中国人中发现一例Charcot-Marie-Tooth病连接蛋白32基因新突变

目的了解中国人进行性腓骨肌萎缩症(Charcot-Marie-Toothdisease,CMT)连接蛋白32(connexin32,Cx32)基因外显子2的突变情况.方法对6例无亲缘关系的、无重复的CMT1患者和10例无亲缘关系的CMT2患者进行SSCP分析,对有异常者进行测序,根据突变点序列设计合适的内切酶,对50名正常对照进行酶切分析.结果在1例CMT1患者发生了Gly21Asp(62G→A)错义突变.用限制性内切酶H-aeⅡ酶切50名正常对照未见异常,表明该突变为致病性突变.结论Gly21Asp是未报道过的新型突变,中国人CMT1患者中CMTX患者可能占一定比例.     

脊肌萎缩症基因缺失的初步研究

目的为了研究中国人进行性脊肌萎缩症（ｓｐｉｎａｌｍｕｓｃｕｌａｒａｔｒｏｐｈｙ，ＳＭＡ）患者中神经元存活基因（ｓｕｒｖｉｖａｌｍｏｔｏｒｎｅｕｒｏｎ，ＳＭＮ）外显子７的缺失情况，进一步证实外显子７缺失与ＳＭＡ疾病的关系，为基因诊断和产前诊断建立切实可行的方法。方法采用ＰＣＲ－ＳＳＣＰ银染技术，对３７个ＳＭＡ患者家系及３０例正常个体的ＳＭＮ基因的外显子７区域进行检测。结果在ＳＭＮ基因的外显子７区域纯合缺失率分别为：ＳＭＡⅠ型８６．７％（１３／１５），Ⅱ型８６．４％（１９／２２），患者父母及正常个体共６７例中除一患者母亲为纯合缺失外，其余均无纯合缺失。结论中国人ＳＭＡ患者中ＳＭＮ基因外显子７的纯合缺失频率高，并与疾病发生密切相关，ＳＳＣＰ银染检测ＳＭＡ基因缺失方法快速安全，结果可靠，可诊断率高，适用于基因诊断和产前诊断     

伴声带麻痹的X连锁腓骨肌萎缩症GJB1基因新突变

目的 报告1个临床可疑的伴声带麻痹的X连锁显性遗传腓骨肌萎缩症(X-1inked Charcot-Marie-Tooth disease,CMTX)家系,并探讨与连接蛋白(gap jurction protein beta 1,GJB1)基因突变的关系.方法 对1个具有声音嘶哑、吞咽困难、致死性呼吸衰竭的临床可疑的CMTX家系进行临床、纤维喉镜、头部MRI及电生理检查,对先证者应用实时PCR排除PMP22基因大片段重复突变后,应用PCR直接测序法对先证者及其家系内其他4例患者、5名有血缘关系的正常家系成员以及50名无血缘关系的正常人进行GJB1基因突变检测.结果 该家系3代共8例患者,先证者临床表现为进行性双下肢胫前肌、腓骨肌严重萎缩、无力;该家系中共4例患者出现声音嘶哑,吞咽困难,经纤维喉镜检查证实为声带麻痹,其中2例双侧声带完全麻痹最终因吞咽困难,呼吸衰竭而死亡;先证者的头部MRI检查正常,神经电生理检查揭示周围神经运动和感觉神经传导速度明显减慢或动作电位消失;分子遗传学结果显示该家系GJB1基因第2外显子存在c.186 C＞G错义突变,该突变与疾病表型共分离.结论 c.186 C＞G突变为GJB1基因新突变,目前国内外尚无报道;因声带麻痹而呼吸衰竭是CMT罕见的严重的临床症状;CMTX具有明显的临床和遗传异质性.     

C1orf194基因Ile122Asn突变致腓骨肌萎缩症的病理机制

目的 初步探讨C1orf194基因内Ile122Asn突变导致常染色体显性遗传腓骨肌萎缩症(CMT)的分子机制.方法 通过PCR克隆构建人野生C1orf194基因和携带Ile122Asn突变的C1orf194基因,分别导入人神经母细胞瘤(SH-SY5Y)细胞内进行表达,通过稳定同位素标记氨基酸免疫沉淀分析和液相色谱确定...     

经典型苯丙酮尿症苯丙氨酸羟化酶基因的新突变鉴定

目的研究经典型苯丙酮尿症(phenylketonuria, PKU)基因突变.方法应用聚合酶链反应,单链构象多态分析和DNA直接测序等技术,对内蒙古地区32个PKU家系苯丙氨酸羟化酶(phenylalanine hydroxylase, PAH)基因第3～12外显子进行了鉴定分析. 结果检出14种PAH基因点突变R243Q (12/64)、Y356X(6/64)、Y204C(5/64)、R261Q(2/64)、Y161S(2/64)、R252Q(1/64)、R111X(2/64)、D282G(1/64)、S303P(1/64)、G239D(1/64)、R413P(1/64)、IVS7nt+2(2/64)、IVS4nt+3(1/64)、IVS9nt+34(2/64),经检索国际PAH基因突变数据统计库(截至到2004年7月),确认IVS4nt+3(G>C)、IVS9nt+34(G>A)为国际首次发现的新突变,S303P(T>C) 、D282G(A>G)为国内首次报道的新突变.结论内蒙古人群苯丙氨酸羟化酶基因存在突变的多样性,R243Q、Y356X、Y204C是PAH基因的突变热点.     

X-linked dominant Charcot-Marie-Tooth disease with connexin 32 (Cx32) mutations in Koreans

X-linked dominant Charcot-Marie-Tooth disease (CMTX) is an inherited peripheral neuropathy, caused mainly by a mutation of connexin 32 (Cx32) gene. We performed a mutation analysis of Cx32 by direct sequencing of the coding sequence, then identified 23 mutations from 28 Korean CMTX families. Nine mutations were not reported previously: Gly5Ser, Ser26fs, Val37Leu, Thr86Ile, Val152fs, Phe153Cys, Asp178X, Ala197Val, and Ile214Asn. The extracellular 2 (EC2) domain of Cx32 protein was the hot spot mutation domain in 44% of Koreans. Transmembrane domain 4 was rarely affected in Koreans (4%), compared with 14% of Europeans. The EC1 and intracellular domain was not affected in Koreans, although they were frequently affected in Europeans. This study revealed that the frequencies of CMTX with Cx32 mutations are specific to different ethnic groups. The frequency of CMTX (5.3%) caused by Cx32 mutation in Koreans is similar to those in Asians but lower than those in Europeans. This study suggests differences between CMTX patients with Cx32 mutations and ethnic background.     

神经丝轻链基因在腓骨肌萎缩症中的突变分析

目的：探讨神经丝轻链基因（neurofilament-light gene,NF-L）在中国人腓骨肌萎缩症（Charcot-Marie-Tooth disease,CMT）中的突变特点。方法：应用聚合酶链反应－单链构象多态性技术结合DNA序列分析方法，对32个来自全国5省汉族的CMT家系先证者进行了NF－L基因的突变分析。结果：32例先证者中只有1例患者出现异常条带，经DNA测序证实该患者在NF－L基因的外显子3发生了1329C→T碱基改变，由于编码的氨基酸未改变，均为酪氨酸（Tyr），为一种同义突变。结论：NF－L基因突变可能在中国人的腓骨肌萎缩症患者中少见。     

A novel mutation in the GDAP1 gene is associated with autosomal recessive Charcot-Marie-Tooth disease in an Amish family

Charcot–Marie–Tooth disease (CMT) constitutes a large group of genetically heterogeneous disorders of the peripheral nervous system. Autosomal recessive forms of CMT are less common in the general population but account for the vast majority of CMT phenotypes in communities with a high prevalence of consanguinity. At least 10 genetic loci cause autosomal recessive forms of CMT. Mutations in the ganglioside-induced differentiation-associated protein 1 ( GDAP1 ) gene are among the most frequent genetic causes of autosomal recessive forms of CMT. To date, 28 mutations in GDAP1 gene have been linked with the disease. Here, we report a novel GDAP1 mutation in an Old Order Amish family with CMT. To ascertain the Amish CMT locus, we performed a genome-wide single nucleotide polymorphism (SNP) analysis on one of three patients from a consanguineous pedigree. Assuming mutation homogeneity, the analysis sought large homozygous SNP blocks that also contained known CMT loci. The largest homozygous SNP block in the patient was localized to chromosome 8q13.1-21.3 and contained the GDAP1 gene. Sequence analysis revealed a novel homozygous mutation, c.692C>T, at codon 231 (p.P231L) in exon 5 of GDAP1 in all patients. Neither the unaffected individuals in the family nor the healthy control samples were homozygous for this mutation. Our findings suggested that this novel mutation in GDAP1 gene is associated with an autosomal recessive form of CMT in Ohio Old Order Amish community.     

Four novel mutations of the connexin 32 gene in four Japanese families with Charcot-Marie-Tooth disease type 1

DNA-based mutation analysis on the connexin 32 gene was performed in 49 families with Charcot-Marie-Tooth disease (CMT) type 1 but without duplication involving the chromosomal region, 17p12-p11.2. Mutations were identified in five of the 49 families, and four of the five mutations were hitherto undescribed: Val37Met, Glu57His, Arg142Glu, Val177Ala. X-linked CMT sometimes lacks evidence for X-linked transmission and cannot be differentiated from CMT type 2, especially in females with mildly decreased nerve conduction velocity. Therefore, molecular analysis is useful for molecular pathology of their disease. Am. J. Med. Genet. 80:352–355, 1998. © 1998 Wiley-Liss, Inc.     

Charcot-Marie-Tooth type X: A novel mutation in the Cx32 gene with central conduction slowing

Charcot-Marie-Tooth disease (CMT) is characterized by distal muscle weakness and wasting, often resulting in foot deformities and gait disturbances, distal sensory impairment and by more or less typical changes in sural nerve biopsy. CMT type 1 is also characterized by reduced nerve conduction velocities. For these demyelinating subtypes, most frequently a 1.5 Mb tandem duplication in chromosome 17p11.2-12 comprising the gene for the peripheral myelin protein 22 (PMP22) is observed (CMT1A), but point mutations in PMP22 have also rarely been reported. X-linked, dominant CMTX1 disease is the second most common type of these hereditary motor and sensory neuropathies (HMSN). Mutations in the X chromosomal gene Connexin32 (Cx32) synonymous gap junction beta-1 (GJB1) are detectable in most X-linked CMT families. We report a novel missense mutation--Tyr65His--in the first extracelullar domain of the Cx32 gene in a Czech CMTX1 family. The mutation was not detectable in 50 healthy controls. The clinical phenotype in both the male proband and his mother was moderate with pronounced peroneal weakness and foot drop. Nerve conduction velocities were intermediately decreased (31-38 m/s) in both patients and slowing of central acoustic conduction (BAEP) was found in both the son and the mother whereas visual central conduction slowing (VEP) was detectable only in the son.     

Molecular analysis in Japanese patients with Charcot-Marie-Tooth disease: DGGE analysis for PMP22, MPZ,and Cx32/GJB1 mutations

Charcot-Marie-Tooth disease (CMT) is a heterogeneous disorder and is traditionally classified into two major types, CMT type 1 (CMT1) and CMT type 2 (CMT2). Most CMT1 patients are associated with the duplication of 17p11.2-p12 (CMT1A duplication) and small numbers of patients have mutations of the peripheral myelin protein 22 (PMP22), myelin protein zero (MPZ), connexin 32 (Cx32/GJB1), and early growth response 2 (EGR2) genes. Some mutations of MPZ and Cx32 were also associated with the clinical CMT2 phenotype. We constructed denaturing gradient gel electrophoresis (DGGE) analysis as a screening method for PMP22, MPZ, and Cx32 mutations and studied 161 CMT patients without CMT1A duplication. We detected 27 mutations of three genes including 15 novel mutations; six of PMP22, three of MPZ, and six of Cx32. We finally identified 21 causative mutations in 22 unrelated patients and five polymorphic mutations. Eighteen of 22 patients carrying PMP22, MPZ, or Cx32 mutations presented with CMT1 and four of them with MPZ or Cx32 mutations presented with the CMT2 phenotype. DGGE analysis was sensitive for screening for those gene mutations, but causative gene mutation was not identified in many of the Japanese patients with CMT, especially with CMT1. Other candidate genes should be studied to elucidate the genetic basis of Japanese CMT patients.     

A recurrent loss-of-function alanyl-tRNA synthetase (AARS) mutation in patients with Charcot-Marie-Tooth disease type 2N (CMT2N)

Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation.     

The first de novo mutation of the connexin 32 gene associated with X linked Charcot-Marie-Tooth disease.

X linked Charcot-Marie-Tooth disease (CMTX) is a hereditary motor and sensory neuropathy caused by mutations in the connexin 32 gene (Cx32). Using the SSCP technique and direct sequencing of PCR amplified genomic DNA fragments of the Cx32 gene from a Moroccan patient and her relatives, we identified the first de novo mutation of the Cx32 gene, consisting of a deletion of a G residue at position 499 in the Cx32 open reading frame. This previously unreported mutation produces a frameshift at position 147 in the protein and introduces a premature stop codon (TAG) at nucleotide 643, which results in the production of a truncated Cx32 molecule. This mutation illustrates the risk of an erroneous diagnosis of autosomal recessive CMT, especially in populations where consanguineous unions are frequent, and its consequences for genetic counselling, which can be avoided by molecular analysis.