Screening for mutations in the peripheral myelin genes PMP22, MPZ and Cx32 (GJB1) in Russian Charcot-Marie-Tooth neuropathy patients

Charcot-Marie-Tooth disease (CMT) and related inherited peripheral neuropathies, including Dejerine-Sottas syndrome, congenital hypomyelination, and hereditary neuropathy with liability to pressure palsies (HNPP), are caused by mutations in three myelin genes: PMP22, MPZ and Cx32 (GJB1). The most common mutations are the 1.5 Mb CMT1A tandem duplication on chromosome 17p11.2-p12 in CMT1 patients and the reciprocal 1.5 Mb deletion in HNPP patients. We performed a mutation screening in 174 unrelated CMT patients and three HNPP families of Russian origin. The unrelated CMT patients included 108 clinically and electrophysiologically diagnosed CMT1 cases, 32 CMT2 cases, and 34 cases with unspecified CMT. Fifty-nine CMT1A duplications were found, of which 58 belonged to the CMT1 patient group. We found twelve distinct mutations in Cx32, six mutations in MPZ, and two mutations in PMP22. Of these respectively, eight, five, and two lead to a CMT1 phenotype. Eight mutations (Cx32: Ile20Asn/Gly21Ser, Met34Lys, Leu90Val, and Phe193Leu; MPZ: Asp134Gly, Lys138Asn, and Thr139Asn; PMP22: ValSer25-26del) were not reported previously. Phenotype-genotype correlations were based on nerve conduction velocity studies and mutation type.     

Mutational analysis and genotype/phenotype correlation in Turkish Charcot-Marie-Tooth Type 1 and HNPP patients

The major Charcot- Marie-Tooth Type 1 (CMT1) locus, CMT1A, and Hereditary neuropathy with liability to pressure palsies (HNPP) cosegregate with a 1.5-Mb duplication and a 1.5-Mb deletion, respectively, in band 17p11.2. Point mutations in peripheral myelin gene 22 (PMP22), myelin protein zero (MPZ), and connexin 32 (Cx32) have been reported in CMT1, and in PMP22 in HNPP patients without deletion. We have screened 54 CMT1 patients, of variable clinical severity, and 25 HNPP patients from Turkey, with no duplication or deletion, for mutations in the PMP22 and Cx32 genes. A novel frameshift mutation affecting the second extracellular domain of PMP22 was found in an HNPP patient, while a point mutation in the second transmembrane domain of the protein was detected in a CMT1 patient. Two point mutations affecting different domains of Cx32 were identified in two CMTX patients. Another patient was found to carry a polymorphism in a non-conserved codon of the Cx32 gene. The clinical phenotypes of the patients correlate well with the effect of the mutation on the protein.     

Novel mutations in the Charcot-Marie-Tooth disease genes PMP22, MPZ,and GJB1

Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous disorder of the peripheral nervous system. CMT type 1 is most frequently caused by a 1.4 Mb tandem duplication in chromosome 17p11.2 comprising the peripheral myelin protein 22 (PMP22) gene. Furthermore sequence variations of PMP22, myelin protein zero (MPZ) and the gap junction protein b 1 gene (GJB1 or Connexin 32) may cause a variety of distinct CMT phenotypes. In this study we screened DNA from 42 unrelated patients for mutations in the PMP22, MPZ and GJB1 genes. Four novel mutations were identified. A Val65Phe amino acid exchange in PMP22 causes CMT type 1 associated with deafness, in GJB1 Tyr7_Thr8delinsSer, Pro172Ala and Ser138Asn are causes of CMTX neuropathies".     

Charcot‐Marie‐Tooth disease type I and related demyelinating neuropathies: Mutation analysis in a large cohort of Italian families

Charcot‐Marie‐Tooth neuropathy type 1 (CMT1), the most common hereditary neurological disorder in humans, is characterized by clinical and genetic heterogeneity. It is caused mainly by a 1.5 Mb duplication in 17p11.2, but also by mutations in the myelin genes PMP22 (peripheral myelin protein 22), MPZ (myelin protein zero), Cx32 (connexin 32; also called GJB1), and EGR2 (early growth response 2). In this study, we have screened 172 index cases of Italian families in which there was at least one subject with a CMT1 diagnosis for the duplication on 17p11.2 and mutations in these genes. Among 170 informative unrelated patients, the overall duplication frequency was 57.6%. A difference could be observed between the duplication frequency in familial cases (71.6%) and that observed in non‐familial cases (36.8%). Among the non‐duplicated patients, 12 were mutated in Cx32, four in MPZ, two in PMP22, and none in the EGR2. In the non‐duplicated cases, the overall point mutation frequency for these genes was 25.0%. We describe the mutations identified, and consider possible genotype–phenotype correlation. Hum Mutat 18:32–41, 2001. © 2001 Wiley‐Liss, Inc.     

Molecular analysis in Japanese patients with Charcot-Marie-Tooth disease: DGGE analysis for PMP22, MPZ,and Cx32/GJB1 mutations

Charcot-Marie-Tooth disease (CMT) is a heterogeneous disorder and is traditionally classified into two major types, CMT type 1 (CMT1) and CMT type 2 (CMT2). Most CMT1 patients are associated with the duplication of 17p11.2-p12 (CMT1A duplication) and small numbers of patients have mutations of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), connexin 32 (Cx32/GJB1), and early growth response 2 (EGR2) genes. Some mutations of MPZ and Cx32 were also associated with the clinical CMT2 phenotype. We constructed denaturing gradient gel electrophoresis (DGGE) analysis as a screening method for PMP22, MPZ, and Cx32 mutations and studied 161 CMT patients without CMT1A duplication. We detected 27 mutations of three genes including 15 novel mutations; six of PMP22, three of MPZ, and six of Cx32. We finally identified 21 causative mutations in 22 unrelated patients and five polymorphic mutations. Eighteen of 22 patients carrying PMP22, MPZ, or Cx32 mutations presented with CMT1 and four of them with MPZ or Cx32 mutations presented with the CMT2 phenotype. DGGE analysis was sensitive for screening for those gene mutations, but causative gene mutation was not identified in many of the Japanese patients with CMT, especially with CMT1. Other candidate genes should be studied to elucidate the genetic basis of Japanese CMT patients.     

Mutational analysis of PMP22, MPZ, GJB1, EGR2 and NEFL in Korean Charcot-Marie-Tooth neuropathy patients

We examined CMT1A duplication of 17p11.2-p12, mutations of PMP22, MPZ (P0), GJB1 (Cx32), EGR2 and NEFL genes in 57 Korean families with patients diagnosed as having Charcot-Marie-Tooth (CMT) disease. The CMT1A duplication was present in 53.6% of 28 CMT type 1 patients. In the 42 CMT families without CMT1A duplication, 10 pathogenic mutations were found in 9 families. The 10 mutations were not detected in 105 healthy controls. Seven mutations (c.318delT (p.Ala106fs) in PMP22, c.352G>A (p.Asp118Asn), c.449-1G>T (3'-splice site), c.706A>G (p.Lys236Glu) in MPZ, c.407T>C (p.Val136Ala)[corrected], c.502T>C (p.Cys168Arg) in GJB1, and c.1001T>C (p.Leu334Pro) in NEFL) were determined to be novel. The mutation frequencies of PMP22 and MPZ were similar to those found in several European populations, however, it appeared that mutations in GJB1 are less frequent in East Asian CMT patients than in Eur opean patients. We described the identified mutations and phenotype-genotype correlations based on nerve conduction studies.     

Charcot-Marie-Tooth type X: A novel mutation in the Cx32 gene with central conduction slowing

Charcot-Marie-Tooth disease (CMT) is characterized by distal muscle weakness and wasting, often resulting in foot deformities and gait disturbances, distal sensory impairment and by more or less typical changes in sural nerve biopsy. CMT type 1 is also characterized by reduced nerve conduction velocities. For these demyelinating subtypes, most frequently a 1.5 Mb tandem duplication in chromosome 17p11.2-12 comprising the gene for the peripheral myelin protein 22 (PMP22) is observed (CMT1A), but point mutations in PMP22 have also rarely been reported. X-linked, dominant CMTX1 disease is the second most common type of these hereditary motor and sensory neuropathies (HMSN). Mutations in the X chromosomal gene Connexin32 (Cx32) synonymous gap junction beta-1 (GJB1) are detectable in most X-linked CMT families. We report a novel missense mutation--Tyr65His--in the first extracelullar domain of the Cx32 gene in a Czech CMTX1 family. The mutation was not detectable in 50 healthy controls. The clinical phenotype in both the male proband and his mother was moderate with pronounced peroneal weakness and foot drop. Nerve conduction velocities were intermediately decreased (31-38 m/s) in both patients and slowing of central acoustic conduction (BAEP) was found in both the son and the mother whereas visual central conduction slowing (VEP) was detectable only in the son.     

Diagnostic algorithms in Charcot–Marie–Tooth neuropathies: experiences from a German genetic laboratory on the basis of 1206 index patients

We present clinical features and genetic results of 1206 index patients and 124 affected relatives who were referred for genetic testing of Charcot–Marie–Tooth (CMT) neuropathy at the laboratory in Aachen between 2001 and 2012. Genetic detection rates were 56% in demyelinating CMT (71% of autosomal dominant (AD) CMT1/CMTX), and 17% in axonal CMT (24% of AD CMT2/CMTX). Three genetic defects (PMP22 duplication/deletion, GJB1/Cx32 or MPZ/P0 mutation) were responsible for 89.3% of demyelinating CMT index patients in whom a genetic diagnosis was achieved, and the diagnostic yield of the three main genetic defects in axonal CMT (GJB1/Cx32, MFN2, MPZ/P0 mutations) was 84.2%. De novo mutations were detected in 1.3% of PMP22 duplication, 25% of MPZ/P0, and none in GJB1/Cx32. Motor nerve conduction velocity was uniformly <38 m/s in median or ulnar nerves in PMP22 duplication, >40 m/s in MFN2, and more variable in GJB1/Cx32, MPZ/P0 mutations. Patients with CMT2A showed a broad clinical severity regardless of the type or position of the MFN2 mutation. Out of 75 patients, 8 patients (11%) with PMP22 deletions were categorized as CMT1 or CMT2. Diagnostic algorithms are still useful for cost‐efficient mutation detection and for the interpretation of large‐scale genetic data made available by next generation sequencing strategies.     

Animal models for inherited peripheral neuropathies

Recent progress in human genetics and neurobiology has led to the identification of various mutations in particular myelin genes as the cause for many of the known inherited demyelinating peripheral neuropathies. Mutations in 3 distinct myelin genes, PMP22, P₀, and connexin 32 cause the 3 major demyelinating subtypes of Charcot-Marie-Tooth (CMT) disease, CMT1A, CMT1B and CMTX, respectively. In addition, a reduction in the gene dosage of PMP22 causes hereditary neuropathy with liability to pressure palsies (HNPP), while particular point mutations in PMP22 and P₀ cause the severe Dejerine-Sottas (DS) neuropathy. A series of spontaneous and genetically engineered rodent mutants for genes for the above-mentioned myelin constituents are now available and their suitability to serve as models for these still untreatable diseases is an issue of particular interest. The spontaneous mutants Trembler-J and Trembler, with point mutations in PMP22, reflect some of the pathological alterations seen in CMT1A and DS patients, respectively. Furthermore, engineered mutants that either over or underexpress particular myelin genes are suitable models for patients who are similarly compromised in the gene dosage of the corresponding genes. In addition, engineered mutants heterozygously or homozygously deficient in the myelin component P₀ show the pathology of distinct CMT1B and DS patients, respectively, while Cx32 deficient mice develop pathological abnormalities similar to those of CMTX patients. Mutants that mimic human peripheral neuropathies might allow the development of strategies to alleviate the symptoms of the diseases, and help to define environmental risk factors for aggravation of the disease. In addition, such mutants might be instrumental in the development of strategies to cure the diseases by gene therapy.     

X-linked dominant Charcot-Marie-Tooth disease with connexin 32 (Cx32) mutations in Koreans

X-linked dominant Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy, caused mainly by a mutation of connexin 32 (Cx32) gene. We performed a mutation analysis of Cx32 by direct sequencing of the coding sequence, then identified 23 mutations from 28 Korean CMTX families. Nine mutations were not reported previously: Gly5Ser, Ser26fs, Val37Leu, Thr86Ile, Val152fs, Phe153Cys, Asp178X, Ala197Val, and Ile214Asn. The extracellular 2 (EC2) domain of Cx32 protein was the hot spot mutation domain in 44% of Koreans. Transmembrane domain 4 was rarely affected in Koreans (4%), compared with 14% of Europeans. The EC1 and intracellular domain was not affected in Koreans, although they were frequently affected in Europeans. This study revealed that the frequencies of CMTX with Cx32 mutations are specific to different ethnic groups. The frequency of CMTX (5.3%) caused by Cx32 mutation in Koreans is similar to those in Asians but lower than those in Europeans. This study suggests differences between CMTX patients with Cx32 mutations and ethnic background.     

Mutations in the peripheral myelin protein zero and connexin32 genes detected by non-isotopic RNase cleavage assay and their phenotypes in Japanese patients with Charcot-Marie-Tooth disease

Mutations of myelin protein zero (MPZ) and connexin32 (Cx32) genes were examined in 70 unrelated Japanese patients with Charcot-Marie-Tooth disease (CMT) without PMP22 gene duplication. A new method, which could detect base pair mismatches with Rnase cleavage on agarose gel electrophoresis, identified 5 and 4 mutations of the MPZ and Cx32 genes, respectively, including one novel mutation (Ser128Ter) of Cx32. This non-isotopic RNase cleavage assay (NIRCA) employed in the present study is very suitable for exploring mutations of MPZ and Cx32 genes in a large number of CMT patients, as the phenotype of patients with CMT is greatly divergent from demyelinating to axonal pathology.     

Infantile Demyelinating Neuropathy Associated with a de novo Point Mutation on Ser72 in PMP22 and Basal Lamina Onion Bulbs in Skin Biopsy

Codon 72 has been designated as a hot spot for distinct missense mutations in the peripheral myelin protein 22 (PMP22) gene. Ser72Leu substitution was associated with Dejerine-Sottas syndrome (DSS) in four patients and with congenital hypomyelination neuropathy (CHN) in one patient. Our objective was to report one other DSS patient with Ser72Leu substitution in PMP22 and to concurrently illustrate how less invasive procedures such as skin biopsy could provide a rapid and reliable alternative to conventional sural nerve biopsy for the characterization of histophenotypic features. A skin biopsy was carried out in a 2⁴/₁₂-year-old girl with muscle atrophy, hypotonia and weakness, as well as generalized areflexia and absent sensory and motor nerve responses. Standard electron microscope techniques were used. PMP22 was screened by automated direct nucleotide sequencing analysis. Morphological examination revealed basal lamina onion bulbs surrounding a de- or hypomyelinated axon in all nerve bundles. Mutation analysis demonstrated a missense point mutation in codon 72 of the PMP22 gene leading to a Ser72Leu substitution. Further genotype-phenotype correlations will have to determine whether morphologically distinct phenotypes can be correlated with specific mutations. For this purpose, cutaneous nerve bundles could serve as an alternative tool to help identify and classify subtypes in this heterogeneous syndrome.     

Mutation frequency for Charcot-Marie-Tooth disease type 1 in the Chinese population is similar to that in the global ethnic patients.

PURPOSE: To investigate the genetic loci/mutations among the Chinese Charcot-Marie-Tooth disease type 1 (CMT1), which accounts for approximately 70% of Charcot-Marie-Tooth; and to study the genetic heterogeneity and mutation frequency. METHODS: CMT1A duplication and mutations at loci of MPZ, Cx32/GJB1, EGR2, and LITAF/SIMPLE were analyzed among 32 clinically diagnosed CMT1 patients of Chinese ancestry. RESULTS: The CMT1A duplication was detected in 62.5% (20/32) CMT1 patients. This duplication accounts for the major mutation for Chinese CMT1. Among 12 cases that have no CMT1A duplication detected, three point mutations including one (3.1%) in MPZ and two (6.3%) in Cx32 were identified. No mutation was detected in genes PMP22, EGR2 and LITAF among the remaining nine (28.1%) CMT1 patients. CONCLUSION: The mutation frequency for the Chinese CMT1 is similar to that seen in the global ethnic population. Molecular testing of the CMT1A duplication, along with the loci of MPZ and Cx32, may detect the majority of Chinese CMT1 patients.     

Infantile demyelinating neuropathy associated with a de novo point mutation on Ser72 in PMP22 and basal lamina onion bulbs in skin biopsy

Codon 72 has been designated as a hot spot for distinct missense mutations in the peripheral myelin protein 22 (PMP22) gene. Ser72Leu substitution was associated with Dejerine-Sottas syndrome (DSS) in four patients and with congenital hypomyelination neuropathy (CHN) in one patient. Our objective was to report one other DSS patient with Ser72Leu substitution in PMP22 and to concurrently illustrate how less invasive procedures such as skin biopsy could provide a rapid and reliable alternative to conventional sural nerve biopsy for the characterization of histophenotypic features. A skin biopsy was carried out in a 2 4/12-year-old girl with muscle atrophy, hypotonia and weakness, as well as generalized areflexia and absent sensory and motor nerve responses. Standard electron microscope techniques were used. PMP22 was screened by automated direct nucleotide sequencing analysis. Morphological examination revealed basal lamina onion bulbs surrounding a de- or hypomyelinated axon in all nerve bundles. Mutation analysis demonstrated a missense point mutation in codon 72 of the PMP22 gene leading to a Ser72Leu substitution. Further genotype-phenotype correlations will have to determine whether morphologically distinct phenotypes can be correlated with specific mutations. For this purpose, cutaneous nerve bundles could serve as an alternative tool to help identify and classify subtypes in this heterogeneous syndrome.     

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

Ten families with X-linked dominant CMT neuropathy (CMTX1) werescreened for point mutations of the connexin32 (Cx32, GJB1)gene. Two families showed missense mutations, respectively anA     

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.     

Phenotypes of X-linked Charcot-Marie-Tooth disease and altered trafficking of mutant Connexin 32 (GJB1)

To clarify the pathomechanism in three patients with X-linked Charcot-Marie-Tooth disease (CMTX) and unique clinical features, we studied three connexin (Cx) 32 (GJB1) mutants with respect to cellular localization in cultured cells. Wild-type Cx32 and three Cx32 mutants (Val63Ile and Glu186Lys, obtained from CMTX patients with hearing impairment; and Arg22Gln, obtained from a CMTX patient with a fair number of onion-bulb formations) were transfected to rat pheochromocytoma cells (PC12). We investigated the expression of Cx32 protein in each clone by immunoblotting and immunohistochemical staining. While Cx32 protein with the Arg22Gln mutation was detectable immunohistochemically only in the cytoplasm, Cx32 protein with the Val63Ile or Glu186Lys mutation was detected in both the plasma membrane and the cytoplasm. Cx32 protein with the wild-type sequence was detected mostly in the plasma membrane, with plaques indicating the existence of active gap junction formation. These three Cx32 mutations associated with CMTX patients with unique clinical and pathological findings caused altered trafficking of the Cx32 protein. These altered expressions indicated loss of active gap junction formation with different expression abnormalities in these CMTX patients. Received: November 27, 2000 / Accepted: February 19, 2001     

Diagnostic yield of targeted sequential and massive panel approaches for inherited neuropathies

Diagnostic yield of genetic studies for Charcot-Marie-Tooth disease (CMT) is little known, with a lack of epidemiological data to build better diagnostic strategies outside the United States and Europe. We aimed to evaluate the performance of two molecular diagnostic strategies for patients with CMT, and to characterize epidemiological findings of these conditions in southern Brazil. We performed a single-center cross-sectional study, in which 94 patients (55 families) with CMT suspicion were evaluated. Overall, the diagnostic yield of the combined strategy of Multiplex-ligation-dependent-probe-amplification (MLPA) of PMP22/GJB1/MPZ and GJB1/MPZ/PMP22 Sanger sequencing was 63.6% (28/44) for index cases with demyelinating/intermediate CMT suspicion (21 CMT1A-PMP22, 5 CMTX1-GJB1 and 2 with probably CMT1B-MPZ diagnosis). Five of the 11 index cases (45.4%) with axonal CMT had at least a possible diagnosis with next generation sequencing (NGS) panel of 104 inherited neuropathies-related genes (one each with CMT1A-PMP22, CMT2A-MFN2, CMT2K-GDAP1, CMT2U-MARS, CMT2W-HARS1). Detailed clinical, neurophysiological and molecular data of families are provided. Sequential molecular diagnosis strategies with MLPA plus target Sanger sequencing for demyelinating/intermediate CMT had high diagnostic yield, and almost half of axonal CMT families had at least a possible diagnosis with the comprehensive NGS panel. Most frequent subtypes of CMT in our region are CMT1A-PMP22 and CMTX1-GJB1.     

Inherited neuropathies: from gene to disease

Inherited disorders of peripheral nerves represent a common group of neurologic diseases. Charcot-Marie-Tooth neuropathy type 1 (CMT1) is a genetically heterogeneous group of chronic demyelinating polyneuropathies with loci mapping to chromosome 17 (CMT1A), chromosome 1 (CMT1B) and to another unknown autosome (CMT1C). CMT1A is most often associated with a tandem 1.5-megabase (Mb) duplication in chromosome 17p11.2-12, or in rare patients may result from a point mutation in the peripheral myelin protein-22 (PMP22) gene. CMT1B is associated with point mutations in the myelin protein zero (P0 or MPZ) gene. The molecular defect in CMT1C is unknown. X-linked Charcot-Marie-Tooth neuropathy (CMTX), which has clinical features similar to CMT1, is associated with mutations in the connexin32 gene. Charcot-Marie-Tooth neuropathy type 2 (CMT2) is an axonal neuropathy, also of undetermined cause. One form of CMT2 maps to chromosome 1p36 (CMT2A), another to chromosome 3p (CMT2B) and another to 7p (CMT2D). Dejerine-Sottas disease (DSD), also called hereditary motor and sensory neuropathy type III (HMSNIII), is a severe, infantile-onset demyelinating polyneuropathy syndrome that may be associated with point mutations in either the PMP22 gene or the P0 gene and shares considerable clinical and pathological features with CMT1. Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant disorder that results in a recurrent, episodic demyelinating neuropathy. HNPP is associated with a 1.5-Mb deletion in chromosome 17p11.2-12 and results from reduced expression of the PMP22 gene. CMT1A and HNPP are reciprocal duplication/deletion syndromes originating from unequal crossover during germ cell meiosis. Other rare forms of demyelinating peripheral neuropathies map to chromosome 8q, 10q and 11q. Hereditary neuralgic amyotrophy (familial brachial plexus neuropathy) is an autosomal dominant disorder causing painful, recurrent brachial plexopathies and maps to chromosome 17q25.