Bi-allelic loss of function variants in COX20 gene cause autosomal recessive sensory neuronopathy.

Sensory neuronopathies are a rare and distinct subgroup of peripheral neuropathies, characterized by degeneration of the dorsal root ganglia neurons. About 50% of sensory neuronopathies are idiopathic and genetic causes remain to be clarified. Through a combination of homozygosity mapping and whole exome sequencing, we linked an autosomal recessive sensory neuronopathy to pathogenic variants in the COX20 gene. We identified eight unrelated families from the eastern Chinese population carrying a founder variant c.41A>G (p.Lys14Arg) within COX20 in either a homozygous or compound heterozygous state. All patients displayed sensory ataxia with a decrease in non-length-dependent sensory potentials. COX20 encodes a key transmembrane protein implicated in the assembly of mitochondrial complex IV. We showed that COX20 variants lead to reduction of COX20 protein in patient's fibroblasts and transfected cell lines, consistent with a loss-of-function mechanism. Knockdown of COX20 expression in ND7/23 sensory neuron cells resulted in complex IV deficiency and perturbed assembly of complex IV, which subsequently compromised cell spare respiratory capacity and reduced cell proliferation under metabolic stress. Consistent with mitochondrial dysfunction in knockdown cells, reduced complex IV assembly, enzyme activity and oxygen consumption rate were also found in patients' fibroblasts. We speculated that the mechanism of COX20 was similar to other causative genes (e.g. SURF1, COX6A1, COA3 and SCO2) for peripheral neuropathies, all of which are functionally important in the structure and assembly of complex IV. Our study identifies a novel causative gene for the autosomal recessive sensory neuronopathy, whose vital function in complex IV and high expression in the proprioceptive sensory neuron further underlines loss of COX20 contributing to mitochondrial bioenergetic dysfunction as a mechanism in peripheral sensory neuron disease.

Genetic spectrum and clinical profiles in a southeast Chinese cohort of Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth (CMT) disease is a heterogeneous group of inherited sensorimotor neuropathies. To clarify the genetic spectrum and clinical profiles in Chinese CMT patients, we enrolled 150 unrelated CMT patients from southeast China. We performed multiplex ligation-dependent probe amplification (MLPA) testing in all patients and next-generation sequencing (NGS) among those patients without PMP22 rearrangements. We identified PMP22 duplications in 40 patients and deletions in 12 patients. In addition, we found 19 novel variants and 36 known mutations in 57 patients. Among these 55 variants, 45 pathogenic or likely pathogenic variants were identified in 48 cases, and 10 variants with uncertain significance were identified in 9 cases. Therefore, we obtained a genetic diagnosis in 63.8% (88/138) of CMT patients and 66.7% (100/150) of all included patients. PMP22, GJB1, and MFN2 are the most common causative genes in CMT1 (demyelinated form), intermediate CMT, and CMT2 (axonal form), respectively. In this study, we identified a higher proportion of intermediate CMT, a relatively high frequency of NDRG1 mutations and clinical features of later onset age in CMT1A patients. Our results broaden the genetic and clinical spectrum of CMT patients, which can help optimize the genetic and clinical diagnosis.

Identification and functional characterization of two missense mutations in NDRG1 associated with Charcot-Marie-Tooth disease type 4D.

Charcot-Marie-Tooth disease type 4D (CMT4D) is an autosomal-recessive demyelinating form of CMT characterized by a severe distal motor and sensory neuropathy. NDRG1 is the causative gene for CMT4D. To date, only four mutations in NDRG1 -c.442C>T (p.Arg148*), c.739delC (p.His247Thrfs*74), c.538-1G>A, and duplication of exons 6-8-have been described in CMT4D patients. Here, using targeted next-generation sequencing examination, we identified for the first time two homozygous missense variants in NDRG1, c.437T>C (p.Leu146Pro) and c.701G>A (p.Arg234Gln), in two Chinese CMT families with consanguineous histories. Further functional studies were performed to characterize the biological effects of these variants. Cell culture transfection studies showed that mutant NDRG1 carrying p.Leu146Pro, p.Arg148*, or p.Arg234Gln variant degraded faster than wild-type NDRG1, resulting in lower protein levels. Live cell confocal microscopy and coimmunoprecipitation analysis indicated that these variants did not disrupt the interaction between NDRG1 and Rab4a protein. However, NDRG1-knockdown cells expressing mutant NDRG1 displayed enlarged Rab4a-positive compartments. Moreover, mutant NDRG1 could not enhance the uptake of DiI-LDL or increase the fraction of low-density lipoprotein receptor on the cell surface. Taken together, our study described two missense mutations in NDRG1 and emphasized the important role of NDRG1 in intracellular protein trafficking.

Inositol-requiring protein 1α signaling pathway is activated in the temporal cortex of patients with mesial temporal lobe epilepsy.

Accumulating evidence suggests that repeated seizures could induce endoplasmic reticulum (ER) stress. Inositol-requiring protein 1α (IRE1α) is a vital pro-apoptotic molecule in ER stress, but it remains unclear whether the signaling pathway mediated by IRE1α is involved in human temporal lobe epilepsy. In this report, we investigated IRE1α-mediated ER stress pro-apoptotic signaling pathway in resected anterior temporal neocortex from 32 patients with intractable mesial temporal lobe epilepsy by immunofluorescence and western blot analysis. Our results indicate that chronic epilepsy induces ER stress, and IRE1α-mediated ER stress apoptotic signaling pathway is involved in brain damage after repeated seizures, which may provide a new therapeutic target to prevent brain damage caused by epilepsy.

Biallelic SORD pathogenic variants cause Chinese patients with distal hereditary motor neuropathy.

Sorbitol dehydrogenase gene (SORD) has been identified as a novel causative gene of recessive forms of hereditary neuropathy, including Charcot-Marie-Tooth disease type 2 and distal hereditary motor neuropathy (dHMN). Our findings reveal two novel variants (c.404 A > G and c.908 + 1 G > C) and one known variant (c.757delG) within SORD in four Chinese dHMN families. Ex vivo cDNA polymerase chain reaction confirmed that c.908 + 1 G > C variant was associated with impaired splicing of the SORD transcript. In vitro cell functional studies showed that c.404 A > G variant resulted in aggregate formation of SORD and low protein solubility, confirming the pathogenicity of SORD variants. We have provided more evidence to establish SORD as a causative gene for dHMN.

A novel frameshift ACTN2 variant causes a rare adult-onset distal myopathy with multi-minicores.

INTRODUCTION: Distal myopathies are a group of rare muscle disorders characterized by selective or predominant weakness in the feet and/or hands. In 2019, ACTN2 gene was firstly identified to be a cause of a new adult-onset distal muscular dystrophy calling actininopathy and another distinctly different myopathy, named multiple structured core disease (MsCD). Thus, the various phenotypes and limited mutations in ACTN2-related myopathy make the genotype-phenotype correlation hard to understand. AIMS: To investigate the clinical features and histological findings in a Chinese family with distal myopathy. Whole exome sequencing and several functional studies were performed to explore the pathogenesis of the disease. RESULTS: We firstly identified a novel frameshift variant (c.2504delT, p.Phe835Serfs*66) within ACTN2 in a family including three patients. The patients exhibited adult-onset distal myopathy with multi-minicores, which, interestingly, was more like a combination of MsCD and actininopathy. Moreover, functional analysis using muscle samples revealed that the variant significantly increased the expression level of α-actinin-2 and resulted in abnormal Z-line organization of muscle fiber. Vitro studies suggested aggregate formations might be involved in the pathogenesis of the disease. CONCLUSION: Our results expanded the phenotypes of ACTN2-related myopathy and provided helpful information to clarify the molecular mechanisms.

Clinical and genetic characteristics of Chinese patients with reducing body myopathy.

Reducing body myopathy (RBM) is a rare myopathy characterized by reducing bodies (RBs) in morphological presentation. The clinical manifestations of RBM present a wide clinical spectrum, varying from infantile lethal form through childhood and adult benign forms. FHL1 gene is the causative gene of RBM. To date, only 6 Chinese RBM patients have been reported. Here, we reported the clinical presentations and genetic findings of 3 Chinese RBM patients from two families. Two novel pathogenic variants, c.395G>A and c.401_402insGAC, were identified by whole exome sequencing. Furthermore, by reviewing previous studies, we revealed that most RBM patients manifested with an early onset, symmetric, progressive limb-girdle and axial muscle weakness with joint contractures, rigid spine or scoliosis except familial female patients who exhibited asymmetric benign muscle involvements. Our results provide insightful information to help better diagnose and understand the disease.

Identification and functional characterization of novel GDAP1 variants in Chinese patients with Charcot-Marie-Tooth disease.

OBJECTIVE: To identify and characterize the pathogenicity of novel variants in Chinese patients with Charcot-Marie-Tooth disease. METHODS: Multiplex ligation-dependent probe amplification (MLPA) and whole-exome sequencing (WES) were performed in 30 unrelated CMT patients. Minigene assay was used to verify the effect of a novel splicing variant (c.694+1G>A) on pre-mRNA. Primary fibroblast cell lines were established from skin biopsies to characterize the biological effects of the novel variants p.L26R and p.S169fs. The mitochondrial structure was observed by an electron microscope. The expression level of protein was analyzed by Western Blotting. Mitochondrial dynamics and mitochondrial membrane potential (MMP, Δψm) were analyzed via immunofluorescence study. Mitochondrial ATP levels were analyzed via bioluminescence assay. The rate of oxygen consumption was measured with a Seahorse Bioscience XF-96 extracellular flux analyzer. RESULTS: We identified 10 pathogenic variants in three known CMT related genes, including three novel variants (p.L26R, p.S169fs, c.694+1G>A) and one known pathogenic variant (p.R120W) in GDAP1. Further, we described the clinical features of patients carrying pathogenic variants in GDAP1 and found that almost all Chinese CMT patients with GDAP1 variants present axonal type. The effect of c.694+1G>A on pre-mRNA was verified via minigene splice assay. Cellular biological effects showed ultrastructure damage of mitochondrial, reduced protein levels, different patterns of mitochondrial dynamics, decreased mitochondrial membrane potential (Δψm), ATP content, and defects in respiratory capacity in the patient carrying p.L26R and p.S169fs in GDAP1. INTERPRETATION: Our results broaden the genetic spectrum of GDAP1 and provided functional evidence for mitochondrial pathways in the pathogenesis of GDAP1 variants.

Functional study and pathogenicity classification of PRRT2 missense variants in PRRT2-related disorders.

AIMS: PRRT2 variants are associated with various paroxysmal disorders. To date, more than 90 PRRT2 variants have been reported in PRRT2-related disorders. Lack of functional study in majority of missense variants makes their pathogenicity uncertain. We aim to evaluate the clinical significance of PRRT2 missense variants by performing in vitro experiments. METHODS: We systematically reviewed PRRT2-related disorders and summarized reported PRRT2 missense variants. Protein expression and subcellular localization of mutant PRRT2 were investigated in mammal cells. American College of Medical Genetics and Genomics (ACMG) guidelines were used to analyze the pathogenicity of PRRT2 missense variants. RESULTS: A total of 29 PRRT2 missense variants were identified in PRRT2-related disorders. Ten variants were observed to affect both subcellular localization and protein level, three variants only affect membrane localization, and two variants only affect protein level. According to ACMG guidelines, 15 variants were finally classified as "likely pathogenic", three as "benign", three as "likely benign", and eight as "uncertain significance" variants. The likely pathogenic variants were concentrated in the C-terminal of PRRT2. CONCLUSIONS: The pathogenicity of eight uncertain significance variants needs further investigation. C-terminal of PRRT2 is crucial for its physiological function.

Associations between neuroanatomical abnormality and motor symptoms in paroxysmal kinesigenic dyskinesia.

INTRODUCTION: The pathophysiologic mechanism of paroxysmal kinesigenic dyskinesia (PKD) is largely unclear. Basal ganglia-thalamo-cortical circuit involvement is thought to underlie PKD pathophysiology. However, microstructural alternations in the motor circuit of PKD require further elucidation. METHODS: Diffusion tensor imaging and high-resolution T1-weighted imaging were performed on 30 PKD patients (15 PRRT2 carriers, 15 PRRT2 non-carriers) and 15 matched healthy controls. Tract-based spatial statistics were conducted on diffusion indices to examine microstructural integrity of white matter. Voxel-based morphometry analysis was used to examine volumetric changes of gray matter. Multiple regression was employed to test the contribution of demography, disease duration, and PRRT2 status to pathological changes in brain structure. RESULTS: Six (including two novel) PRRT2 mutations were identified in PKD patients who exhibited significantly reduced mean diffusivity mainly along the left corticospinal tract, and reduced gray matter volume in pre-supplementary motor area (preSMA) and right opercular part of inferior frontal gyrus (IFGoperc), compared to healthy controls. Both gray matter volume reductions in preSMA and diffusion indices of abnormal white matter negatively correlated with disease duration. Genotype-phenotype analysis revealed that PRRT2 mutation carriers had earlier onset age, longer attacks, and a larger proportion of bilateral symptoms than non-carriers. CONCLUSIONS: We observed that PRRT2 mutations were associated with disease severity, while neuroanatomical abnormality was associated with disease duration in patients with PKD. Aberrant microstructural changes in preSMA and IFG areas, independent of mutation status, point to dysregulated motor inhibition in patients and provide new insights into neurobiological mechanisms underlying motor symptoms of PKD.

Genetic Spectrum and Variability in Chinese Patients with Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by selective impairment of upper and lower motor neurons. We aimed to investigate the genetic spectrum and variability in Chinese patients with ALS. A total of 24 familial ALS (FALS) and 21 early-onset sporadic ALS (SALS) of Chinese ancestry were enrolled. Targeted next-generation sequencing (NGS) was performed in the probands, followed by verification by Sanger sequencing and co-segregation analysis. Clinical features of patients with pathogenic or likely pathogenic variants were present. The mutation frequency of ALS-related genes was then analyzed in Chinese population. In this cohort, 17 known mutations (9 SOD1, 5 FUS, 2 TARDBP and one SETX) were identified in 14 FALS and 6 early-onset SALS. Moreover, 7 novel variants (SOD1 c.112G>C, OPTN c.811C>T, ERBB4 c.965T>A, DCTN1 c.1915C>T, NEFH c.2602G>A, NEK1 c.3622G>A, and TAF15 c.1535G>A) were identified. In southeastern Chinese FALS, the mutation frequency of SOD1, FUS, and TARDBP was 52.9%, 8.8%, 8.8% respectively. In early-onset SALS, FUS mutations were the most common (22.6%). In Chinese ALS cases, p.H47R is most frequent SOD1 mutations, while p.R521 is most common FUS mutation and p.M337V is most common TARDBP mutation. Our results revealed that mutations in SOD1, FUS and TARDBP are the most common cause of Chinese FALS, while FUS mutations are the most common cause of early-onset SALS. The genetic spectrum is different between Chinese ALS and Caucasian ALS.

A De novo Mutation in Dystrophin Causing Muscular Dystrophy in a Female Patient.

BACKGROUND: Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are X-linked recessive neuromuscular diseases resulting from dystrophin (DMD) gene mutations. It has been known that the carrier of DMD mutations may also have symptoms of the disease. While de novo mutation is quite common in BMD/DMD patients, it is rarely reported in the female carriers. METHODS: Two sporadic Chinese patients with progressive muscular dystrophy and their familial members were recruited. The targeted next-generation sequencing (NGS) and the multiplex ligation-dependent probe analysis (MLPA) were performed in the proband. Blood tests, electrocardiography, echocardiography, and electromyography were also evaluated. RESULTS: Two novel mutations of DMD gene were identified, c.7318C>T (p.Q2440*) in the male proband and c.4983dupA (p.A1662Sfs*24) in the female carrier. The MLPA analysis did not detect any large rearrangements. The haplotype analysis indicated that the two mutations were derived from de novo mutagenesis. CONCLUSIONS: We identified two novel de novo mutations of DMD gene in two Chinese pedigrees, one of which caused a female patient with muscular dystrophy. The mutational analysis is important for DMD patients and carriers in the absence of a family history. The NGS can help detect the mutations in MLPA-negative patients.

Mutation Analysis of MR-1, SLC2A1, and CLCN1 in 28 PRRT2-negative Paroxysmal Kinesigenic Dyskinesia Patients.

BACKGROUND: Paroxysmal kinesigenic dyskinesia (PKD) is the most common subtype of paroxysmal dyskinesias and is caused by mutations in PRRT2 gene. The majority of familial PKD was identified to harbor PRRT2 mutations. However, over two-third of sporadic PKD patients did not carry anyPRRT2 mutation, suggesting an existence of additional genetic mutations or possible misdiagnosis due to clinical overlap. METHODS: A cohort of 28 Chinese patients clinically diagnosed with sporadic PKD and excluded PRRT2 mutations were recruited. Clinical features were evaluated, and all subjects were screened for MR-1, SLC2A1, and CLCN1 genes, which are the causative genes of paroxysmal nonkinesigenic dyskinesia (PNKD), paroxysmal exertion-induced dyskinesia, and myotonia congenita (MC), respectively. In addition, 200 genetically matched healthy individuals were recruited as controls. RESULTS: A total of 16 genetic variants including 4 in MR-1 gene, 8 in SLC2A1 gene, and 4 in CLCN1 gene were detected. Among them, SLC2A1 c.363G>A mutation was detected in one case, and CLCN1 c.1205C>T mutation was detected in other two cases. Neither of them was found in 200 controls as well as 1000 Genomes database and ExAC database. Both mutations were predicted to be pathogenic by SIFT and PolyPhen2. The SLC2A1 c.363G>A mutation was novel. CONCLUSIONS: The phenotypic overlap may lead to the difficulty in distinguishing PKD from PNKD and MC. For those PRRT2- negative PKD cases, screening of SLC2A1 and CLCN1 genes are useful in confirming the diagnosis.