Nonsense-mediated decay array analysis of atypical Werner syndrome

非典型维尔纳综合征的无义介导的衰变阵列分析

• 批准号: 7915620
• 负责人: JUNKO OSHIMA
• 金额: $ 15.99万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-15 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7915620
• 关键词: Abbreviations; Adult; Aging; Appearance; Atherosclerosis; Bilateral; Caffeine; Candidate Disease Gene; Cataract; Cell Array Analyses; Complex; Dactinomycin; Diagnosis; Disease; Emetine; Exonuclease; Family; Fibroblasts; Funding; Gene Mutation; Genes; Genetic; Genome Stability; Goals; Hereditary Disease; International; Investigation; Knowledge; Lead; Maintenance; Malignant Neoplasms; Mediating; Messenger RNA; Molecular; Molecular Diagnosis; Mutate; Mutation; Non-Insulin-Dependent Diabetes Mellitus; Nonsense Codon; Nonsense-Mediated Decay; Oligonucleotides; Osteoporosis; Pathogenesis; Pathway interactions; Patients; Phenotype; Phosphotransferases; Ploidies; Proteins; Quality Control; RNA Interference; Recruitment Activity; Registries; Research Project Grants; Reverse Transcriptase Polymerase Chain Reaction; Sampling; Scanning; Syndrome; System; Transcript; Translations; Universities; WRN gene; Washington; Werner Syndrome; age related; aged; base; genetic pedigree; helicase; human WRN protein; inhibitor/antagonist; lymphoblastoid cell line; mRNA Decay; mRNA Transcript Degradation; middle age; mutant; novel; null mutation; prevent; public health relevance; small hairpin RNA

DESCRIPTION (provided by applicant): Werner syndrome (WS) is an "adult-onset" genetic disorder characterized by multiple features consistent with accelerated aging (i.e., a "segmental progeroid syndrome"). An aged appearance and common age-related disorders such as type 2 diabetes mellitus often emerge during the early 20s. Bilateral ocular cataracts are typically extracted during the early 30s, and atherosclerosis and cancers occur by the fifth decade. "Classical" WS is caused by mutations at the WRN gene, which encodes a DNA helicase/exonuclease. Clinically diagnosed or suspected WS cases are referred to our International Registry of Werner Syndrome (University of Washington, Seattle WA) from all over the world for molecular diagnosis. Cases without WRN mutations are operationally categorized as "atypical WS" (AWS). In 2003, we identified LMNA mutations among a small subset of atypical WS cases by a candidate gene approach. As of January 2008, the Werner registry has 107 WS pedigrees with WRN mutations, 6 AWS patients with LMNA mutations, and 41 AWS pedigrees in which we did not find WRN or LMNA mutations. Primary fibroblasts, lymphoblastoid cell lines or both are available from 29 of the 41 atypical cases. The purpose of this study is to identify additional genes responsible for AWS, perhaps revealing a single heretofore unsuspected major pathogenic locus. Our approach is motivated by the knowledge that ~one-third of disease mutations are thought to result in premature termination codons that lead to the rapid degradation of mRNA via the nonsense-mediated mRNA decay (NMD) pathway. A strategy using inhibition of NMD combined with array analysis has been developed to identify disease genes. In this approach, mutated mRNAs that would have undergone NMD are stabilized and their mRNAs detected by the array analysis. Our screens will utilize three different NMD inhibitors, emetine (inhibits initial scanning of truncated proteins), caffeine (inhibits SMG1 kinase in the NMD complex combined with actinomycin D), and an RNAi against UFP1 (a component of the NMD complex) (Aim 1). Stabilization of mutant mRNAs will be confirmed by quantitative PCR (Aim 2), and candidate genes will be sequenced using DNA from the index cases and other AWS cases (Aim 3). Once the genetic evidence is established, further investigations of pathogenesis of will be proposed via separate funding. PUBLIC HEALTH RELEVANCE: Atypical Werner syndrome patients present accelerated aging phenotypes similar to Werner syndrome but do not carry WRN or LMNA mutations. We proposed to identify the genes responsible for atypical WS using the strategy of non-sense mediated mRNA decay inhibition and array analysis.

描述（由申请人提供）：Werner综合征（WS）是一种“成人发病”遗传性疾病，其特征在于与加速衰老一致的多种特征（即，“节段性早老综合征”）。老年人的外表和常见的年龄相关疾病，如2型糖尿病，往往出现在20岁出头。双眼白内障通常在30岁早期摘除，动脉粥样硬化和癌症在50岁时发生。“经典”WS由编码DNA解旋酶/核酸外切酶的WRN基因突变引起。将来自世界各地的临床诊断或疑似WS病例转诊至我们的Werner综合征国际登记处（华盛顿大学，西雅图WA）进行分子诊断。没有WRN突变的病例在操作上被归类为“非典型WS”（AWS）。在2003年，我们通过候选基因的方法在一小部分非典型WS病例中发现了LMNA突变。截至2008年1月，Werner登记处有107个WRN突变的WS家系，6个LMNA突变的AWS患者，41个未发现WRN或LMNA突变的AWS家系。41例非典型病例中有29例可获得原代成纤维细胞、淋巴母细胞样细胞系或两者。这项研究的目的是确定其他基因负责AWS，也许揭示了一个单一的迄今未被怀疑的主要致病基因座。我们的方法的动机是认识到，约三分之一的疾病突变被认为会导致提前终止密码子，导致mRNA通过无义介导的mRNA衰变（NMD）途径快速降解。利用抑制NMD结合阵列分析的策略已经被开发用于识别疾病基因。在这种方法中，已经经历NMD的突变mRNA被稳定化，并且它们的mRNA通过阵列分析检测。我们的筛选将利用三种不同的NMD抑制剂，embrane（抑制截短蛋白的初始扫描），咖啡因（抑制与放线菌素D组合的NMD复合物中的SMG 1激酶）和针对UFP 1（NMD复合物的组分）的RNAi（Aim 1）。将通过定量PCR确认突变mRNA的稳定性（目标2），并使用索引病例和其他AWS病例的DNA对候选基因进行测序（目标3）。一旦遗传证据确立，将通过单独的资金提出进一步的发病机制调查。公共卫生关系：非典型Werner综合征患者表现出与Werner综合征相似的加速衰老表型，但不携带WRN或LMNA突变。我们建议使用无义介导的mRNA衰减抑制和阵列分析的策略来确定负责非典型WS的基因。