New connexin32 mutations associated with X-linked Charcot-Marie-Tooth disease

X-linked Charcot-Marie-Tooth disease (CMTX) is a peripheral neuropathy characterized by progressive distal extremity weakness, atrophy, sensory loss, and areflexia. Males are more severely affected than females, with usual onset of symptoms in adolescence. Nerve conduction velocities range between 25 and 40 m/sec in affected males, whereas heterozygous females have values between 25 and 50 m/sec. Linkage studies and recombination analysis ^[1-6] mapped the CMTX gene to the proximal long arm of the X chromosome at Xq13.1. Connexin32 (Cx32) was also mapped within this region of the X chromosome, ^[7,8] suggesting it as a candidate gene for CMTX. Northern blot analysis demonstrated Cx32 expression in normal peripheral nerve, and we ^[9] identified mutations associated with CMTX by direct sequencing of the coding region of the Cx32 gene.

Cx32 is a 32-kD gap junction protein originally isolated from liver. ^[10-12] It is expressed in kidney, intestine, lung, spleen, stomach, testes, and brain as well as in peripheral nerve. ^[9,13] Gap junctions are intercellular channels made of two hemichannels, each composed of connexin molecules arranged around a central pore. ^[14,15] The pore is thought to be lined by polar residues from the third transmembrane domain ^[16] and allows passage of ions and small molecules of up to 1,000 daltons. The channel diameter is estimated to be 1.2 nm, preventing the passage of larger molecules such as proteins and nucleic acids.

Others have confirmed our finding of Cx32 mutations in CMTX families. ^[17-21] Here, we describe additional mutations in Cx32. Our results show that mutations affect most regions of the molecule, increasing the total number of identified mutations to 33 in 39 families Figure 1.

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Figure 1. Diagram of connexin32 showing amino acid sequence with conserved cysteine residues (triangles), codon position (numbers), and location of CMTX mutations (arrows). New mutations are shown on a black background. There were 33 mutations in 39 families. Abbreviations for amino acid residues: A equals Ala; C equals Cys; D equals Asp; E equals Glu; F equals Phe; G equals Gly; H equals His; I equals Ile; K equals Lys; L equals Leu; M equals Met; N equals Asn; P equals Pro; Q equals Gln; R equals Arg; S equals Ser; T equals Thr; V equals Val; W equals Trp; and Y equals Tyr. References (uncircled letters): B equals Bergoffen et al, 1993 ^[9]; T equals Tan and Ainsworth, 1994 ^[21]; F equals Fairweather et al, 1994 ^[17]; I equals Ionasescu et al, 1994 ^[18]; C equals Cherryson et al, 1994 ^[20]; and O equals Orth et al, 1994. ^[19] Asterisk equals new mutations reported here.

Methods.

Blood samples were obtained from family members under a protocol of informed consent. Genomic DNA was extracted from venous blood by established methods. ^[2]

Two fragments that together encompass the entire Cx32 gene coding region, one of 306 and one of 666 base pairs, were amplified by polymerase chain reaction (PCR) using primers and conditions previously published. ^[9] These fragments were purified with the Geneclean DNA purification kit (BIO 101). The PCR fragments were directly analyzed by automated cycle sequencing with fluorescent tagged terminators on an Applied Biosystems, Inc. sequencer, using the same primers as for amplification plus two additional internal primers that were also previously published. ^[9] Patient sequences were compared with control sequences to reveal mutations within the coding region of the Cx32 gene.

For those mutations that result in changes in restriction sites, as determined by computer analysis of the new sequence, genotypes of individuals were determined by restriction endonuclease digestion of the appropriate PCR fragment. Each reaction was analyzed by electrophoresis in 1 to 5% agarose gels, with ethidium bromide for visualization.

Results.

Direct sequencing of the Cx32 gene in CMTX patients revealed five novel missense mutations and one novel frameshift mutation Table 1. These mutations included a valine right arrow leucine substitution at codon 13 in the amino terminal intracellular domain in patient sample PQ; an isoleucine right arrow asparagine substitution at codon 30 in the middle of the first transmembrane domain in family 266; a tyrosine right arrow cysteine substitution at codon 65 in the first extracellular loop adjacent to the third highly conserved cysteine residue in family 1850; a valine right arrow methionine substitution at codon 95 in the intracellular loop in family 272; and a tryptophan right arrow arginine substitution at codon 133 in the third transmembrane domain in family 261. ^[22] The identified frameshift mutation predicts a truncated 194-amino acid protein with the last 58 amino acids, starting at codon 137, altered from wild type in the VA family.

Three recurrent mutations were also identified during sequencing. Two families (nos. 264 and 265) showed a previously reported nonsense mutation predicting truncation of the protein at amino acid 220. ^[17] One family (no. 69) showed a codon 139 valine right arrow methionine missense mutation, also in the third transmembrane domain. ^[9] Another family (no. 269) showed recurrence of the codon 156 leucine right arrow arginine missense mutation located in the second extracellular loop. ^[9] None of the reported families with the same mutation are known to be related. In most cases, families with the same mutation are from different geographic regions. For example, family 69 is from New York, and the other two families with the identical mutation are from South Dakota and Michigan, with no known New York connection.

Nine families analyzed had no mutation within the coding region of Cx32. Seven of these represent sporadic cases, one has affected members in only two generations, and another is a large, multigenerational kindred previously reported to be X-linked. ^[23] Affected individuals in all these families had features clinically consistent with CMTX.

For seven of the nine mutations, changes in restriction endonuclease sites allowed the analysis of mutation segregation. These seven changes include the gain of a Nla III site in family 69, the gain of a Dde I site in families 264 and 265, the loss of a Mbo II site in family 266, the gain of an Hha I site in family 269, the loss of a BbrPI site in family 272, the gain of a mae III site in family 1850, and the gain of a Dra III site in family VA. Restriction-site changes allowed these mutations to be analyzed in unrelated normal individuals to rule out the possibility that they represent naturally occurring polymorphisms, to be traced through the remainder of the families, and to be used to diagnose carrier status in females. In each case, the putative mutations were not found in a screening of 50 unrelated, normal females.

Discussion.

Cx32 mutations are directly associated with X-linked Charcot-Marie-Tooth disease. The mechanism by which these mutations cause the phenotype remains unknown, but the presence of mutations in almost all regions of the molecule indicates that most of the molecule is important to the function of Cx32 in Schwann cells. Cx32 localizes by immunohistochemistry to the nodes of Ranvier and the Schmidt-Lantermann incisures in peripheral nerve. ^[9] Other forms of Charcot-Marie-Tooth disease, types 1A and 1B, are associated with mutations in myelin genes, peripheral myelin protein 22 kD (PMP-22) in type 1A and protein zero (P0) in type 1B. ^[24] These proteins are in compact myelin, whereas Cx32 is in noncompact myelin. However, the similarities of the resulting disease phenotypes indicate that each of these Schwann cell proteins has an important function in peripheral nerve.

The variety of mutations found suggests that virtually all regions of Cx32 are important in its function. Families linked to the region of the X chromosome where Cx32 is located but without mutations in the open reading frame suggest that mutations in noncoding regions of the gene, such as the promoter and splice sites, may be responsible for causing the disease. ^[9,23] These regions need to be further studied.

Gap junctions allow the passage of ions and small molecules as well as conduction of electrical currents. The loss of junctional conductance in the paired oocyte expression system with three of the originally described mutant Cx32 molecules suggests that these mutations cause a loss of Cx32 function. ^[25] Analysis of these mutants revealed a dominant negative effect for one mutation when co-expressed with connexin 26 but not connexin 40. Depending on the mutation, the mechanism of disease may be either a loss of product effect or a dominant negative effect, as reported in Marfan's syndrome. ^[26] Further identification of mutations may allow a genotype-phenotype correlation, which could aid in defining the CMTX disease mechanism.

Acknowledgments

We are grateful to the families for their cooperation, Dr. Steve Scherer and Suzanne Deschenes for their helpful discussion, Marion Oronzi Scott and Melanie Hartman for technical assistance, the Nucleic Acid/Protein Research Core Facility of the Children's Hospital of Philadelphia for assistance in this project, and Dr. Hank Paulson and Dr. Peter Bingham for review of the manuscript.