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.

Clinical feature, GALC variant spectrum, and genotype-phenotype correlation in Korean Krabbe disease patients: Multicenter experience over 13 years.

Krabbe disease (KD) is an autosomal recessive neurodegenerative disorder caused by deficiency of the galactocerebrosidase (GALC) due to variants in the GALC gene. Here, we provide the first and the largest comprehensive analysis of clinical and genetic characteristics, and genotype-phenotype correlations of KD in Korean in comparison with other ethnic groups. From June 2010 to June 2023, 10 patients were diagnosed with KD through sequencing of GALC. Clinical features, and results of GALC sequencing, biochemical test, neuroimaging, and neurophysiologic test were obtained from medical records. An additional nine previously reported Korean KD patients were included for review. In Korean KD patients, the median age of onset was 2 years (3 months-34 years) and the most common phenotype was adult-onset (33%, 6/18) KD, followed by infantile KD (28%, 5/18). The most frequent variants were c.683_694delinsCTC (23%) and c.1901T>C (23%), while the 30-kb deletion was absent. Having two heterozygous pathogenic missense variants was associated with later-onset phenotype. Clinical features were similar to those of other ethnic groups. In Korean KD patients, the most common phenotype was the adult-onset type and the GALC variant spectrum was different from that of the Caucasian population. This study would further our understanding of KD.

TGFß4 alleviates the phenotype of Charcot-Marie-Tooth disease type 1A.

The duplication of the peripheral myelin protein 22 (PMP22) gene causes a demyelinating type of neuropathy, commonly known as Charcot-Marie-Tooth disease type 1A (CMT1A). Development of effective drugs for CMT1A still remains as an unmet medical need. In the present study, we assessed the role of the transforming growth factor beta 4 (TGFß4)/Nodal axis in the pathogenesis of CMT1A. First, we identified PMP22 overexpression-induced Nodal expression in Schwann cells, which might be one of the downstream effectors in CMT1A. Administration of Nodal protein at the developmental stage of peripheral nerves induced the demyelinating phenotype in vivo. Second, we further isolated TGFß4 as an antagonist that could abolish Nodal-induced demyelination. Finally, we developed a recombinant TGFß4-fragment crystallizable (Fc) fusion protein, CX201, and demonstrated that its application had promyelinating efficacy in Schwann cells. CX201 administration improved the demyelinating phenotypes of CMT1A mouse models at both pre-symptomatic and post-symptomatic stages. These results suggest that the TGFß4/Nodal axis plays a crucial role in the pathogenesis of CMT1A and might be a potential therapeutic target for CMT1A.

Compound Heterozygous Mutations of SACS in a Korean Cohort Study of Charcot-Marie-Tooth Disease Concurrent Cerebellar Ataxia and Spasticity.

Mutations in the SACS gene are associated with autosomal recessive spastic ataxia of Charlevoix-Saguenay disease (ARSACS) or complex clinical phenotypes of Charcot-Marie-Tooth disease (CMT). This study aimed to identify SACS mutations in a Korean CMT cohort with cerebellar ataxia and spasticity by whole exome sequencing (WES). As a result, eight pathogenic SACS mutations in four families were identified as the underlying causes of these complex phenotypes. The prevalence of CMT families with SACS mutations was determined to be 0.3%. All the patients showed sensory, motor, and gait disturbances with increased deep tendon reflexes. Lower limb magnetic resonance imaging (MRI) was performed in four patients and all had fatty replacements. Of note, they all had similar fatty infiltrations between the proximal and distal lower limb muscles, different from the neuromuscular imaging feature in most CMT patients without SACS mutations who had distal dominant fatty involvement. Therefore, these findings were considered a characteristic feature in CMT patients with SACS mutations. Although further studies with more cases are needed, our results highlight lower extremity MRI findings in CMT patients with SACS mutations and broaden the clinical spectrum. We suggest screening for SACS in recessive CMT patients with complex phenotypes of ataxia and spasticity.

PINK1 and Parkin rescue motor defects and mitochondria dysfunction induced by a patient-derived HSPB3 mutant in Drosophila models.

Small heat shock proteins (sHSPs) are ATP-independent molecular chaperones with the α-crystalline domain that is critical to their chaperone activity. Within the sHSP family, three (HSPB1, HSPB3, and HSPB8) proteins are linked with inherited peripheral neuropathies, including distal hereditary motor neuropathy (dHMN) and Charco-Marie-Tooth disease (CMT). In this study, we introduced the HSPB3 Y118H (HSPB3Y118H) mutant gene identified from the CMT2 family in Drosophila. With a missense mutation on its α-crystalline domain, this human HSPB3 mutant gene induced a loss of motor activity accompanied by reduced mitochondrial membrane potential in fly neuronal tissues. Moreover, mitophagy, a critical mechanism of mitochondrial quality control, is downregulated in fly motor neurons expressing HSPB3Y118H. Surprisingly, PINK1 and Parkin, the core regulators of mitophagy, successfully rescued these motor and mitochondrial abnormalities in HSPB3 mutant flies. Results from the first animal model of HSPB3 mutations suggest that mitochondrial dysfunction plays a critical role in HSPB3-associated human pathology.

Neural cell adhesion molecule 1 is a cellular target engaged plasma biomarker in demyelinating Charcot-Marie-Tooth disease.

BACKGROUND AND PURPOSE: Elevated plasma concentrations of neural cell adhesion molecule 1 (NCAM1) and p75 neurotrophin receptor (p75) in patients with peripheral neuropathy have been reported. This study aimed to determine the specificity of plasma concentration elevation of either NCAM1 or p75 in a subtype of Charcot-Marie-Tooth disease (CMT) and its correlation with pathologic nerve status and disease severity. METHODS: Blood samples were collected from 138 patients with inherited peripheral neuropathy and 51 healthy controls. Disease severity was measured using Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), and plasma concentrations of NCAM1 and p75 were analyzed by enzyme-linked immunosorbent assay. Eight sural nerves from CMT patients were examined to determine the relation of histopathology and plasma NCAM1 levels. RESULTS: Plasma concentration of NCAM1, but not p75, was specifically increased in demyelinating subtypes of CMT (median = 7100 pg/mL, p < 0.001), including CMT1A, but not in axonal subtype (5964 pg/mL, p > 0.05), compared to the control (3859 pg/mL). CMT1A patients with mild or moderate severity (CMTNSv2 < 20) showed higher levels of plasma NCAM1 than healthy controls. Immunofluorescent NCAM1 staining for the sural nerves of CMT patients showed that NCAM1-positive onion bulb cells and possible demyelinating Schwann cells might be associated with the specific increase of plasma NCAM1 in demyelinating CMT. CONCLUSIONS: The plasma NCAM1 levels in demyelinating CMT might be a surrogate biomarker reflecting pathological Schwann cell status and disease progression.

INF2 mutations in patients with a broad phenotypic spectrum of Charcot-Marie-Tooth disease and focal segmental glomerulosclerosis.

Mutations in INF2 are associated with the complex symptoms of Charcot-Marie-Tooth disease (CMT) and focal segmental glomerulosclerosis (FSGS). To date, more than 100 and 30 genes have been reported to cause these disorders, respectively. This study aimed to identify INF2 mutations in Korean patients with CMT. This study was conducted with 743 Korean families with CMT who were negative for PMP22 duplication. In addition, a family with FSGS was included in this study. INF2 mutations were screened using whole exome sequencing (WES) and filtering processes. As the results, four pathogenic INF2 mutations were identified in families with different clinical phenotypes: p.L78P and p.L132P in families with symptoms of both CMT and FSGS; p.C104Y in a family with CMT; and p.R218Q in a family with FSGS. Moreover, different CMT types were observed in families with CMT symptoms: CMT1 in two families and Int-CMT in another family. Hearing loss was observed in two families with CMT1. Pathogenicity was predicted by in silico analyses, and considerable conformational changes were predicted in the mutant proteins. Two mutations (p.L78P and p.C104Y) were unreported, and three families showed de novo mutations that were putatively occurred from fathers. This study suggests that patients with INF2 mutations show a broad phenotypic spectrum: CMT1, CMT1 + FSGS, CMTDIE + FSGS, and FSGS. Therefore, the genotype-phenotype correlation may be more complex than previously recognized. We believe that this study expands the clinical spectrum of patients with INF2 mutations and will be helpful in the molecular diagnosis of CMT and FSGS.

Cytokines secreted by mesenchymal stem cells reduce demyelination in an animal model of Charcot-Marie-Tooth disease.

INTRODUCTION: Demyelinating Charcot-Marie-Tooth disease (CMT) is caused by mutations in the genes that encode myelinating proteins or their transcription factors. Our study thus sought to assess the therapeutic effects of cytokines secreted from mesenchymal stem cells (MSCs) on this disease. METHODS: The therapeutic potential of Wharton's jelly MSCs (WJ-MSCs) and cytokines secreted by WJ-MSCs was evaluated on Schwann cells (SCs) exhibiting demyelination features, as well as a mouse model of demyelinating CMT. RESULTS: Co-culture with WJ-MSC protected PMP22-overexpressing SCs from apoptotic cell death. Using a cytokine array, the secretion of growth differentiation factor-15 (GDF-15) and amphiregulin (AREG) was found to be elevated in WJ-MSCs when co-incubated with the PMP22-overexpressing SCs. Administration of both cytokines into trembler-J (Tr-J) mice, an animal model of CMT, significantly enhanced motor nerve conduction velocity compared to the control group. More importantly, this treatment alleviated the demyelinating phenotype of Tr-J mice, as demonstrated by an improvement in the mean diameter and g-ratio of the myelinated axons. CONCLUSIONS: Our findings demonstrated that WJ-MSCs alleviate the demyelinating phenotype of CMT via the secretion of several cytokines. Further elucidation of the underlying mechanisms of GDF-15 and AREG in myelination might provide a robust basis for the development of effective therapies against demyelinating CMT.

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.

CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation.

The endothelial cilium is a microtubule-based organelle responsible for blood flow-induced mechanosensation and signal transduction during angiogenesis. The precise function and mechanisms by which ciliary mechanosensation occurs, however, are poorly understood. Although posttranslational modifications (PTMs) of cytoplasmic tubulin are known to be important in angiogenesis, the specific roles of ciliary tubulin PTMs play remain unclear. Here, we report that loss of centrosomal protein 41 (CEP41) results in vascular impairment in human cell lines and zebrafish, implying a previously unknown pro-angiogenic role for CEP41. We show that proper control of tubulin glutamylation by CEP41 is necessary for cilia disassembly and that is involved in endothelial cell (EC) dynamics such as migration and tubulogenesis. We show that in ECs responding to shear stress or hypoxia, CEP41 activates Aurora kinase A (AURKA) and upregulates expression of VEGFA and VEGFR2 through ciliary tubulin glutamylation, as well as leads to the deciliation. We further show that in hypoxia-induced angiogenesis, CEP41 is responsible for the activation of HIF1α to trigger the AURKA-VEGF pathway. Overall, our results suggest the CEP41-HIF1α-AURKA-VEGF axis as a key molecular mechanism of angiogenesis and demonstrate how important ciliary tubulin glutamylation is in mechanosense-responded EC dynamics.

Variants of aminoacyl-tRNA synthetase genes in Charcot-Marie-Tooth disease: A Korean cohort study.

Charcot-Marie-Tooth disease (CMT) and related diseases are a genetically and clinically heterogeneous group of peripheral neuropathies. Particularly, mutations in several aminoacyl-tRNA synthetase (ARS) genes have been reported to cause axonal CMT (CMT2) or distal hereditary motor neuropathy (dHMN). However, the common pathogenesis among CMT subtypes by different ARS gene defects is not well understood. This study was performed to investigate ARS gene mutations in a CMT cohort of 710 Korean families. Whole-exome sequencing was applied to 710 CMT patients who were negative for PMP22 duplication. We identified 12 disease-causing variants (from 13 families) in GARS1, AARS1, HARS1, WARS1, and YARS1 genes. Seven variants were determined to be novel. The frequency of overall ARS gene mutations was 1.22% among all independent patients diagnosed with CMT and 1.83% in patients negative for PMP22 duplication. WARS1 mutations have been reported to cause dHMN; however, in our patients with WARS1 variants, CMT was associated with sensory involvement. We analyzed genotype-phenotype correlations and expanded the phenotypic spectrum of patients with CMT possessing ARS gene variants. We also characterized clinical phenotypes according to ARS genes. This study will be useful for performing exact molecular and clinical diagnoses and providing reference data for other population studies.

Targeted PMP22 TATA-box editing by CRISPR/Cas9 reduces demyelinating neuropathy of Charcot-Marie-Tooth disease type 1A in mice.

Charcot-Marie-Tooth 1A (CMT1A) is the most common inherited neuropathy without a known therapy, which is caused by a 1.4 Mb duplication on human chromosome 17, which includes the gene encoding the peripheral myelin protein of 22 kDa (PMP22). Overexpressed PMP22 protein from its gene duplication is thought to cause demyelination and subsequently axonal degeneration in the peripheral nervous system (PNS). Here, we targeted TATA-box of human PMP22 promoter to normalize overexpressed PMP22 level in C22 mice, a mouse model of CMT1A harboring multiple copies of human PMP22. Direct local intraneural delivery of CRISPR/Cas9 designed to target TATA-box of PMP22 before the onset of disease, downregulates gene expression of PMP22 and preserves both myelin and axons. Notably, the same approach was effective in partial rescue of demyelination even after the onset of disease. Collectively, our data present a proof-of-concept that CRISPR/Cas9-mediated targeting of TATA-box can be utilized to treat CMT1A.

Farnesol Ameliorates Demyelinating Phenotype in a Cellular and Animal Model of Charcot-Marie-Tooth Disease Type 1A.

Charcot-Marie-Tooth disease (CMT) is a genetically heterogeneous disease affecting the peripheral nervous system that is caused by either the demyelination of Schwann cells or degeneration of the peripheral axon. Currently, there are no treatment options to improve the degeneration of peripheral nerves in CMT patients. In this research, we assessed the potency of farnesol for improving the demyelinating phenotype using an animal model of CMT type 1A. In vitro treatment with farnesol facilitated myelin gene expression and ameliorated the myelination defect caused by PMP22 overexpression, the major causative gene in CMT. In vivo administration of farnesol enhanced the peripheral neuropathic phenotype, as shown by rotarod performance in a mouse model of CMT1A. Electrophysiologically, farnesol-administered CMT1A mice exhibited increased motor nerve conduction velocity and compound muscle action potential compared with control mice. The number and diameter of myelinated axons were also increased by farnesol treatment. The expression level of myelin protein zero (MPZ) was increased, while that of the demyelination marker, neural cell adhesion molecule (NCAM), was reduced by farnesol administration. These data imply that farnesol is efficacious in ameliorating the demyelinating phenotype of CMT, and further elucidation of the underlying mechanisms of farnesol's effect on myelination might provide a potent therapeutic strategy for the demyelinating type of CMT.

Mutations in ATP1A1 Cause Dominant Charcot-Marie-Tooth Type 2.

Although mutations in more than 90 genes are known to cause CMT, the underlying genetic cause of CMT remains unknown in more than 50% of affected individuals. The discovery of additional genes that harbor CMT2-causing mutations increasingly depends on sharing sequence data on a global level. In this way-by combining data from seven countries on four continents-we were able to define mutations in ATP1A1, which encodes the alpha1 subunit of the Na+,K+-ATPase, as a cause of autosomal-dominant CMT2. Seven missense changes were identified that segregated within individual pedigrees: c.143T>G (p.Leu48Arg), c.1775T>C (p.Ile592Thr), c.1789G>A (p.Ala597Thr), c.1801_1802delinsTT (p.Asp601Phe), c.1798C>G (p.Pro600Ala), c.1798C>A (p.Pro600Thr), and c.2432A>C (p.Asp811Ala). Immunostaining peripheral nerve axons localized ATP1A1 to the axolemma of myelinated sensory and motor axons and to Schmidt-Lanterman incisures of myelin sheaths. Two-electrode voltage clamp measurements on Xenopus oocytes demonstrated significant reduction in Na+ current activity in some, but not all, ouabain-insensitive ATP1A1 mutants, suggesting a loss-of-function defect of the Na+,K+ pump. Five mutants fall into a remarkably narrow motif within the helical linker region that couples the nucleotide-binding and phosphorylation domains. These findings identify a CMT pathway and a potential target for therapy development in degenerative diseases of peripheral nerve axons.

Fibulin 5, a human Wharton's jelly-derived mesenchymal stem cells-secreted paracrine factor, attenuates peripheral nervous system myelination defects through the Integrin-RAC1 signaling axis.

In the peripheral nervous system (PNS), proper development of Schwann cells (SCs) contributing to axonal myelination is critical for neuronal function. Impairments of SCs or neuronal axons give rise to several myelin-related disorders, including dysmyelinating and demyelinating diseases. Pathological mechanisms, however, have been understood at the elementary level and targeted therapeutics has remained undeveloped. Here, we identify Fibulin 5 (FBLN5), an extracellular matrix (ECM) protein, as a key paracrine factor of human Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) to control the development of SCs. We show that co-culture with WJ-MSCs or treatment of recombinant FBLN5 promotes the proliferation of SCs through ERK activation, whereas FBLN5-depleted WJ-MSCs do not. We further reveal that during myelination of SCs, FBLN5 binds to Integrin and modulates actin remodeling, such as the formation of lamellipodia and filopodia, through RAC1 activity. Finally, we show that FBLN5 effectively restores the myelination defects of SCs in the zebrafish model of Charcot-Marie-Tooth (CMT) type 1, a representative demyelinating disease. Overall, our data propose human WJ-MSCs or FBLN5 protein as a potential treatment for myelin-related diseases, including CMT.

Texture analysis using T1-weighted images for muscles in Charcot-Marie-Tooth disease patients and volunteers.

OBJECTIVES: To explore whether texture features using T1-weighted images correlate with fat fraction, and whether they differ between Charcot-Marie-Tooth (CMT) disease patients and volunteers. METHODS: The institutional review board approved this retrospective study, and the requirement for informed consent was waived; data of eighteen CMT patients and eighteen healthy volunteers from a previous study was used. Texture features of the muscles including mean, standard deviation (SD), skewness, kurtosis, and entropy of the signal intensity were derived from T1-weighted images. Spearman's correlation analysis was used to assess the relationship between texture features and fat fraction measured by 3D multiple gradient echo Dixon-based sequence. Mann-Whitney U test was used to compare the texture features between CMT patients and volunteers. Intraobserver and interobserver agreements for the texture features were assessed using the intraclass correlation coefficient. RESULTS: The SD (ρ = 0.256, p < 0.001) and entropy (ρ = 0.263, p < 0.001) were significantly and positively correlated with fat fraction; skewness (ρ = - 0.110, p = 0.027) and kurtosis (ρ = - 0.149, p = 0.003) were significantly and inversely correlated with fat fraction. The CMT patients showed a significantly higher SD (63.45 vs. 49.26; p < 0.001), skewness (1.06 vs. 0.56; p < 0.001), kurtosis (4.00 vs. 1.81; p < 0.001), and entropy (3.20 vs. 3.02; p < 0.001) than did the volunteers. Intraobserver and interobserver agreements were almost perfect for mean, SD, and entropy. CONCLUSIONS: Texture features using T1-weighted images correlated with fat fraction and differed between CMT patients and volunteers. KEY POINTS: • Standard deviation and entropy of muscles derived from T1-weighted images were significantly and positively correlated with the muscle fat fraction. • Mean, standard deviation, and entropy were considered highly reliable in muscle analyses. • Texture features may have the potential to diagnose early stage of intramuscular fatty infiltration.

Loss-of-function of EBP50 is a new cause of hereditary peripheral neuropathy: EBP50 functions in peripheral nerve system.

Finding causative genetic mutations is important in the diagnosis and treatment of hereditary peripheral neuropathies. This study was conducted to find new genes involved in the pathophysiology of hereditary peripheral neuropathy. We identified a new mutation in the EBP50 gene, which is co-segregated with neuropathic phenotypes, including motor and sensory deficit in a family with Charcot-Marie-Tooth disease. EBP50 is known to be important for the formation of microvilli in epithelial cells, and the discovery of this gene mutation allowed us to study the function of EBP50 in the nervous system. EBP50 was strongly expressed in the nodal and paranodal regions of sciatic nerve fibers, where Schwann cell microvilli contact the axolemma, and at the growth tips of primary Schwann cells. In addition, EBP50 expression was decreased in mouse models of peripheral neuropathy. Knockout mice were used to study EBP50 function in the peripheral nervous system. Interestingly motor function deficit and abnormal histology of nerve fibers were observed in EBP50+/- heterozygous mice at 12 months of age, but not 3 months. in vitro studies using Schwann cells showed that NRG1-induced AKT activation and migration were significantly reduced in cells overexpressing the I325V mutant of EBP50 or cells with knocked-down EBP50 expression. In conclusion, we show for the first time that loss of function due to EBP50 gene deficiency or mutation can cause peripheral neuropathy.

Human HSPB1 mutation recapitulates features of distal hereditary motor neuropathy (dHMN) in Drosophila.

Distal hereditary motor neuropathies (dHMN) are a group of inherited peripheral nerve disorders characterized by length-dependent motor neuron weakness and subsequent muscle atrophy. Missense mutations in the gene encoding small heat shock protein HSPB1 (HSP27) have been associated with hereditary neuropathies including dHMN. HSPB1 is a member of the small heat shock protein (sHSP) family characterized by a highly conserved α-crystallin domain that is critical to their chaperone activity. In this study, we modeled HSPB1 mutant-induced neuropathies in Drosophila using a human HSPB1S135F mutant that has a missense mutation in its α-crystallin domain. Overexpression of the HSPB1 mutant produced no significant defect in the Drosophila development, however, a partial reduction in the life span was observed. Further, the HSPB1 mutant gene induced an obvious loss of motor activity when expressed in Drosophila neurons. Moreover, suppression of histone deacetylase 6 (HDAC6) expression, which has critical roles in HSPB1 mutant-induced axonal defects, successfully rescued the motor defects in the HSPB1 mutant Drosophila model.

Variation in SIPA1L2 is correlated with phenotype modification in Charcot- Marie- Tooth disease type 1A.

OBJECTIVE: Genetic modifiers in rare disease have long been suspected to contribute to the considerable variance in disease expression, including Charcot-Marie-Tooth disease type 1A (CMT1A). To address this question, the Inherited Neuropathy Consortium collected a large standardized sample of such rare CMT1A patients over a period of 8 years. CMT1A is caused in most patients by a uniformly sized 1.5 Mb duplication event involving the gene PMP22. METHODS: We genotyped DNA samples from 971 CMT1A patients on Illumina BeadChips. Genome-wide analysis was performed in a subset of 330 of these patients, who expressed the extremes of a hallmark symptom: mild and severe foot dorsiflexion strength impairment. SIPA1L2 (signal-induced proliferation-associated 1 like 2), the top identified candidate modifier gene, was expressed in the peripheral nerve, and our functional studies identified and confirmed interacting proteins using coimmunoprecipitation analysis, mass spectrometry, and immunocytochemistry. Chromatin immunoprecipitation and in vitro siRNA experiments were used to analyze gene regulation. RESULTS: We identified significant association of 4 single nucleotide polymorphisms (rs10910527, rs7536385, rs4649265, rs1547740) in SIPA1L2 with foot dorsiflexion strength (p < 1 × 10-7 ). Coimmunoprecipitation and mass spectroscopy studies identified ß-actin and MYH9 as SIPA1L2 binding partners. Furthermore, we show that SIPA1L2 is part of a myelination-associated coexpressed network regulated by the master transcription factor SOX10. Importantly, in vitro knockdown of SIPA1L2 in Schwannoma cells led to a significant reduction of PMP22 expression, hinting at a potential strategy for drug development. INTERPRETATION: SIPA1L2 is a potential genetic modifier of CMT1A phenotypic expressions and offers a new pathway to therapeutic interventions. ANN NEUROL 2019;85:316-330.

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.