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Charcot-Marie-Tooth type 1X (CMT1X) disease is inherited as an X-linked dominant trait. Female CMT1X patients are usually mildly affected or even asymptomatic carriers of mutations in the GJB1 gene coding for a gap junction protein called connexin-32 (Cx32).
In this report, a five-generation CMT1X family is described from which the new mutation in the GJB1 gene Cys179Gly was identified.
The Cys179Gly mutation is located in the highly conservative domain of the Cx32 protein. Previous functional studies performed in the oocyte system have shown that point mutations in the highly conserved Cx32 cysteine residues result in a complete loss of function of the gap junction. However, despite severe biochemical defects, the Cys179Gly mutation segregates with a mild CMT1X phenotype.
This study further documents a discrepancy between biochemical effects of GJB1 mutations and the CMT1X phenotype.
Charcot-Marie-Tooth disease (CMT) is one of the most common hereditary neuromuscular disorders, occurring with a frequency of 1:2500 (1).
Females with CMT1X are usually less severely affected than males. In fact CMT1X clinically manifests in males as early as the first decade of life, while in females the first CMT symptoms appear only in their third decade; some of them remain entirely asymptomatic for most of their lives. As some women are asymptomatic mutation carriers, a phenotype resembling neuropathy with an X-linked recessive mode of inheritance can be recognized. In line with these clinical observations, EMG studies make it clear that both nerve conduction velocities (NCVs) and compound muscle action potentials (CMAPs) experience much more limited impairment in CMT1X-affected females than in males (4).
At the molecular level, CMT1X disease is caused by mutations in the GJB1 gene coding for the gap-junction protein known as connexin 32 (Cx32), with a molecular weight of 32 kDa (5).
Cx32 protein is widely expressed in the myelinating Schwann cells oligomerizing into hemi-channels, forming cell-to-cell gap junctions (6, 7). The whole family of connexins shares a common membrane topography, with two extra-cellular loops, four trans-membrane segments, and three cytoplasmic domains with carboxy- and amino-termini (8).
In the last 14 years, over 300 mutations in the GJB1 gene have been reported in CMT1X families. These are uniformly distributed throughout the Cx32 gene. However, an X-linked inheritance was not characterized for some GJB1 gene mutations, because about 30% of them were identified in patients with sporadic disease only (9).
The vast majority of GJB1 gene mutations segregates with a relatively mild phenotype in CMT1X-affected females.
We report the results of a study on a five-generation CMT1X family in which it was possible to identify a novel Cys179Gly mutation in the GJB1 gene, located in the highly conservative domain of the Cx32 protein.
The family under study originates from what was once the Eastern part of Poland, i.e., the city of Lwów (or today’s Ukrainian L’viv). After the Second World War, family members moved to the Western part of modern Poland. The family for which information is available consists of five generations (Fig. (Fig.1).1). According to the proband (IV:7) indications, her father (III:3), grandmother (II:2) and great-grandfather (I:1) were all affected by polyneuropathy.
Charcot-Marie-Tooth disease (CMT) was diagnosed in the father (III:3) of the proband, who showed his first CMT symptoms at the age of 13. In a neurological examination carried out at 54 years of age, he presented with foot dropping, bilateral pes cavus deformity, wasting of distal muscles in the lower limbs, and wasting of small hand muscles. He died at the age of 67 years with a diagnosis of cerebral tumor in the left hemisphere.
In the proband (IV:7), now 39 years old, a first CMT symptom (pes cavus deformity) was observed at age 13, conservatively treated by an orthopedic surgeon. An examination carried out when she was 31 year old revealed that cognitive function was normal, as the cranial nerves were, except for a slightly flattened left nasal-lip fold and the absence of gag reflexes. The neurological examination showed symmetrical wasting of the hand muscles, bilateral pes cavus deformity, and absence of ankle reflexes. She was unable to walk on her heels and toes. Muscle strength was intact, except in the small hand muscles (Fig. (Fig.22).
A symmetrical impairment of skin sensation up to knee level was found.
Median motor conduction velocity (MCV) was 28.6 m/sec, and distal latencies were prolonged to 5.5 ms. The M amplitude was severely reduced to 0.1 mV. Median SNCV was not recordable, and sural nerve sensory action potential (SAP) was absent. Peroneal MCV was 43 m/s, with markedly prolonged distal latency of 7.5 ms and M amplitude of 0.5 mV. Tibial MCV was reduced to 34 m/s with the M amplitude of 0.1 mV and distal latency prolonged to 8 ms. The results of routine laboratory tests were within the normal range. In conclusion, a typical mild, mixed CMT1X neuropathy was diagnosed in the proband.
A 16-year-old son (V:5) of the proband is also affected by CMT. The first symptoms were observed at the age of 13 years. He was born following a normal full-term pregnancy and delivery. Neurological examination showed that he was unable to walk on his heels and toes, though free of symmetrical distal leg atrophy or pes cavus deformity. The Achilles and knee tendon reflexes were absent. Wasting of distal muscles was limited to the small hand muscles (Fig. (Fig.2),2), and – except for the latter – there was a good muscle strength in the proximal and distal muscles.
Median MCV was 46.8 m/s, distal latency 8.85 ms (normal < 4 ms), and the M amplitude 2.7 mV. Peroneal MCV was 37.3 m/s with a distal latency of 5.85 ms and M amplitude of 0.8 mV. Median SNCV was 38.5 m/s with SAP of 15.1 μV. Sural Sensory Conduction Velocity (SCV) was 43.9 m/s with SAP amplitude of 7.6 μV. Routine hematological and biochemical tests were normal.
The patients gave informed consent to take part in the study which was approved by the local Ethics Committee at Warsaw Medical University. Genomic DNA was extracted from peripheral blood lymphocytes by means of a salting-out procedure.
Duplication of the Peripheral Myelin Protein 22 gene (PMP22) was excluded using the Real Time polymerase chain reaction (RT-PCR) method. The coding sequence of the GJB1 gene was amplified by the PCR reaction with previously reported primers (5). The GJB1 gene sequence was screened for the DNA variants using two approaches i.e., single-strand conformation polymorphism analysis (SSCP) and heteroduplex analysis (HA). The PCR products, obtained by both methods, were separated on a 9% acrylamide gel (37.5:1 acrylamide/bisacrylamide). The gels were silver-stained and dried (Fig. (Fig.3A3A).
The PCR products showing an anomalous pattern of DNA migration were sequenced using a BigDyeTM Terminator Version 3.1 Ready Reaction Cycle Sequencing kit on the ABI 3730/xl Genetic analyzer (Applied Biosystems, Poland). The GJB1 gene sequence was analyzed by comparing it with reference sequences NM_001097642.1 (transcript variant 1) and NM_000166.4 (transcript variant 2) in the Basic Local Alignment Search Tool (Blast NCBI - http://blast.ncbi.nlm.nih.gov/Blast.cgi).
The sequencing of exon 2 of the GJB1 gene revealed a hemi-zygous T to G transversion at nucleotide 535 of the GJB1 gene, resulting (by conceptual translation) in a substitution of cysteine with glycine at codon 179 of the Cx32 protein (Fig. (Fig.3B3B).
To confirm the presence of the c.535 T > G substitution, a restriction fragment length polymorphism (RFLP) analysis with CviKI-1 (New England BioLabs, (UK) Ltd. 75/77 Knowl Piece, Wilbury Way Hitchin, Herts. SG4 0TY,United Kingdom), was performed according to the manufacturer’s instructions. The digested PCR products were separated on a 10% acrylamide gel stained with ethidium bromide, and visualized using UV light (data not shown).
The present data refer to a five-generation CMT1X family of Polish origin, in which a novel Cys179 Gly mutation was identified in the proband (IV:7), and in her son.
Till now, two other substitutions (Cys179Arg and Cys179Tyr) have been reported in codon 179 of the Cx32 protein. However as no clinical or electrophysiological findings were given for them, it was impossible to compare our patients with the CMT1X phenotype associated with these two mutations (9, 10).
The phenotype of the CMT1X family here described is typical, the location of Cys179Gly mutation occurring within a highly-conservative CysX4CysX5Cys motif of the second extra-cellular loop of the Cx32 protein. In fact it was assumed that the change of any one of these cysteine residues into a serine residue will result in an complete loss of function.
Studies performed in the oocyte-pairing system (11) have shown that both Gly179Cys mutated hemi-channels, and a mixture of the wild-type and Cys179Gly protein, result in a loss of function of the Cx32 ion channel (lack of junctional conductance). Thus, in at least functional terms, Gly179Cys could be classified as a loss-of-function mutation, similar to nonsense or deleted variants of GJB1. However this observation seems in contrast with the mild phenotype for CMT1X disease, observed in our family, if compared with the severe phenotype (early-onset neuropathy associated to marked functional disabilities) usually described in patients harboring nonsense mutations of the GJB1 gene (4, 12).
The relationship between the severity of the CMT1X disease and the cell effects of the different GJB1 gene mutations is still controversial (13). A preliminary genotype-phenotype correlation between the type of mutations (frameshifts, deletions and premature truncations) in the GJBJ gene and the severity of clinical course, showed that a more severe phenotype in CMT1X is caused by missense mutations (14). Hahn et al. (12) on the other hand, suggested that missense mutations of the GJB1- located in the cytoplasmic loop and the second trans-membrane domain - segregate with a mild CMT1X phenotype, whereas mutations at all other locations of the connexin 32 result in a severe CMT1X phenotype with an early age at onset and severe disability. Liang et al. (15) did not confirm these data in an extended study on patients with CMT1X carrying the F235C missense mutation. In fact they described this mutation in a severely affected 14-year-old girl with high-degree of scoliosis and markedly reduced motor-nerve conduction velocities (18-20 m/sec), although functional studies showed that F235C mutation results in open hemi-channels affecting cell viability.
The relationship between the clinical severity of CMT1X and the intra-cellular retention of Cx32 mutants was also investigated for a set of GJB1 gene mutations. By this approach, it was possible to assume that Cx32 mutants retained in transfected cells, resulted in a more severe phenotype than those reaching the cell surface (16). This hypothesis was not confirmed in the study of Shy et al. (13) who analyzed a large cohort of CMT1X-affected males harboring 28 different GJB1 gene mutations. They demonstrated that the degree of clinical disability in CMT1X-affected patients does not correlate with specific GJB1 gene mutations, nor with the type of mutation (deletion, frameshift or missense mutation). Furthermore a marked clinical variability was observed even in patients harboring the same mutation (13).
Our results are in agreement with those reported in studies carried out on large groups of CMT1X patients. A loss-of-function mechanism of GJB1 gene mutations has been recently proved in studies on transgenic mice expressing the T55I and R75W point mutations (17). However a larger number of CMT1X Cys179Gly mutated families need to be characterized, at clinical and electrophysiological levels, to determine the spectrum of clinical variability in this disease.
The Authors thank the members of the family for active participation in the research and are grateful to Mrs. Jadwiga Kędzierska for the skillful technical assistance.
The study was supported by grant No. NN 402276336 of Polish Ministry of Science and Higher Education, entitled: The variability of CMT1A clinical course in the light of the studies of PMP22 gene.