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，在约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，因此有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，从而丧失功能突变 必须通过实验来定义。此外，非编码（即调控）基因突变的后果 序列通常高度依赖于特定的细胞类型。因此，功能测定必须在 在适当的CNS细胞类型中。 为了解决搜索空间的挑战，我们建议特别关注非翻译区（UTR）， mRNA。UTR是重要的、保守的调控序列，其通过改变蛋白质水平而深刻地影响蛋白质水平。 特定基因的翻译速率或转录稳定性。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ASD基因突变是偶然的。 UTR突变可能导致疾病。为了解决缺乏用于解释UTR突变的编码，我们 我组建了一个拥有独特专业知识组合的团队， 在体外和体内ASD相关细胞类型中分析来自ASD患者的UTR变体。结合 两个创新但已确立的组成部分：转录后大规模平行报告基因测定和细胞类型 具体的翻译谱，我们的目标是建立一个管道，1）确定UTR突变，导致改变 蛋白质水平，2）对这些变异进行遗传负荷和关联测试，以及3）确定 改变特定突变的蛋白质水平的机制。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.