting in progressive loss of visual acuity, leading in many cases to legal blindness. The OPA1 gene (31 exons) spans ~105 kb of genomic DNA and is located on 3q29, 4.6 Mb from the q- telomere. The P229-B2 probemix contains at least one probe for each exon of the OPA1 gene with the exception of exon 15. Furthermore, the P229-B2 probemix contains a probe which detects the wild type allele of the pathogenic polymorphism c.2708delttag. In addition, 9 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 gene and to detect the presence of the aforementioned mutation 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 this 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 6, 1057 DN Amsterdam, the Netherlands Related SALSA probemixes P219 PAX6: Contains probes for PAX6, SOX2, WT1 genes involved in hereditary ocular malformations. P221 LCA1: Contains probes for AIPL1, CRB1, CRX, RPE65 genes involved in Leber congenital amaurosis. P222 LCA2: Contains probes for GUCY2D, RDH12, RPGRIP1, CEP290 genes involved in Leber congenital amaurosis. P367 VMD2-RDS: Contains probes for VMD2 and RDS genes involved in vitelliform macular dystrophy. P149 CYP4V2: Contains probes for CYP4V2 gene involved in Bietti crystalline corneoretinal dystrophy. References for SALSA probemix P229 OPA1 Fuhrmann N. et al. (2009) Genomic rearrangements in OPA1 are frequent in patients with autosomal dominant optic atrophy. J Med Genet. 46: Fuhrmann N et al. (2010) Solving a 50 year mystery of a missing OPA1 mutation: more insights from the first family diagnosed with autosomal dominant optic atrophy. Mol Neurodegener. 5;25. Almind GJ et al. (2011) Genomic deletions in OPA1 in Danish patients with autosomal dominant optic atrophy. BMC Med Genet. 12:49. SALSA P229 OPA1 probemix Page 1 of 5

2 Data analysis The P229-B2 OPA1 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 (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 by J. Coffa and N. Laddach at. In case the results obtained with this probemix lead to a scientific publication, it would be very much appreciated if the first probemix designer could be made a co-author. Info/remarks/suggestions for improvement: info@mlpa.com. SALSA P229 OPA1 probemix Page 2 of 5

3 Table 1. SALSA MLPA P229-B2 OPA1 probemix Length Chromosomal position SALSA MLPA probe (nt) reference OPA 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 Reference probe L q Reference probe L p OPA1 probe L02704 Exon Reference probe L q OPA1 probe L13646 Exon OPA1 probe L03713 Exon OPA1 probe L14919 Exon OPA1 probe L02695 Exon OPA1 probe L13647 Exon Reference probe L q OPA1 probe L14491 Exon OPA1 probe L14917 Exon OPA1 probe L14918 Exon OPA1 probe L06529 Exon OPA1 probe L02697 Exon OPA1 probe L14493 Exon OPA1 probe L02698 Exon OPA1 probe L02707 Exon Reference probe L q OPA1 probe SP0118-L14687 Exon OPA1 probe L02699 Exon OPA1 probe L14494 Intron OPA1 probe L14495 Exon OPA1 probe L02700 Exon OPA1 probe L06524 Exon OPA1 probe L02701 Exon Reference probe L q OPA1 probe L14701 Intron OPA1 probe L02702 Exon OPA1 probe L14702 Exon OPA1 probe L14497 Exon OPA1 probe L02703 Exon OPA1 probe L02709 Exon OPA1 probe L14498 Exon OPA1 probe L14499 Exon OPA1 probe L14438 Exon OPA1 probe L14439 Exon OPA1 probe L14440 Intron Reference probe L q * Reference probe L q34 * New in version B2 (from lot B onwards). This probe detects the wild type allele of the c.2708delttag polymorphism. The presence of the polymorphisms will result in a decreased probe signal. Note: The OPA1 exon numbering has changed. From description version 10 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the OPA1 gene. This exon numbering might be different as compared to literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. SALSA P229 OPA1 probemix Page 3 of 5

4 Table 2. OPA1 probes arranged according to chromosomal location Length (nt) SALSA MLPA probe OPA1 exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex1) L nt after exon 1 AAATGTGCAGGT-GACTCTCAGGCC 20.7 kb L14440 Intron nt before exon 2 GATTGATTAACT-TCCTTAATTTCC 0.8 kb L CTACGAGACTCT-TAAAACTTCGCT 0.7 kb L GATACCGGACCT-TAGTGAATATAA 1.5 kb L TGACAAGATTGT-TGAAAGCCTTAG 0.6 kb L (4b) 7 nt after exon 5 TTAGGTGTGTAA-ACAGACATTTTT 1.0 kb L (5) AAGGTTCTCCGG-AAGAAACGGCGT 7.2 kb L (5b) GCTCATTCTCTT-ACAACAACAAAT 5.5 kb L (6) ; reverse CTGAGTGTGCAG-AAGTTCTTCCTG 3.9 kb L (7) 16 nt after exon 9 CTAAGTTTGTCT-TGTTTATTCTCA 1.7 kb L (8) ; reverse CGTATTATAACT-GGCATCATAATC 0.8 kb L (9) GAGATGATGACA-CGTTCTCCAGTT 4.7 kb L (10) AGGTGACTCTGA-GTGAAGGTCCTC 0.3 kb L (11) 2 nt after exon 13 AGCCCTGAGGTA-AGGGTTGCAATT 0.3 kb L (12) ; reverse CAACAAGCACCA-TCCTCTGTAGTC 0.6 kb No Probe 15 (13) L (14) CAGACTTGGTCA-GTCAAATGGACC 0.9 kb L14494 Intron nt before exon (14) 17; reverse TTTCACATATGT-TAGAGTTTCTAC 1.1 kb L (16) 203 nt after exon 18; reverse TGCCAGTATGTA-TTGTACCATGTG 1.2 kb L (17) CTTTTGGAAAAT-GGTACGAGAGTC 1.0 kb L (18) TTGAAACTGAAT-GGAAGAATAACT 0.7 kb L (19) 37 nt before exon 21 AATTTACTGTCT-TATGGAAATCTT 6.2 kb L (20) TCATGTGATTGA-AAACATCTACCT 2.2 kb L (21) ACAAGAAGAATT-TTCCCGCTTTAT 1.8 kb L (22) ACAGCAATGGGA-TGCAGCTATTTA 0.6 kb L (23) AACATGGTGGGT-CCAGACTGGAAA 3.4 kb L (24) CCCAGCTTATCT-TGCAAGTGATGA 1.7 kb L (25) 289 nt before exon 27; reverse AGTAGACAGGGT-AGGTATTACCTT 1.7 kb L (26) GTGGTCTTGTTT-TGGCGTATACAG 0.8 kb 283 Ж SP and TCTTTAAACAGT-34 nt spanning 29 (27) L oligo-gaagattttgct 13.4 kb L14701 Intron nt before (27) exon 30; reverse AAGAGGTAGGCA-TCATTTATCACC 11.7 kb L (28) 103 nt after exon 30 CTTGTCACTTAT-GCTGTAATTTCA 2.5 kb L (29) CTTTAACCATCA-GCTGCCTCTCGA stop codon (ex30) This probe detects the wild type allele of the c.2708delttag polymorphism. The presence of the polymorphisms will result in a decreased probe signal. Ж This probe consists of three parts and has two ligation sites. Intron probe. Included to facilitate determination of the extend of a deletion / duplication. The NM_ sequence represents transcript variant 8 and is a reference standard in the NCBI RefSeqGene project. Note: The OPA1 exon numbering has changed. From description version 10 onwards, we have adopted the NCBI exon numbering that is present in the NM_ sequences for the OPA1 gene. This exon numbering might be different as compared to literature! The exon numbering used in previous versions of this product description can be found between brackets in Table 2. Please notify us of any mistakes. Complete probe sequences are available on request: info@mlpa.com. SALSA P229 OPA1 probemix Page 4 of 5

5 SALSA MLPA probemix P229-B2 OPA1 sample picture D y e S i g n a l Size (nt) Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P229-B2 OPA1 (lot B2-0412). Implemented Changes compared to the previous product description version(s). Version January 2015 (54) - Exon number of 283 nt probe in Table 1 corrected. - Data analysis method has been modified: peak areas changed to peak heights. Version 13 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 12 (48) - This product description has been changed to incorporate a new version (version number changed, major changes in Table 1 and Table 2, new picture included). Version 11 (46) - Remark on RefSeqGene standard and transcript variant added below Table 2. - Small correction of chromosomal locations in Table 1. Version 10 (45) - Warning added below Table 1 and 2 that the exon numbering of the OPA1 gene has changed. - Ligation sites updated according to new version of the NM_reference sequence. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. - New references added on page 1. - Small changes of distances to next probe in Table 2. Version 9 (45) - Minor textual changes on page 1 and the data analysis section has been modified. - Exon numbers changed in Table 1: exon 2, exon 15 -> intron 14, exon 2 -> intron 1. Version 8 (44) - This product description has been changed to incorporate a new version (version number changed, major changes in Table 1 and Table 2, new picture included). - Minor textual changes on page 1. SALSA P229 OPA1 probemix Page 5 of 5

MRC-Holland MLPA. Description version 09; 25 April 2017

MRC-Holland MLPA. Description version 09; 25 April 2017 SALSA MLPA probemix P143-C2 MFN2-MPZ Lot C2-0317. As compared to version C1-0813, one reference probe has been removed and two replaced, in addition several probe lengths have been adjusted. This P143