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

 描述（由申请人提供）：人类免疫缺陷病毒（HIV）是一种慢病毒，可感染并破坏携带CD 4的淋巴细胞1，2，最终使宿主的免疫系统无法对即使是常见的无害病原体1，3，4产生足够的反应。艾滋病病毒于20世纪80年代首次被发现，主要是在旧金山弗朗西斯科和纽约的男同性恋者中发现的1，5，6，但从那时起已发展成为一种世界性的流行病，目前感染艾滋病病毒的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，人类对艾滋病毒的反应存在自然变异;有些人从未感染过艾滋病毒，而另一些人在感染后从未发展为获得性免疫缺陷综合征（艾滋病），以及其他可变反应1，8。研究人员认为，人类群体中存在的潜在遗传变异可能是人类对艾滋病毒反应差异的部分原因，因此代表了开发艾滋病毒治疗方法的可能途径。然而，尽管人类遗传学研究已经进行了20多年，包括20世纪90年代的候选基因研究和21世纪后期的全基因组关联研究（GWAS），但仍然没有HIV疫苗1，9并且很少有遗传位点被鉴定为与HIV相关表型显著相关8，10-13。这种缺乏进展的可能解释是，最近的一组研究GWAS以无假设的方法询问了整个基因组的关联，从而产生了大量的多重测试负担，并且几乎没有深入了解哪些位点是最重要的。此外，本发明还 GWAS仅关注常见的遗传变异（次要等位基因频率，MAF，>5%），而已知许多遗传变异存在于低于5%8的MAF处。因此，本提案的目的是集中于克服GWAS方法的这些缺点。首先，AIM 1将鉴定与HIV感染或AIDS进展相关的具有过多罕见变异的候选人HIV靶基因。已经创建了一个包含1，693个具有强有力先验生物学证据的艾滋病毒候选基因的列表，通过进行基因外显子组测序，我们将发现罕见的变异;这些步骤将使我们能够克服缺乏假设的问题，包含大量的多重测试负担，并限于常见变异。其次，AIM 2将通过多个队列中的后续基因组测序/测序和体外分子测定（包括siRNA敲除和CRISPR）的组合来验证来自AIM 1的前25个命中。通过进行这些实验，我们将为AIM 1中发现的关联提供更严格的证据，并开始理解与人类对HIV的反应相关的新宿主生物学以及与潜在药物靶点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.