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.

Transfer of miR-877-3p via extracellular vesicles derived from dental pulp stem cells attenuates neuronal apoptosis and facilitates early neurological functional recovery after cerebral ischemia-reperfusion injury through the Bclaf1/P53 signaling pathway.

Cerebral ischemia-reperfusion injury (I/RI) is one of the principal pathogenic factors in the poor prognosis of ischemic stroke, for which current therapeutic options to enhance neurological recovery are notably insufficient. Dental pulp stem cell-derived extracellular vesicles (DPSC-EVs) have promising prospects in stroke treatment and the specific underlying mechanisms have yet to be fully elucidated. The present study observed that DPSC-EVs ameliorated the degree of cerebral edema and infarct volume by reducing the apoptosis of neurons. Furthermore, the miRNA sequencing and functional enrichment analysis identified that miR-877-3p as a key component in DPSC-EVs, contributing to neuroprotection and anti-apoptotic effects. Following target prediction and dual-luciferase assay indicated that miR-877-3p interacted with Bcl-2-associated transcription factor (Bclaf1) to play a function. The miR-877-3p inhibitor or Bclaf1 overexpression reversed the neuroprotective effects of DPSC-EVs. The findings reveal a novel therapeutic pathway where miR-877-3p, transferred via DPSC-EVs, confers neuroprotection against cerebral I/RI, highlighting its potential in promoting neuronal survival and recovery post-ischemia.

Genotype-phenotype correlations of AR-CMT2S in a cohort of axonal Charcot-Marie-Tooth patients from Central South China.

BACKGROUND AND AIMS: This study aimed to report nine Charcot-Marie-Tooth disease (CMT) families with six novel IGHMBP2 mutations in our CMT2 cohort and to summarize the genetic and clinical features of all AR-CMT2S patients reported worldwide. METHODS: General information, clinical and neurophysiological data of 275 axonal CMT families were collected. Genetic screening was performed by inherited peripheral neuropathy related genes panel or whole exome sequencing. The published papers reporting AR-CMT2S from 2014 to 2023 were searched in Pubmed and Wanfang databases. RESULTS: In our CMT2 cohort, we detected 17 AR-CMT2S families carrying IGHMBP2 mutations and eight were published previously. Among these, c.743 T > A (p.Val248Glu), c.884A > G (p.Asp295Gly), c.1256C > A (p.Ser419*), c.2598_2599delGA (p.Lys868Sfs*16), c.1694_1696delATG (p.Asp565del) and c.2509A > T (p.Arg837*) were firstly reported. These patients prominently presented with early-onset typical axonal neuropathy and without respiratory dysfunction. So far, 56 AR-CMT2S patients and 57 different mutations coming from 43 families have been reported in the world. Twenty-nine of 32 missense mutations were clustered in helicase domain and ATPase region. The age at onset ranged from 0.11to 20 years (Mean ± SD: 3.43 ± 3.88 years) and the majority was infantile-onset (<2 years). The initial symptoms included weakness of limbs (19, 29.7%), delayed milestones (12, 18.8%), gait disturbance (11, 17.2%), feet deformity (8, 12.5%), feet drop (8, 12.5%), etc. INTERPRETATION: AR-CMT2S accounted for 6.2% in our CMT2 cohort. We firstly reported six novel IGHMBP2 mutations which expanded the genotypic spectrum of AR-CMT2S. Furthermore, 17 AR-CMT2S families could provide more resources for natural history study, drug research and development.

Genetic and clinical profile of 15 Chinese families with GDAP1-related Charcot-Marie-Tooth disease and identification of H256R as a frequent mutation.

BACKGROUND AND AIMS: Mutations in ganglioside-induced differentiation-associated protein 1 (GDAP1) cause axonal or demyelinating Charcot-Marie-Tooth disease (CMT) with autosomal dominant or recessive inheritance. In this study, we aim to report the genotypic and phenotypic features of GDAP1-related CMT in a Chinese cohort. METHODS: Clinical, neurophysiological, genetic data, and available muscle/brain imaging information of 28 CMT patients with GDAP1 variants were retrospectively collected. RESULTS: We identified 16 GDAP1 pathogenic variants, among which two novel variants c.980dup(p.L328FfsX25) and c.480+4T>G were first reported. Most patients (16/28) presented with AR or AD CMT2K phenotype. Clinical characteristics in our cohort demonstrated that the AR patients presented earlier onset, more severe phenotype compared with the AD patients. Considerable intra-familial phenotypic variability was observed among three AD families. Muscle atrophy and fatty infiltration in the lower extremity were detected by Muscle magnetic resonance imaging (MRI) scans in four patients. MRI showed two AR patients showed more severe muscle involvement of the posterior compartment than those of the anterolateral compartment in the calf. One patient carrying Q38*/H256R variants accompanied with mild periventricular leukoaraiosis. CONCLUSIONS: In this study, we conducted an analysis of clinical features of the GDAP1-related CMT patients, expanded the mutation spectrum in GDAP1 by reporting two novel variants, and presented the prevalent occurrence of the H256R mutation in China. The screening of GDAP1 should be particularly emphasized in Chinese patients with CMT2, given the incomplete penetrance and pathogenic inheritance patterns involving dominant and recessive modes.

Expanding the genetic and clinical spectrum of SORD-related peripheral neuropathy by reporting a novel variant c.210T>G and evidence of subclinical muscle involvement.

BACKGROUND AND AIMS: Biallelic variants in the sorbitol dehydrogenase (SORD) gene have been identified as the genetic cause of autosomal recessive (AR) peripheral neuropathy (PN) manifesting as Charcot-Marie-Tooth disease type 2 (CMT2) or distal hereditary motor neuropathy (dHMN). We aim to observe the genetic and clinical spectrum of a cohort of patients with SORD-related PN (SORD-PN). METHODS: A total of 107 patients with AR or sporadic CMT2/dHMN underwent molecular diagnosis by whole-exome sequencing and subsequent Sanger sequencing validation. Available phenotypic data for SORD-PN were collected and analyzed. RESULTS: Eleven (10.28%) of 107 patients were identified as SORD-PN, including four with CMT2 and seven with dHMN. The SORD variant c.210 T > G;p.His70Gln in F-d3 was firstly reported and subsequent analysis showed that it resulted in loss of SORD enzyme function. Evidence of subclinical muscle involvement was frequently detected in patients with SORD-PN, including mildly to moderately elevated serum creatine kinase (CK) levels in 10 patients, myogenic electrophysiological changes in one patient, and muscle edema in five patients undergoing lower extremity MRI. Fasting serum sorbitol level was 88-fold higher in SORD-PN patients (9.69 ± 1.07 mg/L) than in healthy heterozygous subjects (0.11 ± 0.01 mg/L) and 138-fold higher than in healthy controls (0.07 ± 0.02 mg/L). INTERPRETATION: The novel SORD variant c.210 T > G;p.His70Gln and evidence of subclinical muscle involvement were identified, which expanded the genetic and clinical spectrum of SORD-PN. Subclinical muscle involvement might be a common but easily overlooked clinical feature. The serum CK and fasting serum sorbitol levels were expected to be sensitive biomarkers confirmed by follow-up cohort study.

Diagnostic value of nerve conduction study in NOTCH2NLC-related neuronal intranuclear inclusion disease.

BACKGROUND AND AIMS: Neuronal intranuclear inclusion disease (NIID) is a rare progressive neurodegenerative disorder mainly caused by abnormally expanded GGC repeats within the NOTCH2NLC gene. Most patients with NIID show polyneuropathy. Here, we aim to investigate diagnostic electrophysiological markers of NIID. METHODS: In this retrospective dual-center study, we reviewed 96 patients with NOTCH2NLC-related NIID, 94 patients with genetically confirmed Charcot-Marie-Tooth (CMT) disease, and 62 control participants without history of peripheral neuropathy, who underwent nerve conduction studies between 2018 and 2022. RESULTS: Peripheral nerve symptoms were presented by 53.1% of patients with NIID, whereas 97.9% of them showed peripheral neuropathy according to electrophysiological examinations. Patients with NIID were characterized by slight demyelinating sensorimotor polyneuropathy; some patients also showed mild axonal lesions. Motor nerve conduction velocity (MCV) of the median nerve usually exceeded 35 m/s, and were found to be negatively correlated with the GGC repeat sizes. Regarding the electrophysiological differences between muscle weakness type (n = 27) and non-muscle weakness type (n = 69) of NIID, nerve conduction abnormalities were more severe in the muscle weakness type involving both demyelination and axonal impairment. Notably, specific DWI subcortical lace sign was presented in only 33.3% of muscle weakness type, thus it was difficult to differentiate them from CMT. Combining age of onset, distal motor latency, and compound muscle action potential of the median nerve showed the optimal diagnostic performance to distinguish NIID from major CMT (AUC = 0.989, sensitivity = 92.6%, specificity = 97.4%). INTERPRETATION: Peripheral polyneuropathy is common in NIID. Our study suggest that nerve conduction study is useful to discriminate NIID.

Genotype and phenotype distribution of 435 patients with Charcot-Marie-Tooth disease from central south China.

BACKGROUND AND PURPOSE: The purpose was to provide an overview of genotype and phenotype distribution in a cohort of patients with Charcot-Marie-Tooth disease (CMT) and related disorders from central south China. METHODS: In all, 435 patients were enrolled and detailed clinical data were collected. Multiplex ligation-dependent probe amplification for PMP22 duplication/deletion and CMT multi-gene panel sequencing were performed. Whole exome sequencing was further applied in the remaining patients who failed to achieve molecular diagnosis. RESULTS: Among the 435 patients, 216 had CMT1, 14 had hereditary neuropathy with pressure palsies (HNPP), 178 had CMT2, 24 had distal hereditary motor neuropathy (dHMN) and three had hereditary sensory and autonomic neuropathy (HSAN). The overall molecular diagnosis rate was 70%: 75.7% in CMT1, 100% in HNPP, 64.6% in CMT2, 41.7% in dHMN and 33.3% in HSAN. The most common four genotypes accounted for 68.9% of molecular diagnosed patients. Relatively frequent causes were missense changes in PMP22 (4.6%) and SH3TC2 (2.3%) in CMT1; and GDAP1 (5.1%), IGHMBP2 (4.5%) and MORC2 (3.9%) in CMT2. Twenty of 160 detected pathogenic variants and the associated phenotypes have not been previously reported. Broad phenotype spectra were observed in six genes, amongst which the pathogenic variants in BAG3 and SPTLC1 were detected in two sporadic patients presenting with the CMT2 phenotype. CONCLUSIONS: Our results provided a unique genotypic and phenotypic landscape of patients with CMT and related disorders from central south China, including a relatively high proportion of CMT2 and lower occurrence of PMP22 duplication. The broad phenotype spectra in certain genes have advanced our understanding of CMT.

Mutations in C1orf194, encoding a calcium regulator, cause dominant Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease is a hereditary motor and sensory neuropathy exhibiting great clinical and genetic heterogeneity. Here, the identification of two heterozygous missense mutations in the C1orf194 gene at 1p21.2-p13.2 with Charcot-Marie-Tooth disease are reported. Specifically, the p.I122N mutation was the cause of an intermediate form of Charcot-Marie-Tooth disease, and the p.K28I missense mutation predominately led to the demyelinating form. Functional studies demonstrated that the p.K28I variant significantly reduced expression of the protein, but the p.I122N variant increased. In addition, the p.I122N mutant protein exhibited the aggregation in neuroblastoma cell lines and the patient's peroneal nerve. Either gain-of-function or partial loss-of-function mutations to C1ORF194 can specify different causal mechanisms responsible for Charcot-Marie-Tooth disease with a wide range of clinical severity. Moreover, a knock-in mouse model confirmed that the C1orf194 missense mutation p.I121N led to impairments in motor and neuromuscular functions, and aberrant myelination and axonal phenotypes. The loss of normal C1ORF194 protein altered intracellular Ca2+ homeostasis and upregulated Ca2+ handling regulatory proteins. These findings describe a novel protein with vital functions in peripheral nervous systems and broaden the causes of Charcot-Marie-Tooth disease, which open new avenues for the diagnosis and treatment of related neuropathies.

[Research advance of underlying pathogenesis and target therapies in Charcot-Marie-Tooth disease type 1A].

Charcot-Marie-Tooth disease (CMT) is the commonest form of inherited neuropathy and has an incidence of 1/2500. CMT1A is the commonest subtype of CMT, which is caused by duplication of peripheral myelin protein 22 (PMP22) gene and accounts for approximately 50% of CMT diagnosed by genetic testing. Duplication of PMP22 may influence the production of PMP22 mRNA and protein, and interfere with the proliferation, differentiation and apoptosis of Schwann cells. In addition, deregulation of NRG1/ErbB pathway and lipid metabolism can also lead to dysfunction of Schwann cells. Such factors may disturb the myelination process, leading to axon degeneration, muscle weakness, and atrophy subsequently. Accordingly, drug therapies for CMT1A are developed by targeting such factors. PXT3003, antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) are supposed to down-regulate the level of PMP22 mRNA, while recombinant human NRG-1 (rhNRG1) and neurotrophin-3 (NT-3) may enhance Schwann cells survival and differentiation. In addition, lipid-supplemented diet may remedy the defect of lipid metabolism and maintain the proper structure of myelin. Other targeting drugs include ascorbic acid, progesterone antagonists, IFB-088, ADX71441, and ACE-083. This review is to sum up the pathogenesis of CMT1A and promising targeting drug therapies for further research.

A novel WARS mutation (p.Asp314Gly) identified in a Chinese distal hereditary motor neuropathy family.

Distal hereditary motor neuropathy (dHMN) is a clinically and genetically heterogeneous group of inherited neuropathies characterized by distal limb muscle wasting and weakness with no or minimal sensory abnormalities. To investigate the clinical and genetic features of dHMN caused by WARS mutations in mainland China, we performed Sanger sequencing of the coding and untranslated region (UTR) regions of WARS in 160 unresolved dHMN and Charcot-Marie-Tooth (CMT) index patients. We detected a novel heterozygous variant c.941A>G (p.Asp314Gly) of WARS in an index patient from an autosomal dominant dHMN family including five affected members over three generations. The variant completely co-segregates with the dHMN phenotype in the family, and it was classified as likely pathogenic according to the American College of Medical Genetics and Genomics standards and guidelines. The clinical features included juvenile to adult onset (15-23 years), distal wasting and weakness, minimal sensory disturbance and length-dependent motor axonal degeneration with CMT examination score ranging from 6 to 10. Our report further confirms the role of WARS in dHMN and indicates that the variant c.941A>G (p.Asp314Gly) of WARS is related to a mild to moderate affected and later onset phenotype of dHMN.

MFN2-related genetic and clinical features in a cohort of Chinese CMT2 patients.

Charcot-Marie-Tooth disease 2A (CMT2A), caused by mutations in the mitofusin 2 gene (MFN2), is the most common CMT2 subtype. The aim of our study is to assess the frequency and summarize the genetic and clinical characteristics of Chinese CMT2A patients. A total of 17 coding exons of MFN2 were detected by direct sequencing in 82 unrelated Chinese families diagnosed as CMT2. Clinical evaluations were analyzed among CMT2A patients. We identified 14 missense variants in 9 sporadic and 6 familial cases, including four novel mutations (T129A, S249F, Q367P, and Q674L), 4 known mutations (R94W, R94Q, T105M, C132Y, M376V and Q751X), and 4 rare missense variants (K120E, C217F, K307E and T356S). A total of 23 patients had early-onset phenotype. Two patients had a CMTNS score of 0 to 10; 16 had a score of 11 to 20; and 7 had a score greater than 20. Five patients were confirmed a de novo origin. Six of 14 variants were located or closed to the GTPase domain. We report four novel mutations and four rare missense variants. MFN2 mutations account for 18% of CMT2 families in mainland China. The common characteristics of Chinese pedigree are early disease onset and moderate phenotypes.

PMP22-Related neuropathies and other clinical manifestations in Chinese han patients with charcot-marie-tooth disease type 1.

INTRODUCTION: Most cases of Charcot-Marie-Tooth (CMT) disease are caused by mutations in the peripheral myelin protein 22 gene (PMP22), including heterozygous duplications (CMT1A), deletions (HNPP), and point mutations (CMT1E). METHODS: Single-nucleotide polymorphism (SNP) arrays were used to study PMP22 mutations based on the results of multiplex ligation-dependent probe amplification (MLPA) and polymerase chain reaction-restriction fragment length polymorphism methods in 77 Chinese Han families with CMT1. PMP22 sequencing was performed in MLPA-negative probands. Clinical characteristics were collected for all CMT1A/HNPP probands and their family members. RESULTS: Twenty-one of 77 CMT1 probands (27.3%) carried duplication/deletion (dup/del) copynumber variants. No point mutations were detected. SNP array and MLPA seem to have similar sensitivity. Fifty-seven patients from 19 CMT1A families had the classical CMT phenotype, except for 1 with concomitant CIDP. Two HNPP probands presented with acute ulnar nerve palsy or recurrent sural nerve palsy, respectively. CONCLUSIONS: The SNP array has wide coverage, high sensitivity, and high resolution and can be used as a screening tool to detect PMP22 dup/del as shown in this Chinese Han population.

Phenotype-driven reanalysis reveals five novel pathogenic variants in 40 exome-negative families with Charcot-Marie-Tooth Disease.

BACKGROUND: To identify genetic causes in 40 whole exome sequencing (WES)-negative Charcot-Marie-Tooth (CMT) families and provide a summary of the clinical and genetic features of the diagnosed patients. METHODS: The clinical information and sequencing data of 40 WES-negative families out of 131 CMT families were collected, and phenotype-driven reanalysis was conducted using the Exomiser software. RESULTS: The molecular diagnosis was regained in 4 families, increasing the overall diagnosis rate by 3.0%. One family with adolescent-onset pure CMT1 was diagnosed [POLR3B: c.2810G>A (p.R937Q)] due to the novel genotype-phenotype association. One infantile-onset, severe CMT1 family with deep sensory disturbance was diagnosed by screening the BAM file and harbored c.1174C>T (p.R392*) and 875_927delinsCTGCCCACTCTGCCCACTCTGCCCACTCTG (p.V292Afs53) of PRX. Two families were diagnosed due to characteristic phenotypes, including an infantile-onset ICMT family with renal dysfunction harboring c.213_233delinsGAGGAGCA (p.S72Rfs34) of INF2 and an adolescent-onset CMT2 family with optic atrophy harboring c.560C>T (p.P187L) and c.616A>G (p.K206E) of SLC25A46. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, the variants of POLR3B and SLC25A46 were classified as likely pathogenic, and the variants of INF2 and PRX were pathogenic. All these variants were first reported worldwide except for p.R392* of PRX. CONCLUSIONS: We identified five novel pathogenic variants in POLR3B, PRX, INF2, and SLC25A46, which broaden their phenotypic and genotypic spectrums. Regular phenotype-driven reanalysis is a powerful strategy for increasing the diagnostic yield of WES-negative CMT patients, and long-term follow-up and screening BAM files for contiguous deletion and missense variants are both essential for reanalysis.

Two novel MPZ mutations in Chinese CMT patients.

To investigate the myelin protein zero (MPZ) gene mutation and related clinical features in Chinese Charcot-Marie-Tooth (CMT) patients, we screened the coding sequence of MPZ in 70 unrelated CMT index patients after excluding the PMP22 duplication, Cx32 and MFN2 mutations. We found four different missense mutations: c.194C>T, c.242A>T, c.371C>T, and c.419C>G. The frequency of MPZ mutation was approximately 4.35% of the total, 3.08% of CMT1, and 6% of CMT2. Mutations c.242A>T and c.419C>G are novel. The mutation c.242A>T exhibited late onset and rapidly progressive CMT2 phenotype. The mutation c.419C>G exhibited relatively late onset and slowly progressive CMT1 phenotype.

[Classification and molecular diagnostic procedure for Chacort-Marie-Tooth disease].

Charcot-Marie-Tooth disease (CMT) is the most common form of hereditary neuropathy with significant clinical and genetic heterogeneity. So far 28 genes have been cloned. The main clinical manifestations of CMT include progressive distal muscle wasting and weakness, impaired distal sensation, and diminishing or loss of tendon reflex. Patients may be classified into demyelinating type (CMT1) and axonal type (CMT2) according to electrophysiological and pathological characteristics. Establishment of a standard diagnostic procedure based on clinical, electrophysiological and pathological findings will enable accurate diagnosis in most CMT patients and provide guidance for gene consulting and prognosis.

[Analysis of CX32 gene mutation and related clinical features in Chinese Han Charcot-Marie-Tooth families].

OBJECTIVE: To analyze the mutation of CX32 gene and related clinical features in Chinese Han patients with Charcot-Marie-Tooth (CMT) disease. METHODS: Thirty-four CMT families, from 2004 to 2011 at Departments of Neurology, Xiangya Hospital, Third Xiangya Hospital and National Key Laboratory of Medical Genetics, were selected for CX32 mutation screening after the exclusion of the PMP22 duplication and male-to-male transmission. Mutation analysis was carried out by polymerase chain reaction (PCR) plus direct sequencing. Analyses of clinical, electrophysiological and pathological features in 11 patients from 6 CMTX1 families were performed by 2 neurologists. RESULTS: Five CX32 gene mutations were detected in 6 CMT families: c.37G > A, c.65G > A, c.246C > G, c.256A > G and c.533A > G. Among them, c.246C > G and c.533A > G were firstly reported. The clinical manifestations included progressive distal muscle atrophy and weakness, areflexia, sensory abnormalities and pes vacus. Nerve conduction velocity ranged from 21.7 to 49.3 m/s. Both demyelination and axonal degeneration were detected in nerve biopsy. CONCLUSIONS: CMT1X has a frequency of around 9% in our study. The male patients tend to have more serious clinical features and their electrophysiological and pathological changes are intermediate. CX32 mutation analysis helps to confirm the genetic diagnosis of CMT so as to provide genetic counseling and reproductive guidance and elucidate its pathogenesis.

[The effect of HSPB8 gene mutation on cell viability in Charcot-Marie-Tooth disease type 2L].

OBJECTIVE: To study the effect of Charcot-Marie-Tooth 2L disease causing gene K141N mutation in heat shock protein B8 gene (HSPB8) on cell viability. METHODS: By using liposome transfection technique, (wt)HSPB8, (K141N)HSPB8 eukaryotic expression vector and green fluorescent protein (GFP) vector were transfected into SHSY-5Y cell, respectively. Twenty-four hours later, the cells were treated with 44 degree centigrade lethal heat shock for 40 minutes. The relative viability of SHSY-5Y cells in each group was tested by using tetrazole blue colorimetric method (methyl thiazolyl tetrazolium, MTT). RESULTS: There were significant differences among the light absorption value of GFP, pEGFP-(wt)HSPB8 and pEGFP-(K141N)HSPB8 transfected groups after heat shock (P<0.05), indicating that the relative viability of cells overexpressed with (wt)HSPB8 and (K141N)HSPB8 was different from that of control cells. The viability of cells overexpressing (wt)HSPB8 was highest, followed by cells overexpressed with (K141N)HSPB8. The viability of cells tranfected with GFP only was the lowest. CONCLUSION: HSPB8 may play an important role in the protection of cells under lethal heat shock treatment, and the K141N mutation can impair the protective effect.

[Cellular expression of (R127W)HSPB1 and its co-localization with neurofilament light chain].

OBJECTIVE: To observe the cellular expression of (R127W) HSPB1 and its influence on neurofilament light chain (NFL) self-assembly and co-localization with NFL. METHODS: Eukaryotic expression vectors pEGFPN1-(wt) HSPB1 and pEGFPN1- (R127W) HSPB1 were constructed. Hela cells were transiently transfected with pEGFPN1-(wt) HSPB1 or pEGFPN1- (R127W) HSPB1 and observed under a confocal microscope. Hela cells were also transiently co-transfected with Pcl-NFL and pEGFPN1-(wt)HSPB1, or pCL-NFL and pEGFPN1-(R127W)HSPB1. The self-assembly of NFL was observed and the co-localization study of HSPB1/ (R127W)HSPB1 with NFL was carried out in these two cell models by immunofluorescence technique. RESULTS: The aggregates formed by EGFP-(R127W)HSPB1 predominantly located around the nucleus, and EGFP-(wt)HSPB1 showed diffusion pattern in Hela cells. When co expressed with EGFP-(wt)HSPB1, NFL formed homogeneous structure in cytosol. When co-expressed with EGFP-(R127W)HSPB1, however, NFL had amorphous staining pattern predominantly consisting of NFL aggregates, and NFL co-localized with (R127W)HSPB1 in these aggregates. CONCLUSION: The R127W mutant of HSPB1 may have reduced capacity to serve as a chaperone to prevent aggregate formation, and fail to correctly organize the neurofilament network. Dysfunction of the axon cytoskeleton and axon transport may be the primary mechanism of R127W mutation of HSPB1 in the pathogenesis of Charcot-Marie-Tooth disease.

One PMP22/MPZ and Three MFN2/GDAP1 Concomitant Variants Occurred in a Cohort of 189 Chinese Charcot-Marie-Tooth Families.

BACKGROUND AND AIMS: Charcot-Marie-Tooth (CMT) disease is a clinically and genetically heterogeneous group of inherited peripheral neuropathies. The wide phenotypic variability may not be completely explained by a single mutation. AIMS AND METHODS: To explore the existence of concomitant variants in CMT, we enrolled 189 patients and performed molecular diagnosis by application of next-generation sequencing combined with multiplex ligation-dependent probe amplification. We conducted a retrospective analysis of patients harboring coinherited variants in different genes. RESULTS: Four families were confirmed to possess variants in two genes, accounting for 2.1% (4/189) of the total in our cohort. One CMT1 patient with PMP22 duplication and MPZ variant (c.286A>C, p.K96Q) exhibited moderate neuropathy with infantile onset, while her father possessing MPZ variant was mildly affected with adolescence onset. A CMT2 patient with heterozygous variants in MFN2 (c.613_622delGTCACCACAG, p.V205Sfs*26) and GDAP1 (c.713G>T, p.W238L) exhibited childhood onset mild phenotype, while his mother with MFN2 variant developed bilateral pes cavus only. A CMT2 patient with heterozygous variants in MFN2 (c.839G>A, p.R280H) and GDAP1 (c.3G>T, p.M1?) presented infantile onset and rapid progression, while her father with MFN2 variant presented with absence of deep tendon reflexes. One sporadic CMT2 patient with early onset was confirmed harboring de novo MFN2 variant (c.1835C>T, p.S612F) and heterozygous GDAP1 variant (c.767A>G, p.H256R). CONCLUSION: Our results suggest that the possibility of concomitant variants was not uncommon and should be considered when significant intrafamilial clinical heterogeneity is observed.