Intended use: The SALSA MLPA probemix P033 CMT1 is an in vitro diagnostic (IVD)¹ or a research use only (RUO) assay for the detection of deletions or duplications in the human PMP22 and KIF1b genes in order to confirm a potential cause and clinical diagnosis for Charcot-Marie-Tooth disease (CMT). This assay can also be used for the detection of deletions in the human PMP22 gene in order to confirm a potential cause and clinical diagnosis for hereditary neuropathy with liability to pressure palsies (HNPP). This assay is for use with human DNA extracted from peripheral blood or saliva. This product can also be used for molecular genetic testing of at-risk family members.

Deletions or duplications obtained with the P033 CMT1 probemix must be confirmed by another technique. In particular, deletions or duplications detected by only a single probe always require validation by another method. Not all exons of KIF1b are covered. Some defects in PMP22 and all CMT-related defects in KIF1b are point mutations, none of which will be detected by MLPA. It is therefore recommended to use this SALSA MLPA probemix in combination with sequence analysis of PMP22 and KIF1b. This assay is not intended to be used as a standalone assay for clinical decisions. The results of this test must 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 at the end of this product description. 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 to be between 1:2500 and 1:1214. Clinical symptoms of CMT include distal muscle weakness and atrophy, sensory loss, depressed tendon reflexes and high-arched feet (pes cavus). At present, more than 80 genes are known to be associated with 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.
CMT type 2 shows extensive clinical overlap with CMT1. However, CMT2 patients tend to be less disabled and have milder sensory loss than CMT1 patients. At present, more than 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).

Another subtype of CMT disease is hereditary neuropathy with liability to pressure palsies (HNPP), which is characterized by repeated focal pressure neuropathies such as carpal tunnel syndrome and peroneal palsy with foot drop. PMP22 is the only gene known to be associated with HNPP. A contiguous gene deletion of chromosome 17p11.2 that includes PMP22 is present in approximately 80% of affected individuals; the remaining 20% have a pathogenic variant in PMP22. The prevalence of HNPP is estimated at two to five cases per 100.000 individuals. However, the majority of individuals with HNPP probably remain undiagnosed due to the mild phenotype.

More information on CMT and HNPP can be found on:
Clinical Utility Gene Card: http://www.nature.com/ejhg/journal/v18/n9/full/ejhg201075a.html
Gene Reviews: http://www.ncbi.nlm.nih.gov/books/NBK1358/,
https://www.ncbi.nlm.nih.gov/books/NBK1392/.

Table 1: Overview of the probemixes and genes related to CMT.

Probemix	Genes and coverage	Condition	Remarks*
P033	PMP22: all exons KIF1b: 2 probes	CMT1A and HNPP CMT2A1	PMP22 probes are all identical to P405, plus there is one exon 5 probe extra in P033.
P405	PMP22 MPZ GJB1	CMT1A and HNPP CMT1B CMTX1	PMP22 probes are all identical to P033, plus there is one exon 5 probe extra in P033. MPZ probes are all identical to P143. GJB1 probes are all identical to P129, plus there is one exon 2 probe extra in P129.
P143	MFN2 MPZ	CMT2A CMT1B	MPZ probes are all identical to P405.
P129	GJB1	CMTX1	GJB1 probes are all identical to P405, plus there is one exon 2 probe extra in P129.

*Probes are identical in sequence; they can differ in length.

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 17p12 region, two flanking probes, two probes in the KIF1b gene and 18 reference probes detecting autosomal chromosomes. The identity of the genes detected by the reference probes is available online (www.mlpa.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 121 nt: four DNA Quantity Fragments (Q-fragments), two DNA Denaturation Fragments (D-fragments), one benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at www.mpla.com.