Function and polymorphisms of complement receptor 1 (CR1) in Alzheimer's disease

描述(由申请人提供)：最近的全基因组关联研究(Gwas)发现，CR1的单核苷酸多态(SNPs)与阿尔茨海默病(AD)的遗传风险之间的关联排名第三，仅次于ApoE和ApoJ。三项不重叠的GWAS努力证实了CR1 SNP与AD风险之间存在高度显著的关联。本项目的总体目标是填补全球气候变化网络报告留下的两个主要空白。1)在AD中CR1的功能是如何改变的？2)CR1基因多态性在多大程度上解释了这些功能改变？在我们的提案中，这两个问题通过反向工程战略联系在一起，该战略将首先评估AD中的CR1功能变化，然后使用该信息来确定负责的CR1 SNP。具体目的1.确定AD和ND患者CR1蛋白分子量、表达、结合或功能的异常。尽管CR1与AD风险有关，但实际上CR1在大脑中的表达非常低。相反，它的主要作用是调节自发的补体激活和清除病原体和免疫复合体(IC)-这两者几乎都只发生在外周循环中。因此，目的1将使用研究人员已有文献记载的其他技术，评估300名AD和300名ND患者的血液样本中CR1的结构、表达、结合、补体调节和病原体/IC清除。SNPs本身不会引起疾病风险；SNPs引起的功能变化会引起疾病风险。因此，检测CR1在AD中的功能机制是理解AD相关CR1基因多态性的全部意义的必要的第一步。特定目的2.检测目标1中发现的一个或多个CR1蛋白异常与特定CR1基因多态的关联程度。以前的Gwas分析只能对CR1中已知的476个SNP中的一小部分进行采样，他们发现的CR1 SNPs位于非编码区。然而，阐明精确的、增加风险的SNPs涉及许多众所周知的挑战和数千个样本。因此，我们选择了更温和的目标--寻找功能相关性。使用Aim 1样本，将使用RainDance富集法选择CR1基因组区域，然后在Illumina GA HiSeq仪器上进行条码下一代测序。然后，将在受试者中测试CR1 SNP是否与AIM 1中发现的任何CR1蛋白变化有关。一个潜在的捷径在于，已有近12个CR1 SNP与其他疾病背景下的特定CR1蛋白异常有关。候选SNPs将通过定点突变进一步评估，以确定它们中的哪一个重现了目标1 CR1蛋白缺陷。最终目标将是在更大的样本人群中进行验证和真正的风险评估，这项工作将通过我们事先识别导致特定CR1功能改变的CR1 SNP来保证和促进这一工作。

DESCRIPTION (provided by applicant): A recent genome-wide association study (GWAS) found that single nucleotide polymorphisms (SNPs) in CR1 exhibited the third highest association with genetic risk for Alzheimer's disease (AD), following only ApoE and ApoJ. Three non-overlapping GWAS efforts have confirmed a highly significant CR1 SNP association with AD risk. The overall goal of the present project is to fill in two major gaps left by the GWAS reports. 1) How is CR1 functionally altered in AD? and 2) To what extent do CR1 polymorphisms account for the functional alterations? These two issues are linked in our proposal by a reverse-engineering strategy that will first assess functional CR1 changes in AD, then use that information to identify the responsible CR1 SNPs. Specific Aim 1. Identify abnormalities in CR1 protein molecular weight, expression, binding, or function in AD and ND subjects. Despite its association with AD risk, CR1 is actually very poorly expressed in brain. Instead, its primary roles are regulation of spontaneous complement activation and clearance of pathogens and immune complexes (ICs)-both of which occur almost exclusively in the peripheral circulation. Aim 1 will therefore evaluate CR1 structure, expression, binding, complement regulation, and pathogen/IC clearance in blood samples from 300 AD and 300 ND subjects using ELISA, Western blot, complement activation and other techniques with which the investigators have documented expertise. SNPs themselves do not cause disease risk; functional changes induced by SNPs do. Testing mechanisms of CR1 function in AD is therefore an essential first step in understanding the full significance of AD-related CR1 polymorphisms. Specific Aim 2. Test the extent to which the CR1 protein abnormality or abnormalities identified in Aim 1 are linked to specific CR1 gene polymorphisms. Previous GWAS analyses were able to sample only a small fraction of the 476 known SNPs in CR1, and the CR1 SNPs they identified turn out to be in non- coding regions. The elucidation of exact, risk-enhancing SNPs, however, entails many well known challenges and thousands of samples. We have therefore chosen the more modest goal of seeking functional correlates. Using the Aim 1 samples, a RainDance enrichment approach will be employed to select for the CR1 genomic region, followed by bar-coded next generation sequencing on the Illumina GA HiSeq instrument. CR1 SNPs will then be tested, within subjects, for association with any CR1 protein alterations found in Aim 1. A potential shortcut lies in the fact that nearly a dozen CR1 SNPs have already been linked to specific CR1 protein abnormalities in other disease contexts. Candidate SNPs will be further evaluated by site-directed mutagenesis to determine which of them recapitulates Aim 1 CR1 protein defects. An ultimate goal will be to perform validation and true risk assessment in a much larger sample population, an undertaking that would be warranted and facilitated by our prior identification of CR1 SNPs that induce specific CR1 functional alterations.