Functional effects of ion channel mutations found via exome sequencing

神经系统疾病患者的基因组分析变得越来越普遍，提供了大量与患者DNA变化相关的基因组数据。然而，基因突变的识别率并没有与理解这些突变对靶蛋白的功能影响的进展相匹配。对于大多数基因，已知的疾病相关的错义突变远远超过功能数据存在的突变。因此，我们所知道的突变与我们对单个突变如何影响蛋白质功能的理解之间存在着巨大的鸿沟。随着测序步伐的加快，这一鸿沟越来越大。虽然存在预测哪些错义突变可能有害的计算方法，但这些算法是功能数据的不良替代品。因此，迫切需要一种手段，通过这种手段，面向临床的实验室可以获得对突变影响的功能性洞察。该R24提案旨在填补配体门控谷氨酸受体基因家族的突变和功能之间的差距，该基因家族包括分别由GRIA、GRIK和GRIN基因编码的AMPA、红藻氨酸和NMDA受体。将重点放在一个基因家族上的决定是与NIH协商后做出的，并受到该资助机会的预算的推动。在编码NMDA受体亚基的这个基因家族中，已知突变的数量正在快速增长，仅在过去的18个月中就描述了100多个新的疾病相关突变，还有更多已知但未发表的突变。因此，虽然我们将关注所有谷氨酸受体突变，但我们预计大多数突变将在GRIN家族中发现。对谷氨酸受体突变感兴趣的研究人员将分享氨基酸的变化，将其引入商业cDNA中用于异源表达系统，以获得描述突变影响的功能数据。资源中心将与临床实验室共享此功能信息，并努力促进数据的发布。这一努力将填补文献中的一个关键空白，促进我们对神经系统疾病的理解，并为未来的治疗模式提供新的思路。 三项活动将得到R24资源赠款的支持。首先，我们将联系至少100个进行全外显子组/基因组测序的实验室，告知他们这一资源。其次，对于已经鉴定GRIN突变并希望获得功能分析的临床或研究者，我们将突变引入市售的人cDNA中，并功能性地评估突变对激动剂效力、调节剂敏感性、响应时间过程和受体表面表达的影响。第三，我们将在网络上开发一个可搜索的数据库，以存档该项目的结果，并将我们的结果与几个现有的数据库（ClinVar，EGI等）连接起来。

Genomic analysis of patients with neurological conditions is becoming increasingly more common, providing a great deal of genomic data relating changes in patient DNA to neurological disease. However, the rate at which genetic mutations are identified has not been matched by advances in understanding the functional effects of these mutations on target proteins. For most genes, known disease-associated missense mutations far outnumber mutations for which functional data exists. Thus, a large gulf exists between the mutations we know about and our understanding of how individual mutations impact protein function. This gulf is growing wider with the accelerating pace of sequencing. While computational methods exist that predict which missense mutations may be harmful, these algorithms are a poor substitute for functional data. Thus, there is a critical need for a means by which clinically-oriented laboratories can obtain functional insight into the effects of mutations. This R24 proposal seeks to fill the gap between mutations and function for the ligand-gated glutamate receptor gene family, which includes AMPA, kainate, and NMDA receptors encoded by GRIA, GRIK, and GRIN genes, respectively. The decision to focus efforts on one gene family was made in consultation with NIH, and was driven by the budget for this funding opportunity. The number of known mutations in this family of genes encoding NMDA receptor subunits is growing at a fast pace, with well over a hundred novel disease- associated mutations described in just the past 18 months, and many more known but unpublished. Thus, while we will focus on all glutamate receptor mutations, we expect most mutations identified will be in the GRIN family. Interested investigators with glutamate receptor mutations will share the amino acid change, which will be introduced into commercial cDNAs for use in heterologous expressions systems to obtain functional data describing the effects of the mutation. The Resource Center will share this functional information with the clinical lab, and work to facilitate publication of the data. This effort will fill a critical gap in the literature, advance our understanding of neurological disease, and provide new ideas about future treatment paradigms. Three activities will be supported by this R24 resource grant. First, we will contact at least 100 laboratories performing whole exome/genome sequencing to inform them of this resource. Second, for clinics or investigators who have identified GRIN mutations and would like to obtain functional analysis, we will introduce the mutation into commercially available human cDNAs and functionally evaluate the effects of the mutation on agonist potency, modulator sensitivity, response time course, and receptor surface expression. Third, we will develop a searchable database on the web to archive results from this project, and connect our results with several existing databases (ClinVar, EGI, etc.).