Exome re-sequencing candidate loci for familial essential tremor

描述（由申请人提供）：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%。ET在大多数家族性病例中是以不完全显性遗传的方式遗传的，但复杂的多基因传播是可能的。在染色体3p13.1（ETM1）、2p24（ETM2）和6p23（ETM3）上存在三个家族性ET的遗传易感基因座，但与这些基因座连锁的家族中的致病基因尚未鉴定。16个冰岛家庭的多态性ETM1基因座和23个法国家庭的多巴胺受体D3基因的变体与ET表型相关。ETM2基因座与四个美国家庭中的疾病等位基因连锁。进一步的研究表明，在美国、新加坡和韩国人群中，ETM2和ET表型之间存在紧密的等位基因关联。两个北美家族与ETM3基因座连锁，其中最大的家族显示出除了ET之外还包括肌张力障碍的混合表型。由于存在表型模仿、不完全遗传和ET的高患病率而导致的遗传模式不清楚，阻碍了对致病基因的研究。固相微阵列和仪器的进步通过允许大规模DNA测序而部分缓解了这些障碍。基因组生物信息学的进展和经验证的规范性人群数据库（例如1000个基因组SNP数据库、dbSNP和HapMap）的可用性提供了从推定突变中筛选遗传变异的能力。通过使用这项技术，我们最近确定了一个新的基因，猫白血病病毒亚群C受体1基因，错义突变的原因孟德尔氏紊乱，后柱共济失调和视网膜色素变性，在三个不相关的家庭的4.2兆碱基（Mb）的候选区域的高密度测序。这一发现证实了我们在开展拟议遗传研究所需的所有特定技术方面的专业知识，并为Specific Aim 1提供了原理证明，以使用高通量重新测序技术在与ETM 2和ETM 3基因座相关的两个大型、信息丰富的家族中识别导致ET的基因。我们设计了一个诱饵平铺文库，使用120个碱基对诱饵长度的24.6 Mb ETM2（48，054诱饵）和14.4 Mb ETM3（30，796诱饵）基因座，包括所有的调控区，外显子，剪接位点，增强子，和保守的基因间区域。功效分析估计，以95%置信度检测突变所需的样本量是来自与ETM2和ETM3基因座连锁的每个家族的10个具有疾病单倍型的受影响个体和10个不具有疾病单倍型的未受影响个体。桑格测序将再次确认来自两个家族的20名受影响个体中鉴定的ETM2和ETM3突变。这些候选基因将在另外73个不相关的ET家族中进行桑格测序，以鉴定其他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基因的鉴定将提高诊断的准确性，完善震颤的分类，是寻找有效治疗方法的关键一步。研究突变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认识。

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.