Studies Of Hereditary Neurological Disease: Disease Gene Identification

遗传性神经疾病的研究：疾病基因鉴定

• 批准号: 8342220
• 负责人: Kenneth Fischbeck
• 金额: $ 84.49万
• 依托单位: NATIONAL INSTITUTE OF NEUROLOGICAL DISORDERS AND STROKE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8342220
• 关键词: 12q24.11; 13q13.3; 19p13.11; 3' Untranslated Regions; Adult; Affect; Africa; Alleles; American; Amino Acid Substitution; Ankyrin Repeat; Atrophic; Autoimmune Process; Biology; Candidate Disease Gene; Cations; Cells; Charcot-Marie-Tooth Disease; Chromosomes; Chromosomes, Human, Pair 19; Consanguinity; Degenerative Disorder; Dideoxy Chain Termination DNA Sequencing; Disease; Distal; Employee Strikes; Evaluation; Family; Gene Proteins; Genes; Genetic; Genetic Polymorphism; Genetic Predisposition to Disease; Genetic Transcription; Goals; Hereditary Spastic Paraparesis; Hereditary Spastic Paraplegia; Heterozygote; Individual; Inherited; Laryngeal muscle structure; Limb structure; Lower Extremity; Mali; Maps; Missense Mutation; Muscle Weakness; Mutation; Myasthenia; Myasthenia Gravis; NADH oxidase; Names; Neurodegenerative Disorders; Neuromuscular Diseases; Neuropathy; Nucleotides; Patients; Peripheral Nerves; Proteins; Recombinant Proteins; Relative (related person); Reporting; Research; Respiratory Diaphragm; Reverse Transcriptase Polymerase Chain Reaction; Siblings; Single Nucleotide Polymorphism; Single Nucleotide Polymorphism Map; Single-Gene Defect; Sister; Site; Spastic Paraplegia; Toxic effect; Transcript; Upper Extremity; Variant; base; effective therapy; exome; genetic regulatory protein; genome-wide; kindred; mRNA Stability; member; mutant; nervous system disorder; novel; outreach program; programs; protein protein interaction; skeletal dysplasia

The purpose of this research program is to investigate the causes of hereditary neurological diseases, with the goal of developing effective treatments for these disorders. A genetic outreach program allows the identification and characterization of patients and families with hereditary neurological diseases. Specific research accomplishments in the past year include (1) the evaluation of a candidate gene for familial autoimmune myasthenia gravis, (2) Charcot-Marie-Tooth disease type 2C (CMT2C), and (3) a new form of hereditary spastic paraplegia mapped to chromosome 19. Myasthenia gravis is usually sporadic. However, familial cases suggest a genetic predisposition. We sought to identify a causative mutation in a previously reported Italian-American kindred with parental consanguinity and five out of ten siblings affected by adult-onset autoimmune myasthenia gravis. We performed genome-wide homozygosity mapping, and sequenced all genes in the one region of extended homozygosity. Quantitative and allele specific RT-PCR were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles. A region of shared homozygosity at chromosome 13q13.3-13q14.11 was found in four affected subjects and one unaffected individual. A homozygous single nucleotide variant was found in the 3'-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that the expression of ENOX1 is decreased to about 20% normal levels in lymphoblastoid cells from individuals who are homozygous for the variant and to about 50% in two unaffected heterozygotes. Allele specific RT-PCR showed a 55-60% reduction in the transcript level of the variant in heterozygote cells due to reduced mRNA stability. These results indicate that this sequence variant in ENOX1 may contribute to the late onset myasthenia in these patients. To our knowledge, this is the first single-gene defect to be associated with autoimmune myasthenia. CMT2C is an autosomal dominant neuropathy characterized by limb, diaphragm, and laryngeal muscle weakness. Two unrelated families with CMT2C showed significant linkage to chromosome 12q24.11. All genes in this region were sequenced and heterozygous missense mutations were identified in the TRPV4 gene causing the amino acid substitutions R269C and R269H. TRPV4 is a well known member of the TRP superfamily of cation channels. In TRPV4-transfected cells, the CMT2C mutations caused marked cellular toxicity and increased constitutive and activated channel currents. Mutations in TRPV4 were previously associated with skeletal dysplasias. Our findings indicate that TRPV4 mutations can also cause a degenerative disorder of peripheral nerves. The CMT2C mutations lie in a distinct region of the TRPV4 ankyrin repeats, suggesting that this striking phenotypic variability may be due to differential effects on regulatory protein-protein interactions. We subsequently investigated interacting proteins and used exome-sequencing looked for a causative mutation in a CMT2C family in which we had found no TRPV4 mutation. The exome analysis revealed a novel TRPV4 mutation in this family, R186Q, which had been missed by Sanger sequencing due to a rare polymorphism at the primer site. We identified a family in Mali with two sisters affected by spastic paraplegia. In addition to spasticity and weakness of the lower limbs, the patients had marked atrophy of the distal upper extremities. Homozygosity mapping using single nucleotide polymorphism arrays showed that the sisters shared a region of extended homozygosity at chromosome 19p13.11-q12 that was not shared by controls. These findings indicate a clinically and genetically distinct form of hereditary spastic paraplegia with amyotrophy, designated SPG43. We subsequently evaluated candidate genes in the critical region by exome sequencing, and identified one gene, C19orf12, with a sequence variant not found in controls. The variant is associated with aberrant localization of the recombinant protein in transfected cells. Mutation in the protein product of this gene, which we have named senfaganin, may be a novel cause of hereditary spastic paraparesis.

该研究计划的目的是调查遗传性神经系统疾病的原因，目的是为这些疾病开发有效的治疗方法。遗传外展计划允许识别和描述遗传性神经系统疾病的患者和家庭。过去一年的具体研究成果包括（1）家族性自身免疫性重症肌无力候选基因的评估，（2）Charcot-Marie-Tooth疾病2C型（CMT 2C），以及（3）定位于19号染色体的遗传性痉挛性截瘫的新形式。 重症肌无力通常是散发性的。然而，家族性病例表明有遗传倾向。我们试图确定一个致病突变，在以前报道的意大利裔美国人的亲属与父母的血缘关系和五分之十的兄弟姐妹受成人发病的自身免疫性重症肌无力。我们进行了全基因组纯合性作图，并对一个扩展纯合性区域中的所有基因进行了测序。对候选基因进行定量和等位基因特异性RT-PCR，以确定受影响同胞和对照的RNA表达水平以及野生型和突变等位基因的相对丰度。在4名受影响的受试者和1名未受影响的个体中发现了染色体13q13.3-13q14.11的共享纯合性区域。在胞外NADH氧化酶1基因（ENOX 1）的3 '非翻译区发现了纯合单核苷酸变体。在该区域或其他地方的基因中没有发现其他可能致病的变异。在764例对照中未发现ENOX 1序列变异。定量RT-PCR显示，ENOX 1的表达降低到约20%的正常水平，在淋巴母细胞样细胞的个体谁是纯合子的变体和约50%，在两个未受影响的杂合子。等位基因特异性RT-PCR显示，由于mRNA稳定性降低，杂合子细胞中该变体的转录水平降低了55-60%。这些结果表明，ENOX 1的这种序列变异可能有助于这些患者中的迟发性肌无力。据我们所知，这是第一个与自身免疫性肌无力相关的单基因缺陷。 CMT 2C是一种常染色体显性遗传性神经病，以肢体、膈肌和喉肌无力为特征。两个CMT 2C家系与染色体12q24.11存在显著连锁。对该区域的所有基因进行测序，并在TRPV 4基因中鉴定出杂合错义突变，导致氨基酸取代R269 C和R269 H。TRPV 4是阳离子通道TRP超家族的一个众所周知的成员。在TRPV 4转染的细胞中，CMT 2C突变引起显著的细胞毒性和增加的组成性和激活的通道电流。TRPV 4的突变以前与骨骼发育不良有关。我们的研究结果表明，TRPV 4突变也可能导致周围神经退行性疾病。CMT 2C突变位于TRPV 4锚蛋白重复序列的一个不同区域，表明这种显著的表型变异可能是由于对调节蛋白-蛋白相互作用的差异效应。我们随后研究了相互作用的蛋白质，并使用外显子组测序在CMT 2C家族中寻找致病突变，其中我们没有发现TRPV 4突变。外显子组分析揭示了该家族中一种新的TRPV 4突变R186 Q，由于引物位点的罕见多态性，该突变被桑格测序遗漏。 我们在马里发现了一个家庭，他们的两个姐妹篇都患有痉挛性截瘫。除了下肢痉挛和无力外，患者上肢远端明显萎缩。利用单核苷酸多态性阵列进行的纯合性作图显示，这对姐妹篇在染色体19p13.11-q12处共享一个扩展纯合性区域，而对照组则没有。这些发现表明一种临床和遗传上不同的遗传性痉挛性截瘫伴肌萎缩，命名为SPG 43。我们随后通过外显子组测序评估了关键区域的候选基因，并确定了一个基因C19 orf 12，其序列变异在对照中未发现。该变体与重组蛋白在转染细胞中的异常定位有关。这个基因的蛋白质产物突变，我们将其命名为senfaganin，可能是遗传性痉挛性下肢轻瘫的一个新原因。