Exome re-sequencing candidate loci for familial essential tremor

描述(申请人提供)：特发性震颤(ET)是人类最常见的神经系统疾病之一，患病率几乎达到4%。在大多数家族性病例中，ET是遗传的显性性状，具有不完全外显性，但复杂的多基因传递是可能的。在染色体3p13.1(ETM1)、2p24(ETM2)和6p23(ETM3)上存在3个家族性ET的遗传易感基因，但在与这些基因连锁的家系中尚未发现致病基因。16个冰岛家系中的ETM1基因多态和23个法国家系中的多巴胺受体D3基因变异与ET表型相关。在四个美国家庭中，ETM2基因座与一种疾病等位基因有关。进一步的研究表明，在美国、新加坡和韩国的人群中，ETM2和ET表型之间存在紧密的等位基因关联。两个北美家系与ETM3基因座连锁，其中最大的一个家系表现出除ET外还包括肌张力障碍的混合表型。由于表型的存在、不完全的外显和ET的高患病率，遗传模式不明阻碍了对致病基因的寻找。通过允许大规模DNA测序，固相微阵列和仪器的进步在一定程度上缓解了这些障碍。基因组生物信息学的进步和有效的标准种群数据库(如1000基因组SNP数据库、数据库SNP和HapMap)提供了从假定的突变中筛选遗传变异的能力。利用这项技术，我们最近通过4.2兆碱基(Mb)候选区域的高密度测序，在三个无关的家系中发现了一个新基因-猫白血病病毒C亚群受体1基因的错义突变，该基因是导致孟德尔病、后柱性共济失调和视网膜色素变性的原因。这一发现证实了我们在开展拟议的基因研究所需的所有特定技术方面的专业知识，并为特定目标1利用高通量重测序技术在两个与ETM2和ETM3基因座相关联的信息丰富的大型家族中识别导致ET的基因提供了原则证据。我们已经为24.6Mb的ETM2(48,054个诱饵)和14.4Mb的ETM3(30,796个诱饵)设计了一个长度为120个碱基对的诱饵平铺文库，包括所有的调节区、外显子、剪接位点、增强子和保守的基因间隔区。一项能力分析估计，检测突变所需的样本量为10个具有疾病单倍型的患者和10个未受影响的非患者，每个家系中与ETM2和ETM3基因座相关联的个体。桑格测序将重新确认在这两个家庭的20名受影响个人中发现的ETM2和ETM3突变。这些候选基因将在另外73个与ET无关的家庭中进行桑格测序，以确定其他ETM2和ETM3突变。未来使用体外和体内模型的实验将分析ET基因突变在衰老和发育过程中的功能后果。 公共卫生相关性：ET基因的识别将提高诊断的准确性，完善震颤的分类，是找到有效治疗方法的关键一步。研究突变的ET基因的不良影响将增加我们对ET和其他运动障碍(如帕金森病)的了解。

DESCRIPTION (provided by applicant): Essential tremor (ET) is one of the most common neurological disorders in humans with a prevalence reaching almost four percent. ET is inherited as a dominant trait with incomplete penetrance in most familial cases but complex multi-genic transmission is possible. Three genetic susceptibility loci for familial ET exist on chromosomes 3p13.1 (ETM1), 2p24 (ETM2), and 6p23 (ETM3), but the causal genes have not been identified in the families linked to these loci. Polymorphic ETM1 loci in 16 Icelandic families and a variant in the dopamine receptor D3 gene in 23 French families are associated with an ET phenotype. ETM2 loci are linked to a disease allele in four American families. Further studies suggest a tight allelic association between ETM2 and the ET phenotype in populations from the United States, Singapore, and Korea. Two North American families are linked to ETM3 loci with the largest family showing a mixed phenotype that includes dystonia in addition to ET. An unclear pattern of inheritance due to the presence of phenocopies, incomplete penetrance, and the high prevalence of ET, hinder the search for causal genes. Advances in solid- phase microarrays, and instrumentation have partially alleviated these barriers by permitting large scale DNA sequencing. Advances in genome bioinformatics and the availability of validated normative population databases (e.g. 1000 Genomes SNP database, dbSNP, and HapMap) provide the capability to filter genetic variants from putative mutations. By using this technology, we recently identified missense mutations in a novel gene, feline leukemia virus subgroup C receptor 1 gene, as the cause of the Mendelian disorder, posterior column ataxia and retinitis pigmentosa, in three unrelated families by high density sequencing of the 4.2 megabase (Mb) candidate region. This discovery confirms our expertise in all of the specific technologies required to carry out the proposed genetic studies and provides proof of principle for Specific Aim 1 to identify the genes that cause ET in two large, informative families linked to the ETM2 and ETM3 loci using high- throughput resequencing technology. We have designed a bait tiling library using 120 base-pair bait lengths for the 24.6 Mb ETM2 (48,054 baits) and the 14.4 Mb ETM3 (30,796 baits) loci to include all regulatory regions, exons, splice sites, enhancer, and conserved intergenic regions. A power analysis estimates that the sample size required to detect mutations with 95% confidence are 10 affected individuals with a disease haplotype and 10 unaffected individuals without the disease haplotype from each family linked to the ETM2 and ETM3 loci. Sanger sequencing will reconfirm the ETM2 and ETM3 mutations identified in the 20 affected individuals from the two families. These candidate genes will be Sanger sequenced in 73 additional, unrelated families with ET to identify other ETM2 and ETM3 mutations. Future experiments using in vitro and in vivo models will analyze the functional consequences of ET gene mutations during aging and development. PUBLIC HEALTH RELEVANCE: The identification of ET genes will improve diagnostic accuracy, refine the classification of tremor, and is a pivotal step toward finding effective treatments. Studying the untoward effects of mutant ET genes will increase our knowledge of ET and other movement disorders such as Parkinson disease.