Product Description SALSA MLPA probemix P033-B4 CMT1 To be used with the MLPA General Protocol.

Transcription

1 Product Description SALSA probemix P033-B4 CMT1 To be used with the MLPA General Protocol. Version B4. As compared to version B3, three reference probes have been replaced. For complete product history see page 8. Catalogue numbers: P R: SALSA probemix P033 CMT1, 25 reactions. P R: SALSA probemix P033 CMT1, 50 reactions. P R: SALSA probemix P033 CMT1, 100 reactions. To be used in combination with SALSA reagent kit EK1 (100 reactions) or EK5 (500 reactions). MLPA reagent kits are available with different fluorescent dyes: EK1-FAM, EK5-FAM (Applied Biosystems sequencers) and EK1-Cy5.0, EK5-Cy5.0 (Beckman sequencers). Certificate of Analysis: Information regarding storage conditions, quality tests, and a sample electropherogram from the current sales lot is available at Precautions and warnings: For professional use only. Always consult the most recent product description AND the MLPA General Protocol before use: It is the responsibility of the user to be aware of the latest scientific knowledge of the application before drawing any conclusions from findings generated with this product. Intended use: This SALSA probemix P033 CMT1 is an in vitro diagnostic (IVD) 1 or research use only (RUO) assay for the detection of copy number changes in the human PMP22 and surrounding genes located in the CMT/HNPP region on chromosome 17p12, and in the KIF1b gene on chromosome 1p36, as a potential cause and confirmation of diagnosis for Charcot-Marie-Tooth disease (CMT) or hereditary neuropathy with liability to pressure palsies (HNPP). This assay can be used with human DNA extracted from peripheral blood or saliva. In the majority of patients, defects in the CMT/HNPP region are duplications. However, CMT may also be caused by point mutations in the PMP22 gene, which are not detected by MLPA. The majority of CMT-related KIF1b defects are point mutations. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of these genes. Copy number changes detected by only a single probe always require validation by another method. This probemix is not intended to be used as a standalone assay for clinical decisions. The results of this test should be interpreted by a clinical molecular geneticist or equivalent. 1 Please note that this probemix is for In Vitro Diagnostic use (IVD) in the countries specified on the Certificate of Analysis, which is available at In all other countries, the product is for Research Use Only (RUO). Clinical background: Inherited peripheral neuropathies are among the most common genetic neuromuscular disorders worldwide. The most common form is Charcot-Marie-Tooth (CMT) disease. Prevalence of CMT and related disorders has been estimated between 1:2500 and 1:1214. Clinical symptoms of CMT include distal muscle weakness and atrophy, sensory loss, depressed tendon reflexes, and higharched feet (pes cavus). Up to now, more than 70 genes are known to be associated with the different types of CMT. The disease can be inherited in an autosomal dominant, autosomal recessive, or X-linked manner. CMT type 1 (CMT1) is a demyelinating peripheral neuropathy which is usually slowly progressive. Several subtypes exist. CMT1A accounts for ~70-80% of all CMT1 cases and this subtype is mainly caused by a ~1.5 Mb duplication on chromosome 17p, including the peripheral myelin protein 22 (PMP22) gene and flanking regions. The de novo rate of PMP22 duplications in CMT1 patients is ~10-20%. Furthermore, CMT1A can be caused by activating point mutations in this gene. Increased PMP22 gene dosage leads to altered nerve conduction velocity, which is the main cause of the clinical manifestations in CMT1A. SALSA Probemix P033 CMT1 Page 1 of 9

2 A reciprocal deletion of the PMP22 gene leads to hereditary neuropathy with liability to pressure palsies (HNPP), which is another subtype of CMT disease. However, the majority of individuals with the HNPP deletion probably remain undiagnosed due to the mild phenotype. CMT type 2 shows extensive clinical overlap with CMT1. However, CMT2 patients tend to be less disabled and have less sensory loss than CMT1 patients. Until now, >15 subtypes of CMT2 have been described, each involving a different gene or chromosomal locus. Haploinsufficiency for the KIF1b gene is suggested to be responsible for CMT type 2A1 (Zhao et al. 2001; Drew et al. 2015). Information was obtained from: Clinical Utility Gene Card: Gene Reviews: Gene structure and transcript variants: The PMP22 gene (5 exons) spans ~35.5 kb of genomic DNA and is located on chromosome 17p12, ~15.2 Mb from the p-telomere. An LRG sequence for this gene is available on Five protein coding transcript variants have been described ( Transcript variant 1 (NM_ , 1861 nt, coding sequence ) is the main transcript encoding the PMP22 protein. This sequence is a reference standard in the NCBI RefSeq Gene project. Transcript variants 2, 3, 4 and 5 differ in the 5 UTR sequence, but encode the same protein as transcript variant 1. The KIF1b gene (47 exons) spans ~167 kb on chromosome 1p36.22 and is located ~10 Mb from the p- telomere. An LRG sequence for this gene is available on Two protein coding transcript variants have been described ( Transcript variant 1 (NM_ , nt, coding sequence ) is the longest transcript and encodes the longest isoform. This sequence is a reference standard in the NCBI RefSeq Gene project. Exon numbering: The exon numbering for the PMP22 and KIF1b genes used in this P033-B4 CMT1 product description and in the P033-B4 CMT1 Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_ and NG_ reference sequences, respectively. These sequences are identical to the LRG sequences. P033-B4 probemix content: This SALSA MLPA probemix P033 CMT1 contains 38 MLPA probes with amplification products between 130 and 436 nt: 16 probes located in the CMT/HNPP region, 2 flanking probes, 2 probes in the KIF1b gene and 18 reference probes detecting autosomal chromosomes (Table 1 and Table 2). The identity of the genes detected by the reference probes is available online ( This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity Fragments (Q-fragments), three DNA Denaturation Fragments (D-fragments), and one chromosome X and one chromosome Y-specific fragment (Table 1). The Q-fragments are only visible when less than 100 ng sample DNA is used. Low signal of the 88 or 96 nt fragment indicates incomplete DNA denaturation. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol. MLPA technique: The principles of the MLPA technique (Schouten et al. 2002) are described in the MLPA General Protocol ( MLPA technique validation: Internal validation of the MLPA technique using 16 DNA samples from healthy individuals is required, in particular when using MLPA for the first time, or when changing the sample handling procedure, DNA extraction method or instruments used. This validation experiment should result in a standard deviation <0.10 for all probes over the experiment. Required specimens: Purified DNA from peripheral blood or saliva, free from impurities known to affect MLPA reactions. For more information please refer to the section on DNA sample treatment found in the MLPA General Protocol. SALSA Probemix P033 CMT1 Page 2 of 9

3 Reference samples: Reference DNA samples should be derived from the same tissue type, handled using the same procedure, and prepared using the same DNA extraction method as the patient samples. Reference samples should be derived from unrelated individuals who are from families without a history of Charcot-Marie-Tooth disease. More information regarding the selection and use of reference samples can be found in the MLPA General Protocol. Positive control DNA samples: cannot provide positive DNA samples. Inclusion of a positive sample in each experiment is recommended. Performance characteristics: In approximately two-third of all patients with a CMT phenotype, the molecular cause has been established. More than 90% of CMT cases in which a molecular diagnosis has been reached are associated with changes in four genes (PMP22, GJB1, MFN2 and MPZ). About 30-50% of all diagnosed CMT/HNPP patients have a rearrangement in the PMP22 gene. In some distinct populations, the percentage of PMP22 rearrangements among diagnosed CMT patients is ~65%. Rare cases of CMT type 2A caused by a mutation in the KIF1b gene have been described. So far, copy number changes involving this gene have not been reported. Performance can be compromised by: SNPs or other polymorphisms (e.g. indels) in the DNA target sequence, impurities in the DNA sample, incomplete DNA denaturation, the use of insufficient or too much sample DNA, the use of insufficient or unsuitable reference samples, problems with capillary electrophoresis or a poor data normalisation procedure and other technical errors. The MLPA General Protocol contains technical guidelines and information on data evaluation/normalisation. Data analysis: Coffalyser.Net software must be used for data analysis in combination with the appropriate lot-specific MLPA Coffalyser sheet. For both, the latest version should be used which are freely downloadable at Use of other non-proprietary software may lead to inconclusive or false results. The analysis method used for P033-B4 is block normalisation. Normalisation of results should be performed within one experiment. For more details on MLPA quality control and data analysis, see the Coffalyser.Net Manual. Interpretation of results: The expected results for the PMP22- and KIF1b-specific probes are allele copy numbers of 2 (normal), 1 (heterozygous deletion) or 3 (heterozygous duplication). In rare cases, copy numbers of 0 (homozygous deletion) or 4 (heterozygous triplication/homozygous duplication) may be obtained. The standard deviation of all probes in the reference samples should be <0.10 and the dosage quotient (DQ) of the reference probes in the patient samples should be between 0.80 and When these criteria are fulfilled, the following cut-off values for the DQ of the probes can be used to interpret MLPA results: Copy Number status Dosage quotient Normal 0.80 < DQ < 1.20 Homozygous deletion DQ = 0 Heterozygous deletion 0.40 < DQ < 0.65 Heterozygous duplication 1.30 < DQ < 1.65 Heterozygous triplication/ Homozygous duplication 1.75 < DQ < 2.15 Ambiguous copy number All other values - Arranging probes according to chromosomal location facilitates interpretation of the results and may reveal more subtle changes such as those observed in mosaic cases. Analysis of parental samples may be necessary for correct interpretation of complex results. - False positive results: Please note that abnormalities detected by a single probe (or multiple consecutive probes) still have a considerable chance of being a false positive result. Incomplete DNA denaturation (e.g. due to salt contamination) can lead to a decreased probe signal, in particular for probes located within a CpG island in or near the CMT/HNPP region or KIF1b gene. The use of an additional purification step or an alternative DNA extraction method may resolve such cases. SALSA Probemix P033 CMT1 Page 3 of 9

4 - False positive duplication results: Contamination of DNA samples with cdna or PCR amplicons of individual exons can lead to false positive duplication results (Varga et al. 2012). Analysis of an independently collected secondary DNA sample can exclude these kinds of contamination artefacts. - Normal copy number variation in healthy individuals is described in the database of genomic variants: Users should always consult the latest update of the database and scientific literature when interpreting their findings. - Not all abnormalities detected by MLPA are pathogenic. In some genes, intragenic deletions are known that result in very mild or no disease (Schwartz et al. 2007). For many genes, more than one transcript variant exists. Copy number changes of exons that are not present in all transcript variants may not have clinical significance. Duplications that include the first or last exon of a gene (e.g. exons 1-3) might in some cases not result in inactivation of that gene copy. - Copy number changes detected by reference probes are unlikely to have any relation to the condition tested for. Notes on results obtained with the P033 CMT1 probemix: - Several types and subtypes of Charcot-Marie-Tooth disease exist, each involving a different gene or locus. Furthermore, there is a large overlap in clinical phenotype between the different types of CMT. Other disorders with comparable clinical features have been described, including hereditary neuralgic amyotrophy (HNA), amyloid neuropathies, Krabbe disease and hereditary ataxias. Therefore, molecular diagnosis may be complicated for certain patients. - Please note that recurrent duplications have been described which can be detected by the two TEKT3 probes, but not by any of the PMP22 probes. These duplications may cause CMT through an unknown mechanism affecting PMP22 expression (Weterman et al. 2010; Zhang et al. 2010). Limitations of the procedure: - Point mutations in the CMT/HNPP region or KIF1b gene will not be detected by using SALSA MLPA probemix P033 CMT1. - MLPA cannot detect any changes that lie outside the target sequence of the probes and will not detect copy number neutral inversions or translocations. Even when MLPA did not detect any aberrations, the possibility remains that biological changes in that gene or chromosomal region do exist but remain undetected. - Sequence changes (e.g. SNPs, point mutations, small indels) in the target sequence detected by a probe can cause false positive results. Mutations/SNPs (even when >20 nt from the probe ligation site) can reduce the probe signal by preventing ligation of the probe oligonucleotides or by destabilising the binding of a probe oligonucleotide to the sample DNA. Confirmation of results: Copy number changes detected by only a single probe always require confirmation by another method. An apparent deletion detected by a single probe can be due to e.g. a mutation/polymorphism that prevents ligation or destabilises the binding of probe oligonucleotides to the DNA sample. Sequence analysis can establish whether mutations or polymorphisms are present in the probe target sequence. The finding of a heterozygous mutation or polymorphism indicates that two different alleles of the sequence are present in the sample DNA and that a false positive MLPA result was obtained. Copy number changes detected by one or more than one consecutive probe should be confirmed by another independent technique such as long range PCR, qpcr, array CGH or Southern blotting, whenever possible. Deletions/duplications of more than 50 kb in length can often be confirmed by FISH. Inherited Peripheral Neuropathies Mutation Database (IPNMDB): We strongly encourage users to deposit positive results in the IPNMDB database. Recommendations for the nomenclature to describe deletions/duplications of one or more exons can be found on Please report copy number changes detected by the reference probes, false positive results due to SNPs and unusual results (e.g., a duplication of PMP22 exons 2 and 4 but not exon 3) to : info@mlpa.com. SALSA Probemix P033 CMT1 Page 4 of 9

5 Table 1. SALSA probemix P033-B4 CMT1 probemix Length Chromosomal position (a) SALSA MLPA probe (nt) Reference KIF1b PMP22 / 17p Q-fragments (Only visible with <100 ng sample DNA) D-fragments (Low signal of 88 or 96 fragment indicates incomplete denaturation) 100 X-fragment (X chromosome specific) 105 Y-fragment (Y chromosome specific) 130 Reference probe L q Reference probe L q PMP22 probe L04039 Exon PMP22 probe L04461 Exon KIF1b probe L p36 exon «Reference probe L p PMP22 probe L04041 Exon PMP22 probe L04463 Exon Reference probe L q PMP22 probe L02158 Upstream PMP Reference probe L p TEKT3 probe L p12, within CMT1 region 211 Reference probe L q KIF1b probe L p36 exon PMP22 probe L00926 Exon PMP22 probe L04464 Exon Reference probe L q PMP22 probe L00927 Exon * Reference probe L q ± DRC3 probe L p11.2, outside the CMT1 region 283 Reference probe L p TEKT3 probe L p12, within CMT1 region 302 Reference probe L p PMP22 probe L01641 Exon Reference probe L q Reference probe L q PMP22 probe L00930 Exon ELAC2 probe L p12, outside the CMT1 region 355 PMP22 probe L02157 Upstream PMP Reference probe L q PMP22 probe L02156 Upstream PMP * Reference probe L q COX10 probe L p12, within CMT1 region 400 * Reference probe L q Reference probe L q COX10 probe L p12, within CMT1 region 427 Reference probe L q Reference probe L p15 (a) The exon numbering for the PMP22 and KIF1b genes used in this P033-B4 CMT1 product description and in the P033-B4 CMT1 Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_ and NG_ reference sequences, respectively. These sequences are identical to the LRG sequences. * New in version B4 (from lot B onwards). + Defects in the KIF1b gene have been implicated in Charcot-Marie-Tooth disease type 2A1. ± The name of the LRRC48 gene has been changed to DRC3; the sequence detected by this probe has not been changed. «Probe located within, or near a CpG island. A low signal can be caused by salt contamination in the DNA sample leading to incomplete DNA denaturation, especially of CG rich regions. Flanking probe. Included to help determine the extent of a deletion/duplication. Copy number alterations outside of the PMP22 gene are unlikely to be related to the condition tested. The 310 nt probe signal may be influenced by presence of the PMP22: c.260t>c sequence variant. The 202 nt probe signal may be influenced by presence of a 9 nt deletion including the ligation site. SALSA Probemix P033 CMT1 Page 5 of 9

6 Table 2. P033 probes arranged according to chromosomal location Table 2a. CMT/HNPP region Length (nt) SALSA MLPA probe L00917 ELAC L00924 COX L00925 COX10 Gene/ exon (a) Ligation site (b) Partial sequence (d) (24 nt adjacent to ligation site) NM_ ; TTGGTCCTGAAT-GAGAACTGTGCC 1231 Start of common chromosome 17p12 deletion/duplication region NM_ ; CTCCTGGCAGTT-TCCTCATTTCAA 1173 NM_ ; CCATCAATGCGT-ACATCTCCTACC 1376 Distance to next probe kb 0.2 kb kb PMP22 NM_ stop codon (exon 5) L04464 Exon ATCTATGTGATC-TTGCGGAAACGC 0.1 kb L00930 Exon CTCAACTCGGAT-TACTCCTACGGT 8.5 kb L01641 Exon CTTCTGCCAACT-CTTCACCCTCAC 0.1 kb L04463 Exon reverse TGGTGGCCTGGA-CAGACTGCAGCC 19.5 kb L04041 Exon TCCACCACTGTT-TCTCATCATCAC 0.1 kb L04461 Exon reverse GAGATCAGTTGC-GTGTCCATTGCC 1.5 kb L00927 Exon TGTTGCTGAGTA-TCATCGTCCTCC 4.5 kb start codon (exon 2) L00926 Exon TTAACATCCCTT-GCATTTGGCTGC 0.1 kb L04039 Exon ACCACCAGGGAA-CATCTCGGGGAG 2.3 kb L02157 Upstream 2.2 kb before PMP22 GGTGCTAGAAAT-AGCCAGTCTCAT 4.2 kb L02156 Upstream 6.4 kb before PMP22 GCCTCCATGGTT-AGAGACTAGAAT 5.9 kb L02158 Upstream 12.4 kb before PMP22 TGAAGAGCCCTT-GGATACGGAAGG 26.4 kb L00921 TEKT3 NM_ ; TACACGAGGTTG-ACGACACCATCC 27.3 kb L02155 TEKT3 NM_ ; CCCACTCCAATT-TGACCCATAGCC kb End of common chromosome 17p12 deletion/duplication region L00936 DRC3 NM_ ; CGGATCTCCAAG-ATCGACTCCCTG Table 2b. KIF1b gene Length (nt) SALSA MLPA probe KIF1b exon (a) Ligation site (c) NM_ Partial sequence (d) (24 nt adjacent to ligation site) Distance to next probe start codon (exon 2) L04462 Exon CTCAGTGAAGGT-GGCTGTCCGGGT kb L04060 Exon CGTGGGGTCCTT-TTGCAGGCCCTC stop codon (exon 47) (a) The exon numbering for the PMP22 and KIF1b genes used in this P033-B4 CMT1 product description and in the P033-B4 CMT1 Coffalyser.Net analysis sheet is the exon numbering from the Genbank NG_ and NG_ reference sequences, respectively. These sequences are identical to the LRG sequences. (b) Ligation sites of the P033 CMT1 MLPA probes in the PMP22 gene are indicated according to RefSeq sequence NM_ containing 5 exons. (c) Ligation sites of the P033 CMT1 MLPA probes in the KIF1b gene are indicated according to RefSeq sequence NM_ containing 47 exons. (d) Only partial probe sequences are shown. Complete probe sequences are available at Please notify us of any mistakes: info@mlpa.com. Flanking probe. Included to help determine the extent of a deletion/duplication. Copy number alterations outside of the PMP22 gene are unlikely to be related to the condition tested. The 310 nt probe signal may be influenced by presence of the PMP22: c.260t>c sequence variant. The 202 nt probe signal may be influenced by presence of a 9 nt deletion including the ligation site. SALSA Probemix P033 CMT1 Page 6 of 9

7 Related SALSA probemixes P129 GJB1: Contains probes for the GJB1 gene, involved in X-linked CMT. P143 MFN2-MPZ: Contains probes for the MFN2 and MPZ genes, involved in CMT type 2A and 1B. P307 SEPT9: Contains probes for almost all exons of the SEPT9 gene involved in Hereditary neuralgic amyotrophy (HNA). P369 Smith Magenis syndrome: Contains 21 probes in the 17p11.2 Smith-Magenis region, which is immediately adjacent to the CMT/HNPP chromosomal region. P405 CMT1: Contains probes for the CMT/HNPP region, as well as probes for the GJB1 and MPZ genes. References Drew AP et al. (2015). Improved inherited peripheral neuropathy genetic diagnosis by whole-exome sequencing. Mol Genet Genomic Med. 3(2): Schouten JP et al. (2002). Relative quantification of 40 nucleic acid sequences by multiplex ligationdependent probe amplification. Nucleic Acids Res. 30:e57. Schwartz M et al. (2007). Deletion of exon 16 of the dystrophin gene is not associated with disease. Hum Mutat. 28:205. Varga RE et al. (2012). MLPA-based evidence for sequence gain: pitfalls in confirmation and necessity for exclusion of false positives. Anal Biochem. 421: Weterman MA et al. (2010). Copy number variation upstream of PMP22 in Charcot-Marie-Tooth disease. Eur J Hum Genet. 18(4): Zhang F et al. (2010). Mechanisms for Nonrecurrent Genomic Rearrangements Associated with CMT1A or HNPP: Rare CNVs as a Cause for Missing Heritability. Am J Hum Genet. 86: Zhao C et al. (2001). Charcot-Marie-Tooth disease type 2A caused by mutation in a microtubule motor KIF1Bbeta. Cell. 105(5): Selected publications using SALSA Probemix P033 CMT1 Bergamin G et al. (2014). Mutation analysis of MFN2, GJB1, MPZ and PMP22 in Italian patients with axonal charcot-marie-tooth disease. Neuromol Med. 16(3): Brown SB and Bunyan DJ. (2014) Point Mutation Analysis of PMP22 in Patients Referred for Hereditary Neuropathy with Liability to Pressure Palsies. Open J Genet. 4(6): Casasnovas C et al. (2012). A novel small deletion in PMP22 causes a mild hereditary neuropathy with liability to pressure palsies phenotype. Muscle Nerve. 45(1): Cho SM et al. (2014) Partial Gene Deletions of PMP22 Causing Hereditary Neuropathy with Liability to Pressure Palsies. Case Rep Genet. 2014: DiVincenzo C et al. (2014). The allelic spectrum of Charcot-Marie-Tooth disease in over 17,000 individuals with neuropathy. Mol Genet Genomic Med. 2(6): Hung CC et al. (2008). Identification of deletion and duplication genotypes of the PMP22 gene using PCR- RFLP, competitive multiplex PCR, and multiplex ligation-dependent probe amplification: a comparison. Electrophoresis. 29(3): Matejas V et al. (2006) Identification of Alu elements mediating a partial PMP22 deletion. Neurogenetics. 7(2): Miltenberger-Miltenyi G et al. (2009) Identification and in silico analysis of 14 novel GJB1, MPZ and PMP22 gene mutations. Eur J Hum Genet. 17(9): Park CH et al. (2014). Molecular characterization of near-complete trisomy 17p syndrome from inverted duplication in association with cryptic deletion of 17pter. Gene. 537(2): Rudnik-Schoneborn S et al. (2015) Diagnostic algorithms in Charcot-Marie-Tooth neuropathies: experiences from a German genetic laboratory on the basis of 1206 index patients. Clin Genet Shahrizaila N et al. (2014). X-linked Charcot-Marie-Tooth disease predominates in a cohort of multiethnic Malaysian patients. Muscle Nerve. 49(2): Slater H et al. (2004) Improved testing for CMT1A and HNPP using multiplex ligation-dependent probe amplification (MLPA) with rapid DNA preparations: comparison with the interphase FISH method. Hum Mutat. 24(2): Stangler Herodež S et al. (2009) Molecular diagnosis of PMP22 gene duplications and deletions: comparison of different methods. J Int Med Res. 37(5): Stangler Herodež S et al. (2005) MLPA method for PMP22 gene analysis. Acta Chim Slov. 52: Zhan Y et al. (2015). PMP22-Related neuropathies and other clinical manifestations in Chinese han patients with charcot-marie-tooth disease type 1. Muscle Nerve. 52(1): SALSA Probemix P033 CMT1 Page 7 of 9

8 P033 Product history Version Sold from Modification B Three reference probes have been replaced. B Three reference probes and the 88 and 96 nt control fragments have been replaced. B Two reference probes at 283 and 301 nt have been replaced. In addition, four extra control fragments have been added at nt. B Probemix has been completely redesigned. Several reference and target probes have been replaced, and more target probes in the CMT/HNPP region have been added. A First release. Implemented changes in the product description Version B June 2016 (02) - Product description restructured and adapted to a new template. - Various minor textual and layout changes. - Ligation sites of the probes targeting the PMP22 gene updated according to new version of the NM_ reference sequence. - Small changes of probe lengths in Table 1 in order to better reflect the true lengths of the amplification products. - The name of the LRRC48 gene (detected by the 274 nt probe) has been changed to DRC3. Version September 2015 (55) - Product description adapted to a new version (lot number added, small changes in Table 1 and Table 2a and 2b, new picture included). - Table 2b for KIF1b gene added. Version March 2015 (54) - Warning on PMP22 exon numbering removed from table. - Picture with old MLPA buffer removed. - Extra references added. - Peak area replaced with peak height. - Updated link for Database of Genomic Variants. Version December 2012 (48) - Electropherogram pictures using the new MLPA buffer (introduced in December 2012) added. Version September 2012 (48) - Reference Berciano et al added on page 1. Version February 2012 (48) - Product description adapted to a new version (lot number added, small changes in Table 1 and Table 2, new picture included). - Various minor textual and layout changes. - Ligation sites and partial sequences have been added to table 2. - Remark on RefSeqGene standard and transcript variant added below Table 2. - Small correction of chromosomal locations in Table 1. - Data analysis method has been modified. SALSA Probemix P033 CMT1 Page 8 of 9

9 More information: bv; Willem Schoutenstraat DL, Amsterdam, The Netherlands info@mlpa.com (information & technical questions); order@mlpa.com (orders) Phone SALSA Probemix P033 CMT1 Page 9 of 9