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

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1 SALSA MLPA probemix P143-C2 MFN2-MPZ Lot C 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 MFN2-MPZ probemix can be used to detect copy number changes in the MFN2 gene resulting in Charcot-Marie-Tooth disease (CMT) type 2A and the MPZ gene resulting in CMT type 1B. Charcot-Marie- Tooth disease constitutes a clinically and genetically heterogeneous group of hereditary motor and sensory neuropathies, affecting approximately 1 in every 2500 individuals. On the basis of electrophysiological criteria, CMT is divided into 2 major types. Type 1, the demyelinating form is characterised by a slow motor median nerve conduction velocity. Type 2, the axonal form, has normal or slightly reduced nerve conduction velocity. The MFN2 gene (19 exons) spans ~33 kb of genomic DNA and is located on 1p36.22, 12 Mb from the p- telomere. Mitofusins, such as MFN2, mediate the fusion of mitochondria and thereby contribute to the dynamic balance between fusion and fission that determines mitochondrial morphology. Mutations in MFN2 have been detected in affected members of several families with Charcot-Marie-Tooth disease type 2A (CMT2A). The Myelin protein zero (MPZ) gene (6 exons) spans ~5 kb of genomic DNA and is located on 1q23.3, ~161 Mb from the p-telomere. Myelin protein zero is the major structural protein of peripheral myelin. Mutations in the MPZ gene are associated with the autosomal dominant form of Charcot-Marie-Tooth disease type 1 (CMT1B) which is characterised by progressive slowing of nerve conduction and hypertrophy of Schwann cells. Mutations in MPZ can also produce the more severe polyneuropathies, Dejerine-Sottas syndrome (DSS) and congenital hypomyelinating neuropathy (CHN), as well as several types of axonal CMT2. The P143-C2 probemix contains one probe for each exon of the MFN2 gene and two probes for exon 3. Furthermore it contains one probe for each exon of the MPZ gene and two probes for exon 1. Two probes upstream of MFN2, recognising the PLOD1 gene, are included. Please note that in Ehlers-Danlos syndrome VIA (kyphoscoliotic type) duplications of PLOD1 exons are found (Giunta et al., 2005). 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(s) 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 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 P143 MFN2-MPZ probemix Page 1 of 5

2 Related SALSA probemixes P033 CMT1/HNPP region: Charcot-Marie-Tooth disease genes included: PMP22, COX, and TEKT3. P129 GJB1: X-linked Charcot-Marie-Tooth disease gene included: GJB1. P405 CMT1: Charcot-Marie-Tooth disease (CMT) and hereditary neuropathy with liability to pressure palsies (HNPP) genes included: MPZ, GJB1, 17p12 region (including PMP22). References Carr, AS et al., MFN2 deletion of exons 7 and 8: founder mutation in the UK population. J Peripher Nerv Syst 20.2: Høyer, H et al., Copy number variations in a population-based study of Charcot-Marie-Tooth disease. BioMed Res Int Ostern, R et al., Diagnostic laboratory testing for Charcot Marie Tooth disease (CMT): the spectrum of gene defects in Norwegian patients with CMT and its implications for future genetic test strategies. BMC Med Genet. 14:94. Sivera, R et al., Charcot-Marie-Tooth disease: Genetic and clinical spectrum in a Spanish clinical series. Neurology 81: Polke, JM et al., Recessive axonal Charcot-Marie-Tooth disease due to compound heterozygous mitofusin 2 mutations. Neurology 77: Data analysis The P143-C2 MFN2-MPZ probemix contains 38 MLPA probes with amplification products between 137 and 427 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 P143 MFN2-MPZ probemix Page 2 of 5

3 Table 1. SALSA MLPA P143-C2 MFN2-MPZ probemix Chromosomal position SALSA MLPA probe Reference MFN2 MPZ 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 137 Reference probe L p Reference probe L q «MFN2 probe L05353 Exon MPZ probe L04279 Exon * Reference probe L p MFN2 probe L04261 Exon «MFN2 probe L04271 Exon MPZ probe L04280 Exon MFN2 probe L29924 Exon MFN2 probe L05581 Exon «MFN2 probe L04272 Exon MPZ probe L04281 Exon * Reference probe L q MFN2 probe L04263 Exon «MFN2 probe L29925 Exon MPZ probe L29923 Exon MPZ probe L08284 Exon MFN2 probe L29700 Exon Reference probe L q «MFN2 probe L05354 Exon MPZ probe L04283 Exon Reference probe L q MFN2 probe L04265 Exon «MFN2 probe L04275 Exon MPZ probe L04284 Exon PLOD1 probe L kb upstream of MFN2 320 MFN2 probe L04266 Exon «MFN2 probe L04276 Exon PLOD1 probe L kb upstream of MFN2 346 MFN2 probe L04267 Exon «MFN2 probe L04277 Exon Reference probe L q MFN2 probe L04268 Exon «MFN2 probe L04278 Exon Reference probe L p «MFN2 probe L24212 Exon MFN2 probe L29926 Exon Reference probe L p14 * 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. 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 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 P143 MFN2-MPZ probemix Page 3 of 5

4 Table 2. P143 probes arranged according to chromosomal location Table 2a. MFN2 probes SALSA MLPA probe L L04064 MFN2 Exon PLOD1 gene Exon 10 PLOD1 gene Exon 16 Ligation site in NM_ NM_ NM_ Partial sequence (24 nt adjacent to ligation site) GCATGGCAGCGA-GTACCAGTCTGT CCATCTTCACGG-AGGTGGCCTGTG Distance to next probe 6.3 kb 13.2 kb start codon (exon 3) L29700 Exon GAGTCCGAGCCT-CTGCGTCGTCCG 1.8 kb L04261 Exon CAGTCAATCAAT-AGCCAACCTCAA 7.2 kb L29924 Exon TCTCTCGATGCA-ACTCTATCGTCA 0.1 kb L29926 Exon reverse GGTGGCGCTCTC-CTGGATGTAGGC 3.3 kb L04263 Exon GACGTCAAAGGT-TACCTATCCAAA 3.6 kb L05581 Exon GCACCGTGATCA-ATGCCATGCTCT 1.2 kb L04265 Exon GCCCAACTCTAA-GTGCCCACTTCT 1.4 kb L04266 Exon ACAGAGCTGGAC-AGCTGGATTGAC 0.3 kb L04267 Exon ACAACCGCTGGG-ATGCATCTGCCT 2.4 kb L04268 Exon TTCTTTGTGTCT-GCTAAGGAGGTG 0.3 kb 410 «20882-L24212 Exon CGCAGAAGGCTT-TCAAGTGAGGAT 0.3 kb 148 «04886-L05353 Exon GAGGCGGTTCGA-CTCATCATGGAC 2.0 kb 172 «04887-L04271 Exon GCTCAAGACTAT-AAGCTGCGAATT 0.5 kb 196 «04888-L04272 Exon CTGGTGGACGAT-TACCAGATGGAC 0.3 kb 220 «04889-L29925 Exon CACATAGAGGAA-GGACTGGGTCGA 1.0 kb 256 «04890-L05354 Exon ATGCTGGTGAAT-AGGTTCCTGGGC 0.7 kb 292 «04891-L04275 Exon CAGGGCTCGCTC-ACCCAGGAGGAG 0.6 kb 328 «04892-L04276 Exon AGCTTGTCATCA-GCTACACTGGCT 2.5 kb 355 «04893-L04277 Exon GGAGCAGGAAAT-TGCCGCCATGAA 1.9 kb 382 «04894-L04278 Exon AGCTCAACATGT-TCACACACCAGT stop codon (exon 19) «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 probes are unlikely to be related to the condition tested. The NM_ sequence (MFN2) represents transcript variant 1 and is a reference standard in the NCBI RefSeqGene project. The NM_ sequence (PLOD1) is a reference standard in the NCBI RefSeqGene project. Table 2b. MPZ probes SALSA MLPA probe MPZ Exon Ligation site in NM_ Partial sequence (24 nt adjacent to ligation site) Distance to next probe start codon (exon 1) L29923 Exon 1 81 nt before exon 1 CTGCACATGCCA-GGCTGCAATTGG 0.2 kb L04279 Exon CTCCCTCATCCA-GCCCCAGCCCTA 2.6 kb L04280 Exon TGCACTGCTCCT-TCTGGTCCAGTG 0.5 kb L04281 Exon CCCTCGCTGGAA-GGATGGCTCCAT 0.4 kb L08284 Exon CGGGGTCGTTCT-GGGAGCTGTGAT 0.3 kb L04283 Exon TTGCACAAGCCA-GGAAAGGACGCG 0.2 kb L04284 Exon ATGCAATGCTGG-ACCACAGCAGAA stop codon (exon 6) The NM_ sequence represents transcript variant 1. 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 P143 MFN2-MPZ probemix Page 4 of 5

5 SALSA MLPA probemix P143-C2 MFN2-MPZ sample picture Figure 1. Capillary electrophoresis pattern from a sample of approximately 50 ng human male control DNA analysed with SALSA MLPA probemix P143-C2 MFN2-MPZ (lot C2-0317). Implemented Changes compared to the previous product description version(s). Version April 2017 (55) - Various minor textual changes on pages 1 and 2. - New references added on page 1. - Small changes of probe lengths in Table 1 and 2 in order to better reflect the true lengths of the amplification products. Version 08 (52) Version 07 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version 6 (47) - Ligation sites of the probes targeting the MPZ gene updated according to new version of the NM_reference sequence. - New reference added on page 1. - Various minor textual changes. - Various minor layout changes. - Remark on RefSeqGene standard and transcript variant added below Table 2. Version 5 (45) - Product name changed from CMT2A/1B to MFN2-MPZ - 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; tables have been numbered. - Data analysis section modified. Sentence when only small numbers of samples are tested, visual comparison of peak profiles should be sufficient removed from data analysis section SALSA P143 MFN2-MPZ probemix Page 5 of 5

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

MRC-Holland MLPA. Description version 14; 21 January 2015 SALSA MLPA probemix P229-B2 OPA1 Lot B2-0412. 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