Mutations of the noncoding region of the connexin32 gene in X-linked dominant Charcot-Marie-Tooth neuropathy

X-linked Charcot-Marie-Tooth (CMTX) neuropathy may be inherited as dominant or recessive. The dominant form of CMTX1 makes up 90% of all CMTX, whereas the recessive CMTX2 and CMTX3 ^[1] forms occur with a combined frequency of only 10%. The CMTX1 gene has been mapped to the proximal long arm of the X chromosome at Xq13.1 by genetic linkage studies. ^[2-9] There are recent reports of families with mutations in the coding region of the connexin32 gene (CX32) that cosegregrate with the CMTX1 phenotype. ^[10-12]

Five of the families diagnosed with CMTX1 by us on the basis of linkage analysis ^[11] did not show point mutations of the CX32 gene coding region. We suggested that their phenotypes might be due to mutations in the CX32 promoter, splice sites, or untranslated regions (UTRs). The present study of two CMTX1 families is the first report of mutations in the noncoding region of the CX32 gene. The mutations occur near the nerve-specific promoter P2 of the CX32 gene.

Methods.

Results.

Clinical data.

The pedigrees of the two families studied by us are shown in Figure 1. Family 1 has a small size with four affected members and only two still alive. Family 2 has 10 affected members with 9 still alive. The proband of family 1 was aged 48 years, and the proband of family 2 was aged 31 years. In the clinical diagnosis we distinguish three CMT phenotypes: (1) a mild CMT phenotype showing relatively good muscle strength, which is compatible with normal gait and ability to climb stairs; (2) a moderate CMT phenotype that is characterized by weakness of the tibialis anterior and peroneals, requiring ankle foot orthoses (braces), and weakness of palmar and dorsal interossei; and (3) a severe CMT phenotype that shows some proximal limb weakness in addition to distal weakness. The patients with this phenotype require canes or wheelchairs, and some of them have triple arthrodesis of the feet.

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Figure 1. (A) The pedigree of family 1 showing no male-to-male transmission, consistent with X-linked inheritance. (B) Pedigree of family 2. Arrows indicate the proband.

Based on these definitions, patients in both families were diagnosed with moderate CMTX1. The onset of the disease was between the ages of 12 and 14 years, with pes cavus, hammer toes, moderate to severe weakness of feet extensors (tibialis anterior) and evertors (peroneals), weakness of palmar and dorsal interossei, absence of deep tendon reflexes, distal hypesthesia (proprioceptive, pallesthetic, tactile), positive Romberg's sign, slow progressivity. Male patients were more severely affected than female patients. The probands of both families wore ankle foot orthoses. Motor nerve conduction velocities showed slowing (20 to 30 m/sec) in both male and female patients, and their EMGs were normal.

Molecular genetic studies.

The results of genetic linkage analysis of family 1 were inconclusive because of the small family size. Analysis of family 2 revealed positive lod scores with maximal values of 1.50 with markers of the Xq13.1 region. Neither family showed any point mutations in the coding region of the CX32 gene. To look for possible promoter or splice site mutations, or both, the nervespecific promoter P2 region Figure 2 (Neuhaus et al., Bioscience Reports, in press) of the CX32 gene was analyzed by PCR amplification of DNA from male patients. Direct sequence analysis of the PCR products revealed that patients of family 1 had a T-to-G transversion at position -528 in relation to the translation start codon, and patients of family 2 had a C-to-T transition at position -458. The latter mutation destroys the HhaI restriction endonuclease site, which was used to analyze a large number of members of this family. Both mutations occurred in male and female (heterozygous) patients and were not present in the nonaffected members of the families. In addition, over 100 unrelated control individuals, both males and females, lacked the two mutations, making it unlikely that the mutations represent polymorphisms without phenotype.

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Figure 2. (a) The human connexin32 gene with its two tissue-specific promoters. Alternative splicing of mRNA is indicated by thin lines. (b) Sequence of the promoter P2 region of the human connexin32 gene. The locations of the two mutations found in families 1 and 2 are shown in boxes above the normal sequence. The transcription start site (Neuhaus et al., Bioscience Reports, in press) and normal donor splice site are indicated.

Discussion.

Dominantly inherited diseases are usually caused by structural protein defects. Genes that encode for a protein subunit of a membrane, receptor, or organelle structure will be responsible for dominant diseases as distinct from the recessive disorders, which are caused by enzyme protein defects. ^[15] CMTX1 is associated with mutations in the gene encoding CX32, a member of the family of proteins forming intercellular channels. The CX32 mutations identified in CMTX1 patients are dominant and are considered to produce the clinical phenotype by single amino acid substitutions (missense mutations), a codon deletion, or frame shifts resulting in truncated proteins (nonsense mutations). ^[10-12] Nonsense mutations are very convincing regarding causality for the disease because of a complete loss of function of the protein.

Female patients (manifesting carriers) have milder phenotypes than male patients with CMTX1, and the suggested explanation is that they are heterozygotes for the mutant gene and therefore mosaics for CX32 mutation.

New studies have shown that CMTX1 mutants selectively act as dominant inhibitors of intercellular communications because they are devoid of channel activity. These results demonstrate a functional loss and are consistent with null mutations of CX32 in patients with CMTX1 neuropathy. ^[16]

The rat CX32 gene consists of three exons that are transcribed from two alternative promoters and spliced alternatively in different tissues. Exon Ia forms most of the 5 prime-UTR of mRNA present in liver and pancreas, whereas exon Ib forms most of the 5 prime-UTR of mRNA present in nervous tissue. Exon II is shared by both mRNA species and contains the remainder of the 5 prime-UTR, the entire coding region and the 3 prime-UTR. ^[17] The structure of the human CX32 gene is analogous. Promoter P1 is used exclusively in liver and pancreas, whereas promoter P2 is functional in nervous tissue (Neuhaus et al., Bioscience Reports, in press). The regulation of expression of the CX32 gene is similar to that of the peripheral myelin protein 22 gene in neural and non-neural tissue that also involves the use of two alternative promoters producing mRNAs with distinct 5 prime-UTRs. ^[18] This suggests that the translation machinery in nervous tissue requires specific 5 prime-UTRs for the efficient translation of mRNA.

The mutation in family 1 is located in the nervespecific promoter P2 of the CX32 gene 16 base pairs upstream of the TATA box. This mutation may directly affect the rate of initiation of transcription. In contrast, the mutation in family 2 is located in a region that codes for the 5 prime-UTR of the mRNA found in nervous tissue. The mutation creates a potential donor splice site, showing only a one-base deviation from the consensus sequence, and thus might cause incorrect splicing of the CX32 transcript. This would produce an mRNA with a shortened 5 prime-UTR. Alternatively, the GTG codon, which is created by this mutation, represents a potential translation initiation site that may be used as a translation start codon and thereby compete with the initiation from the correct AUG located downstream from it. There is a similar mutation in some patients with beta-thalassemia in which the mutation created a new ATG site that led to the initiation of a very short peptide chain. ^[19] Furthermore, Davidson et al. described a mutation of the AUG initiation codon of the human HPRT gene creating a GUG codon that functioned as a translation start codon. ^[20] Similarly, the CMTX1 mutation in family 2 would lead to the initiation of a polypeptide chain of only 12 amino acids because of a stop codon located only 37 bases downstream of the newly created GTG initiation codon. The precise effects of these mutations will have to be studied in transfection experiments or in transgenic mice.

The present study is the first report of mutations in the CX32 gene that are not located in the coding region. The location of these mutations near the nerve-specific promoter of the gene would not affect the expression of the gene in liver or pancreas where a different promoter is used (Neuhaus et al., Bioscience Reports, in press). The association of these mutations with the CMT phenotype does not by itself prove that they cause the disease. However, taken together with the CX32 coding region mutations that were found associated with the CMT phenotype, they make a strong argument that the CX32 protein is essential for nerve function and that mutations in its gene cause CMT neuropathy.