Intended purpose
The SALSA MLPA Probemix P033 CMT1 is an in vitro diagnostic (IVD)¹ or research use only (RUO) semi-quantitative assay² for the detection of deletions or duplications in the human PMP22 and KIF1b genes in genomic DNA isolated from human peripheral whole blood specimens or buccal swabs. P033 CMT1 is intended to confirm a potential cause for and clinical diagnosis of Charcot-Marie-Tooth disease (CMT) or hereditary neuropathy with liability to pressure palsies (HNPP) and for molecular genetic testing of at-risk family members.

Copy number variations (CNVs) detected with P033 CMT1 should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Not all exons of KIF1b are covered. Some defects in PMP22 and most defects in KIF1b are point mutations, none of which will be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.

This device is not intended to be used for standalone diagnostic purposes, pre-implantation or prenatal testing, population screening, or for the detection of, or screening for, acquired or somatic genetic aberrations.

¹Please note that this probemix is for In Vitro Diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for Research Use Only (RUO).
²To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Clinical background
Charcot-Marie-Tooth disease (CMT), with a worldwide incidence of 1 in 2500, is the most common hereditary sensorimotor neuropathy, comprising a group of clinically and genetically heterogeneous peripheral neuropathies. CMT is characterized by progressive distal muscle atrophy and weakness, sensory disturbance, the absence of deep tendon reflexes, and pes cavus deformity of the foot. More than 80 different genes are associated with CMT (GeneReviews: http://www.ncbi.nlm.nih.gov/books/NBK1358/). Subtypes related to the genes PMP22, GJB1, MPZ and MFN2 are the most common ones, being responsible for up to 95% of CMT cases with a final diagnosis (Padilha et al. 2020). The disease can be inherited in an autosomal dominant, autosomal recessive or X-linked manner. Table 1 provides an overview of the different genes involved in the CMT subtypes and the probemixes that cover these genes.

The most frequent form, CMT1A is a dominantly inherited, childhood-onset, slowly progressive motor and sensory neuropathy due to a duplication of PMP22 on chromosome 17. CMT type 2 is an axonal peripheral neuropathy which shows extensive clinical overlap with CMT1. However, in general, the phenotype of CMT2 patients is less severe. 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 2A (Zhao et al. 2001; Drew et al. 2015).

Hereditary neuropathy with liability to pressure palsies (HNPP) is characterized by repeated focal pressure neuropathies such as carpal tunnel syndrome and peroneal palsy with foot drop. Recovery from acute neuropathy is often complete; when recovery is not complete, the resulting disability is usually mild. Some affected individuals also have signs of a mild to moderate peripheral neuropathy. The prevalence of HNPP is estimated at 7 to 16 cases per 100,000 population. Penetrance is 100% but expressivity is highly variable even within the same family. Approximately 6% to 23% of individuals diagnosed with HNPP have an asymptomatic affected parent. 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 (GeneReviews: 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 CMT1 (IVD)	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.
PP405 CMT1 (IVD)	PMP22: all exons MPZ: all exons GJB1: all exons	CMT1A and HNPP CMT1B CMTX	PMP22 probes are all identical to P033, plus one extra exon 4 and exon 5 probe in P033. MPZ probes are all identical to P143.
P143 MFN2-MPZ (RUO)	MFN2: all exons MPZ: all exons	CMT2A CMT1B	MPZ probes are all identical to P405.

*Probes are identical in sequence; they can differ in length. IVD: in vitro diagnostic. RUO: research use only.

Probemix content
The SALSA MLPA Probemix P033-B4 CMT1 contains 38 MLPA probes with amplification products between 130 and 436 nucleotides (nt). This includes 16 probes for the 17p12 chromosomal region, 2 flanking probes, and 2 probes for the KIF1b gene. In addition, 18 reference probes are included that detect autosomal chromosomal locations. Complete probe sequences and the identity of the genes detected by the reference probes are available online (www.mrcholland.com).

This probemix contains nine quality control fragments generating amplification products between 64 and 105 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.mrcholland.com.