Whole genome sequencing increases the diagnostic rate in Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is one of the most common and genetically heterogeneous inherited neurological diseases, with more than 130 disease-causing genes. Whole genome sequencing (WGS) has improved diagnosis across genetic diseases, but the diagnostic impact in CMT is yet to be fully reported. We present the diagnostic results from a single specialist inherited neuropathy centre, including the impact of WGS diagnostic testing. Patients were assessed at our specialist inherited neuropathy centre from 2009 to 2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome sequencing and WGS and, latterly, WGS through the UK National Health Service. Variants were assessed using the American College of Medical Genetics and Genomics and Association for Clinical Genomic Science criteria. Excluding patients with hereditary ATTR amyloidosis, 1515 patients with a clinical diagnosis of CMT and related disorders were recruited. In summary, 621 patients had CMT1 (41.0%), 294 CMT2 (19.4%), 205 intermediate CMT (CMTi, 13.5%), 139 hereditary motor neuropathy (HMN, 9.2%), 93 hereditary sensory neuropathy (HSN, 6.1%), 38 sensory ataxic neuropathy (2.5%), 72 hereditary neuropathy with liability to pressure palsies (HNPP, 4.8%) and 53 'complex' neuropathy (3.5%). Overall, a genetic diagnosis was reached in 76.9% (1165/1515). A diagnosis was most likely in CMT1 (96.8%, 601/621), followed by CMTi (81.0%, 166/205) and then HSN (69.9%, 65/93). Diagnostic rates remained less than 50% in CMT2, HMN and complex neuropathies. The most common genetic diagnosis was PMP22 duplication (CMT1A; 505/1165, 43.3%), then GJB1 (CMTX1; 151/1165, 13.0%), PMP22 deletion (HNPP; 72/1165, 6.2%) and MFN2 (CMT2A; 46/1165, 3.9%). We recruited 233 cases to the UK 100 000 Genomes Project (100KGP), of which 74 (31.8%) achieved a diagnosis; 28 had been otherwise diagnosed since recruitment, leaving a true diagnostic rate of WGS through the 100KGP of 19.7% (46/233). However, almost half of the solved cases (35/74) received a negative report from the study, and the diagnosis was made through our research access to the WGS data. The overall diagnostic uplift of WGS for the entire cohort was 3.5%. Our diagnostic rate is the highest reported from a single centre and has benefitted from the use of WGS, particularly access to the raw data. However, almost one-quarter of all cases remain unsolved, and a new reference genome and novel technologies will be important to narrow the 'diagnostic gap'.

A recurrent missense variant in ITPR3 causes demyelinating Charcot-Marie-Tooth with variable severity.

Charcot-Marie-Tooth (CMT) disease is a neuromuscular disorder affecting the peripheral nervous system. The diagnostic yield in demyelinating CMT (CMT1) is typically ∼80-95%, of which at least 60% is due to the PMP22 gene duplication. The remainder of CMT1 is more genetically heterogeneous. We used whole exome and whole genome sequencing data included in the GENESIS database to investigate novel causal genes and mutations in a cohort of ∼2,670 individuals with CMT neuropathy. A recurrent heterozygous missense variant p.Thr1424Met in the recently described CMT gene ITPR3, encoding IP3R3 (inositol 1,4,5-trisphosphate receptor 3) was identified. This previously reported p.Thr1424Met change was present in 33 affected individuals from nine unrelated families from multiple populations, representing an unusual recurrence rate at a mutational hotspot, strengthening the gene-disease relationship (GnomADv4 allele frequency 1.76e-6). Sanger sequencing confirmed the co-segregation of the CMT phenotype with the presence of the mutation in autosomal dominant and de novo inheritance patterns, including a four-generation family with multiple affected second-degree cousins. Probands from all families presented with slow nerve conduction velocities, matching the diagnostic category of CMT1. Remarkably, we observed a uniquely variable clinical phenotype for age at onset and phenotype severity in p.Thr1424Met carrying patients, even within families. Finally, we present data supportive of a dominant-negative effect of the p.Thr1424Met mutation with associated changes in protein expression in patient-derived cells.

Intermediate conduction velocity in two cases of Charcot-Marie-Tooth disease type 1A.

BACKGROUND AND PURPOSE: Charcot-Marie-Tooth disease type 1A (CMT1A) is the most prevalent hereditary neuropathy worldwide and classically has slow nerve conduction velocity (NCV), in most cases below 38 m/s. Two unrelated patients with motor NCVs in the upper limbs above 38 m/s are reported. METHOD: Case report. RESULTS: Two genetically confirmed CMT1A patients are presented, from two unrelated families (one of British origin and the other of Brazilian origin). Both individuals had upper limb motor NCVs above 38 m/s, with values ranging from 41.9 to 45 m/s in the median nerve and from 42 to 42.3 m/s in the ulnar nerve. They presented with a very mild phenotype, with CMT Neuropathy Score version 2 (CMTNSv2) of 6 and 5, respectively. In contrast, affected family members within both kinships exhibited a classical phenotype with more severe disease manifestation (CMTNSv2 ranging from 12 to 20) and motor NCVs below 30 m/s. CONCLUSION: These cases, although very rare, highlight the importance of testing PMP22 duplication in patients with intermediate conduction velocities.

Mutations in alpha-B-crystallin cause autosomal dominant axonal Charcot-Marie-Tooth disease with congenital cataracts.

BACKGROUND AND PURPOSE: Mutations in the alpha-B-crystallin (CRYAB) gene have initially been associated with myofibrillar myopathy, dilated cardiomyopathy and cataracts. For the first time, peripheral neuropathy is reported here as a novel phenotype associated with CRYAB. METHODS: Whole-exome sequencing was performed in two unrelated families with genetically unsolved axonal Charcot-Marie-Tooth disease (CMT2), assessing clinical, neurophysiological and radiological features. RESULTS: The pathogenic CRYAB variant c.358A>G;p.Arg120Gly was segregated in all affected patients from two unrelated families. The disease presented as late onset CMT2 (onset over 40 years) with distal sensory and motor impairment and congenital cataracts. Muscle involvement was probably associated in cases showing mild axial and diaphragmatic weakness. In all cases, nerve conduction studies demonstrated the presence of an axonal sensorimotor neuropathy along with chronic neurogenic changes on needle examination. DISCUSSION: In cases with late onset autosomal dominant CMT2 and congenital cataracts, it is recommended that CRYAB is considered for genetic testing. The identification of CRYAB mutations causing CMT2 further supports a continuous spectrum of expressivity, from myopathic to neuropathic and mixed forms, of a growing number of genes involved in protein degradation and chaperone-assisted autophagy.

Post-transcriptional microRNA repression of PMP22 dose in severe Charcot-Marie-Tooth disease type 1.

Copy number variation (CNV) may lead to pathological traits, and Charcot-Marie-Tooth disease type 1A (CMT1A), the commonest inherited peripheral neuropathy, is due to a genomic duplication encompassing the dosage-sensitive PMP22 gene. MicroRNAs act as repressors on post-transcriptional regulation of gene expression and in rodent models of CMT1A, overexpression of one such microRNA (miR-29a) has been shown to reduce the PMP22 transcript and protein level. Here we present genomic and functional evidence, for the first time in a human CNV-associated phenotype, of the 3' untranslated region (3'-UTR)-mediated role of microRNA repression on gene expression. The proband of the family presented with an early-onset, severe sensorimotor demyelinating neuropathy and harboured a novel de novo deletion in the PMP22 3'-UTR. The deletion is predicted to include the miR-29a seed binding site and transcript analysis of dermal myelinated nerve fibres using a novel platform, revealed a marked increase in PMP22 transcript levels. Functional evidence from Schwann cell lines harbouring the wild-type and mutant 3'-UTR showed significantly increased reporter assay activity in the latter, which was not ameliorated by overexpression of a miR-29a mimic. This shows the importance of miR-29a in regulating PMP22 expression and opens an avenue for therapeutic drug development.

Charcot-Marie-Tooth disease type 2CC due to NEFH variants causes a progressive, non-length-dependent, motor-predominant phenotype.

OBJECTIVE: Neurofilaments are the major scaffolding proteins for the neuronal cytoskeleton, and variants in NEFH have recently been described to cause axonal Charcot-Marie-Tooth disease type 2CC (CMT2CC). METHODS: In this large observational study, we present phenotype-genotype correlations on 30 affected and 3 asymptomatic mutation carriers from eight families. RESULTS: The majority of patients presented in adulthood with motor-predominant and lower limb-predominant symptoms and the average age of onset was 31.0±15.1 years. A prominent feature was the development of proximal weakness early in the course of the disease. The disease progressed rapidly, unlike other Charcot-Marie-Tooth disease (CMT) subtypes, and half of the patients (53%) needed to use a wheelchair on average 24.1 years after symptom onset. Furthermore, 40% of patients had evidence of early ankle plantarflexion weakness, a feature which is observed in only a handful of CMT subtypes. Neurophysiological studies and MRI of the lower limbs confirmed the presence of a non-length-dependent neuropathy in the majority of patients.All families harboured heterozygous frameshift variants in the last exon of NEFH, resulting in a reading frameshift to an alternate open reading frame and the translation of approximately 42 additional amino acids from the 3' untranslated region (3'-UTR). CONCLUSIONS: This phenotype-genotype study highlights the unusual phenotype of CMT2CC, which is more akin to spinal muscular atrophy rather than classic CMT. Furthermore, the study will enable more informative discussions on the natural history of the disease and will aid in NEFH variant interpretation in the context of the disease's unique molecular genetics.

A multicenter retrospective study of charcot-marie-tooth disease type 4B (CMT4B) associated with mutations in myotubularin-related proteins (MTMRs).

OBJECTIVE: Charcot-Marie-Tooth (CMT) disease 4B1 and 4B2 (CMT4B1/B2) are characterized by recessive inheritance, early onset, severe course, slowed nerve conduction, and myelin outfoldings. CMT4B3 shows a more heterogeneous phenotype. All are associated with myotubularin-related protein (MTMR) mutations. We conducted a multicenter, retrospective study to better characterize CMT4B. METHODS: We collected clinical and genetic data from CMT4B subjects in 18 centers using a predefined minimal data set including Medical Research Council (MRC) scores of nine muscle pairs and CMT Neuropathy Score. RESULTS: There were 50 patients, 21 of whom never reported before, carrying 44 mutations, of which 21 were novel and six representing novel disease associations of known rare variants. CMT4B1 patients had significantly more-severe disease than CMT4B2, with earlier onset, more-frequent motor milestones delay, wheelchair use, and respiratory involvement as well as worse MRC scores and motor CMT Examination Score components despite younger age at examination. Vocal cord involvement was common in both subtypes, whereas glaucoma occurred in CMT4B2 only. Nerve conduction velocities were similarly slowed in both subtypes. Regression analyses showed that disease severity is significantly associated with age in CMT4B1. Slopes are steeper for CMT4B1, indicating faster disease progression. Almost none of the mutations in the MTMR2 and MTMR13 genes, responsible for CMT4B1 and B2, respectively, influence the correlation between disease severity and age, in agreement with the hypothesis of a complete loss of function of MTMR2/13 proteins for such mutations. INTERPRETATION: This is the largest CMT4B series ever reported, demonstrating that CMT4B1 is significantly more severe than CMT4B2, and allowing an estimate of prognosis. ANN NEUROL 2019.

Cryptic Amyloidogenic Elements in the 3' UTRs of Neurofilament Genes Trigger Axonal Neuropathy.

Abnormal protein aggregation is observed in an expanding number of neurodegenerative diseases. Here, we describe a mechanism for intracellular toxic protein aggregation induced by an unusual mutation event in families affected by axonal neuropathy. These families carry distinct frameshift variants in NEFH (neurofilament heavy), leading to a loss of the terminating codon and translation of the 3' UTR into an extra 40 amino acids. In silico aggregation prediction suggested the terminal 20 residues of the altered NEFH to be amyloidogenic, which we confirmed experimentally by serial deletion analysis. The presence of this amyloidogenic motif fused to NEFH caused prominent and toxic protein aggregates in transfected cells and disrupted motor neurons in zebrafish. We identified a similar aggregation-inducing mechanism in NEFL (neurofilament light) and FUS (fused in sarcoma), in which mutations are known to cause aggregation in Charcot-Marie-Tooth disease and amyotrophic lateral sclerosis, respectively. In summary, we present a protein-aggregation-triggering mechanism that should be taken into consideration during the evaluation of stop-loss variants.

Development of MRC Centre MRI calf muscle fat fraction protocol as a sensitive outcome measure in Hereditary Sensory Neuropathy Type 1.

OBJECTIVES: Hereditary sensory neuropathy type 1 (HSN1) is a rare, slowly progressive neuropathy causing profound sensory deficits and often severe motor loss. L-serine supplementation is a possible candidate therapy but the lack of responsive outcome measures is a barrier for undertaking clinical trials in HSN1. We performed a 12-month natural history study to characterise the phenotype of HSN1 and to identify responsive outcome measures. METHODS: Assessments included Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), CMTNSv2-Rasch modified, nerve conduction studies, quantitative sensory testing, intraepidermal nerve fibre density (thigh), computerised myometry (lower limbs), plasma 1-deoxysphingolipid levels, calf-level intramuscular fat accumulation by MRI and patient-based questionnaires (Neuropathic Pain Symptom Inventory and 36-Short Form Health Survey version 2 [SF-36v2]). RESULTS: 35 patients with HSN1 were recruited. There was marked heterogeneity in the phenotype mainly due to differences between the sexes: males generally more severely affected. The outcome measures that significantly changed over 1 year and correlated with CMTNSv2, SF-36v2-physical component and disease duration were MRI determined calf intramuscular fat accumulation (mean change in overall calf fat fraction 2.36%, 95% CI 1.16 to 3.55, p=0.0004), pressure pain threshold on the hand (mean change 40 kPa, 95% CI 0.7 to 80, p=0.046) and myometric measurements of ankle plantar flexion (median change -0.5 Nm, IQR -9.5 to 0, p=0.0007), ankle inversion (mean change -0.89 Nm, 95% CI -1.66 to -0.12, p=0.03) and eversion (mean change -1.61 Nm, 95% CI -2.72 to -0.51, p=0.006). Intramuscular calf fat fraction was the most responsive outcome measure. CONCLUSION: MRI determined calf muscle fat fraction shows validity and high responsiveness over 12 months and will be useful in HSN1 clinical trials.

Genetic and clinical characteristics of NEFL-related Charcot-Marie-Tooth disease.

OBJECTIVES: To analyse and describe the clinical and genetic spectrum of Charcot-Marie-Tooth disease (CMT) caused by mutations in the neurofilament light polypeptide gene (NEFL). METHODS: Combined analysis of newly identified patients with NEFL-related CMT and all previously reported cases from the literature. RESULTS: Five new unrelated patients with CMT carrying the NEFL mutations P8R and N98S and the novel variant L311P were identified. Combined data from these cases and 62 kindreds from the literature revealed four common mutations (P8R, P22S, N98S and E396K) and three mutational hotspots accounting for 37 (55%) and 50 (75%) kindreds, respectively. Eight patients had de novo mutations. Loss of large-myelinated fibres was a uniform feature in a total of 21 sural nerve biopsies and 'onion bulb' formations and/or thin myelin sheaths were observed in 14 (67%) of them. The neurophysiological phenotype was broad but most patients with E90K and N98S had upper limb motor conduction velocities <38 m/s. Age of onset was ≤3 years in 25 cases. Pyramidal tract signs were described in 13 patients and 7 patients were initially diagnosed with or tested for inherited ataxia. Patients with E90K and N98S frequently presented before age 3 years and developed hearing loss or other neurological features including ataxia and/or cerebellar atrophy on brain MRI. CONCLUSIONS: NEFL-related CMT is clinically and genetically heterogeneous. Based on this study, however, we propose mutational hotspots and relevant clinical-genetic associations that may be helpful in the evaluation of NEFL sequence variants and the differential diagnosis with other forms of CMT.

MFN2 deletion of exons 7 and 8: founder mutation in the UK population.

Mitofusin 2 (MFN2) mutations are the most common cause of axonal Charcot-Marie-Tooth disease (CMT2). The majority are inherited in an autosomal dominant manner but recessive and semi-dominant kindreds have also been described. We previously reported a deletion of exons 7 and 8 resulting in nonsense-mediated decay, segregating with disease when present in trans with another pathogenic MFN2 mutation. Detailed clinical and electrophysiological data on a series of five affected patients from four kindreds and, when available, their parents and relatives were collected. MFN2 Sanger sequencing, multiplex ligation probe amplification, and haplotype analysis were performed. A severe early-onset CMT phenotype was seen in all cases: progressive distal weakness, wasting, and sensory loss from infancy or early childhood. Optic atrophy (four of five) and wheelchair dependency in childhood were common (four of five). All were compound heterozygous for a deletion of exons 7 and 8 in MFN2 with another previously reported pathogenic mutation (Phe216Ser, Thr362Met, and Arg707Trp). Carrier parents and relatives were unaffected (age range: 24-82 years). Haplotype analysis confirmed that the deletion had a common founder in all families.

Pain and small fiber function in Charcot-Marie-Tooth disease type 1A.

INTRODUCTION: Charcot-Marie-Tooth (CMT) disease type 1A is the most common form of CMT. The main clinical features are distal weakness, sensory loss, and skeletal deformities. Although pain is a frequent complaint, small fiber involvement in CMT1A has not been studied extensively. METHODS: We assessed pain and small fiber involvement in 49 CMT1A patients using a variety of pain scales, pain questionnaires, and thermal thresholds. RESULTS: Forty-three of 49 patients (88%) complained of pain. The pain was localized to the feet in 61% of patients. Only 18% of patients had neuropathic pain. Cold and warm detection thresholds were elevated in 53% and 12% of patients, respectively. CONCLUSIONS: Our findings confirm that CMT1A patients have significant pain, which is more likely to be multifactorial in origin and suggests that a proportion of patients have small fiber dysfunction affecting mainly thinly myelinated Aδ fibers.

Charcot-Marie-Tooth disease: frequency of genetic subtypes and guidelines for genetic testing.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is a clinically and genetically heterogeneous group of diseases with approximately 45 different causative genes described. The aims of this study were to determine the frequency of different genes in a large cohort of patients with CMT and devise guidelines for genetic testing in practice. METHODS: The genes known to cause CMT were sequenced in 1607 patients with CMT (425 patients attending an inherited neuropathy clinic and 1182 patients whose DNA was sent to the authors for genetic testing) to determine the proportion of different subtypes in a UK population. RESULTS: A molecular diagnosis was achieved in 62.6% of patients with CMT attending the inherited neuropathy clinic; in 80.4% of patients with CMT1 (demyelinating CMT) and in 25.2% of those with CMT2 (axonal CMT). Mutations or rearrangements in PMP22, GJB1, MPZ and MFN2 accounted for over 90% of the molecular diagnoses while mutations in all other genes tested were rare. CONCLUSION: Four commonly available genes account for over 90% of all CMT molecular diagnoses; a diagnostic algorithm is proposed based on these results for use in clinical practice. Any patient with CMT without a mutation in these four genes or with an unusual phenotype should be considered for referral for an expert opinion to maximise the chance of reaching a molecular diagnosis.

A novel p.Gln175X [corrected] premature stop mutation in the C-terminal end of HSP27 is a cause of CMT2.

Mutations in the gene HSPB1, encoding the small heat shock protein 27 (HSP27), are a cause of distal hereditary motor neuropathy (dHMN) and axonal Charcot-Marie-Tooth disease (CMT2). dHMN and CMT2 are differentiated by the presence of a sensory neuropathy in the latter although in the case of HSPB1 this division is artificial as CMT2 secondary to HSPB1 mutations is predominantly a motor neuropathy with only minimal sensory involvement. A recent study in mice has suggested that mutations in the C-terminus result in a motor only phenotype resembling dHMN, whereas mutations at the N-terminus result in a CMT2-like phenotype. However, we present a family with a novel mutation in the C-terminus of HSP27 (p.Gln175X) [corrected] with a motor predominant distal neuropathy but with definite sensory involvement compatible with CMT2. This case highlights the artificial distinction between patients with motor predominant forms of CMT2 and dHMN and argues against the hypothesis that mutations in the C-terminus have no sensory involvement.

A novel mutation in the nerve-specific 5'UTR of the GJB1 gene causes X-linked Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMT1X) is the second most common cause of CMT, and is usually caused by mutations in the gap junction protein beta 1 (GJB1) gene which codes for connexin 32 (CX32). CX32 has three tissue-specific promoters, P1 which is specific for liver and pancreas, P1a specific for liver, oocytes and embryonic stem cells, and P2 which is nerve-specific. Over 300 mutations have been described in GJB1, spread throughout the coding region. We describe two families with X-linked inheritance and a phenotype consistent with CMT1X who did not have mutations in the GJB1 coding region. The non-coding region of GJB1 was sequenced and an upstream exon-splicing variant found at approximately - 373G>A which segregated with the disease in both families and was not present in controls. This substitution is located at the last base of the nerve-specific 5'UTR and thus may disrupt splicing of the nerve-specific transcript. Online consensus splice-site programs predict a reduced score for the mutant sequence vs. the normal sequence. It is likely that other mutations within the GJB1 non-coding regions account for the CMT1X families who do not have coding region mutations.

Clinical, pathological and genetic characterization of hereditary sensory and autonomic neuropathy type 1 (HSAN I).

Hereditary sensory and autonomic neuropathy type I (HSAN I) is the most frequent type of hereditary neuropathy that primarily affects sensory neurons. The genetic locus for HSAN I has been mapped to chromosome 9q22.1-22.3 and recently the gene was identified as SPTLC1, encoding serine palmitoyltransferase, long chain base subunit-1. Sequencing in HSAN I families have previously identified mutations in exons 5, 6 and 13 of this gene. We analysed the SPTLC1 gene for mutations in 8 families with HSAN I, 60 individuals with sporadic sensory neuropathy, 6 HSAN II families, 20 Charcot-Marie-Tooth type I families and 20 families with Charcot-Marie-Tooth type II. Six HSAN I families and a single sporadic neuropathy case had an identical SPTLC1 mutation. No mutations were found in the other groups. Genetic haplotyping across the HSAN I critical region in 5 families and the sporadic case suggested a common founder. Several characteristics, previously not widely recognized were identified, including lack of penetrance of the SPTLC1 mutation in some individuals, variability in age of onset along with an earlier age of onset in younger generations, in some patients surprisingly early and often severe motor involvement and an earlier onset characterized by motor involvement with demyelinating features in males compared to females in 4 families. The sensory findings were often disassociated with prominent pain and temperature loss. Neurophysiology mainly showed a sensory axonal neuropathy but in many individuals there was electrical evidence of demyelination. Sural nerve biopsies from six affected individuals and the post-mortem findings in 1 case showed mainly axonal loss. This in depth study on the phenotype of HSAN I in 6 families and a single sporadic case with a common founder identifies a number of poorly recognized features in this disorder and highlights the clinical heterogeneity both within and between families suggesting the influence of other genetic and acquired factors.

Pilot phenotype and natural history study of hereditary neuropathies caused by mutations in the HSPB1 gene.

Mutations in HSPB1 are one of the commonest causes of distal Hereditary Motor Neuropathy (dHMN). Transgenic mouse models of the disease have identified HDAC6 inhibitors as promising treatments for the condition paving the way for human trials. A detailed phenotype and natural history study of HSPB1 neuropathy is therefore required in order to inform the duration and outcome measures of any future trials. Clinical and neurophysiological data and lower limb muscle MRI were collected both prospectively and retrospectively from patients with mutations in HSPB1. The natural history was assessed by recording the weighted Charcot-Marie-Tooth Examination Score (CMTES) at annual intervals in a subset of patients. 20 patients from 14 families were recruited into the study. The average age of onset was in the 4th decade. Patients presented with a length dependent neuropathy but with early ankle plantar flexion weakness. Neurophysiology confirmed a motor neuropathy but also showed sensory nerve involvement in most patients. Cross sectional muscle MRI revealed soleus and medial gastrocnemius fat infiltration as an early signature of mutant HSPB1 disease. In this study neither semi quantitative muscle MRI, the CMTES nor neurophysiology were able to detect disease progression in HSPB1 neuropathy over 1 or 2 years. Further studies are therefore required to identify a suitable biomarker before clinical trials in HSPB1 neuropathy can be undertaken.

Mutations in noncoding regions of GJB1 are a major cause of X-linked CMT.

OBJECTIVE: To determine the prevalence and clinical and genetic characteristics of patients with X-linked Charcot-Marie-Tooth disease (CMT) due to mutations in noncoding regions of the gap junction ß-1 gene (GJB1). METHODS: Mutations were identified by bidirectional Sanger sequence analysis of the 595 bases of the upstream promoter region, and 25 bases of the 3' untranslated region (UTR) sequence in patients in whom mutations in the coding region had been excluded. Clinical and neurophysiologic data were retrospectively collected. RESULTS: Five mutations were detected in 25 individuals from 10 kindreds representing 11.4% of all cases of CMTX1 diagnosed in our neurogenetics laboratory between 1996 and 2016. Four pathogenic mutations, c.-17G>A, c.-17+1G>T, c.-103C>T, and c.-146-90_146-89insT were detected in the 5'UTR. A novel mutation, c.*15C>T, was detected in the 3' UTR of GJB1 in 2 unrelated families with CMTX1 and is the first pathogenic mutation in the 3'UTR of any myelin-associated CMT gene. Mutations segregated with the phenotype, were at sites predicted to be pathogenic, and were not present in the normal population. CONCLUSIONS: Mutations in noncoding DNA are a major cause of CMTX1 and highlight the importance of mutations in noncoding DNA in human disease. Next-generation sequencing platforms for use in inherited neuropathy should therefore include coverage of these regions.