Genetic and Functional Analysis of Nested AD Risk Genes CTNNA3 and LRRTM3

 描述（由申请人提供）：晚发性阿尔茨海默病（LOAD）的全基因组关联研究（GWAS）确定了候选遗传位点，但无法明确揭示疾病基因或变体。此外，GWAS变异不能解释疾病的全部遗传性。这种“缺失遗传性”的一个重要因素可能是GWAS遗漏的具有较大效应大小的罕见功能性疾病变异。事实上，最近通过测序在TREM 2中鉴定出罕见和强AD风险变体支持了这一假设。因此，在LOAD中存在下一代测序（NGS）的努力，然而NGS具有其自身的一系列挑战。首先，从NGS中鉴定出的大量变体将需要优先考虑下游复制和功能研究。其次，需要适当的测定来测试这些变体的功能后果。最后，大量样本的NGS仍然是成本高昂的，排除了变体的快速功能评估。在这个探索性的R21中，我们提出了一种具有成本效益的新的替代方法，并计划将其应用于两个有趣的嵌套候选AD基因：CTNNA 3和LRRTM 3。我们将利用现有的和公开可用的全外显子组和基因组序列（WES，WGS）数据来识别变体，以在我们的约11，000名受试者的病例对照队列中进行测试（目标1），并通过体外功能测定评估最有希望的优先变体（目标2）。我们关于AD GWAS基因ABCA 7的初步数据，以及TREM 2的发现，为我们方法的可行性提供了强有力的支持。CTNNA 3和LRRTM 3位于AD风险和淀粉样蛋白水平的连锁区域，由其他人和co-PI独立鉴定。在我们发表的数据中，它们具有相反的转录方向和强烈相关的基因表达水平，表明功能相互作用。这两种基因都与突触发生有关。我们鉴定了CTNNA 3中的内含子变体，这些变体解释了我们的A β连锁信号。LRRTM 3影响APP处理。我们显示了LRRTM 3、APP和BACE 1之间的蛋白质相互作用，并在此证明了在长时程增强中的作用。总之，嵌套CTNNA 3/LRRTM 3是在APP加工和/或突触生理学中具有潜在作用的优秀候选AD基因。我们和其他人报道了这两个基因的变异与AD风险的相关性，但复制一直不一致，这可能部分是由于缺乏对罕见变异的评估。我们的目标是：1）测试从3个NIH资助的NGS数据中鉴定的CTNNA 3/LRRTM 3中的罕见变体，EVS（WES 4300名高加索人，2203名非洲裔美国人），ADNI（WGS 246名对照，359名轻度认知障碍，184名AD），ADSP（来自111个AD家族的584名受试者），在我们的11，000名受试者中进行AD风险关联。2)为了测试CTNNA 3/LRRTM 3在APP加工、细胞毒性和突触完整性中的推定功能变体。这种探索性的R21将能够对两个有趣的候选AD基因进行彻底的和基于假设的研究，并且还将建立我们的成本效益方法作为范式转变的替代方案，以评估AD和其他复杂疾病中提名的大量基因。

 DESCRIPTION (provided by applicant): Genome-wide association studies (GWAS) in late-onset Alzheimer's disease (LOAD) identified candidate genetic loci, however cannot unequivocally uncover the disease gene or variants. Further, GWAS variants do not explain the full disease heritability. An important factor underlying this "missing heritability" may be rare functional disease variants of larger effect size that are missed by GWAS. Indeed, the recent identification of rare and strong AD risk variants in TREM2 via sequencing supports this hypothesis. Consequently, there are efforts for next-generation sequencing (NGS) in LOAD, however NGS comes with its own set of challenges. First, the large number of variants identified from NGS will require prioritization for downstream replication and functional studies. Second, appropriate assays are needed to test the functional consequences of these variants. Finally, NGS of large number of samples are still cost-prohibitive, precluding rapid functional assessment of variants. In this exploratory R21, we propose a cost-effective, novel alternative approach and plan to apply it to two intriguing nested candidate AD genes: CTNNA3 and LRRTM3. We will take advantage of existing and publicly available whole exome and genome sequence (WES, WGS) data to identify variants to test in our case-control cohort of ~11,000 subjects (Aim 1) and to assess the most promising, prioritized variants by in-vitro functional assays (Aim 2). Our preliminary data on the AD GWAS gene ABCA7, generated by this novel paradigm, as well as TREM2 findings, provide strong support for the feasibility of our approach. CTNNA3 and LRRTM3 reside in a linkage region of AD risk and amyloid ß levels identified independently by others and the co-PIs. They have opposite transcriptional orientation and strongly correlated gene expression levels in our published data, suggesting functional interactions. Both genes are implicated in synaptogenesis. We identified intronic variants in CTNNA3 that account for our Aß linkage signal. LRRTM3 influences APP processing. We showed protein interactions between LRRTM3, APP and BACE1 and herein demonstrate a role in long term potentiation. In summary, the nested CTNNA3/LRRTM3 are excellent candidate AD genes with potential roles in APP processing and/or synaptic physiology. We and others reported association of variants in both genes with AD risk, but replication has been inconsistent, which may in part be due to the lack of an assessment of rare variants. Our aims are: 1) To test rare variants in CTNNA3/LRRTM3 identified from 3 NIH funded NGS data, EVS (WES 4300 Caucasians, 2203 African-Americans), ADNI (WGS 246 Controls, 359 mild cognitive impairment, 184 AD), ADSP (584 subjects from 111 AD families), for AD risk association in our 11,000 subjects. 2) To test putative functional variants in CTNNA3/LRRTM3 in APP processing, cell toxicity and synaptic integrity. This exploratory R21 will enable a thorough and hypothesis-based study of two intriguing candidate AD genes and will also establish our cost-effective approach as a paradigm-shifting alternative to assess the deluge of genes being nominated in AD and other complex diseases.