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

Abstract

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.

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

“…The male patient carried p.R904X, p.D475V, and p.A1237V in SH3TC2 had a later AAO of 30 years, and the symptoms of weakness and atrophy were involved in both lower and upper proximal limbs. Besides two unrelated probands with NDRG1 mutations previously reported by our group, we identified an additional patient with a novel NDRG1 homozygous splicing variant (c.595‐2A>G), which presented a classic early‐onset CMT phenotype. The 15‐year‐old patient with a novel homozygous FGD4 variant (c.2379+1G>C) manifested a recurrent tumble and impairing gallop ability at onset when he was 11 years old.…”

“…One hundred and fifty unrelated CMT patients consisting of 96 males and 54 females were recruited from Second Affiliated Hospital of Zhejiang University School of Medicine and Huashan Hospital of Fudan University between December 2007 and August 2018. Patients were evaluated and diagnosed by at least two senior neurologists according to the strategy described in our previous reports . This study was approved by the local Ethics Committees for medical research.…”

“…7,8 Owing to CMT's clinical and genetic heterogeneity, it is expensive and time-consuming to screen all the possible causative genes using conventional Sanger sequencing. However, highthroughput targeted next-generation sequencing (NGS) has been employed successfully in CMT 9,10 and other neurogenetic diseases such as amyotrophic lateral sclerosis (ALS), [11][12][13] hereditary spastic paraplegia (HSP), 14,15 and hereditary ataxia (HA). 16,17 Consequently, nearly 90 genes associated with CMT and other inherited peripheral neuropathies have been assembled in a gene panel, and parallel sequencing can be performed to interrogate these genes.…”

“…Patients were evaluated and diagnosed by at least two senior neurologists according to the strategy described in our previous reports. 9,10 This study was approved by the local Ethics Committees for medical research. Written informed consents were obtained from participants or their guardians.…”

Genetic and clinical features of Chinese patients with mitochondrial ataxia identified by targeted next‐generation sequencing

“…For those subjects without mutations in hereditary ataxia genes, mtDNA and nDNA genes were screened with a panel covering mitochondrial 16 569 base pairs (NC_012920.1) and 56 nuclear genes involved in mitochondrial disorders (Table S2). The targeted NGS and data analysis were performed using our previously reported protocol . Sanger sequencing was used to validate the variants that passed the filtering criteria in the patients and their family members.…”