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，目标是开发针对这些疾病的有效治疗方法。遗传外展计划可以识别和表征患有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）夏科-玛丽-图思病2C型（CMT2C），以及（3）绘制了一种新型遗传性痉挛性截瘫的图谱至19号染色体。 重症肌无力通常是散发性的。然而，家族病例表明存在遗传倾向。我们试图在先前报道的一个意大利裔美国人亲属中找出致病突变，该亲属具有血亲关系，并且十分之五的兄弟姐妹患有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，以确定受影响的兄弟姐妹和对照中的 RNA 表达水平以及野生型和突变等位基因的相对丰度。在四名受影响的受试者和一名未受影响的个体中发现了染色体 13q13.3-13q14.11 上的共享纯合性区域。在 ecto-NADH 氧化酶 1 基因 (ENOX1) 的 3'-非翻译区发现纯合单核苷酸变体。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 个对照中未发现 ENOX1 序列变异。定量 RT-PCR 显示，在该变异纯合子个体的类淋巴母细胞中，ENOX1 的表达降低至正常水平的 20% 左右，而在两个未受影响的杂合子中，ENOX1 的表达降低至正常水平的 50% 左右。等位基因特异性 RT-PCR 显示，由于 mRNA 稳定性降低，杂合子细胞中变异体的转录水平降低了 55-60%。这些结果表明 ENOX1 中的这种序列变异可能导致这些患者迟发性肌无力。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。 CMT2C 是一种常染色体显性神经病，其特征是肢体、膈肌和喉肌无力。两个不相关的 CMT2C 家族显示出与染色体 12q24.11 的显着连锁。对该区域的所有基因进行了测序，并在 TRPV4 基因中鉴定出导致氨基酸取代 R269C 和 R269H 的杂合错义突变。 TRPV4 是阳离子通道 TRP 超家族中众所周知的成员。在 TRPV4 转染的细胞中，CMT2C 突变引起明显的细胞毒性并增加了组成型和激活型通道电流。 TRPV4 突变以前被认为与骨骼发育不良有关。我们的研究结果表明 TRPV4 突变也会导致周围神经退行性疾病。 CMT2C 突变位于 TRPV4 锚蛋白重复序列​​的不同区域，表明这种显着的表型变异可能是由于对调节蛋白-蛋白相互作用的不同影响所致。随后，我们研究了相互作用的蛋白质，并使用外显子组测序在 CMT2C 家族中寻找致病突变，但我们没有发现 TRPV4 突变。外显子组分析揭示了该家族中的一个新的 TRPV4 突变 R186Q，由于引物位点上罕见的多态性，桑格测序遗漏了该突变。 我们发现马里的一个家庭有两个姐妹患有痉挛性截瘫。患者除下肢痉挛、无力外，上肢远端明显萎缩。使用单核苷酸多态性阵列进行的纯合性作图表明，姐妹俩在染色体 19p13.11-q12 上共享一个扩展纯合性区域，而对照则不共享该区域。这些发现表明一种临床和遗传上独特的遗传性痉挛性截瘫伴肌萎缩症，命名为 SPG43。随后，我们通过外显子组测序评估了关键区域的候选基因，并鉴定了一个基因 C19orf12，其序列变异在对照中未发现。该变体与转染细胞中重组蛋白的异常定位有关。该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截瘫的新原因。