MRC-Holland MLPA. Description version 09; 31 March 2016

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1 SALSA MLPA probemix P071-B1 LMNB1-PLP1-NOTCH3 Lot B Compared to previous lot A2-0112, three reference probes have been replaced, and the NOTCH3 exon 22 probe has been removed. Leukodystrophies are a heterogeneous group of rare hereditary disorders affecting the central nervous system. They are characterised by the imperfect development or maintenance of the white matter (myelin sheath covering nerve fibers in the brain). The age of onset is typically during infancy or childhood as in the case of Pelizaeus-Merzbacher disease (PMD) and leukoencephalopathy. Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive, neurological disorder characterized by symmetrical widespread myelin loss in the CNS. The ADLD phenotype is in some respects similar to that of chronic progressive multiple sclerosis (MS). Autosomal dominant hereditary leukodystrophy has been associated with duplications of the nuclear lamin protein lamin B1 gene (LMNB1). Hereditary leukodystrophies are also associated with the PMD disease. PMD is caused by mutations in, or a duplication of the myelin proteolipid protein (PLP1) gene. Mutations in the NOTCH3 gene are a cause for cerebral arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). The P071-B1 LMNB1-PLP1-NOTCH3 probemix contains probes for each of the 11 exons (2 probes for exon 1) of the LMNB1 gene which spans 60 kb of genomic DNA on chromosome 5q23.2. In addition, the probemix contains probes for each of the 8 coding exons of the PLP1 gene (except for exon 1), which spans 16 kb on chromosome Xq22.2. Three probes are included for the NOTCH3 gene which spans 41 kb on chromosome 19p13.12, comprising 33 exons. Furthermore, four probes are included in this probemix flanking PLP1. Finally, the P071-B1 probemix contains 10 reference probes 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 autosomal probe recognition sequences should give a 35-50% reduced relative peak height of the amplification product of that probe. Deletions of a probe s recognition sequence on the X-chromosome will lead to a complete absence of the corresponding probe amplification product in males, whereas female heterozygotes are recognizable by a 35-50% reduction in relative peak height. 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 SALSA probemix P071 LMNB1-PLP1-NOTCH3 Page 1 of 6

2 Related SALSA probemixes P022 PLP1: Contains probes for the PLP1 gene, involved in Pelizaeus-Merzbacher disease. Reference for SALSA probemix P071 Brussino et al., A family with autosomal dominant leukodystrophy linked to 5q23.2-q23.3 without lamin B1 mutations. Eur J Neurol. 17: Data analysis The P071-B1 probemix contains 36 probes with amplification products between 122 and 415 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 be normalised intra-sample by dividing the peak height of each amplification product by the total peak height of only the reference probes in the 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 that no changes occurred in the genomic regions targeted by the reference probes. It is strongly recommended to use reference and patient samples of the same sex to minimize variation, as intersex comparison makes analysis more difficult. Sex determination can also be done by visual examination of the electropherogram. 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 P071 LMNB1-PLP1-NOTCH3 Page 2 of 6

3 Table 1. SALSA MLPA probemix P071-B1 LMNB1-PLP1-NOTCH3 probemix Chromosomal position SALSA MLPA probe reference LMNB1 NOTCH3 PLP 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 122 Reference probe L q ± AR probe L00423 Xq Reference probe L q PLP1 probe L11144 Exon PLP1 probe L11199 Exon PLP1 probe L00828 Exon Reference probe L q * Reference probe L p LMNB1 probe L09539 Exon LMNB1 probe L09542 Exon LMNB1 probe L10868 Exon ± NOTCH3 probe L10869 Exon LMNB1 probe L09541 Exon ± NOTCH3 probe L11200 Exon LMNB1 probe L09535 Exon PLP1 probe L11519 Exon FRMD7 probe L22041 Xq LMNB1 probe L09540 Exon LMNB1 probe L09543 Exon PLP1 probe L11204 Exon LMNB1 probe L11205 Exon Reference probe L q LMNB1 probe L09544 Exon LMNB1 probe L09534 Exon ± NOTCH3 probe L09547 Exon LMNB1 probe L09537 Exon LMNB1 probe L09545 Exon Reference probe L q * Reference probe L p Reference probe L q PLP1 probe L00830 Exon PLP1 probe L00965 Exon PAK3 probe L03178 Xq HNRNPH2 probe L04554 Xq * Reference probe L q Reference probe L q26 * New in version B1 (from lot B onwards). Changed in version B1 (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 can be due to incomplete sample DNA denaturation, e.g. due to the presence of salt in the sample DNA. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. Note: Exon numbering might be different as compared to literature! Please notify us of any mistakes. The identity of the genes detected by the control probes as well as the complete probe sequences are available on request:: info@mlpa.com. SALSA probemix P071 LMNB1-PLP1-NOTCH3 Page 3 of 6

4 Table 2. P071 probes arranged according to chromosomal location Table 2a. LMNB1 SALSA MLPA probe LMNB1 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) L09534 Exon CAAAGTGCTGCG-AGCAGGAGACGG 0.7 kb L09535 Exon CAAGCTGCAGAT-CGAGCTGGGCAA 27.1 kb L11205 Exon CTGCACTTGGTG-ACAAAAAAAGTT 0.7 kb L09537 Exon AAAAAACAGTTA-GCAGATGAAACT 4.6 kb L10868 Exon CGCGCTTGGTAG-AGGTGGATTCTG 1.6 kb L09539 Exon ATGAATACTTCT-ACTGTCAACAGT 7.2 kb L09540 Exon CAGACAAAGAGA-GAGAGATGGCGG 2.0 kb L09541 Exon GAGGCGAGTAGT-AGTGTTAGCATC 1.8 kb L09542 Exon AAATATACCTCA-AGATATGTGCTG 3.2 kb L09543 Exon GTGAAGGTTATA-TTGAAAAATTCT 6.7 kb L09544 Exon GGAGGAGGAGGA-AGAAGCAGCTGG 4.1 kb L09545 Exon AGCACTCTGGAT-GATGGATTCCAC stop codon (ex 11) The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2b. NOTCH3 SALSA MLPA probe NOTCH3 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (ex 1) 206 ± L10869 Exon AATGGAGGTCGT-TGCACCCAGCTG 8.1 kb 310 ± L09547 Exon GATGACGCCTGT-GTCAGCAACCCC 19.3 kb 220 ± L11200 Exon CAGACTGGATGG-ACACAGAGTGCC stop codon (ex 33) ± 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. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. Table 2c. PLP1 SALSA MLPA probe PLP1 Exon Ligation site NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe 129 ± L00423 AR CATGCAACTCCT-TCAGCAACAGCA kb L04554 HNRNPH2 GTCAGAGCAGTA-TGAGTGGATATG kb start codon (ex 2) No probe Exon L11199 Exon AGCCACAAAGCA-GACTAGCCAGCC 8.7 kb L11519 Exon ACTGGATTGTGT-TTCTTTGGGGT 1.0 kb L11204 Exon CATCAAGCTCAT-TCTTTGGAGCGG 1.2 kb L11144 Exon TGCCTGTGTACA-TTTACTTCAACA 0.6 kb L00828 Exon TTCCCTGGCAAG-GTTTGTGGCTCC 0.9 kb L00965 Exon GTTTATTGCTGC-ATTTGTGGGGGC 1.2 kb L00830 Exon GCCACTTACAAC-TTTGCCGTCCTT kb stop codon (ex 8) L03178 PAK3 CGGGATTCTTCA-GCACTCAACCAC kb L22041 FRMD7 ATTCTGCAGCCA-TTCTGGAAATAA ± 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. Flanking probe. Included to facilitate the determination of the extent of a deletion/duplication. Copy number alterations of flanking and reference probes are unlikely to be related to the condition tested. The NM_ sequence is a reference standard in the NCBI RefSeqGene project. SALSA probemix P071 LMNB1-PLP1-NOTCH3 Page 4 of 6

5 SALSA MLPA probemix P071-B1 LMNB1-PLP1-NOTCH3 sample pictures Figure 1. Capillary electrophoresis pattern of a sample of approximately 50 ng human male control DNA analysed with SALSA probemix P071-B1 LMNB1-PLP1-NOTCH3 (lot B1-0316). Figure 2. Capillary electrophoresis pattern of a sample of approximately 50 ng human female control DNA analysed with SALSA probemix P071-B1 LMNB1-PLP1-NOTCH3 (lot B1-0316). SALSA probemix P071 LMNB1-PLP1-NOTCH3 Page 5 of 6

6 Implemented Changes compared to the previous product description version(s). Version 09 (55) - 31 March 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). - Ligation sites in PLP1 gene adjusted. - Leukodystrophy removed from probemix name. Version 08 (53) - Various textual and lay-out changes. - Updated link for Database of genomic variants. - Peak area replaced with peak height. Version 07 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 06 (48) - Various minor textual changes. Version 05 (48) - Ligation sites of the probes targeting the PLP1 gene updated according to new version of the NM_reference sequence. - Various minor textual changes. - Remark on RefSeqGene standard and transcript variant added below Table 2. - Small correction of chromosomal locations in Table 1 and 2. Version 04 (46) - Warning added in Table 1, 129 nt probe L Related probemixes section added. - Exon numbering of the PLP1 gene has been changed on page 3 and 4. - Ligation sites of the probes targeting the LMNB1 gene updated according to new version of the NM_reference sequence. - Partial probe sequences extended to 24 nt. - 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 changes on page 1. Version 03 (44) - Various minor textual changes on page 1. - Minor changes in the data analysis section on page 2. - Tables have been numbered. SALSA probemix P071 LMNB1-PLP1-NOTCH3 Page 6 of 6