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 2009-2023. Genetic testing was performed using single gene testing, next-generation sequencing targeted panels, research whole exome and whole genome sequencing (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. 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'.

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.

Digenic FLNA and UCHL1 variants resulting in a complex phenotype.

AIM: X-linked variants in Filamin A (FLNA) are associated with the Ehlers-Danlos-syndrome-variant form of periventricular heterotopia, and autosomal dominant variants in ubiquitin C-terminal hydrolase L1 (UCHL1) are associated with a late-onset spastic ataxia, peripheral neuropathy and optic atrophy. Here we present a rare case involving both a novel heterozygous whole-gene deletion of UCHL1 and a heterozygous frameshift variant in the FLNA gene resulting in a complex phenotype. METHODS: A 67-year-old female with a confirmed pathogenic variant in the FLNA gene, resulting in an enlarged aorta and joint pains, presented with a 4-year history of severe sensory ataxia, upper motor neuron signs, eye movement abnormalities and severe sensory loss. RESULTS: Neurophysiology including Somatosensory-evoked potentials confirmed the sensory loss as predominantly preganglionic with denervation. Genetic testing revealed a digenic cause of her complex presentation, confirming a pathogenic frameshift variant in the FLNA gene and a heterozygous loss of function deletion in the UCHL1 gene. CONCLUSIONS: To the best of our knowledge, this is the first case with concomitant pathogenic variants in the FLNA and UCHL1 genes which explain the complex phenotype. The severe preganglionic sensory loss is also a rare finding and expands the phenotype of UCHL1 variants.

Genetic analysis and natural history of Charcot-Marie-Tooth disease CMTX1 due to GJB1 variants.

Charcot-Marie-Tooth disease (CMT) due to GJB1 variants (CMTX1) is the second most common form of CMT. It is an X-linked disorder characterized by progressive sensory and motor neuropathy with males affected more severely than females. Many reported GJB1 variants remain classified as variants of uncertain significance (VUS). In this large, international, multicentre study we prospectively collected demographic, clinical and genetic data on patients with CMT associated with GJB1 variants. Pathogenicity for each variant was defined using adapted American College of Medical Genetics criteria. Baseline and longitudinal analyses were conducted to study genotype-phenotype correlations, to calculate longitudinal change using the CMT Examination Score (CMTES), to compare males versus females, and pathogenic/likely pathogenic (P/LP) variants versus VUS. We present 387 patients from 295 families harbouring 154 variants in GJB1. Of these, 319 patients (82.4%) were deemed to have P/LP variants, 65 had VUS (16.8%) and three benign variants (0.8%; excluded from analysis); an increased proportion of patients with P/LP variants compared with using ClinVar's classification (74.6%). Male patients (166/319, 52.0%, P/LP only) were more severely affected at baseline. Baseline measures in patients with P/LP variants and VUS showed no significant differences, and regression analysis suggested the disease groups were near identical at baseline. Genotype-phenotype analysis suggested c.-17G>A produces the most severe phenotype of the five most common variants, and missense variants in the intracellular domain are less severe than other domains. Progression of disease was seen with increasing CMTES over time up to 8 years follow-up. Standard response mean (SRM), a measure of outcome responsiveness, peaked at 3 years with moderate responsiveness [change in CMTES (ΔCMTES) = 1.3 ± 2.6, P = 0.00016, SRM = 0.50]. Males and females progressed similarly up to 8 years, but baseline regression analysis suggested that over a longer period, females progress more slowly. Progression was most pronounced for mild phenotypes (CMTES = 0-7; 3-year ΔCMTES = 2.3 ± 2.5, P = 0.001, SRM = 0.90). Enhanced variant interpretation has yielded an increased proportion of GJB1 variants classified as P/LP and will aid future variant interpretation in this gene. Baseline and longitudinal analysis of this large cohort of CMTX1 patients describes the natural history of the disease including the rate of progression; CMTES showed moderate responsiveness for the whole group at 3 years and higher responsiveness for the mild group at 3, 4 and 5 years. These results have implications for patient selection for upcoming clinical trials.

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.

Expanding the FDXR-Associated Disease Phenotype: Retinal Dystrophy Is a Recurrent Ocular Feature.

Purpose: The purpose of this study was to report retinal dystrophy as a novel clinical feature and expand the ocular phenotype in patients harboring biallelic candidate FDXR variants. Methods: Patients carrying biallelic candidate FDXR variants were identified by whole genome sequencing (WGS) as part of the National Institute for Health Research BioResource rare-disease and the UK's 100,000 Genomes Project (100KGP) with an additional case identified by exome sequencing. Retrospective clinical data were collected from the medical records. Haplotype reconstruction was performed in families harboring the same missense variant. Results: Ten individuals from 8 unrelated families with biallelic candidate variants in FDXR were identified. In addition to bilateral optic atrophy and variable extra-ocular findings, 7 of 10 individuals manifested retinal dystrophy comprising dysfunction and degeneration of both rod and cone photoreceptors. Five of 10 subjects had sensorineural hearing loss. The previously unreported missense variant (c.1115C > A, p.(Pro372His)) was found in 5 of 8 (62.5%) study families. Haplotype reconstruction using WGS data demonstrated a likely ancestral haplotype. Conclusions: FDXR-associated disease is a phenotypically heterogeneous disorder with retinal dystrophy being a major clinical feature observed in this cohort. In addition, we hypothesize that a number of factors are likely to drive the pathogenesis of optic atrophy, retinal degeneration, and perhaps the associated systemic manifestations.

Targeted next-generation sequencing panels in the diagnosis of Charcot-Marie-Tooth disease.

OBJECTIVE: To investigate the effectiveness of targeted next-generation sequencing (NGS) panels in achieving a molecular diagnosis in Charcot-Marie-Tooth disease (CMT) and related disorders in a clinical setting. METHODS: We prospectively enrolled 220 patients from 2 tertiary referral centers, one in London, United Kingdom (n = 120), and one in Iowa (n = 100), in whom a targeted CMT NGS panel had been requested as a diagnostic test. PMP22 duplication/deletion was previously excluded in demyelinating cases. We reviewed the genetic and clinical data upon completion of the diagnostic process. RESULTS: After targeted NGS sequencing, a definite molecular diagnosis, defined as a pathogenic or likely pathogenic variant, was reached in 30% of cases (n = 67). The diagnostic rate was similar in London (32%) and Iowa (29%). Variants of unknown significance were found in an additional 33% of cases. Mutations in GJB1, MFN2, and MPZ accounted for 39% of cases that received genetic confirmation, while the remainder of positive cases had mutations in diverse genes, including SH3TC2, GDAP1, IGHMBP2, LRSAM1, FDG4, and GARS, and another 12 less common genes. Copy number changes in PMP22, MPZ, MFN2, SH3TC2, and FDG4 were also accurately detected. A definite genetic diagnosis was more likely in cases with an early onset, a positive family history of neuropathy or consanguinity, and a demyelinating neuropathy. CONCLUSIONS: NGS panels are effective tools in the diagnosis of CMT, leading to genetic confirmation in one-third of cases negative for PMP22 duplication/deletion, thus highlighting how rarer and previously undiagnosed subtypes represent a relevant part of the genetic landscape of CMT.

Clinical, ophthalmological, imaging and genetic features in Brazilian patients with ARSACS.

BACKGROUND: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is an important form of inherited ataxia with a varied clinical spectrum. Detailed studies of phenotype and genotype are necessary to improve diagnosis and elucidate this disorder pathogenesis. OBJECTIVE AND METHODS: To investigate the clinical phenotype, retinal architecture, neuroimaging features and genetic profile of Brazilian patients with ARSACS, we performed neurological and ophthalmological evaluation in thirteen Brazilian patients with molecularly confirmed ARSACS, and examined their mutation profiles. Optical coherence tomography protocol (OCT) consisted in peripapillary retinal nerve fiber layer (RNFL) measurement and qualitative analysis of perifoveal scans. Neuroimaging protocol accessed the frequency of atrophy in cerebellum, corpus callosum and parietal lobe, brainstem signal abnormalities, and posterior fossa arachnoid cysts. We reviewed the literature to delineate the ARSACS phenotype in the largest series worldwide. RESULTS: All patients had ataxia and spasticity, and 11/13 had peripheral neuropathy. Macular microcysts were present in two patients. Peripapillary striations, dentate appearance of inner retina and papillomacular fold were found in eleven cases. All individuals exhibited thickening of RNFL in OCT. The most frequent radiological signs were cerebellar atrophy (13/13), biparietal atrophy (12/13), and linear pontine hypointensities (13/13). Genetic analysis revealed 14 different SACS variants, of which two are novel. CONCLUSION: Macular microcysts, inner retina dentate appearance and papillomacular fold are novel retinal imaging signs of ARSACS. Ophthalmological and neuroimaging changes are common findings in Brazilian patients. The core clinical features of ARSACS are ataxia, spasticity and peripheral neuropathy with onset predominantly in the first decade of life.

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.

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.

Structure and denaturation of 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1.

The secondary structure of the trimeric protein 4-chlorobenzoyl coenzyme A dehalogenase from Arthrobacter sp. strain TM-1, the second of three enzymes involved in the dechlorination of 4-chlorobenzoate to form 4-hydroxybenzoate, has been examined. E(mM) for the enzyme was 12.59. Analysis by circular dichroism spectrometry in the far uv indicated that 4-chlorobenzoyl coenzyme A dehalogenase was composed mostly of alpha-helix (56%) with lesser amounts of random coil (21%), beta-turn (13%) and beta-sheet (9%). These data are in close agreement with a computational prediction of secondary structure from the primary amino acid sequence, which indicated 55.8% alpha-helix, 33.7% random coil and 10.5% beta-sheet; the enzyme is, therefore, similar to the 4-chlorobenzoyl coenzyme A dehalogenase from Pseudomonas sp. CBS-3. The three-dimensional structure, including that of the presumed active site, predicted by computational analysis, is also closely similar to that of the Pseudomonas dehalogenase. Study of the stability and physicochemical properties revealed that at room temperature, the enzyme was stable for 24 h but was completely inactivated by heating to 60 degrees C for 5 min; thereafter by cooling at 1 degrees C min(-1) to 45 degrees C, 20.6% of the activity could be recovered. Mildly acidic (pH 5.2) or alkaline (pH 10.1) conditions caused complete inactivation, but activity was fully recovered on returning the enzyme to pH 7.4. Circular dichroism studies also indicated that secondary structure was little altered by heating to 60 degrees C, or by changing the pH from 7.4 to 6.0 or 9.2. Complete, irreversible destruction of, and maximal decrease in the fluorescence yield of the protein at 330-350 nm were brought about by 4.5 M urea or 1.1 M guanidinium chloride. Evidence was obtained to support the hypothetical three-dimensional model, that residues W140 and W167 are buried in a non-polar environment, whereas W182 appears at or close to the surface of the protein. At least one of the enzymes of the dehalogenase system (the combined 4-chlorobenzoate:CoA ligase, the dehalogenase and 4-hydroxybenzoyl coenzyme A thioesterase) appears to be capable of association with the cell membrane.

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1.

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.