X-linked Charcot-Marie-Tooth disease with connexin 32 mutations: clinical and electrophysiologic study.

OBJECTIVE: To relate X-linked Charcot-Marie-Tooth disease (CMTX) phenotypes to gender and type of neuropathy by the study of a large series of CMTX patients with proven Cx32 point mutations. BACKGROUND: CMTX is an X-linked form of Charcot-Marie-Tooth disease, caused by mutations in the connexin 32 gene. Males are usually more severely affected and have slower nerve conduction velocities than females. METHODS: Forty-eight patients from 10 families with Cx32 mutations were examined clinically and electrophysiologically. Mutations were characterized in index cases by automatic sequencing and detected in at-risk individuals by polymerase chain reaction (PCR)-restriction or single strand conformation polymorphism (SSCP) analysis. Two patients from different families had light and electron microscopy examination of a sural nerve biopsy. RESULTS: Males (n = 21) were more severely affected than females (n = 27), although six of the females were severely disabled. In the majority of males, the median motor nerve conduction velocity (MNCV) was between 30 and 40 m/s, whereas in females it ranged from 30 to normal values. Two children with mutation, a 6-year-old boy and a 7-year-old girl, were normal clinically and electrophysiologically. In most patients, the amplitude of motor nerve compound muscle action potentials (CMAP) was reduced in all nerves tested. MNCV was reduced as a function of the degree of axonal loss. A significant correlation was found between the decrease in CMAP amplitude and MNCV in the median, ulnar, and peroneal nerves. Sural nerve biopsies in one patient with a missense and one with a nonsense mutation both showed axonal neuropathy. CONCLUSION: Electrophysiologic and histologic findings support primary axonal neuropathy in CMTX with Cx32 mutations. Clinical and electrophysiologic data in males with different missense mutations in the of Cx32 gene differed significantly. Furthermore, males with a nonsense mutation (Arg22Stop) had earlier onset and a more severe phenotype than males with missense mutations.

The first de novo mutation of the connexin 32 gene associated with X linked Charcot-Marie-Tooth disease.

X linked Charcot-Marie-Tooth disease (CMTX) is a hereditary motor and sensory neuropathy caused by mutations in the connexin 32 gene (Cx32). Using the SSCP technique and direct sequencing of PCR amplified genomic DNA fragments of the Cx32 gene from a Moroccan patient and her relatives, we identified the first de novo mutation of the Cx32 gene, consisting of a deletion of a G residue at position 499 in the Cx32 open reading frame. This previously unreported mutation produces a frameshift at position 147 in the protein and introduces a premature stop codon (TAG) at nucleotide 643, which results in the production of a truncated Cx32 molecule. This mutation illustrates the risk of an erroneous diagnosis of autosomal recessive CMT, especially in populations where consanguineous unions are frequent, and its consequences for genetic counselling, which can be avoided by molecular analysis.

Charcot-Marie-Tooth disease with intermediate motor nerve conduction velocities: characterization of 14 Cx32 mutations in 35 families.

Charcot-Marie-Tooth disease can be inherited either autosomal dominantly or recessively or linked to the X chromosome. X-linked dominant Charcot-Marie-Tooth disease (CMTX) is a sensorimotor peripheral neuropathy in which males have usually more severe clinical symptoms and decreased nerve conduction velocities than do females. CMTX is usually associated with mutations in exon 2 of the connexin 32 (Cx32) gene. DNA from 35 unrelated CMT patients, without the 17p11.2 duplication, but with median nerve conduction between 30 and 40 m/s, were tested for the presence of Cx32 mutations. The entire coding sequence of the Cx32 gene was explored using a rapid nonradioactive technique to detect single-strand conformation polymorphisms (SSCP) on large PCR fragments. Thirteen abnormal SSCP profiles were detected and characterized by sequencing. In addition, systematic sequencing of the entire Cx32 coding region in the remaining index cases revealed another mutation that was not detected by SSCP. A total of 14 mutations were found, five of which were not previously reported. These results demonstrate the high frequency (40%) of mutations in the coding region of the Cx32 gene in CMT patients with intermediate MNCV, without 17p11.2 duplications. Most of these mutations (93%) can be detected by SSCP.

Five unknown mutations in the LR pyruvate kinase gene associated with severe hereditary nonspherocytic haemolytic anaemia in France.

A survey of PK-deficient patients by molecular biology techniques has been performed in France in 26 unrelated families, in which at least one mutation has been characterized. The patients, of European or North African origin, exhibited approximatively 10% of PK activity. Among the PK-R mutants described, mutation G1529-->A (Arg-509-->Gln) was the most frequent. The strategy followed for the description of PK mutants in France firstly involves determination of this mutation by PCR amplification and restriction enzyme digestion and, secondly, the sequencing of the gene for negative samples. Study of the mutation at residue 509 in 26 unrelated families indicated that 10/52 defective alleles possessed this mutation. Our study described seven different mutations; five of these have not as yet been documented. Two frameshift mutations were found: the deletion of one G base in a repetition of four Gs in position 1231-1234 (PK Mondor), del C-1527 (PK Rouen), and three missense mutations: G382-->C (Ala-114-->Pro) (PK Val-de-Marne), C398-->T (Ser-119-->Phe) (PK Beaujon), A1217-->G (Asn-392-->Ser) (PK Paris). Two mutations which were detected have been reported previously: C760-->T (Glu-240-->End) and G1529-->A (Arg-509-->Gln.

PK Mondor: prenatal diagnosis of a frameshift mutation in the LR pyruvate kinase gene associated with severe hereditary non-spherocytic haemolytic anaemia.

A mutant form of pyruvate kinase (PK) from the red blood cells of a consanguineous family with severe non-spherocytic haemolytic anaemia has been characterized by polymerase chain reaction (PCR) amplification and sequencing. The variant enzyme was named PK Mondor, according to the recommendations of the International Committee for Standardisation in Haematology. The propositus lacked PK activity and the low level of PK activity found resulted more likely from PK-M2 (fetal isozyme) expression in the red blood cells of the propositus. PK Mondor corresponds to a frameshift mutation with deletion of one G in a repetition of four Gs in positions 1231-1234. This family, whose first child was stillborn and whose second was homozygous for the frameshift mutation, requested prenatal diagnosis during the third pregnancy. Diagnosis was made after chorionic biopsy by a specific approach combining PCR amplification and restriction enzyme digestion.

Genetic associations with human longevity at the APOE and ACE loci.

In an effort to dissect the genetic components of longevity, we have undertaken case-control studies of populations of centenarians (n = 338) and adults aged 20-70 years at several polymorphic candidate gene loci. Here we report results on two genes, chosen for their impact on cardiovascular risk, encoding apolipoprotein E (ApoE), angiotensin-converting enzyme (ACE). We find that the epsilon 4 allele of APOE, which promotes premature atherosclerosis, is significantly less frequent in centenarians than in controls (p < 0.001), while the frequency of the epsilon 2 allele, associated previously with type III and IV hyperlipidemia, is significantly increased (p < 0.01). A variant of ACE which predisposes to coronary heart disease is surprisingly more frequent in centenarians, with a significant increase of the homozygous genotype (p < 0.01). These associations provide examples of genetic influences on differential survival and may point to pleiotropic age-dependent effects on longevity.