Identification of Genetic Factors Associated with Infectious Diseases

这个项目的重点是继续对全球健康产生巨大影响的两种传染病。人类免疫缺陷病毒（HIV）是大流行病，乙型肝炎病毒（HBV）感染和HBV相关的肝细胞癌（HCC）在东亚普遍存在，影响全球数百万人，并且无法治愈。该项目的目标是确定导致这些以及其他潜在传染病发生和发展的宿主因素。我们的目标是确定影响宿主先天限制或易感性的宿主遗传因素，这些因素在病毒病原体的获取、复制、发病机制和致癌作用中，其机制尚不完全清楚。鉴定参与病毒复制、先天免疫或获得性免疫或致癌途径的宿主蛋白将为合理开发抗病毒药物和有效疫苗提供重要见解。我们的策略是寻找对感染率或发病过程有不同影响的遗传变异，从而确定含有变异的基因是否对感染或进展有限制或易感性。我们正在使用靶向基因和基因组广泛关联研究（GWAS）方法，包括Illumina和Affymetrix芯片技术，来发现与HIV-1、HBV感染和HBV相关肝癌相关的基因。1) HIV-1抗性APOBEC3 （A3）基因。7个A3基因中的6个（包括A3B、A3G和A3F）是人类先天耐药因子，通过引起病毒RNA的高突变赋予HIV-1抗性。之前我们发现APOBEC3G和CUL5通路中的遗传因素影响艾滋病的进展。7个APOBEC3基因（a - h）聚集在22号染色体100 kb的区域。我们评估了所有APOBEC3基因（A-H）的遗传变异和单倍型对HIV-1感染和病程的影响。除了我们之前在A3G和A3B中发现的关联外，我们还发现了A3F基因变异可以延缓艾滋病的进展。在体外，与APOBEC3G不同，APOBEC3F强烈抑制HIV-1，并部分抵抗HIV-1 vif。我们发现密码子改变变体rs5750728（不同亚型的内含子或密码子改变的78 V/A）和231 I/V与防止欧美人发展为艾滋病有关。我们发现rs5750728改变HeLa细胞与核蛋白的结合，表明其具有调节作用。与约翰霍普金斯大学的研究人员合作，评估了APOBEC3F与HIV-1 vif的相互作用；携带改变密码子的SNP 231 I/V被证明对降解A3F的vif具有更强的抗性，可能导致更丰富的A3F抑制HIV-1。有趣的是，在A3F和vif序列的比对中，携带231 I/V的A3F区域似乎被HIV-1 vif模仿或与HIV-1 vif共同进化。这可能揭示了一种机制，允许HIV-1通过适应宿主A3F序列来逃避A3F。2) ZNRD1与HIV-1感染。我们在5个美国HIV-1纵向队列中研究了ZNRD1区域snp对HIV-1感染和进展的影响。ZNRD1基因的单倍型显示与宿主限制HIV-1获得显著相关。相关变异的功能相关性在基因报告试验中得到证实，其中一个启动子变异（rs3132130）增加了ZNRD1基因的表达。此外，在dna -蛋白质相互作用分析中，该变体还赋予与核蛋白的差异等位基因特异性结合。我们的发现为ZRND1在调节HIV/AIDS中的重要作用提供了新的证据。这项研究发表在2014年6月的《传染病杂志》上。3) HBV和HCC的GWAS。HBV感染在中国极为普遍（2006年HBsAg携带者率为7%）。许多HBV感染患者发展为肝硬化和肝细胞癌（HCC）。MGE正在与北京大学第一医院曾铮博士合作，对中国乙型肝炎病毒感染队列进行遗传研究。该HBV队列由MGE发起，是中国13个主要研究机构与MGE合作的项目。HBV队列包括HBV结局的全谱：HBV耐药性、HBV清除率、慢性肝炎、肝硬化和HCC，使其成为涵盖自然HBV结局的最全面的样本集。我们使用affymetrix SNP6.0芯片对1200个样本进行了GWAS。我们完成了基因分型呼叫的质量控制，并进行了GWAS关联分析。使用第二次TaqMan试验验证了6个顶部相关snp的基因型，并在同一队列的扩展样本集中验证了相关信号。GWAS发现HLA-DPA1和HLA-DPB1区域是HBV清除和感染的主要遗传因素，并通过了全基因组显著阈值。这些结果与其他组的GWAS结果以及我们之前发表的发现一致，其中HLA-DPA1 rs3077不仅与降低HBV感染风险有关，而且与HBV清除有关。我们检测到HLA-DQ1区域与宿主对HBV感染的抗性相关，且具有全基因组意义（p5e-8）。在两项已发表的全基因组QTL研究中，顶部的HLA-DQ1 SNP是与HLA-DQ表达水平相关的eQTL。5)我们将来自HIV/AIDS GWAS的基因型-表型数据提供给国际HIV基因组学，以确定与HIV获得相关的常见变异。发表在《公共科学图书馆·病原体》上的这项研究的结论是，基因对艾滋病毒感染的影响要么很少，要么影响很小。6)我们启动了一项关于不同疾病阶段hiv感染者pbmc差异甲基化的纵向研究，将每个受试者的感染前样本与同一受试者在疾病后期的样本进行比较。我们在Illumina Infinium 450甲基化芯片上对来自DCG队列的82个样本进行了检测。我们正在与CCR生物信息学小组（CCR- ifx）合作分析芯片数据。甲基化数据预测个体CD4计数的能力证实了读数的质量，正如测量的CD4计数所证实的那样。初步结果显示，在校正年龄、CD4计数和其他混杂变量后，感染前和感染后样本中大约有150个位点甲基化差异。7)艾滋病眼部并发症的纵向研究（LSOCA）包括2393名艾滋病患者，并有详细的随访临床数据，以监测艾滋病眼部并发症的发生率，并确定抗逆转录病毒治疗对免疫功能对眼部疾病风险的影响。我们对该队列进行了15个与年龄相关性黄斑变性相关的候选基因和与视网膜小动脉损伤和肾脏疾病相关的APOL1变异的基因分型；分析正在进行中。

This project's focus is on two infectious diseases that continue to have tremendous impact on global health. Human Immunodeficiency Virus (HIV) is pandemic and Hepatitis B Virus (HBV) infection and HBV-associated hepatocellular carcinoma (HCC) is prevalent in East Asia, globally affecting millions of people and having no cure. The objective of this project is to identify host factors that contribute to the occurrence and development of these, and potentially other, infectious diseases. We aim to identify host genetic factors that affect host innate restriction or susceptibility in acquisition, replication, and pathogenesis of viral pathogens, and carcinogenesis, the mechanisms of which are not fully understood. The identification of host proteins involved in viral replication, in innate or acquired immunity, or in carcinogenesis pathways will provide critical insights for the rational development of antiviral drugs and effective vaccines. Our strategy is to search for genetic variants that differentially affect rates of infection, or the course of pathogenesis, and which thereby identify the variant-containing gene as conferring restriction or susceptibility to infection or progression. We are using both targeted gene and genome wide association study (GWAS) approaches, including Illumina and Affymetrix chip technologies, to discover genes associated with HIV-1, HBV infection and HBV-associated liver cancer. Accomplishments 1) HIV-1 resistant APOBEC3 (A3) genes. Six of the seven A3 genes (including A3B, A3G and A3F) are human innate resistance factors that confer resistance to HIV-1 by causing hypermutation of viral RNA. Previously we discovered genetic factors in the APOBEC3G and CUL5 pathway that affect AIDS progression. The seven APOBEC3 genes (A-H) are clustered in a 100 kb region in chromosome 22. We assessed the influence of the genetic variants and haplotype in all APOBEC3 genes (A-H) on HIV-1 infection and disease courses. Besides the associations we previous found in A3G and A3B, we have identified A3F genetic variants that retard AIDS progression. In vitro, APOBEC3F strongly inhibits HIV-1 and is partially resistant to HIV-1 vif, unlike APOBEC3G. We found that codon-changing variants rs5750728 (intronic or codon-changing 78 V/A in different isoforms) and 231 I/V were associated with protection from progression to AIDS in European Americans. We found that rs5750728 alters binding to nuclear proteins in HeLa cells, suggesting a regulator role. In collaboration with researchers at Johns Hopkins University, the interaction of the APOBEC3F with HIV-1 vif was evaluated; the carrying codon-changing SNP 231 I/V was shown to confer stronger resistance to vif that degrades A3F, possibly leading to more abundant A3F to inhibit HIV-1. It is of interest that in the alignment of A3F and vif sequences, the A3F region carrying 231 I/V appears to either be mimicked by or has co-evolved with HIV-1 vif. This may reveal a mechanism allowing HIV-1 to evade A3F by adapting to host A3F sequences. 2) ZNRD1 and HIV-1 infection. We investigated the effect of the SNPs in the ZNRD1 region on HIV-1 infection and progression in five U.S-based HIV-1 longitudinal cohorts. A haplotype in the ZNRD1 gene showed significant association with host restriction to HIV-1 acquisition. The functional relevance of the associated variant was demonstrated in a gene reporter assay that showed one promoter variant (rs3132130) increased the ZNRD1 gene expression. Further, in a DNA-protein interaction assay, the variant also confers differential allele-specific binding to nuclear proteins. Our findings provide novel evidence of significant roles of ZRND1 in modulating HIV/AIDS. This work has been published in Journal of Infectious Diseases, June 2014. 3) GWAS of HBV and HCC. HBV infection is extremely prevalent in China (HBsAg carrier rate was 7% in 2006). Many HBV infected patients develop cirrhosis and hepatocellular carcinoma (HCC). MGE has an ongoing collaboration with Dr. Zeng Zheng, Beijing University First Hospital, for genetic studies in a Chinese Hepatitis B virus infection cohort. This HBV cohort, initiated by MGE, is collaboration between 13 major institutes in China and MGE. The HBV cohort comprises the full spectrum of the HBV outcomes: HBV resistance, HBV clearance, chronic hepatitis, cirrhosis and HCC, making this the most comprehensive sample set covering natural HBV outcomes available. Our GWAS was performed on 1200 samples using the affymetrix SNP6.0 chip. We have completed quality control of genotyping calls and performed GWAS association analyses. The genotypes from 6 top associated SNPs were verified using a second TaqMan assay, and the association signals were validated in expanded sample sets in the same cohort. The GWAS identified HLA-DPA1 and HLA-DPB1 region as major genetic factors for HBV clearance and infection that passed the genome wide significance threshold. These results are in agreement of GWAS results from other groups as well as our previous published finding, in which HLA-DPA1 rs3077 was associated with not only reduced risk to HBV infection but also HBV clearance. We detected an association with host resistance to HBV infection in the HLA-DQ1 region that passed the genome wide significance (p5e-8). The top HLA-DQ1 SNP is an eQTL associated with HLA-DQ expression level in two published genome wide QTL studies. 5) We contributed genotype-phenotype data from the HIV/AIDS GWAS to the International for the Genomics of HIV to identify common variants associated with HIV acquisition. The conclusion of the study, published in PLoS Pathogens, was that genetic influences on HIV acquisition are either rare or have small effects. 6) We have initiated a longitudinal study of differential methylation in PBMCs from HIV-infected subjects at different stages of disease, comparing pre-infection samples from each subject with samples from the same subject at later stages of disease. We ran 82 samples from the DCG cohort on the Illumina Infinium 450 methylation chip. We are collaborating with the CCR bioinformatics group (CCR-IFX) to analyze the chip data. Quality of the reads was confirmed by the ability of the methylation data to predict the CD4 count of individuals, as confirmed by measured CD4 counts. Initial results show approximately 150 sites differentially methylated between pre- and post-infection samples, after correcting for age, CD4 count, and other confounding variables. 7) The Longitudinal Studies of Ocular Complications of AIDS (LSOCA) comprises 2393 patients with AIDS and with detailed follow-up clinical data to monitor incidence of ocular complications of AIDS and to determine the effect of antiretroviral treatment on immune function on the risk of ocular disease. We have genotyped this cohort for 15 candidate genes associated with age-related macular degeneration and for APOL1 variants for association with retinal arteriole injury and renal disease; analysis is ongoing.