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Charcot-Marie Tooth (CMT) is a clinically and genetically heterogeneous group of diseases with rough genotype–phenotype correlation, so the final diagnosis requires extensive clinical and electrophysiological examination, family data, and gene mutation analysis. Although there is a common pattern of genetic basis of CMT, there could be some population differences that should be taken into account to facilitate analyses. Here we present the algorithm for genetic testing in Serbian patients with demyelinating CMT, based on their genetic specificities: in cases of no PMP22 duplication, and if -X-linked CMT (CMTX) is not contraindicated by pattern of inheritance (male-to-male transmission), one should test for c.94A>G GJB founder mutation, first. Also, when a patient is of Romani ethnicity, or if there is an autosomal recessive inheritance in a family and unclear ethnicity, c.442C>T mutation in NDRG1 should be tested.
Hereditary motor and sensory neuropathies, also called Charcot-Marie-Tooth (CMT) disease, comprise a group of inherited peripheral neuropathies. CMT affects one in 2,500 people (Skre, 1974) and is the most common inherited neurological disorder (Kedlaya, 2012).
Demyelinating CMT or CMT1, the most common form of the disease, accounts for more than 60% CMT cases (Numakura et al., 2003), and is characterized by lowered nerve conduction velocity (NCV) both in sensory and motor nerves. The other major form is axonal CMT or CMT2, distinguished by normal or mildly reduced NCVs.
CMT is a clinically and genetically heterogeneous group of diseases with rough genotype–phenotype correlation, so the final diagnosis requires extensive clinical and electrophysiological examination, family data, and gene mutation analysis. To date, about 30 genes and 50 loci have been associated with CMT (Inherited Peripheral Neuropathies Mutation Database - IPNMD, 2011). Among them, mutations in the PMP22, MPZ, and GJB1 genes are responsible for the vast majority of patients with CMT, while mutations in other genes are often present in rare, even individual cases (Mersiyanova et al., 2000; Mostacciuolo et al., 2001; Numakura et al., 2002; Keckarevic Markovic et al., 2009).
PMP22 and MPZ are associated with autosomal dominant forms of CMT1 (and CMT2 for MPZ), while GJB1 is responsible for X-linked CMT (IPNMD, 2011). Autosomal recessive (AR) forms are rare, but could be over-represented in small populations, due to a founder effect. Therefore, a founder mutation in the NDRG1 gene is responsible for CMT1 ARD (CMT4D), also known as CMT Lom, the most common peripheral neuropathy among Romani people (Kalaydieva, 2002).
The genetic basis of CMT varies among populations, but the pattern is the same: the most frequent mutation in CMT1 is PMP22 duplication, then GJB1, MPZ, and PMP22 mutations. For CMT2, the gene mostly affected by mutations is MFN2, then GJB1, and MPZ. Therefore, the main algorithm for genetic testing of patients with CMT worldwide would be the same, suggesting genetic analyses in the order mentioned above. GJB1 gene analyses should not be done if there is male-to-male transmission of the disease.
Despite that, in general, the given algorithm is the best way to genetically evaluate most CMT cases, there could be some population differences that should be taken into account to facilitate analyses, to make them faster, cheaper, and easier.
Here, we present the algorithm for genetic testing in Serbian patients with demyelinating CMT, based on their genetic specificities.
A total of 98 patients with unrelated demyelinating CMT, 23 family members and 100 healthy controls participated in this study. CMT was diagnosed in patients based on the clinical and electrophysiological findings: distal muscle weakness and atrophy, absent or reduced deep tendon reflexes, sensory loss, and reduced NCVs. The study was approved by the Ethics Committee of the Institute for Neurology and Psychiatry for Children and Youth, and informed consents were obtained.
DNA was isolated from blood samples and buccal swabs. 17p11.2 duplication/deletion screening was performed by polymerase chain reaction (PCR)/EcoRI restriction digestion (Stronach et al., 1999) and by microsatellite analysis using six polymorphic markers located in the involved region (Mersiyanova et al., 2000). The coding regions, exon-adjacent sequences, and some promoters of GJB1, MPZ, PMP22, EGR2, and LITAF/SIMPLE were amplified using PCR, and then sequenced. The primer sequences are available upon request. Sequencing variations were confirmed by sequencing both strands. NDRG1 c.442C>T mutation detection was performed by PCR/Taq1 restriction digestion (Kalaydjieva et al., 2000).
Microsatellite markers surrounding GJB1 were analyzed using fluorescent-labeled primers. The marker positions and primers are available upon request.
The PMP22 duplication was detected in 37/98 patients. Six different GJB mutations were detected in 10/98 patients, five of them detected for the first time in the Serbian population. Haplotype analysis showed that one of them, c.94A>G, present in five patients, was a founder mutation. Three mutations were found in MPZ (one identified for the first time), one in PMP22, while no mutations were found in EGR2 and LITAF/SIMPLE. Mutation c.442C>T in NDRG1 was identified in 5/98 patients, all of them were of Romani descent.
Results of mutation analysis are shown in Table 1.
According to the results obtained, an algorithm for genetic testing of patients with demyelinating CMT in a population of Serbia was constructed, as shown in Figure 1.
The results of genetic analyses in Serbian patients with CMT showed no major differences compared to other populations studied earlier (Keckarevic-Markovic et al., 2009). PMP22 duplication was found to be the most frequent CMT mutation, followed by GJB1, MPZ, and PMP22 mutations. EGR2 and LITAF/SIMPLE mutations were not found. However, six out of 10 mutations identified in this study have never been reported before. A large contribution of newly identified mutations confirmed the necessity for sequencing analyses of gene candidates, which is often time consuming and expensive.
One newly identified mutation in GJB1, c.94A>G, was found to be a founder mutation, and is present in 50% of patients with genetically confirmed CMTX, with overall contribution of 5.1%. Also, there are about 400,000–500,000 gypsies in Serbia (around 5% population), and CMT4D is the most frequent peripheral neuropathy among them, due to a c.442C>T founder mutation in NDRG1. In the Serbian CMT population, CMT4D accounts for another 5.1%. In both cases, restriction digestion analysis or allele-specific PCR, fast and inexpensive compared to gene sequencing, should be the methods of choice.
An algorithm for genetic testing of patients with demyelinating CMT in a population of Serbia was constructed regarding a respectable proportion of Serbian patients with CMT (~3,6%), considering that there are 7,000,000 people in Serbia and that the estimated frequency of CMT is 1 in 2,500. According to a given guideline, in cases of no PMP22 duplication, and if CMTX is not contraindicated by the pattern of inheritance (male-to-male transmission), one should test for the c.94A>G GJB mutation, first (Fig. 1). No, especially, frequent mutation was outlined in the guidelines for genetic testing by Saporta et al. (2011) and Murphy et al. (2012), implicating that the high frequency of that c.94A>G GJB mutation is a consequence of a genetic homogeneity of the Serbian population.
Also, when a patient is of Romani ethnicity, or if there is an AR inheritance in a family and unclear ethnicity, a c.442C>T mutation in NDRG1 should be tested. Compared to other published algorithms (Saporta et al., 2011; Murphy et al., 2012) in Serbian patients with CMT, a greater percentage of genetically confirmed AR CMT is expected, due to a large proportion of Romani people in Serbia, bearing a founder recessive mutation in the NDRG1 gene combined with consanguinity.
The given algorithm should provide an overall guidance to genetic testing of Serbian patients with CMT with lowered nerve conduction velocities. In cases of specific information, the order of analyses could be changed and adjusted to a specific situation.
This work was supported by the Serbian Ministry of Education and Science, grant no. 173016.
No competing financial interests exist.