The CMT2D locus: refined genetic position and construction of a bacterial clone-based physical map.

Charcot-Marie-Tooth (CMT) disease is a progressive neuropathy of the peripheral nervous system, typically characterized by muscle weakness of the distal limbs. CMT is noted for its genetic heterogeneity, with four distinct loci already identified for the axonal form of the disease (CMT2). In 1996, linkage analysis of a single large family revealed the presence of a CMT2 locus on chromosome 7p14 (designated CMT2D). Additional families have been linked subsequently to the same genomic region, including one with distal spinal muscular atrophy (dSMA) and one with mixed features of dSMA and CMT2; symptoms in both of these latter families closely resemble those seen in the original CMT2D family. There is thus a distinct possibility that CMT2 and dSMA encountered in these families reflect allelic heterogeneity at a single chromosome 7 locus. In the study reported here, we have performed more detailed linkage analysis of the original CMT2D family based on new knowledge of the physical locations of various genetic markers. The region containing the CMT2D gene, as defined by the original family, overlaps with those defined by at least two other families with CMT2 and/or dSMA symptoms. Both yeast artificial chromosome (YAC) and bacterial clone-based [bacterial artificial chromosome (BAC) and P1-derived artificial chromosome (PAC)] contig maps spanning approximately 3.4 Mb have been assembled across the combined CMT2D critical region, with the latter providing suitable clones for systematic sequencing of the interval. Preliminary analyses have already revealed at least 28 candidate genes and expressed-sequence tags (ESTs). The mapping information reported here in conjunction with the evolving sequence data should expedite the identification of the CMT2D/dSMA gene or genes.

Fine mapping of de novo CMT1A and HNPP rearrangements within CMT1A-REPs evidences two distinct sex-dependent mechanisms and candidate sequences involved in recombination.

The molecular mechanism resulting in the duplication or deletion of a 1.5 Mb region of 17p11.2-p12, associated, respectively, with Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP), has been proposed to be an unequal crossing-over during meiosis between the two chromosome 17 homologues generated by misalignment of the proximal and distal CMT1A-REP repeats, two homologous sequences flanking the 1.5 Mb CMT1A/HNPP monomer unit. In a recent study of a large series of de novo cases of CMT1A and HNPP, two distinct sex-dependent mechanisms were identified. Rearrangements of paternal origin, essentially duplications, were indeed generated by unequal meiotic crossing-over between the two chromosome 17 homologues, but duplications and deletions of maternal origin resulted from an intrachromosomal process, either unequal sister chromatid exchange or, in the case of deletion, excision of an intrachromatidal loop. In order to determine how these recombinations occur, 24 de novo crossover breakpoints were localized within the 1.7 kb rearrangement hot spot by comparing the sequences of the parental CMT1A-REPs with the chimeric copy in affected offspring. Nineteen out of 21 paternal crossovers were found in a 741 bp hot spot. All the breakpoints of maternal origin (n = 3), however, were located outside this interval, but in closely flanking sequences, supporting the hypothesis that two distinct sex-dependent mechanisms are involved. Several putative recombination promoting sequences in the hot spot, which are rare or absent in the surrounding 7.8 kb, were identified.

X-linked Charcot-Marie-Tooth disease and connexin32.

We studied 29 families with X-linked dominant CMT (CMTX1) neuropathy. Twenty-five families showed mutations in the coding region of the connexin32 (Cx32) gene. The mutations included five nonsense mutations, 17 missense mutations, two medium size deletions and one insertion. Most missense mutations showed a mild clinical phenotype and slowing of motor nerve conduction velocities. All five nonsense mutations, the larger deletion and the insertion showed severe clinical phenotype. Four CMTX1 families with mild clinical phenotype showed no point mutations of the Cx32 gene coding region. Two mutations of the non-coding region were identified. The first mutation was located in the nerve specific Cx32 promoter, the second mutation was located in the 5' untranslated region of the mRNA.

Severe Charcot-Marie-Tooth neuropathy type 1A with 1-base pair deletion and frameshift mutation in the peripheral myelin protein 22 gene.

A 27-year-old man with negative family history and both parents with normal neurological evaluation and motor nerve conduction velocities (MNCVs) showed onset of severe weakness of feet at 4 years of age. Subsequently he developed left equinovarus deformity, thoracic scoliosis, ulnar nerve enlargement, areflexia, distal hypesthesia and slowing of MNCVs for median and ulnar nerves (15-25 m/sec). Molecular genetic studies showed deletion of one nucleotide (G330) (codon 94) in exon 3 of the PMP22 gene associated with frameshift mutation.

Dejerine-Sottas neuropathy in mother and son with same point mutation of PMP22 gene.

We studied a 25-year-old black woman with healthy parents and her 2-year, 11-month-old son. Her motor development was delayed and she started to walk with support when she was 6 years old. She never walked independently and had always used a wheelchair. Neurological evaluation showed severe weakness and atrophy of her feet, legs, and hands, bilateral pes cavus and hammertoes, corrected scoliosis, hypesthesia for proprioception and vibration sense in both feet and ankles, and areflexia. She had normal intelligence. Her son also had delayed motor milestones and was still unable to stand and walk independently at almost 3 years. Neurological evaluation revealed diffuse muscle hypotonia and weakness with generalized areflexia and normal intelligence. No muscle atrophies or feet deformities were noticed. Nerve conduction velocities showed significant slowing (less than 5 m/s) with prolonged distal latencies (above 30 ms). Compound motor action potential amplitudes were markedly reduced. Electromyography revealed polyphasic motor unit potentials. Molecular genetic studies indicated a Trembler type missense point mutation of exon 4 of the peripheral myelin protein 22 gene that led to the substitution of a spartic acid for glycine in both the mother and her son. Her parents showed normal DNA studies.

Dejerine-Sottas disease with sensorineural hearing loss, nystagmus, and peripheral facial nerve weakness: de novo dominant point mutation of the PMP22 gene.

A 32 year old woman with Dejerine-Sottas disease and negative family history is reported. Clinical onset of her condition was with congenital weakness of her distal four extremities, accompanied by peripheral facial nerve weakness, deafness, and nystagmus. She has used a wheelchair all her life. Sural nerve biopsy showed proliferation of Schwann cells, extensive endoneural fibrosis, axon loss, and demyelination. MNCVs showed marked slowing. MRI of the brain was normal. Molecular genetic studies indicated a de novo dominant missense point mutation of exon 3 of the peripheral myelin protein 22 gene at nucleotide 264 causing replacement of serine with leucine.

Autosomal dominant Charcot-Marie-Tooth axonal neuropathy mapped on chromosome 7p (CMT2D).

Clinical, electrophysiological and genetic linkage studies were performed on a large autosomal dominant family with Charcot-Marie-Tooth axonal neuropathy type 2 (CMT2) with 38 members of which 14 were affected. Onset of the disease was between 16 and 30 years of age with weakness and atrophy of the hands more severe than of the feet with slow progressive course in 12 patients. Deep tendon reflexes were absent in the upper extremities and decreased in the lower extremities. There was distal hypesthesia for touch, proprioception and vibration sense for the hands more than for the feet. Motor nerve conduction velocities showed normal values (48-53 M/s) with normal latencies (2-3 msec) and electromyography revealed signs of denervation. Genetic linkage analysis used 167 short tandem repeat markers (STRPs) spaced throughout the 22 autosomes. Linkage to the short arm of chromosome 7 at 7p14 was found using the marker D7S435 (Z = 4.83 at theta = 0). Flanking markers were D7S1808 and D7S1806 and the genetic distance between them was 6.8 cM. The multipoint linkage analysis gave a peek multipoint lod score of 6.89 between the markers D7S1808 and D7S435. Linkage analysis showed significantly negative lod scores (with values less than -2) with markers of chromosomes 1 and 3 where CMT axonal forms have been previously mapped. PFGE analysis indicated the absence of the CMT1A duplication. Our findings are consistent with a new genetic type of axonal CMT neuropathy designated by us as CMT2D. Potential candidate genes are multiple T-cell gamma receptor genes which map to the same cytogenetic interval as CMT2D neuropathy.

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

We studied two families with X-linked dominant Charcot-Marie-Tooth neuropathy. The clinical findings included onset around age 14 years, with moderate weakness of feet extensors and palmar and dorsal interossei, areflexia, distal hypesthesia, and slow progressivity. Motor nerve conduction velocities showed slowing (20 to 30 m/sec) and EMGs were normal. Genetic linkage analysis revealed positive lod scores with the markers of the Xq13.1 region in family 2, but was noninformative in family 1. There were no point mutations in the connexin32 gene coding region. Instead, family 1 revealed a T-to-G transversion at position -528 relative to the ATG start codon, whereas family 2 showed a C-to-T transition at position -458. The first mutation is located in the nerve-specific connexin32 promoter just upstream of the transcription start site, the second is located in the 5' untranslated region of the mRNA.

The human connexin32 gene is transcribed from two tissue-specific promoters.

The connexin32 (cx32) gene codes for the gap junction protein found in liver, pancreas and nervous tissue. Recently mutations in the coding region of this gene have been associated with the dominant X-linked form of Charcot-Marie-Tooth (CMTX1) neuropathy. Since some CMTX1 patients show no mutations in their cx32 gene coding region, it was speculated that these patients carry mutations in the promoter region of the gene. This paper describes the organization of the human cx32 gene and its tissue-specific transcription. The gene consists of three exons that are alternatively spliced to produce mRNAs with different 5'-untranslated regions (UTRs). Transcription is initiated from two tissue-specific promoters. In liver and pancreas, promoter P1, located more than 8 kb upstream of the translation start codon, is used, and the transcript is processed to remove a large intron. In contrast, in nerve cells, transcription is initiated from promoter P2, located 497 bp upstream from the translation start codon, and the transcript is processed to remove a small 355-pb intron. The downstream exon, which includes the entire coding sequence, is shared by both mRNAs. CMTX1 patients with a normal cx32 coding region are expected to have mutations in this newly described promoter P2 rather than the known promoter P1.

Correlation between connexin 32 gene mutations and clinical phenotype in X-linked dominant Charcot-Marie-Tooth neuropathy.

We studied the relationship between the genotype and clinical phenotype in 27 families with dominant X-linked Charcot-Marie-Tooth (CMTX1) neuropathy. Twenty-two families showed mutations in the coding region of the connexin32 (cx32) gene. The mutations include four nonsense mutations, eight missense mutations, two medium size deletions, and one insertion. Most missense mutations showed a mild clinical phenotype (five out of eight), whereas all nonsense mutations, the larger of the two deletions, and the insertion that produced frameshifts showed severe phenotypes. Five CMTX1 families with mild clinical phenotype showed no point mutations of the cx32 gene coding region. Three of these families showed positive genetic linkage with the markers of the Xq13.1 region. The genetic linkage of the remaining two families could not be evaluated because of their small size.

A Dejerine-Sottas neuropathy family with a gene mapped on chromosome 8.

The Patient is a 55-year-old black male who belongs to a large family with 9 affected relatives with autosomal dominant Dejerine-Sottas neuropathy (DSN). Onset of his condition was at 2 years of age with steppage gait followed by severe progressive weakness, atrophy, and sensory loss of his legs and hands accompanied by areflexia and thoracolumbar kyphoscoliosis. The patient became wheelchair confined at age 38. At around age 42, the left shoulder became dislocated and the humeral head underwent aseptic necrosis (Charcot joint). Nerve conduction studies showed absent motor and sensory responses for all major nerves tested. Genetic linkage suggested mapping of this DSN gene on chromosome 8qter. A younger brother with similar neurological findings also demonstrated Charcot joints with bone destruction of the joints of the fourth and fifth fingers.

Dejerine-Sottas disease with de novo dominant point mutation of the PMP22 gene.

We studied a 33-year-old woman with a negative family history. Both of her parents were examined clinically by nerve conduction velocities (NCVs) and EMG, with normal results. The clinical onset of her condition was at 24 months, with severe weakness and atrophy of her feet and hands, but the proximal muscles were relatively spared. She had bilateral pes cavus, distal weakness and hypesthesia for touch and proprioception, areflexia, claw hands, and severe thoracolumbar kyphoscoliosis. NCVs showed absent motor and sensory responses and EMG revealed diffuse fibrillation potentials. Molecular genetic studies indicated a de novo dominant missense point mutation of exon 3 of the peripheral myelin protein 22 gene at nucleotide 264 that caused the replacement of serine with leucine.

New point mutations and deletions of the connexin 32 gene in X-linked Charcot-Marie-Tooth neuropathy.

The purpose of this study was the identification of new mutations of the connexin 32 (CX32) gene in CMTX families. We report six new mutations of the CX32 gene including two medium sized (29 and 18 bp) deletions. The clinical phenotype is consistent with CMT peripheral neuropathy in all patients. Four families show both male and female patients, with more severe symptoms in males. The disease is asymptomatic in females in two families. The clinical deficit in CMTX families Nos 1, 2 and 4 with missense mutations of the CX32 gene was mild or moderate. Severe weakness of the feet and hands was present in CMTX family No. 5 with a G insertion and family No. 6 with a 29 bp deletion in the carboxyl terminal region of the CX32 gene. Most likely the severe clinical impact in those families was related to frame shift and premature termination of the protein.

Charcot-Marie-Tooth neuropathies: from clinical description to molecular genetics.

Ninety-five families with Charcot-Marie-Tooth (CMT) neuropathies were studied clinically, electrophysiologically (MNCVs and EMGs), and by molecular genetics. Fifty-four families (56.8%) were type 1A mapped at 17p11.2-p12 and DNA duplication was present in 50 (92.6% of CMT1A families). One family with type 1B (1.1%) mapped at 1q22-q23 showed a point mutation of the myelin P0 gene. Eighteen families (18.9%) were type CMT2 based on electrophysiological studies. Molecular genetics was not yet conclusive. Twenty CMT families were with X-linked dominant inheritance (CMTX1) (21.1%) mapped at Xq13.1 and connexin 32 (CX32) point mutations were present in 15 families (75%) (five nonsense mutations, eight missense mutations, two deletions). Two CMT families (2.1%) with X-linked recessive inheritance showed no point mutations of CX32 and their mapping was different from CMTX1, respectively at Xp22.2 for CMTX2 and at Xq26 for CMTX3.

Intestinal pseudo-obstruction in adult spinal muscular atrophy.

A 42-year-old woman with negative family history had the insidious onset of weakness in her lower extremities 8 years before, in 1983. The disorder slowly progressed to include cramps and muscle twitches. The diagnosis of adult spinal muscular atrophy (SMA) was made when electromyography showed large rapidly firing motor unit-potentials, positive waves, and fibrillation potentials, and when muscle biopsy of the quadriceps revealed severe alterations consistent with neurogenic atrophy. The patient also had severe chronic constipation for many years. More recently she had developed unremitting diarrhea. Gastrointestinal studies showed no evidence of peristaltic contractions in the rectum, delayed gastric emptying, and abnormal jejunal manometry with altered propagation of the migrating myoelectrical complex.

X-linked spastic paraplegia (SPG1), MASA syndrome and X-linked hydrocephalus result from mutations in the L1 gene.

X-linked hydrocephalus, spastic paraplegia type I and MASA syndrome are related disorders with loci in subchromosomal region Xq28. We have previously shown that X-linked hydrocephalus is caused by mutations in the gene for neural cell adhesion molecule L1 (L1CAM), an axonal glycoprotein involved in neuronal migration and differentiation. Here we report mutations of the L1 gene in MASA syndrome and SPG1, in addition to HSAS families. Two of the HSAS mutations would abolish cell surface expression of L1 and represent the first functional null mutations in this disorder. Our results indicate that these three syndromes from part of a clinical spectrum resulting from a heterogeneous group of mutations in the L1 gene.

Clinical and morphologic features of a myopathy associated with a point mutation in the mitochondrial tRNA(Pro) gene.

We studied a 9-year-old girl with progressive weakness of her extremities for two years. Her neurologic evaluation showed weakness of proximal muscles but no ophthalmoparesis. With the exception of elevated serum lactic acid, the general blood screen, EMG, nerve conduction velocity tests, and ECG were normal. Light and electron microscopy of a muscle biopsy showed proliferation of mitochondria containing disorganized cristae. Activities of respiratory chain enzymes containing mitochondrial DNA (mtDNA)-encoded subunits were significantly impaired in muscle homogenates. A G-->A transition at position 15990 previously detected in this patient's muscle was not present in peripheral blood cells of her mother or sister. However, the patient's WBCs appeared to contain a very small percentage of mutant mtDNAs, indicating that the mutation may have originated during early embryogenesis.

Point mutations of the connexin32 (GJB1) gene in X-linked dominant Charcot-Marie-Tooth neuropathy.

Ten families with X-linked dominant CMT neuropathy (CMTX1) were screened for point mutations of the connexin32 (Cx32, GJB1) gene. Two families showed missense mutations, respectively an A-->G transition at amino acid 102 (glutamate to glycine) and a C-->T transition at amino acid 142 (arginine to tryptophan). Three families showed nonsense mutations, respectively a C-->T transition at amino acid 22 (arginine to stop) a G-->T transversion at amino acid 186 (glutamate to stop), and a T-->A transversion at amino acid 217 (cysteine to stop). Five CMTX1 neuropathy families showed no evidence of point mutations of the connexin32 coding sequence. These findings suggest that the CMTX1 neuropathy genotype is heterogeneous or the result of promoter mutations, 3'-untranslated region mutations or exon/intron splice site mutations. Four of the reported mutations created or destroyed restriction enzyme sites: a HaeIII restriction enzyme site was destroyed by the mutation at amino acid position 22, a HpaII site was eliminated at amino acid position 142, a Bfal restriction site was created by the mutation at amino acid 186 and a Ddel restriction site was created by the mutation at amino acid 217. These changes allowed us to test family members for the mutations and observe the segregation of the disease with the mutations.

Ocular pathology of MELAS syndrome with mitochondrial DNA nucleotide 3243 point mutation.

PURPOSE: The authors describe the clinical, histopathologic, and ultrastructural findings in two eyes obtained at autopsy from a 21-year-old woman with mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS syndrome). METHODS: The eyes were obtained immediately after death. The right eye was fixed in 10% neutral-buffered formalin and processed for standard histologic examination. The left eye was fixed in a neutral-buffered 2.5% glutaraldehyde solution and processed for transmission electron microscopic examination. The authors compared the histologic and ultrastructural findings with the clinical features recorded photographically. RESULTS: The main clinical ophthalmologic features were bilateral ptosis, chronic external ophthalmoplegia, diffuse choroidal atrophy, atypical pigmentary retinopathy with macular involvement, and patchy atrophy of the iris stroma. Molecular genetic analysis detected a tRNA Leu (UUR) point mutation at position 3243 of mitochondrial DNA (MELAS genotype). Results of histologic and ultrastructural examination showed ragged-red fibers in the rectus muscles, degeneration of photoreceptor outer segments in the macula, hyperpigmentation and atrophy of the retinal pigment epithelium of the macula, atrophy of the iris stroma, early posterior subcapsular cataract, and optic atrophy. The retinal pigment epithelium, inner segments of the photoreceptors, smooth muscle cells of the choroidal and retinal vessels, the dilator and sphincter muscle of the iris, cornea, lens epithelium, and ciliary epithelium all contained many, often enlarged, structurally abnormal mitochondria with occasional paracrystalline inclusions and circular cristae. CONCLUSIONS: The MELAS-associated mitochondrial DNA nucleotide 3243 point mutation can cause a spectrum of ocular signs and symptoms that may be dependent on the patient's age and the amount of mutant mitochondrial DNA in the tissue. MELAS syndrome should be considered in the differential diagnosis of bilateral ptosis, external ophthalmoplegia, and atypical pigmentary retinopathy with macular involvement.