Rare-variant analysis to validate HIV-target genes affecting human HIV-response

 描述(申请人提供)：人类免疫缺陷病毒(HIV)是一种慢病毒，它感染和破坏携带CD4的淋巴细胞1，2，最终使宿主的免疫系统无法对通常无害的病原体1，3，4做出足够的反应。HIV于20世纪80年代首次发现，主要发生在旧金山和纽约的男同性恋者1，5，6中，但此后发展成为一种世界性流行病，目前感染HIV的3500万人中有70%生活在发展中国家1，4，7。然而，人们很快注意到，人类对HIV的反应存在自然变异；一些人永远不会感染艾滋病毒，而另一些人在感染后从未出现获得性免疫缺陷综合症(艾滋病)，以及其他可变反应1，8研究人员此后认为，人类群体中存在的潜在基因变异可能是人类对艾滋病毒反应的这些差异的部分原因，因此是开发艾滋病毒治疗方法的一种可能方法。然而，尽管人类遗传学研究已有20多年，包括1990年代的候选基因研究和2000年代末的全基因组关联研究，但仍然没有针对HIV1、9的疫苗，也很少有被确定与艾滋病毒相关表型显著相关的遗传位点8、10-13。对这种缺乏进展的可能解释是，最近的一组研究Gwas以一种无假设的方法询问了整个基因组的关联，因此产生了大量的多重测试负担，并且几乎没有对哪些基因座最重要的洞察力。另外， GWAS只关注常见的遗传变异(微小等位基因频率，MAF，&gt；5%)，而众所周知，在MAF低于5%8的情况下，存在大量的遗传变异。因此，这项建议的目的是克服GWAS方法的这些缺点。首先，目标1将是识别与艾滋病毒感染或艾滋病进展相关的具有过多罕见变异的候选人类艾滋病毒靶基因。已经创建了具有强大先验生物学证据的1,693个候选艾滋病毒基因的名单，通过进行基因外显子组测序，我们将发现罕见的变异；这些步骤加在一起将使我们能够克服缺乏假设、包含巨大的多重测试负担以及仅限于常见变异的问题。其次，AIM 2将通过多个队列中的后续基因配对/测序以及包括siRNA敲除和CRISPR在内的体外分子分析来验证AIM 1的前25个命中。通过进行这些实验，我们将为在AIM 1中发现的关联提供更严格的证据，并开始了解与人类对艾滋病毒的反应相关的新宿主生物学以及与潜在药物靶标和疫苗开发相关的过程。

 DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) is a lentivirus that infects and destroys CD4-bearing lymphocytes1,2, eventually rendering the host's immune system incapable of mounting sufficient responses to even commonly- innocuous pathogens1,3,4. HIV was first discovered in the 1980s predominantly among gay men in San Francisco and New York1,5,6, but since then has developed into a world-wide epidemic, with 70% of the 35 million individuals currently infected with HIV living in the developing world1,4,7. t was quickly noted however that humans contain natural variation in their response to HIV; some individuals never become infected with HIV, while others upon infection never develop Acquired Immunodeficiency Syndrome (AIDS), among other variable responses1,8. Researchers have since believed that the underlying genetic variation present among human populations may be partially responsible for these differences in human response to HIV, thus representing a possible way to develop a treatment for HIV. However, despite 20+ years of human genetics research, including candidate gene studies in the 1990s and genome-wide association studies (GWAS) in the late 2000s, there is still no vaccine for HIV1,9 and very few genetic loci that have been identified as significantly associated with HIV-related phenotypes8,10-13. Possible explanations for this lack of progress is that the most recent set of studies, GWAS, interrogated the entire genome for association in a hypothesis-free approach, thus producing a large multiple-testing burden and giving little insight into which loci are most important. Additionally, GWAS only focused on common genetic variation (minor allele frequency, MAF, >5%), whereas it is known that much genetic variation is present at MAFs below 5%8. Therefore, the aims of this proposal are focused on overcoming these drawbacks of the GWAS approach. First, AIM 1 will be to identify candidate human HIV-target genes with an excess of rare variation in association with either HIV-infection or AIDS- progression. A list of 1,693 HIV-candidate genes with strong a priori biological evidence has already been created and by conducting gene-exome sequencing we will discover rare variants; taken together these steps will allow us to overcome the issues of lacking a hypothesis, containing a large multiple-testing burden and being limited to common variation. Second, AIM 2 will be to validate the top 25 hits from AIM 1 by a combination of follow-up genoptying/sequencing in multiple cohorts and in vitro molecular assays, including siRNA knockdowns and CRISPRs. By conducting these experiments we will provide more rigorous evidence for the associations discovered in AIM 1 as well as begin the process of understanding new host-biology relevant to human response to HIV and relevant to potential drug targets and vaccine development.