Intended purpose
The SALSA MLPA Probemix P405 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, MPZ and GJB1 genes in genomic DNA isolated from human peripheral whole blood specimens or buccal swabs. P405 CMT1 is intended to confirm a potential cause for and clinical diagnosis of Charcot-Marie-Tooth disease type 1 (CMT1), X-linked CMT (CMTX) 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 P405 CMT1 should be confirmed with a different technique. In particular, CNVs detected by only a single probe always require confirmation by another method. Most defects in MPZ and GJB1, and some defects in PMP22 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.

¹ Please 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).
² To 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 (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, accounts for as much as 50% of all CMT cases. CMT1A is a dominantly inherited, childhood-onset, slowly progressive motor and sensory neuropathy due to a duplication of PMP22 on chromosome 17. CMT1B accounts for an additional ~10% of CMT1 patients. The gene involved in CMT1B is myelin protein zero (MPZ). CMT1A and 1B are clinically indistinguishable; classification is based solely on molecular findings.

X-linked CMT (CMTX) is characterized by moderate to severe motor and sensory neuropathy in affected males and usually mild to no symptoms in carrier females. Subtype CMTX1 accounts for about 90% of X-linked CMT, and is caused by mutations in the gap-junction protein beta 1 (GJB1) gene (https://www.ncbi.nlm.nih.gov/books/NBK1374/).

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 7-16 in 100,000. The penetrance is 100% but expressivity is highly variable even within the same family. Approximately 6-23% of individuals diagnosed with HNPP have an asymptomatic affected parent. A contiguous gene deletion of chromosome 17p12 that includes PMP22 is present in approximately 80% of affected individuals; the remaining 20% have a pathogenic variant in PMP22 (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-B4 CMT1 (IVD)	PMP22: all exons KIF1b: 2 probes	CMT1A and HNPP CMT2A1	PMP22 probes in P033-B4 have the same ligation site as PMP22 probes in P405-B1 except for one exon 1 probe and one exon 4 probe. There is one additional PMP22 exon 5 probe present in P033-B4.
P405-B1 CMT1 (IVD)	PMP22: all exons MPZ: all exons GJB1: all exons	CMT1A and HNPP CMT1B CMTX	PMP22 probes in P405-B1 have the same ligation sites as PMP22 probes in P033-B4 except for one exon 1 probe and one exon 4 probe. There is one additional PMP22 exon 5 probe present in P033-B4. MPZ probes in P405-B1 have the same ligation sites as MPZ probes in P143-C3.
P143-C3 MFN2-MPZ (RUO)	MFN2: all exons MPZ: all exons	CMT2A CMT1B	MPZ probes in P143-C3 have the same ligation sites as MPZ probes in P405-B1.

*IVD: in vitro diagnostic. RUO: research use only.

Probemix content
The SALSA MLPA Probemix P405-B1 CMT1 contains 42 MLPA probes with amplification products between 130 and 445 nucleotides (nt). This includes 15 probes for the common 17p12 deletion/duplication region, two flanking probes for genes outside the common 17p12 deletion/duplication region, seven probes for the MPZ gene, and five probes for the GJB1 gene. In addition, 10 ten reference probes are included that detect autosomal chromosomal locations and three probes detecting locations on the X-chromosome. 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.