Molecular genetics and population studies of the KIR and HLA gene complexes

KIR 和 HLA 基因复合物的分子遗传学和群体研究

• 批准号: 10262153
• 负责人: Mary N. Carrington
• 金额: $ 49.07万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262153
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Affinity; African American; Alleles; Amino Acids; Antibodies; Arginine; Binding; Biology; CD8-Positive T-Lymphocytes; Cell surface; Cells; Characteristics; Chromosome 14; Complement Activation; Complex; Cytotoxic T-Lymphocytes; Data; Data Set; Dependence; Dideoxy Chain Termination DNA Sequencing; Disease; Disease Outcome; Dissociation; Endoplasmic Reticulum; European; Exhibits; Exons; Frequencies; GEM gene; Gene Family; Genes; Genetic; Genetic Polymorphism; Genetic Population Study; Genome; Genomics; Genotype; Goals; HIV; HIV-1; HLA-A gene; Half-Life; Histidine; Human; Human Chromosomes; IgG1; IgG2; IgG3; IgG4; Immune; Immune Response Genes; Immune response; Immunoglobulin Constant Region; Immunoglobulin G; Individual; Laboratories; Laboratory Study; Length; Malignant Neoplasms; Mediating; Methods; Modeling; Molecular; Molecular Genetics; Multiprotein Complexes; Nature; Nucleotides; Pathogenesis; Peptides; Phagocytosis; Population; Population Group; Positioning Attribute; Predisposition; Process; Property; Proteome; Regulation; Resistance; Serological; South Africa; Structure; Surface; T cell response; Testing; Vaccines; Variant; Viral; Viral Load result; Virus; Work; adaptive immune response; base; causal variant; cohort; cytotoxic CD8 T cells; cytotoxicity; exome sequencing; gene product; genome-wide analysis; genomic locus; human disease; interest; member; method development; neoplastic cell; novel; pathogen; peptide I; preference; protein complex; tapasin; trait; whole genome

The classical HLA class I molecules, HLA-A, -B and -C, present antigenic peptides to CD8+ T cells, eliciting an adaptive immune response. The genes encoding these molecules are highly polymorphic, resulting in extensive diversity of the peptide repertoire, both within individuals and at the population level. Peptide loading of HLA class I molecules takes place primarily in the endoplasmic reticulum within the peptide loading complex (PLC). Tapasin is a critical component of the PLC, which performs its peptide "editing" function by association with peptide-empty HLA class I, stabilizing its structure, and promoting dissociation of low affinity peptides. HLA class I allotypes vary in level of cell surface expression in the absence of tapasin. Some allotypes are expressed at very low levels on the surface of tapasin-deficient cells (tapasin-dependent allotypes), while others exhibit normal expression on these cells (tapasin-independent allotypes). The exact molecular determinants of tapasin dependence (TD) remain unknown, although amino acids in the peptide binding groove near the peptide C terminus appear to contribute most to this phenomenon. For example, a single amino acid change (D116Y) located in this region distinguishes the highly tapasin-dependent B4402 allotype from the tapasin-independent B4405 allotype. Tapasin function can be targeted by viruses as a means of downmodulating HLA class I and evading cytotoxic CD8+ T cell (CTL) responses. Similarly, loss of tapasin expression has been observed in various human cancers. Thus, tapasin-independent HLA class I allotypes may be advantageous in terms of eliciting CTL responses against virally infected cells or tumor cells when tapasin function has been diminished. Allotype-specific regulation of the peptide repertoire by tapasin may also affect the quality of CTL responses. We have quantified the level of TD across all common HLA allotypes present in European and African Americans and tested the functional significance of differential HLA class I TD and its impact on disease. Ex vivo examination of cytotoxic T cell responses to the entire HIV-1 proteome from infected subjects indicates that tapasin-dependent allotypes present a more limited set of distinct peptides than do tapasin-independent allotypes, data supported by computational predictions. This suggests that variation in tapasin dependence may impact the strength of the immune responses by altering peptide repertoire size. In support of this model, we observed that individuals carrying HLA class I genotypes characterized by greater tapasin independence progress more slowly to AIDS and maintain lower viral loads, presumably due to increased breadth of peptide presentation. Thus, tapasin dependence level, like HLA zygosity, may serve as a means to restrict or expand breadth of the HLA-I peptide repertoire across humans, ultimately influencing immune responses to pathogens and vaccines. The development of methods for assessing the nature and extent of KIR genomic diversity has been limited by the complexity of the region. Members of this gene family share a high amount of sequence similarity. Also, there is remarkable diversity with respect to KIR gene content at the genomic level. Therefore, it is extremely challenging to determine the absence or presence of KIR genes based on short-read sequencing data. Currently, we are developing a computational approach that allows predictions for KIR gene content based on either whole-genome or whole-exome sequencing data. This approach utilizes same-length sequence fragments (k-mers) that are unique to individual KIR genes. Preliminary results are promising and show that high-confidence predictions of KIR genes based on sequencing data is feasible. This method will allow us to interrogate publicly available datasets for disease association analyses. IgG subclasses, IgG1, IgG2, IgG3, and IgG4, display distinct functional properties due to the differences in their constant H chains, encoded by individual genetic loci, IGHG1, IGHG2, IGHG3, and IGHG4, respectively. These genes form a cluster spanning approximately 150 kb region within the Ig H chain constant (IGHC) locus on human chromosome 14. The Fc portion of the constant region (CH2 and CH3 domains) mediates antibody stability and its effector functions, such as cytotoxicity, phagocytosis, and complement activation. Therefore, polymorphism in this region may directly affect immune responses. For example, the naturally occurring change from arginine to histidine at position 435 in IgG3 causes a dramatic increase in the antibody half-life. Variation in the constant regions has been characterized primarily by serological methods (Gm-Am allotypes) with limited information on nucleotide diversity. This region of the genome is not well covered in genome-wide studies due to high homology between the IGHG genes. We have developed a genotyping method based on Sanger sequencing, which covers all exons of IGHG1, IGHG2, and IGHG3 genes. In addition, we can distinguish hinge exon copy number, which is variable for IGHG3. The method has been applied to several population groups, including healthy whites, HIV infected whites and blacks from the US, as well as healthy blacks from South Africa. We have observed striking differences in frequency distributions among these groups.

经典的HLA I类分子HLA-A、-B和-C将抗原肽呈递给CD 8 + T细胞，引发适应性免疫应答。编码这些分子的基因是高度多态性的，导致在个体内和群体水平上肽库的广泛多样性。HLA I类分子的肽装载主要发生在肽装载复合物（PLC）内的内质网中。Tapasin是PLC的关键组分，其通过与肽空的HLA I类结合来执行其肽“编辑”功能，稳定其结构，并促进低亲和力肽的解离。HLA I类同种异型在缺乏tapasin的情况下在细胞表面表达水平上变化。一些同种异型在tapasin缺陷细胞的表面上以非常低的水平表达（tapasin依赖性同种异型），而其他同种异型在这些细胞上表现出正常表达（tapasin非依赖性同种异型）。Tapasin依赖性（TD）的确切分子决定因素仍然未知，尽管肽C末端附近的肽结合沟中的氨基酸似乎对这种现象贡献最大。例如，位于该区域的单个氨基酸变化（D116 Y）将高度tapasin依赖性B4402同种异型与tapasin非依赖性B4405同种异型区分开。Tapasin功能可以被病毒靶向作为下调HLA I类和逃避细胞毒性CD 8 + T细胞（CTL）应答的手段。类似地，已经在各种人类癌症中观察到tapasin表达的丧失。因此，当tapasin功能减弱时，tapasin非依赖性HLA I类同种异型在引发针对病毒感染细胞或肿瘤细胞的CTL应答方面可能是有利的。Tapasin对肽库的同种异型特异性调节也可能影响CTL应答的质量。我们已经量化了欧洲人和非洲裔美国人中所有常见HLA同种异型的TD水平，并测试了差异HLA I类TD的功能意义及其对疾病的影响。对来自感染受试者的整个HIV-1蛋白质组的细胞毒性T细胞应答的离体检查表明，Tapasin依赖的同种异型比Tapasin独立的同种异型呈现更有限的一组不同的肽，数据得到计算预测的支持。这表明tapasin依赖性的变化可能通过改变肽库大小来影响免疫应答的强度。为了支持这一模型，我们观察到携带HLA I类基因型的个体，其特征在于具有更大的tapasin独立性，进展更慢，并保持较低的病毒载量，这可能是由于肽呈递的宽度增加。因此，tapasin依赖性水平，如HLA接合性，可以作为限制或扩大人类HLA-I肽库宽度的手段，最终影响对病原体和疫苗的免疫应答。评估KIR基因组多样性的性质和程度的方法的发展受到该地区的复杂性的限制。该基因家族的成员具有高度的序列相似性。此外，在基因组水平上KIR基因含量存在显著的多样性。因此，基于短读段测序数据来确定KIR基因的存在或不存在是极具挑战性的。目前，我们正在开发一种计算方法，允许基于全基因组或全外显子组测序数据预测KIR基因含量。这种方法利用单个KIR基因所特有的相同长度的序列片段（k-mer）。初步结果是有希望的，并表明基于测序数据的KIR基因的高置信度预测是可行的。这种方法将使我们能够查询公开可用的数据集进行疾病关联分析。IgG亚类IgG 1、IgG 2、IgG 3和IgG 4由于其恒定H链的差异而显示出不同的功能特性，所述恒定H链分别由个体遗传基因座IGHG 1、IGHG 2、IGHG 3和IGHG 4编码。这些基因在人14号染色体上的IG H链恒定（IGHC）基因座内形成跨越约150 kb区域的簇。恒定区的Fc部分（CH 2和CH 3结构域）介导抗体稳定性及其效应子功能，如细胞毒性、吞噬作用和补体激活。因此，该区域的多态性可能直接影响免疫应答。例如，IgG 3中435位从精氨酸到组氨酸的天然变化导致抗体半衰期的显著增加。恒定区的变异主要通过血清学方法（Gm-Am同种异型）进行表征，核苷酸多样性信息有限。由于IGHG基因之间的高度同源性，基因组的该区域在全基因组研究中没有很好地覆盖。我们开发了一种基于桑格测序的基因分型方法，该方法涵盖了IGHG 1、IGHG 2和IGHG 3基因的所有外显子。此外，我们可以区分铰链外显子拷贝数，这对于IGHG 3是可变的。该方法已被应用于几个人群，包括健康的白人，艾滋病毒感染的白人和黑人从美国，以及健康的黑人从南非。我们观察到这些群体之间的频率分布存在显著差异。