Highly parallel analysis of 5' and 3' UTR variants in Autism Spectrum Disorders

目前正在进行大量投资对自闭症谱系障碍家庭的基因组进行测序 (ASD)。然而，在~1%的蛋白质编码序列之外识别疾病突变是具有挑战性的 因为1)搜索空间要大得多，因此更多的突变是偶然发生的，2) 没有简单的编码来识别非编码序列中的有害突变，从而失去功能突变 必须通过实验来定义。此外，非编码突变的后果(即受调控的) 序列通常高度依赖于特定的细胞类型。因此，功能分析必须在 体内，在适当的中枢神经系统细胞类型中。 为了解决搜索空间的挑战，我们建议专门关注的未翻译区域(UTR MRNAs。UTRs是重要的、保守的调控序列，通过改变蛋白质水平而深刻地影响蛋白质水平 特定基因的翻译率或转录本稳定性。重要的是，在ASD病例中， 已知ASD基因的UTR突变率比预期的要高，这表明大约有一半的人 非编码区突变可能导致疾病。为了解决缺乏解释UTR突变的代码的问题，我们 已经组建了一支拥有独特专业知识组合的团队，以执行大规模并行功能 分析ASD患者体内和体外ASD相关细胞类型的非编码区变异。组合 两个创新但成熟的组成部分：转录后大规模平行报告分析和细胞类型 具体的翻译图谱，我们的目标是建立一条管道，以1)识别导致改变的UTR突变 蛋白质水平，2)对这些变异进行遗传负担和关联测试，以及3)定义分子 针对特定突变改变蛋白质水平的机制。这条管道将利用现有的大量投资 在ASD基因组测序中，通过定义在一类序列中引起突变的个体非编码疾病 到目前为止，这还不是疾病研究的重点。

Substantial investments are being made to sequence the genomes of families with Autism Spectrum Disorder (ASD). However, identifying disease mutations outside the ~1% of protein coding sequences is challenging because 1) the ‘search space’ is so much larger, and thus many more mutations occur by chance, and 2) there is no simple code to identify deleterious mutations in non-coding sequence, and thus loss of function mutations must be defined experimentally. In addition, the consequences of mutations in non-coding (i.e. regulatory) sequences are often highly dependent on the specific cell type. Thus functional assays must be conducted in vivo, in the appropriate CNS cell types. To address the search space challenge, we propose to focus specifically on the untranslated regions (UTRs) of mRNAs. UTRs are important, conserved regulatory sequences that profoundly impact protein levels by altering translation rates or transcript stability for specific genes. Importantly, in ASD cases there is a 2-fold greater rate of UTR mutations in known ASD genes than expected by chance, indicating that roughly half of these UTR mutations may contribute to disease. To address the lack of a code for interpreting UTR mutations, we have assembled a team with a unique combination of expertise to conduct massively parallel functional analysis of UTR variants from ASD patients, in ASD-relevant cell types in vitro and in vivo. Combining two innovative but established components: post-transcriptional massively parallel reporter assays, and cell type specific translational profiling, we aim to establish a pipeline to 1) Identify UTR mutations that result in altered protein levels, 2) conduct genetic burden and association testing on these variants, and 3) define the molecular mechanisms altering protein levels for specific mutations. This pipeline will leverage the existing large investment in ASD genome sequencing by defining individual non-coding disease causing mutations in a class of sequences that has, so far, not been the focus of disease studies.