X-linked dominant Charcot–Marie–Tooth neuropathy: clinical, electrophysiological, and morphological phenotype in four families with different connexin32 mutations1

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Abstract

The sensorimotor neuropathy of the Charcot–Marie–Tooth type (CMT) is the most common hereditary disorder of the peripheral nervous system. The X-linked dominant form of CMT (CMTX) is associated with mutations in the gene for the gap junction protein connexin32. We examined four CMTX pedigrees two of which had potentially novel mutations in the only coding exon of connexin32. One previously unreported missense mutation, Ala39Val, was found in a family displaying a CMT phenotype with additional upper limb postural tremor reminiscent of a Roussy–Lévy syndrome. A novel single base insertion, 679insT, is among the first mutations found in the fourth transmembrane domain of connexin32. Frameshift and premature stop of translation are supposed to result in a non-functional carboxy-terminus. Two further families had the known missense mutations Arg15Trp and Arg22Gln. Several female carriers were found normal on clinical presentation, however, the genotype was paralleled by decreased nerve conduction velocities (NCV) and slowed central conduction of brain stem auditory evoked responses (BAER). Median motor NCVs showed mild (in women) to intermediate (in males) reduction, indicating a peripheral neuropathy with a predominating axonal component. Nerve biopsy findings were consistent with the electrophysiological data showing a marked loss of large myelinated fibres and clusters of regenerating axons. Electron microscopy revealed various alterations of the axoglial attachment zone. This suggests defective axon-Schwann cell interactions which may induce the axonopathy in CMTX.

Introduction

Charcot–Marie–Tooth neuropathy (CMT) or hereditary motor and sensory neuropathy (HMSN) comprises hereditary disorders of the peripheral nervous system with autosomal as well as X-linked patterns of inheritance. Patients have progressive distal muscle weakness and atrophy, foot deformities, steppage gait, distal sensory loss, and decreased or absent tendon reflexes. Type 1, the demyelinating form of CMT, can be distinguished from type 2, the axonal form, by measurement of nerve conduction velocities (NCV) and histopathological examination of a nerve biopsy. The first presents with markedly decreased NCV and segmental degeneration of peripheral nerve myelin, the second with not or only slightly slowed NCV and loss of nerve fibres [1]. A heterozygous duplication of the locus for the peripheral myelin protein 22 (PMP22) on chromosome 17p11.2 is found in about 70% of CMT1 patients (CMT1A) [2], [3]. Occasionally CMT1A is also associated with point mutations in PMP22 [4] while CMT1B is caused by mutations in the myelin protein zero gene (P0, MPZ, locus: 1q22–q23) [5]. Recently, mutations in the early growth response 2 gene (EGR2, locus: 10q21–q22) were found in patients with severe, early onset peripheral neuropathies [6].

X-linked dominant CMT (CMTX) shows both demyelinating and axonal aspects of peripheral nerve pathology [7], [8], [9], [10], thus causing difficulties in categorising CMTX as a subtype of CMT1 or CMT2. Due to the X-linked pattern of inheritance, males tend to be earlier and more severely affected than women and show more severely decreased NCV. Female carriers may even appear asymptomatic, however, they can usually be identified on electrophysiological investigation [11], [12]. The causative role of mutations in the gap junction protein connexin32 (Cx32, locus: Xq13.1) in CMTX has been established by linkage and mutation analysis [13], [14]. Connexin32 mutations are now considered to be the second most common molecular cause of hereditary sensorimotor neuropathy accounting for at least 10% of families with CMT. Mutations reported so far are spread throughout the protein with scarcity in the fourth transmembrane domain ( see [15], [16] and references therein). Connexin32 has been localised in non-compact peripheral myelin probably forming gap junctions between adjacent myelin wraps of the same Schwann cell [13], [17], [18]. While various other tissues including central nervous system (CNS) glia express connexin32 [18], the only clinically affected one seems to be peripheral nerve. However, slowed CNS conduction of brain stem auditory evoked responses (BAER) in CMTX patients [19] suggest the involvement of central pathways.

CMTX can be confused with other types of CMT on electrophysiological and morphological investigation, or with sporadic male cases when the mutation is inherited by an asymptomatic female heterozygote. Hence, genetic testing is required for establishing a firm diagnosis which is of peculiar interest considering family planning and prognosis, especially of female patients. We have begun a regular screening of suspected CMT patients for connexin32 mutations when X-linked inheritance of the neuropathy seems likely. We have so far detected a total of four different mutations two of which are potentially novel. In this work, we analysed segregation of these mutations and phenotypic expression of the disease in the respective pedigrees. Some of the findings in the index cases of the families with the known mutations Arg15Trp and Arg22Gln have already been reported elsewhere documenting that archival, paraffin-embedded nerve biopsies allow the identification of connexin32 mutations [20].

Section snippets

Patients

We studied the segregation of connexin32 mutations in four unrelated German families with X-linked dominant CMT. For identification of index cases, DNA was extracted from archival paraffin-embedded sural nerve biopsy specimens of suspected CMT patients and screened to search for the presence of mutations within the connexin32 gene. Extending our previous study on 30 patients with a CMT2 phenotype [20], we performed genetic testing in 15 cases diagnosed with unspecified CMT, however, with a

Results

We were able to study four families with CMTX, each with a different mutation in the connexin32 gene. The families showed complete cosegregation between the mutation and the clinical phenotype, with the exception of eight female carriers who had no clinical symptoms of CMT. On electrophysiological investigation, however, five of these patients revealed signs of peripheral nerve lesion. The mutations could neither be detected in healthy relatives nor in any of the 78 normal chromosomes derived

Discussion

We were able to study four unrelated CMT families with mutations in the coding region of the connexin32 gene. Two of these mutations are potentially novel, one of which causing frameshift and premature stop of translation. Molecular analysis and clinical investigation displayed complete segregation of the mutated alleles with the CMT phenotype or transmission of the disease. Nerve biopsy samples from the index cases were indicative of a predominating axonal type of neuropathy with only minor

Acknowledgements

The technical assistance of A. Knischewski and H. Mader is gratefully acknowledged. The authors wish to thank Professor H. Buchner for reviewing the manuscript and offering helpful suggestions. This work was partially supported by grants from the Deutsche Forschungsgemeinschaft (SCHR 195/17-1), from the Wilhelm-Sander-Stiftung to Dr S. Quasthoff, and from the START programme of the Medical Faculty, RWTH Aachen.

References (66)

  • P.J. Dyck et al.

    Hereditary motor and sensory neuropathies

  • L.J. Valentijn et al.

    Identical point mutations of PMP22 in Trembler-J mouse and Charcot–Marie–Tooth disease type 1A

    Nat Genet

    (1992)
  • K. Hayasaka et al.

    Charcot–Marie–Tooth neuropathy type 1B is associated with mutations of the myelin P0 gene

    Nat Genet

    (1993)
  • L.E. Warner et al.

    Mutations in the early growth response 2 (EGR2) gene are associated with hereditary myelinopathies

    Nat Genet

    (1998)
  • A.F. Hahn

    Hereditary motor and sensory neuropathy: HMSN type II (neuronal type) and X-linked HMSN

    Brain Pathol

    (1993)
  • M.L. Mostacciuolo et al.

    X-linked Charcot–Marie–Tooth disease. A linkage study in a large family by using 12 probes of the pericentromeric region

    Hum Genet

    (1991)
  • M.P. Rozear et al.

    Hereditary motor and sensory neuropathy, X-linked: a half century follow-up

    Neurology

    (1987)
  • S. Sander et al.

    Charcot–Marie–Tooth disease: histopathological features of the peripheral myelin protein (PMP22) duplication (CMT1A) and connexin32 mutations (CMTX1)

    Muscle Nerve

    (1998)
  • G.A. Nicholson et al.

    Intermediate nerve conduction velocities define X-linked Charcot–Marie–Tooth neuropathy families

    Neurology

    (1993)
  • G.A. Nicholson et al.

    Efficient neurophysiological selection of X-linked Charcot–Marie–Tooth families

    Neurology

    (1998)
  • J. Bergoffen et al.

    Connexin mutations in X-linked Charcot–Marie–Tooth disease

    Science

    (1993)
  • A. Gal et al.

    X-linked dominant Charcot–Marie–Tooth disease: suggestion of linkage with a cloned DNA sequence from the proximal Xq

    Hum Genet

    (1985)
  • P. De Jonghe et al.

    Charcot–Marie–Tooth disease and related peripheral neuropathies

    J Peripher Nerv Syst

    (1997)
  • E. Nelis et al.

    Mutations in the peripheral myelin genes and associated genes in inherited peripheral neuropathies

    Hum Mutat

    (1999)
  • C. Sandri et al.

    Membrane morphology of the vertebrate nervous system

    Prog Brain Res

    (1982)
  • S.S. Scherer et al.

    Connexin32 is a myelin-related protein in the PNS and CNS

    J Neurosci

    (1995)
  • G.A. Nicholson et al.

    Slowing of central conduction in X-linked Charcot–Marie–Tooth neuropathy shown by brain stem auditory evoked responses

    J Neurol Neurosurg Psychiatry

    (1996)
  • J. Senderek et al.

    X-linked dominant Charcot–Marie–Tooth disease: nerve biopsies allow morphological assessment and identification of connexin32 mutations (Arg15Trp, Arg22Gln)

    Acta Neuropathol

    (1998)
  • J.M. Schröder

    Peripheral neuropathies. Correlation between moleculargenetic and fine structural diagnosis of inherited peripheral neuropathies

    Brain Pathol

    (1997)
  • R. Thiex et al.

    PMP-22 gene duplications and deletions identified in archival, paraffin-embedded sural nerve biopsies: correlation to structural changes

    Acta Neuropathol

    (1998)
  • N.M. Kumar et al.

    Cloning and characterization of human and rat liver cDNAs coding for a gap junction protein

    J Cell Biol

    (1986)
  • R. Bruzzone et al.

    Connections with connexins: the molecular basis of direct intercellular signaling

    Eur J Biochem

    (1996)
  • L.C. Milks et al.

    Topology of the 32-kD liver gap junction protein determined by site-directed antibody localizations

    EMBO J

    (1988)
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    Presented in part at the 7th Annual Symposium of the European Charcot–Marie–Tooth Consortium, 3–5 July 1998, in Antwerp, Belgium.

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