Diverse CMT2 neuropathies are linked to aberrant G3BP interactions in stress granules.

Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.

Monoallelic variation in DHX9, the gene encoding the DExH-box helicase DHX9, underlies neurodevelopment disorders and Charcot-Marie-Tooth disease.

DExD/H-box RNA helicases (DDX/DHX) are encoded by a large paralogous gene family; in a subset of these human helicase genes, pathogenic variation causes neurodevelopmental disorder (NDD) traits and cancer. DHX9 encodes a BRCA1-interacting nuclear helicase regulating transcription, R-loops, and homologous recombination and exhibits the highest mutational constraint of all DDX/DHX paralogs but remains unassociated with disease traits in OMIM. Using exome sequencing and family-based rare-variant analyses, we identified 20 individuals with de novo, ultra-rare, heterozygous missense or loss-of-function (LoF) DHX9 variant alleles. Phenotypes ranged from NDDs to the distal symmetric polyneuropathy axonal Charcot-Marie-Tooth disease (CMT2). Quantitative Human Phenotype Ontology (HPO) analysis demonstrated genotype-phenotype correlations with LoF variants causing mild NDD phenotypes and nuclear localization signal (NLS) missense variants causing severe NDD. We investigated DHX9 variant-associated cellular phenotypes in human cell lines. Whereas wild-type DHX9 was restricted to the nucleus, NLS missense variants abnormally accumulated in the cytoplasm. Fibroblasts from an individual with an NLS variant also showed abnormal cytoplasmic DHX9 accumulation. CMT2-associated missense variants caused aberrant nucleolar DHX9 accumulation, a phenomenon previously associated with cellular stress. Two NDD-associated variants, p.Gly411Glu and p.Arg761Gln, altered DHX9 ATPase activity. The severe NDD-associated variant p.Arg141Gln did not affect DHX9 localization but instead increased R-loop levels and double-stranded DNA breaks. Dhx9-/- mice exhibited hypoactivity in novel environments, tremor, and sensorineural hearing loss. All together, these results establish DHX9 as a critical regulator of mammalian neurodevelopment and neuronal homeostasis.

Histidine supplementation can escalate or rescue HARS deficiency in a Charcot-Marie-Tooth disease model.

Aminoacyl-tRNA synthetases are essential enzymes responsible for charging amino acids onto cognate tRNAs during protein synthesis. In histidyl-tRNA synthetase (HARS), autosomal dominant mutations V133F, V155G, Y330C and S356N in the HARS catalytic domain cause Charcot-Marie-Tooth disease type 2 W (CMT2W), while tRNA-binding domain mutation Y454S causes recessive Usher syndrome type IIIB. In a yeast model, all human HARS variants complemented a genomic deletion of the yeast ortholog HTS1 at high expression levels. CMT2W associated mutations, but not Y454S, resulted in reduced growth. We show mistranslation of histidine to glutamine and threonine in V155G and S356N but not Y330C mutants in yeast. Mistranslating V155G and S356N mutants lead to accumulation of insoluble proteins, which was rescued by histidine. Mutants V133F and Y330C showed the most significant growth defect and decreased HARS abundance in cells. Here, histidine supplementation led to insoluble protein aggregation and further reduced viability, indicating histidine toxicity associated with these mutants. V133F proteins displayed reduced thermal stability in vitro, which was rescued by tRNA. Our data will inform future treatment options for HARS patients, where histidine supplementation may either have a toxic or compensating effect depending on the nature of the causative HARS variant.

Clinically relevant mouse models of Charcot-Marie-Tooth type 2S.

Charcot-Marie-Tooth disease is an inherited peripheral neuropathy that is clinically and genetically heterogenous. Mutations in IGHMBP2, a ubiquitously expressed DNA/RNA helicase, have been shown to cause the infantile motor neuron disease spinal muscular atrophy with respiratory distress type 1 (SMARD1), and, more recently, juvenile-onset Charcot-Marie-Tooth disease type 2S (CMT2S). Using CRISPR-cas9 mutagenesis, we developed the first mouse models of CMT2S [p.Glu365del (E365del) and p.Tyr918Cys (Y918C)]. E365del is the first CMT2S mouse model to be discovered and Y918C is the first human CMT2S allele knock-in model. Phenotypic characterization of the homozygous models found progressive peripheral motor and sensory axonal degeneration. Neuromuscular and locomotor assays indicate that both E365del and Y918C mice have motor deficits, while neurobehavioral characterization of sensory function found that E365del mutants have mechanical allodynia. Analysis of femoral motor and sensory nerves identified axonal degeneration, which does not impact nerve conduction velocities in E365del mice, but it does so in the Y918C model. Based on these results, the E365del mutant mouse, and the human allele knock-in, Y918C, represent mouse models with the hallmark phenotypes of CMT2S, which will be critical for understanding the pathogenic mechanisms of IGHMBP2. These mice will complement existing Ighmbp2 alleles modeling SMARD1 to help understand the complex phenotypic and genotypic heterogeneity that is observed in patients with IGHMBP2 variants.

A weakened interface in the P182L variant of HSP27 associated with severe Charcot-Marie-Tooth neuropathy causes aberrant binding to interacting proteins.

HSP27 is a human molecular chaperone that forms large, dynamic oligomers and functions in many aspects of cellular homeostasis. Mutations in HSP27 cause Charcot-Marie-Tooth (CMT) disease, the most common inherited disorder of the peripheral nervous system. A particularly severe form of CMT disease is triggered by the P182L mutation in the highly conserved IxI/V motif of the disordered C-terminal region, which interacts weakly with the structured core domain of HSP27. Here, we observed that the P182L mutation disrupts the chaperone activity and significantly increases the size of HSP27 oligomers formed in vivo, including in motor neurons differentiated from CMT patient-derived stem cells. Using NMR spectroscopy, we determined that the P182L mutation decreases the affinity of the HSP27 IxI/V motif for its own core domain, leaving this binding site more accessible for other IxI/V-containing proteins. We identified multiple IxI/V-bearing proteins that bind with higher affinity to the P182L variant due to the increased availability of the IxI/V-binding site. Our results provide a mechanistic basis for the impact of the P182L mutation on HSP27 and suggest that the IxI/V motif plays an important, regulatory role in modulating protein-protein interactions.

Homozygous MFN2 variants causing severe antenatal encephalopathy with clumped mitochondria.

Pathogenic variants in the MFN2 gene are commonly associated with autosomal dominant (CMT2A2A) or recessive (CMT2A2B) Charcot-Marie-Tooth disease, with possible involvement of the CNS. Here, we present a case of severe antenatal encephalopathy with lissencephaly, polymicrogyria and cerebellar atrophy. Whole genome analysis revealed a homozygous deletion c.1717-274_1734 del (NM_014874.4) in the MFN2 gene, leading to exon 16 skipping and in-frame loss of 50 amino acids (p.Gln574_Val624del), removing the proline-rich domain and the transmembrane domain 1 (TM1). MFN2 is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial fusion, shaping large mitochondrial networks within cells. In silico modelling showed that the loss of the TM1 domain resulted in a drastically altered topological insertion of the protein in the mitochondrial outer membrane. Fetus fibroblasts, investigated by fluorescent cell imaging, electron microscopy and time-lapse recording, showed a sharp alteration of the mitochondrial network, with clumped mitochondria and clusters of tethered mitochondria unable to fuse. Multiple deficiencies of respiratory chain complexes with severe impairment of complex I were also evidenced in patient fibroblasts, without involvement of mitochondrial DNA instability. This is the first reported case of a severe developmental defect due to MFN2 deficiency with clumped mitochondria.

Morc2a variants cause hydroxyl radical-mediated neuropathy and are rescued by restoring GHKL ATPase.

Mutations in the Microrchidia CW-type zinc finger 2 (MORC2) GHKL ATPase module cause a broad range of neuropathies, such as Charcot-Marie-Tooth disease type 2Z; however, the aetiology and therapeutic strategy are not fully understood. Previously, we reported that the Morc2a p.S87L mouse model exhibited neuropathy and muscular dysfunction through DNA damage accumulation. In the present study, we analysed the gene expression of Morc2a p.S87L mice and designated the primary causing factor. We investigated the pathological pathway using Morc2a p.S87L mouse embryonic fibroblasts and human fibroblasts harbouring MORC2 p.R252W. We subsequently assessed the therapeutic effect of gene therapy administered to Morc2a p.S87L mice. This study revealed that Morc2a p.S87L causes a protein synthesis defect, resulting in the loss of function of Morc2a and high cellular apoptosis induced by high hydroxyl radical levels. We considered the Morc2a GHKL ATPase domain as a therapeutic target because it simultaneously complements hydroxyl radical scavenging and ATPase activity. We used the adeno-associated virus (AAV)-PHP.eB serotype, which has a high CNS transduction efficiency, to express Morc2a or Morc2a GHKL ATPase domain protein in vivo. Notably, AAV gene therapy ameliorated neuropathy and muscular dysfunction with a single treatment. Loss-of-function characteristics due to protein synthesis defects in Morc2a p.S87L were also noted in human MORC2 p.S87L or p.R252W variants, indicating the correlation between mouse and human pathogenesis. In summary, CMT2Z is known as an incurable genetic disorder, but the present study demonstrated its mechanisms and treatments based on established animal models. This study demonstrates that the Morc2a p.S87L variant causes hydroxyl radical-mediated neuropathy, which can be rescued through AAV-based gene therapy.

A new mouse model of Charcot-Marie-Tooth 2J neuropathy replicates human axonopathy and suggest alteration in axo-glia communication.

Myelin is essential for rapid nerve impulse propagation and axon protection. Accordingly, defects in myelination or myelin maintenance lead to secondary axonal damage and subsequent degeneration. Studies utilizing genetic (CNPase-, MAG-, and PLP-null mice) and naturally occurring neuropathy models suggest that myelinating glia also support axons independently from myelin. Myelin protein zero (MPZ or P0), which is expressed only by Schwann cells, is critical for myelin formation and maintenance in the peripheral nervous system. Many mutations in MPZ are associated with demyelinating neuropathies (Charcot-Marie-Tooth disease type 1B [CMT1B]). Surprisingly, the substitution of threonine by methionine at position 124 of P0 (P0T124M) causes axonal neuropathy (CMT2J) with little to no myelin damage. This disease provides an excellent paradigm to understand how myelinating glia support axons independently from myelin. To study this, we generated targeted knock-in MpzT124M mutant mice, a genetically authentic model of T124M-CMT2J neuropathy. Similar to patients, these mice develop axonopathy between 2 and 12 months of age, characterized by impaired motor performance, normal nerve conduction velocities but reduced compound motor action potential amplitudes, and axonal damage with only minor compact myelin modifications. Mechanistically, we detected metabolic changes that could lead to axonal degeneration, and prominent alterations in non-compact myelin domains such as paranodes, Schmidt-Lanterman incisures, and gap junctions, implicated in Schwann cell-axon communication and axonal metabolic support. Finally, we document perturbed mitochondrial size and distribution along MpzT124M axons suggesting altered axonal transport. Our data suggest that Schwann cells in P0T124M mutant mice cannot provide axons with sufficient trophic support, leading to reduced ATP biosynthesis and axonopathy. In conclusion, the MpzT124M mouse model faithfully reproduces the human neuropathy and represents a unique tool for identifying the molecular basis for glial support of axons.

Charcot-Marie-Tooth type 2A in vivo models: Current updates.

Charcot-Marie-Tooth type 2A (CMT2A) is an inherited sensorimotor neuropathy associated with mutations within the Mitofusin 2 (MFN2) gene. These mutations impair normal mitochondrial functioning via different mechanisms, disturbing the equilibrium between mitochondrial fusion and fission, of mitophagy and mitochondrial axonal transport. Although CMT2A disease causes a significant disability, no resolutive treatment for CMT2A patients to date. In this context, reliable experimental models are essential to precisely dissect the molecular mechanisms of disease and to devise effective therapeutic strategies. The most commonly used models are either in vitro or in vivo, and among the latter murine models are by far the most versatile and popular. Here, we critically revised the most relevant literature focused on the experimental models, providing an update on the mammalian models of CMT2A developed to date. We highlighted the different phenotypic, histopathological and molecular characteristics, and their use in translational studies for bringing potential therapies from the bench to the bedside. In addition, we discussed limitations of these models and perspectives for future improvement.

How T118M peripheral myelin protein 22 predisposes humans to Charcot-Marie-Tooth disease.

Data from gnomAD indicate that a missense mutation encoding the T118M variation in human peripheral myelin protein 22 (PMP22) is found in roughly one of every 75 genomes of western European lineage (1:120 in the overall human population). It is unusual among PMP22 variants that cause Charcot-Marie-Tooth (CMT) disease in that it is not 100% penetrant. Here, we conducted cellular and biophysical studies to determine why T118M PMP22 predisposes humans to CMT, but with only incomplete penetrance. We found that T118M PMP22 is prone to mistraffic but differs even from the WT protein in that increased expression levels do not result in a reduction in trafficking efficiency. Moreover, the T118M mutant exhibits a reduced tendency to form large intracellular aggregates relative to other disease mutants and even WT PMP22. NMR spectroscopy revealed that the structure and dynamics of T118M PMP22 resembled those of WT. These results show that the main consequence of T118M PMP22 in WT/T118M heterozygous individuals is a reduction in surface-trafficked PMP22, unaccompanied by formation of toxic intracellular aggregates. This explains the incomplete disease penetrance and the mild neuropathy observed for WT/T118M CMT cases. We also analyzed BioVU, a biobank linked to deidentified electronic medical records, and found a statistically robust association of the T118M mutation with the occurrence of long and/or repeated episodes of carpal tunnel syndrome. Collectively, our results illuminate the cellular effects of the T118M PMP22 variation leading to CMT disease and indicate a second disorder for which it is a risk factor.

Charcot-Marie-tooth disease type 2A: An update on pathogenesis and therapeutic perspectives.

Mutations in the gene encoding MFN2 have been identified as associated with Charcot-Marie-Tooth disease type 2A (CMT2A), a neurological disorder characterized by a broad clinical phenotype involving the entire nervous system. MFN2, a dynamin-like GTPase protein located on the outer mitochondrial membrane, is well-known for its involvement in mitochondrial fusion. Numerous studies have demonstrated its participation in a network crucial for various other mitochondrial functions, including mitophagy, axonal transport, and its controversial role in endoplasmic reticulum (ER)-mitochondria contacts. Considerable progress has been made in the last three decades in elucidating the disease pathogenesis, aided by the generation of animal and cellular models that have been instrumental in studying disease physiology. A review of the literature reveals that, up to now, no definitive pharmacological treatment for any CMT2A variant has been established; nonetheless, recent years have witnessed substantial progress. Many treatment approaches, especially concerning molecular therapy, such as histone deacetylase inhibitors, peptide therapy to increase mitochondrial fusion, the new therapeutic strategies based on MF1/MF2 balance, and SARM1 inhibitors, are currently in preclinical testing. The literature on gene silencing and gene replacement therapies is still limited, except for a recent study by Rizzo et al.(Rizzo et al., 2023), which recently first achieved encouraging results in in vitro and in vivo models of the disease. The near-future goal for these promising therapies is to progress to the stage of clinical translation.

Boosting BDNF in muscle rescues impaired axonal transport in a mouse model of DI-CMTC peripheral neuropathy.

Charcot-Marie-Tooth disease (CMT) is a genetic peripheral neuropathy caused by mutations in many functionally diverse genes. The aminoacyl-tRNA synthetase (ARS) enzymes, which transfer amino acids to partner tRNAs for protein synthesis, represent the largest protein family genetically linked to CMT aetiology, suggesting pathomechanistic commonalities. Dominant intermediate CMT type C (DI-CMTC) is caused by YARS1 mutations driving a toxic gain-of-function in the encoded tyrosyl-tRNA synthetase (TyrRS), which is mediated by exposure of consensus neomorphic surfaces through conformational changes of the mutant protein. In this study, we first showed that human DI-CMTC-causing TyrRSE196K mis-interacts with the extracellular domain of the BDNF receptor TrkB, an aberrant association we have previously characterised for several mutant glycyl-tRNA synthetases linked to CMT type 2D (CMT2D). We then performed temporal neuromuscular assessments of YarsE196K mice modelling DI-CMT. We determined that YarsE196K homozygotes display a selective, age-dependent impairment in in vivo axonal transport of neurotrophin-containing signalling endosomes, phenocopying CMT2D mice. This impairment is replicated by injection of recombinant TyrRSE196K, but not TyrRSWT, into muscles of wild-type mice. Augmenting BDNF in DI-CMTC muscles, through injection of recombinant protein or muscle-specific gene therapy, resulted in complete axonal transport correction. Therefore, this work identifies a non-cell autonomous pathomechanism common to ARS-related neuropathies, and highlights the potential of boosting BDNF levels in muscles as a therapeutic strategy.

Distinct roles for the Charcot-Marie-Tooth disease-causing endosomal regulators Mtmr5 and Mtmr13 in axon radial sorting and Schwann cell myelination.

The form of Charcot-Marie-Tooth type 4B (CMT4B) disease caused by mutations in myotubularin-related 5 (MTMR5; also called SET binding factor 1, SBF1) shows a spectrum of axonal and demyelinating nerve phenotypes. This contrasts with the CMT4B subtypes caused by MTMR2 or MTMR13 (SBF2) mutations, which are characterized by myelin outfoldings and classic demyelination. Thus, it is unclear whether MTMR5 plays an analogous or distinct role from that of its homolog, MTMR13, in the peripheral nervous system (PNS). MTMR5 and MTMR13 are pseudophosphatases predicted to regulate endosomal trafficking by activating Rab GTPases and binding to the phosphoinositide 3-phosphatase MTMR2. In the mouse PNS, Mtmr2 was required to maintain wild-type levels of Mtmr5 and Mtmr13, suggesting that these factors function in discrete protein complexes. Genetic elimination of both Mtmr5 and Mtmr13 in mice led to perinatal lethality, indicating that the two proteins have partially redundant functions during embryogenesis. Loss of Mtmr5 in mice did not cause CMT4B-like myelin outfoldings. However, adult Mtmr5-/- mouse nerves contained fewer myelinated axons than control nerves, likely as a result of axon radial sorting defects. Consistently, Mtmr5 levels were highest during axon radial sorting and fell sharply after postnatal day seven. Our findings suggest that Mtmr5 and Mtmr13 ensure proper axon radial sorting and Schwann cell myelination, respectively, perhaps through their direct interactions with Mtmr2. This study enhances our understanding of the non-redundant roles of the endosomal regulators MTMR5 and MTMR13 during normal peripheral nerve development and disease.

MITOL-mediated DRP1 ubiquitylation and degradation promotes mitochondrial hyperfusion in a CMT2A-linked MFN2 mutant.

Mutations in mitofusin 2 (MFN2) that are associated with the pathology of the debilitating neuropathy Charcot-Marie-Tooth type 2A (CMT2A) are known to alter mitochondrial morphology. One such abundant MFN2 mutation, R364W, results in the generation of elongated, interconnected mitochondria. However, the mechanism leading to this mitochondrial aberration remains poorly understood. Here, we show that mitochondrial hyperfusion in the presence of R364W-MFN2 is due to increased degradation of DRP1 (also known as DNM1L). The E3 ubiquitin ligase MITOL (also known as MARCHF5) is known to ubiquitylate both MFN2 and DRP1. Interaction with and subsequent ubiquitylation by MITOL is stronger in the presence of wild-type MFN2 than with R364W-MFN2. This differential interaction of MITOL with MFN2 in the presence of R364W-MFN2 renders the ligase more available for DRP1 ubiquitylation. Multi-monoubiquitylation and proteasomal degradation of DRP1 in R364W-MFN2 cells in the presence of MITOL eventually leads to mitochondrial hyperfusion. Here, we provide a mechanistic insight into mitochondrial hyperfusion, while also reporting that MFN2 can indirectly modulate DRP1 - an effect not shown previously. This article has an associated First Person interview with the first author of the paper.

Alternative splicing expands the clinical spectrum of NDUFS6-related mitochondrial disorders.

PURPOSE: We describe 3 families with Charcot-Marie-Tooth neuropathy (CMT), harboring a homozygous NDUFS6 NM_004553.6:c.309+5G>A variant previously linked to fatal Leigh syndrome. We aimed to characterize clinically and molecularly the newly identified patients and understand the mechanism underlying their milder phenotype. METHODS: The patients underwent extensive clinical examinations. Exome sequencing was done in 4 affected individuals. The functional effect of the c.309+5G>A variant was investigated in patient-derived EBV-transformed lymphoblasts at the complementary DNA, protein, and mitochondrial level. Alternative splicing was evaluated using complementary DNA long-read sequencing. RESULTS: All patients presented with early-onset, slowly progressive axonal CMT, and nystagmus; some exhibited additional central nervous system symptoms. The c.309+5G>A substitution caused the expression of aberrantly spliced transcripts and negligible levels of the canonical transcript. Immunoblotting showed reduced levels of mutant isoforms. No detectable defects in mitochondrial complex stability or bioenergetics were found. CONCLUSION: We expand the clinical spectrum of NDUFS6-related mitochondrial disorders to include axonal CMT, emphasizing the clinical and pathophysiologic overlap between these 2 clinical entities. This work demonstrates the critical role that alternative splicing may play in modulating the severity of a genetic disorder, emphasizing the need for careful consideration when interpreting splice variants and their implications on disease prognosis.

Disease Progression in Charcot-Marie-Tooth Disease Related to MPZ Mutations: A Longitudinal Study.

OBJECTIVE: The paucity of longitudinal natural history studies in MPZ neuropathy remains a barrier to clinical trials. We have completed a longitudinal natural history study in patients with MPZ neuropathies across 13 sites of the Inherited Neuropathies Consortium. METHODS: Change in Charcot-Marie-Tooth Examination Score (CMTES) and Rasch modified CMTES (CMTES-R) were evaluated using longitudinal regression over a 5-year period in subjects with MPZ neuropathy. Data from 139 patients with MPZ neuropathy were examined. RESULTS: The average baseline CMTES and CMTES-R were 10.84 (standard deviation [SD] = 6.0, range = 0-28) and 14.60 (SD = 7.56, range = 0-32), respectively. A mixed regression model showed significant change in CMTES at years 2-5 (mean change from baseline of 0.87 points at 2 years, p = 0.008). Subgroup analysis revealed greater change in CMTES at 2 years in subjects with axonal as compared to demyelinating neuropathy (mean change of 1.30 points [p = 0.016] vs 0.06 points [p = 0.889]). Patients with a moderate baseline neuropathy severity also showed more notable change, by estimate, than those with mild or severe neuropathy (mean 2-year change of 1.14 for baseline CMTES 8-14 [p = 0.025] vs -0.03 for baseline CMTES 0-7 [p = 0.958] and 0.25 for baseline CMTES ≥ 15 [p = 0.6897]). The progression in patients harboring specific MPZ mutations was highly variable. INTERPRETATION: CMTES is sensitive to change over time in adult patients with axonal but not demyelinating forms of MPZ neuropathy. Change in CMTES was greatest in patients with moderate baseline disease severity. These findings will inform future clinical trials of MPZ neuropathies. ANN NEUROL 2023;93:563-576.

Heterozygous Seryl-tRNA Synthetase 1 Variants Cause Charcot-Marie-Tooth Disease.

OBJECTIVE: Despite the increasing number of genes associated with Charcot-Marie-Tooth (CMT) disease, many patients currently still lack appropriate genetic diagnosis for this disease. Autosomal dominant mutations in aminoacyl-tRNA synthetases (ARSs) have been implicated in CMT. Here, we describe causal missense mutations in the gene encoding seryl-tRNA synthetase 1 (SerRS) for 3 families affected with CMT. METHODS: Whole-exome sequencing was performed in 16 patients and 14 unaffected members of 3 unrelated families. The functional impact of the genetic variants identified was investigated using bioinformatic prediction tools and confirmed using cellular and biochemical assays. RESULTS: Combined linkage analysis for the 3 families revealed significant linkage (Zmax LOD = 6.9) between the genomic co-ordinates on chromosome 1: 108681600-110300504. Within the linkage region, heterozygous SerRS missense variants segregated with the clinical phenotype in the 3 families. The mutant SerRS proteins exhibited reduced aminoacylation activity and abnormal SerRS dimerization, which suggests the impairment of total protein synthesis and induction of eIF2α phosphorylation. INTERPRETATION: Our findings suggest the heterozygous SerRS variants identified represent a novel cause for autosomal dominant CMT. Mutant SerRS proteins are known to impact various molecular and cellular functions. Our findings provide significant advances on the current understanding of the molecular mechanisms associated with ARS-related CMT. ANN NEUROL 2023;93:244-256.

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.

Genetic diversity in hereditary axonal neuropathy: Analyzing 53 Brazilian children.

BACKGROUND AND AIMS: The genetic epidemiology of inherited neuropathies in children remains largely unknown. In this study, we specifically investigated the genetic profile of a Brazilian cohort of pediatric patients with pure or complex axonal neuropathies, a crucial knowledge in the near future for establishing treatment priorities and perspectives for this group of patients. METHODS: Fifty-three pediatric patients who were assessed prior to reaching the age of 20, and who had clinical diagnoses of axonal hereditary neuropathy or presented with axonal neuropathy as the primary clinical feature, were included in the study. The recruitment of these cases took place from January 1, 2018, to December 31, 2020. The diagnosis was based on clinical and electrophysiological data. A molecular assessment was made using target-gene panel or whole-exome sequencing. Subsequently, segregation analysis was performed on available family members, and all candidate variants found were confirmed through Sanger. RESULTS: A molecular diagnosis was reached in 68% of the patients (n = 36/53), considering only pathogenic and probably pathogenic variants. Variants in MFN2 (n = 15) and GJB1 (n = 3) accounted for half of the genetically confirmed patients (50%; n = 18/36). The other 18 genetically diagnosed patients had variants in several less common genes. INTERPRETATION: Apart from MFN2 and GJB1 genes, universally recognized as a frequent cause of axonal neuropathies in most studied population, our Brazilian cohort of children with axonal neuropathies showed an important genetic heterogeneity, probably reflecting the multi ethnicity of the Brazilian population. Diagnostic, counseling, and future interventions should consider this characteristic.