Xiaohong Zi

A SIGMAR1 splice-site mutation causes distal hereditary motor neuropathy

Objective: To identify the underlying genetic cause in a consanguineous Chinese family segregating distal hereditary motor neuropathy (dHMN) in an autosomal recessive pattern. Methods: We used whole-exome sequencing and homozygosity mapping to detect the genetic variant in 2 affected individuals of the consanguineous Chinese family with dHMN. RNA analysis of peripheral blood leukocytes and immunofluorescence and immunoblotting of stable cell lines were performed to support the pathogenicity of the identified mutation. Results: We identified 3 shared novel homozygous variants in 3 shared homozygous regions of the affected individuals. Sequencing of these 3 variants in family members revealed the c.151+1G>T mutation in SIGMAR1 gene, which located in homozygous region spanning approximately 5.3 Mb at chromosome 9p13.1-p13.3, segregated with the dHMN phenotype. The mutation causes an alternative splicing event and generates a transcript variant with an in-frame deletion of 60 base pairs in exon 1 (c.92_151del), and results in an internally shortened protein σ1R31_50del. The proteasomal inhibitor treatment increased the intracellular amount of σ1R31_50del and led to the formation of nuclear aggregates. Stable expressing σ1R31_50del induced endoplasmic reticulum stress and enhanced apoptosis. Conclusion: The homozygous c.151+1G>T mutation in SIGMAR1 caused a novel form of autosomal recessive dHMN in a Chinese consanguineous family. Endoplasmic reticulum stress may have a role in the pathogenesis of dHMN.

A novel genetic locus for benign familial infantile seizures maps to chromosome 1p36.12‐p35.1

To the Editor: Benign familial infantile seizures (BFIS), formerly named benign familial infantile convulsions, is an autosomal dominant idiopathic epilepsy syndrome characterized by afebrile partial seizures with or without secondary generalized tonic-clonic seizures, age at onset from3 to 10 months, a benign course and normal psychomotor development. Patients affected with BFIS usually have a normal neurological examination and no changes in biochemical and neuroradiological investigations (1, 2). To date, three susceptibility loci of BFIS have been identified, 19q12-q13.1, 16p12-q12 and 2q24. Further investigations have indicated that other loci may exist (3–5). We report here a family of Chinese origin affected with BFIS and mapped to a different causative locus from previously established studies. All patients and unaffected members of the family gave informed consent to the clinical and genetic studies. Eight individuals (four female and four male) of the family were diagnosed as having BFIS. Neurological examination, serum biochemical determination (total protein, electrolytes, glucose, lactic dehydrogenase, uric acid, etc.), electroencephalogram (EEG), brain computed tomography (CT) and/or magnetic resonance imaging (MRI) were performed for all existing familymembers. No demonstrable underlying pathologywas observed.Age at seizure onset ranged from 3 to 10 months of life. The proband (Fig. 1, III:6), 31 years old at the timeof our study, was born at full term after normal pregnancy. At age 6 months, he experienced afebrile seizures, including staring, eye deviation, focal clonus of the face, followed by secondarily generalized phase with hypertonia, extremities clonus, cyanosis and urinary incontinence. The seizures, lasting from 30 s to 2 min in one attack, occurred mainly in clusters with 3–10 episodes per day. Interictal EEG, brain CT and MRI were classified as normal. Serum biochemical determination and chromosome karyotype were normal. Treatment with sodium valproate was effective. His seizures spontaneously remitted by 3 years of age. Blood samples were collected from available family members. Karyotype analysis of peripheral lymphocytes by G-banding was normal. Genomic DNA samples were extracted from peripheral blood leukocytes and tested first with microsatellite markers situated at chromosome 19q12-13.1, 16p12-q12 and 2q24. Linkage analysis showed that two-point logarithm of odds (LOD) scores were negative (Table 1), thereby excluding this BFIS family from linkage to the three known susceptibility loci. Genome-wide scan was subsequently performed using 382 fluorescencelabeled microsatellite markers of the ABI Prism Linkage Mapping Set, version 2 (Applied Biosystems, Foster City, CA). At recombination fraction y 1⁄4 0, a maximum two-point LOD score of 3.14 was obtained at marker D1S2674 (Table 1). Multipoint linkage analyses with markers D1S2702-D1S2674-D1S2830 on the sexaveraged linkage map confirmed the linkage, with a maximum LOD score of 3.27 at D1S2674. A particular single haplotype was identified to cosegregate with the disease phenotype in all affected individuals (Fig. 1). Based on recombination events, the underlying causative gene was mapped to a 12.4-cM interval extending from D1S2864 to D1S2830, corresponding to 1p36.12-p35.1 by referring to NCBI Map Viewer Build 34. In 1996,Auburger et al. (6) reportedanautosomal dominant form of paroxysmal choreo-athetosis/ spasticity,withonset age ranging from2 to15 years, precipitated by factors such as physical exercise and emotional stress. It was alsomapped to 1p, between D1S443 and D1S197, but about 11.07 cM distant to locus 1p36.12-p35.1. Differences in onset age, clinical features and genetic mapping indicate that they are likely to be two different disorders. The Chinese family affected with pure BFIS is identified to map to 1p36.12-p35.1, a novel locus

Mutation Detection in Candidate Genes for Benign Familial Infantile Seizures on a Novel Locus

ABSTRACT Benign familial infantile seizures (BFIS) is an autosomal dominant epileptic syndrome characterized by afebrile partial seizures with or without secondary generalized tonic-clonic seizures beginning at three to ten months of age. Genetic studies have revealed three susceptibility chromosomal loci on 19q12-q13.1, 16p12-q12 and 2q24. Previously we described the novel locus on 1p36.12-p35.1 for a Chinese family affected with BFIS, and below is a subsequent mutation analysis of candidate genes for the mapped chromosome region. Forty-five genes were selected and subjected to mutation analysis. Thirty-six nucleotide variants were found, none of which led to pathogenic changes, thereby were identified as nucleotide polymorphisms. The analyses suggest those candidate genes that were detected might not be involved in the epileptogenesis of pure BFIS, at least in the Chinese family we studied.

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.

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.

Phenotypes and cellular effects of GJB1 mutations causing CMT1X in a cohort of 226 Chinese CMT families

The aim of this study is to explore the phenotypic and genotypic features of X‐linked Charcot–Marie‐Tooth (CMT) disease in the mainland of China and to study the cellular effects of six novel Gap junction protein beta‐1 variants. We identified 25 missense and 1 non‐sense mutations of GJB1 in 31 unrelated families out of 226 CMT families. The frequency of GJB1 mutations was 13.7% of the total and 65% of intermediate CMT. Six novel GJB1 variants (c.5A>G, c.8G>A, c.242T>C, c.269T>C, c.317T>C and c.434T>G) were detected in six unrelated intermediate CMT families. Fluorescence revealed that HeLa cells transfected with EGFP‐GJB1‐V74M, EGFP‐GJB1‐L81P or EGFP‐GJB1‐L90P had diffuse endoplasmic reticulum staining, HeLa cells transfected with EGFP‐GJB1‐L106P had diffuse intracellular staining, and HeLa cells transfected with EGFP‐GJB1‐N2S had cytoplasmic and nuclear staining. The distribution of Cx32 in HeLa cells transfected with EGFP‐GJB1‐F145C was similar to that of those transfected with wild‐type (WT). These six variants resulted in a higher percentage of apoptosis than did WT as detected by flow cytometry and Hoechst staining. In conclusion, mutation screening should be first performed in intermediate CMT patients, especially those with additional features. The novel GJB1 variants c.5A>G, c.8G>A, c.242T>C and c.269T>C are considered pathogenic, and c.317T>C and c.434T>G are classified as probably pathogenic.

A novel transgenic mouse model of Chinese Charcot-Marie-Tooth disease type 2L.

We previously found that the K141N mutation in heat shock protein B8 (HSPB8) was responsible for Charcot-Marie-Tooth disease type 2L in a large Chinese family. The objective of the present study was to generate a transgenic mouse model bearing the K141N mutation in the human HSPB8 gene, and to determine whether this K141NHSPB8 transgenic mouse model would manifest the clinical phenotype of Charcot-Marie-Tooth disease type 2L, and consequently be suitable for use in studies of disease pathogenesis. Transgenic mice overexpressing K141NHSPB8 were generated using K141N mutant HSPB8 cDNA cloned into a pCAGGS plasmid driven by a human cytomegalovirus expression system. PCR and western blot analysis confirmed integration of the K141NHSPB8 gene and widespread expression in tissues of the transgenic mice. The K141NHSPB8 transgenic mice exhibited decreased muscle strength in the hind limbs and impaired motor coordination, but no obvious sensory disturbance at 6 months of age by behavioral assessment. Electrophysiological analysis showed that the compound motor action potential amplitude in the sciatic nerve was significantly decreased, but motor nerve conduction velocity remained normal at 6 months of age. Pathological analysis of the sciatic nerve showed reduced myelinated fiber density, notable axonal edema and vacuolar degeneration in K141NHSPB8 transgenic mice, suggesting axonal involvement in the peripheral nerve damage in these animals. These findings indicate that the K141NHSPB8 transgenic mouse successfully models Charcot-Marie-Tooth disease type 2L and can be used to study the pathogenesis of the disease.

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. Muscle Nerve 52: 69–75, 2015

IGHMBP2-related clinical and genetic features in a cohort of Chinese Charcot–Marie–Tooth disease type 2 patients

IGHMBP2 mutations had been exclusively associated with spinal muscular atrophy with respiratory distress type I. However, increasing AR-CMT2S cases without respiratory failure caused by IGHMBP2 mutations have been reported in the past two years. We detected IGHMBP2 mutations in a cohort of Chinese CMT2 patients using genes panel testing, polymerase chain reaction and Sanger sequencing. We found four families with autosomal recessive IGHMBP2 mutations, and the frequency of IGHMBP2 mutations is 6.5% in CMT2 without dominant inheritance. We detected a homozygous variant c.1235 + 3A > G in Family 1, compound heterozygous variants c.1737C > A and c.2597_2598delAG in Family 2, compound heterozygous variants c.1489G > A and c.2356delG in Family 3, compound heterozygous variants c.1909C > T and c.1061-2A > G in Family 4. According to the standards and guidelines of the American College of Medical Genetics and Genomics, all the above variants were classified as pathogenic. Four mutations, c.1489G > A, c.2356delG, c.2597_2598delAG and c.1061-2A > G, are reported for the first time. The novel splice acceptor site mutation c.1061-2A > G resulted in deletion of 175 bp, and it was predicted to lead to a frameshift after codon 354 with a premature termination at codon 364. In conclusion, mutation screening of IGHMBP2 should be especially considered in AR-CMT2 and sporadic CMT2 patients.

Screening for SH3TC2, PMP2, and BSCL2 Variants in a Cohort of Chinese Patients with Charcot-Marie-Tooth

Background: SH3TC2, PMP2, and BSCL2 genes are related to autosomal recessive (AR) Charcot-Marie-Tooth (CMT) disease type 1, autosomal dominant (AD)-CMT1, and AD-CMT2, respectively. Pathogenic variants in these three genes were not well documented in Chinese CMT patients. Therefore, this study aims to detect SH3TC2, PMP2, and BSCL2 pathogenic variants in a cohort of 315 unrelated Chinese CMT families. Methods: A total of 315 probands from 315 unrelated Chinese CMT families were recruited from the Department of Neurology of Third Xiangya Hospital and Xiangya Hospital. We screened for SH3TC2 pathogenic variants in 84 AR or sporadic CMT probands, PMP2 pathogenic variants in 39 AD or sporadic CMT1 probands, and BSCL2 pathogenic variants in 50 AD or sporadic CMT2 probands, using polymerase chain reaction and Sanger sequencing. All these patients were out of 315 unrelated Chinese CMT families and genetically undiagnosed after exclusion of pathogenic variants of PMP22, MFN2, MPZ, GJB1, GDAP1, HSPB1, HSPB8, EGR2, NEFL, and RAB7. Candidate variants were analyzed based on the standards and guidelines of American College of Medical Genetics and Genomics (ACMG). Clinical features were reevaluated. Results: We identified three novel heterozygous variants such as p.L95V (c.283C>G), p.L1048P (c.3143T>C), and p.V1105M (c.3313G>A) of SH3TC2 gene and no pathogenic variants of PMP2 and BSCL2 genes. Although evaluation in silico and screening in the healthy control revealed that the three SH3TC2 variants were likely pathogenic, no second allele variants were discovered. According to the standards and guidelines of ACMG, the heterozygous SH3TC2 variants such as p.L95V, p.L1048P, and p.V1105M were considered to be of uncertain significance. Conclusions: SH3TC2, PMP2, and BSCL2 pathogenic variants might be rare in Chinese CMT patients. Further studies to confirm our findings are needed.