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Charcot-Marie-Tooth disease (CMT) and related peripheral neuropathies together also termed hereditary motor and sensory neuropathies (HSMN) represent a clinically and genetically heterogeneous group of disorders affecting the peripheral nervous system.
CMT in general is the most common inherited peripheral neuropathy, with an estimated prevalence of 1 in 2500 individuals (Lupski et al., 1991a; Skre, 1974). It is found world wide in people of all races and ethnic groups except for some rare subtypes (Emery et al., 1991). CMT is characterized by slowly progressive weakening and atrophy of distal muscles and distal sensory impairment, which usually starts in the lower limbs and progrades to the upper limbs in advanced stages of disease. Additional symptoms are skeletal deformities and decreased or absent tendon reflexes. CMT is commonly further divided into three main groups based on electrophysiological findings, i.e. nerve conduction velocity (NCV) in peripheral nerves: CMT1, CMT2 and CMT with intermediate NCV. Demyelinating neuropathies with markedly slowed motor nerve conduction velocity (NCV lower than 38 m/s) belong into the CMT1 group. Axonal neuropathies with normal or slightly lowered motor nerve conduction velocity (NCV higher than 38 m/s) belong into the CMT2 group. The intermediate form gathers CMT types with non-homogeneous electrophysiological findings, that is, clinical entities with a phenotype where NCVs can be both above or below the 38 m/s criterion value in different patients from one family (Carter et al., 2004; Nicholson et al., 2006). The current classification combines these basic criteria with inheritance pattern and genetic background to further subdivide these main groups and comprises more than 40 clinical entities. The most common subtype is CMT 1A responsible for 43% of all CMT cases. It is a demyelinating neuropathy with autosomal dominant inheritance. Patients show general symptoms of CMT as well as symptoms specific for CMT1, which are decreased NCV and structures called onion-bulbs visible on sural nerve biopsies arising due to demyelination and remyelination of axons (Lupski et al., 1991a; Dyck et al., 1993). The clinical features of this non-lethal disease have a broad range, causing mild to moderate disability in patients. Variable severity of symptoms can presumably be addressed to different underlying mutations but the study of Garcia et al. (1995) showed that also non-genetic factors may be responsible.
Hereditary neuropathy with liability to pressure palsies (HNPP) is one of the diseases belonging to CMT related peripheral neuropathies. The prevalence of HNPP was originally estimated to be 2-5 cases per 100 000 individuals (Nelis et al., 1996), but a study by Meretoja et al. (1997) which revealed the prevalence of 16 cases per 100 000 individuals pointed out that it is a rather common disease. This difference was probably caused by under diagnosis due to the mild phenotype of the disease. HNPP is an autosomal dominant disorder characterized by recurrent episodes of nerve palsy resulting in motor and sensory neuropathy. The palsy, which always affects a single nerve, is caused by minor compression or trauma to superficial nerves, and is followed by numbness, muscle weakness and atrophy (Verhagen et al., 1993; Gouider et al., 1995; Pareyson et al., 1996). Sural biopsy findings of compression sites often show sausage like thickenings of the myelin sheath called tomaculae, which are the consequences of demyelination of the affected fibers (Meier and Moll 1982). The recurrent palsies usually dissipate over a period of many years, but patients with a single episode and even asymptomatic individuals have been reported. In the majority of cases patients recover from palsies completely, otherwise they develop mild disability (Felice et al., 1994).
CMT1A and HNPP are both caused by distinct mutations of the gene encoding peripheral myelin protein 22 (PMP22). The major cause of CMT1A and HNPP is an unequal meiotic crossing over event in the region 17p11.2-p12 encompassing PMP22. The consequent chromosomes carry a 1.5 Mb duplication and deletion respectively. Owing to the dosage sensitivity of peripheral myelin protein 22 (Pmp22) not only point mutations in its gene but also altered gene copy number leads to peripheral neuropathy. Thus three copies of PMP22 cause CMT1A, only one copy causes HNPP and other mutations in the gene give rise to CMT1A, HNPP, Dejerine-Sottas syndrome (DSS), congenital hypomyelinating neuropathy (CHN) and CMT type 2 (Fig. 1).
PMP22 is a gene highly expressed by myelinating Schwann cells of the PNS, but expression in other tissues like brain, intestine, lung and heart have also been reported (Patel et al., 1992; Baechner et al., 1995). Its expression is differentially regulated during early stages of development and later during maintenance of the PNS (De Leon et al., 1991) and interestingly, despite its pleiotropic expression during development, the pathogenic effect of either dosage differences or other mutations in PMP22 seems to be restricted to the PNS (Nelis et al., 1999b). The gene encodes a major structural membrane protein of the compact myelin, where it comprises 2-5% of the total protein content (Snipes et al., 1992). Pmp22 is involved in the regulation of myelin thickness and stability and in the maintenance of the myelin sheath (D'Urso et al., 1997); however its exact function still remains elusive.
Validation of the real time PCR method optimized for PMP22 copy number determination was the initial step of our study, prior to testing of patients. Validation has been accomplished by evaluation of 13 samples with confirmed CMT1A duplication, 2 samples with confirmed HNPP deletion as well as 110 healthy individuals. Obtained value ranges for the three groups of samples were as follows: 0.624-0.626 for samples with deletion, 0.758-1.24 for samples of healthy individuals and 1.356-1.546 for samples with duplication (Tab. 1.). On the basis of these results values below 0.65 were assigned to HNPP, values between 0.75-1.25 to healthy individuals and values above 1.35 to CMT1A. The value ranges of distinct groups were not overlapping, showing the feasibility of the method to determine PMP22 copy number.
After method validation, 121 families with suspected CMT and 2 families with suspected HNPP were tested for duplication or deletion. During testing also Ct values outside of the designated diagnostic ranges occurred, altogether 4.76% of analyzed samples. In these cases the real time assay was repeated using other calibrator samples - always giving unambiguous results. Individuals in 41 families from among 121 showed to have duplication of PMP22, of which 22 were familial cases and 19 were isolated cases or families with only the proband's sample provided. In addition, 7 families of the 121 with suspected CMT showed to have PMP22 deletion. Among the two families with suspected HNPP the deletion was identified in one. After PMP22 copy number determination, chosen samples from families without duplication or deletion were further screened for other causative mutations in the PMP22 gene. Mutation screen was performed by dHPLC analysis of amplicons which included exons 1A, 2, 3, 4 and 5. Amplicons with aberrant dHPLC profiles were sequenced, revealing two mutations and three polymorphisms. One of the identified mutations was a heterozygous nonsense mutation c.327C>A (Cys109X) in exon 5 already described by Abe et al. (2004). The second mutation was a heterozygous missense mutation c.172C>T (Pro58Ser) in exon 3, at present not listed in the Inherited Peripheral Neuropathies Mutation Database (Nov. 2009) (http://www.molgen.ua.ac.be/CMTMutations/Home/Default.cfm). The polymorphisms found were c.-244C>T in the amplicon including exon 1A, c. 319+33C>T in the amplicon including exon 4 and c.353C>T (Thr118Met) in exon 5.
Altogether, mutation analysis of PMP22 revealed 40.65% of underlying mutations of Charcot-Marie-Tooth disease and related peripheral neuropathies in the Slovak population.
According to our best knowledge only a few published papers discuss the frequency of 17p11.2-p12 duplication and deletion in the total population of CMT-affected patients (KabziÅ„ska et al. 2009). The majority of studies assess the frequency of duplication among CMT1 patients; even though they usually also mention the overall number of CMT patients examined, thus frequencies of duplication in CMT can be calculated. Other studies show only data acquired from strictly defined sets of CMT1 patients. The results of these studies can be extrapolated for the whole CMT population based on the frequency of CMT1, which is estimated to account for 50-70% of CMT cases (Szigeti et al. 2006, Shy et al. 2005). Duplication frequencies in diverse populations are presented in Table 2.
Quantitative real time PCR analysis of PMP22 copy number in our sample set identified the duplication in 35.96% of families (41/114). This outcome correlates well with results of other studies (Tab. 2). Nevertheless, authentic frequency of duplication in the Slovak population may be higher due to the possibility of misdiagnosis of CMT in some families, taking into account the 7 CMT families with indentified PMP22 deletion. In these cases we also considered the possibility of mutations causing CMT phenotype in hemizygous state, but this alternative has been ruled out by sequencing which didn't confirm the presence of such mutations.
Familial occurrence of duplication was approved in 78.05% of cases (32/41), while in 19.51% of cases (8/41) only the probands sample was available and the family records had no indication of CMT phenotype in relatives. In one family de novo origin of duplication was confirmed. If we consider the 8 families with only the probands sample provided as isolated cases, the minimum share of de novo duplication on isolated cases is 11.1% (1/9).
The frequencies of duplication and deletion of the 1.5 Mb region mapping to chromosome 17p11.2-12 should be equal according to the fact that these mutations are reciprocal end products of the same crossing over event. A large epidemiological study of HNPP by Meretoja et al. (1997) already confirmed the equal prevalence of the two mutations; however most studies inform about considerably fewer cases of deletion than duplication and anticipate it to be caused by under diagnosis of deletion due to its mild phenotype. In our sample set PMP22 copy number determination identified altogether 8 families with deletion, thus the ratio of deletion to duplication is roughly 1:5. This difference in mutation frequencies is in agreement with literature, which