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: 9556365
• 负责人: Mary N. Carrington
• 金额: $ 29.32万
• 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/9556365
• 关键词: 5' Untranslated Regions; Affect; African American; Alleles; American; Arginine; Autoimmune Diseases; B-Lymphocytes; Binding; Binding Sites; Biology; Cell Line; Cell surface; Cells; Characteristics; Code; Complex; Data; Data Set; Dependence; Disease; Disease Outcome; Drug Hypersensitivity; ERp57; Epigenetic Process; European; Foundations; Frequencies; Gene Frequency; Genes; Genetic; Genetic Polymorphism; Genetic Population Study; Genotype; Goals; HIV; HLA Class I Genes; Health; Human Genome; Immune; Immune Response Genes; Immune response; Immunoglobulin Genes; Individual; Infection; Inflammatory; Innate Immune Response; Laboratories; Link; Linkage Disequilibrium; Malignant Neoplasms; Measures; Mediating; Messenger RNA; Michigan; Modification; Molecular; Molecular Genetics; Nucleotides; Pattern; Peptides; Phenotype; Phylogenetic Analysis; Positioning Attribute; Predisposition; Promoter Regions; Proteins; Quantitative Trait Loci; Regulation; Reporting; Resistance; Role; Series; Site; Statistical Data Interpretation; Structure; Testing; Threonine; Time; Transcription Initiation Site; Trees; Universities; Variant; Viral; Viral Load result; base; cohort; differential expression; experimental study; gene product; genetic regulatory protein; genetic variant; genome wide association study; genome-wide; human disease; interest; mRNA Expression; nervous system disorder; novel; peptide I; promoter; reconstitution; simulation; tapasin

The MHC is the most polymorphic locus in the human genome, and it is also the richest segment genome wide in terms of disease associations, including bacterial, viral, inflammatory, and autoimmune diseases, cancer, neurologic disorders, and drug hypersensitivity. Assigning the mechanism for these disease associations is confounded by the extensive polymorphism, dense gene content with strong linkage disequilibrium between pairs of genes, and functional similarity of the molecules encoded by genes within the MHC. The regulation of HLA class I expression is mediated by a series of core promoter motifs, which are located within 500 bp upstream of the start site and are relatively conserved. Binding sites for regulatory proteins further upstream from the core promoter have also been identified and it is likely that others exist, some of which may be polymorphic. Indeed, genetic variants and epigenetic modifications located within the 3 and 5 untranslated regions (UTRs), as well as the coding region, have been shown to alter HLA allele expression levels, but so far, these mechanisms account for only a small percentage of the expression variation found within the respective genes. Differential HLA expression levels have recently been shown to associate with a number of diseases. We determined sequences extending nearly 2 kb upstream of the transcription start site for 68 alleles from 57 major lineages of classical HLA class I genes. The nucleotide diversity within this promoter segment roughly follows that seen within the coding regions, with HLA B showing the highest (approx.1.9%), followed by HLA A (approx.1.8%), and HLA C showing the lowest diversity (approx. 0.9%). Despite its greater diversity, HLA B mRNA expression levels determined in 178 European Americans do not vary in an allele or lineage specific manner, unlike the differential expression levels of HLA A or HLA C reported previously. Close proximity of promoter sequences in phylogenetic trees is roughly reflected by similarity of expression pattern for most HLA A and C loci. Although promoter sequence divergence might impact promoter activity, we observed no clear link between the phylogenetic structures as represented by pairwise nucleotide differences in the promoter regions with estimated differences in mRNA expression levels for the classical class I loci. Further, no pair of class I loci showed coordinated expression levels, suggesting that distinct mechanisms across loci determine their expression level under nonstimulated conditions. These data serve as a foundation for more in depth analysis of the functional consequences of promoter region variation within the classical HLA class I loci. HLA class I peptide loading involves several proteins including tapasin, whose function is editing the peptide repertoire presented by HLA class I. Experimental data demonstrate differential dependence of HLA class I allotypes on tapasin to form stable peptide complex. In tapasin deficient cell lines, some HLA class I allotypes can be expressed at normal levels on the cell surface, whereas others are almost undetectable. Remarkably, two alleles, representing protective and susceptible alleles in HIV disease demonstrate opposite phenotypes: tapasin dependent B*57:01 and tapasin independent B*35:01, respectively. Given the importance of tapasin in assembly of certain HLA allotypes and its potential influence on peptide repertoire, we hypothesized that genetically defined variation in tapasin/HLA interactions may influence HIV disease outcomes. Recently, a set of 27 HLA B alleles were tested in Dr. Malini Raghavan's lab at the University of Michigan, in the tapasin-deficient M553 cell line, which demonstrated a wide range of expression levels. These expression levels can be used as characteristics of tapasin dependence of the HLA class I alleles and serve as variables in statistical analysis. To optimize the analysis, we expanded the Raghavan dataset to 42 HLA B alleles,which represent all HLA B alleles with frequencies more than 0.5 percent in either white or black populations. The alleles were expressed in the 721.220 tapasin negative B cell line using lentiviral constructs and the expression levels were measured by FACS. Each HLA class I allele was assigned tapasin dependence value based on these expression levels (the higher expression the more independent the corresponding allele is). Variation in the gene encoding tapasin, includes one non-synonymous variation (rs2071888) corresponding to two versions of protein with arginine and threonine at position 240 of a mature peptide. The variants are almost equally presented among whites, whereas Arg is more common in blacks (72% allelic frequency). Molecular dynamic simulations demonstrate that the variation at pos 240 may influence interactions with ERp57, another component of the peptide loading complex, and HLA class I. We have genotyped rs2071888 in our HIV cohorts and our preliminary analyses indicate that Thr240 associates with lower viral load among individuals with tapasin dependent alleles. We are planning to expand the genotyping dataset to increase the statistical power. In addition, we will characterize further tapasin dependence by reconstituting expression of both versions of tapasin (Thr/Arg240) in 721.220 cells. We will also perform similar experiments in M553 cell line to see how different cells can influence tapasin dependence.

MHC是人类基因组中最具多态性的基因座，也是基因组范围内与疾病相关性最丰富的区段，包括细菌性、病毒性、炎症性和自身免疫性疾病、癌症、神经系统疾病和药物超敏反应。这些疾病关联的机制被广泛的多态性、密集的基因内容与基因对之间的强连锁不平衡以及MHC内基因编码的分子的功能相似性所混淆。HLA I类基因表达的调控是由一系列核心启动子基序介导的，这些基序位于起始位点上游500 bp以内，并且相对保守。核心启动子上游调控蛋白的结合位点也已确定，很可能存在其他位点，其中一些可能是多态性的。事实上，位于3和5个非翻译区（UTR）以及编码区的遗传变异和表观遗传修饰已被证明会改变HLA等位基因表达水平，但到目前为止，这些机制仅占相应基因内发现的表达变异的一小部分。最近已显示差异HLA表达水平与许多疾病相关。我们确定了从57个主要谱系的经典HLA I类基因的68个等位基因的转录起始位点上游延伸近2kb的序列。该启动子区段内的核苷酸多样性大致与编码区内所见的一致，HLA B显示最高（约1.9%），其次是HLA A（约1.8%），HLA C显示最低多样性（约1.8%）。0.9%）。尽管其更大的多样性，HLA B mRNA表达水平在178个欧洲裔美国人确定不改变等位基因或谱系特异性的方式，不像以前报道的HLA A或HLA C的差异表达水平。在系统发育树中，启动子序列的紧密接近大致反映在大多数HLA A和C基因座的表达模式的相似性上。虽然启动子序列的差异可能会影响启动子活性，我们观察到没有明确的联系之间的系统发育结构所代表的成对的核苷酸差异的启动子区域的mRNA表达水平的经典I类基因座的估计差异。此外，没有一对I类基因座表现出协调的表达水平，这表明在非刺激条件下，基因座之间的不同机制决定了它们的表达水平。这些数据作为一个基础，更深入地分析在经典的HLA I类基因座内的启动子区变异的功能后果。HLA I类肽加载涉及几种蛋白质，包括tapasin，其功能是编辑由HLA I类呈递的肽库。实验数据表明HLA I类同种异型对tapasin形成稳定肽复合物的差异依赖性。在tapasin缺陷的细胞系中，一些HLA I类同种异型可以在细胞表面上以正常水平表达，而其他的几乎检测不到。值得注意的是，代表HIV疾病中的保护性和易感性等位基因的两个等位基因表现出相反的表型：分别为tapasin依赖性B*57：01和tapasin非依赖性B*35：01。鉴于tapasin在某些HLA同种异型组装中的重要性及其对肽库的潜在影响，我们假设tapasin/HLA相互作用中的遗传定义变异可能影响HIV疾病的结果。最近，在密歇根大学的Malini Raghavan博士的实验室中，在塔帕辛缺陷的M553细胞系中测试了一组27个HLA B等位基因，其表现出广泛的表达水平。这些表达水平可用作HLA I类等位基因的tapasin依赖性的特征，并用作统计分析中的变量。为了优化分析，我们将Raghavan数据集扩展到42个HLA B等位基因，这些等位基因代表了白色或黑色人群中频率超过0.5%的所有HLA B等位基因。使用慢病毒构建体在721.220 tapasin阴性B细胞系中表达等位基因，并通过FACS测量表达水平。基于这些表达水平，为每个HLA I类等位基因分配tapasin依赖性值（表达越高，相应的等位基因越独立）。编码tapasin的基因中的变异包括一个非同义变异（rs 2071888），其对应于在成熟肽的位置240处具有精氨酸和苏氨酸的两种形式的蛋白质。这些变体在白人中几乎同样存在，而Arg在黑人中更常见（72%等位基因频率）。分子动力学模拟表明，位置240处的变化可能影响与ERp 57（肽加载复合物的另一种组分）和HLA I类的相互作用。我们在我们的HIV队列中对rs 2071888进行了基因分型，我们的初步分析表明，在具有tapasin依赖性等位基因的个体中，Thr 240与较低的病毒载量相关。我们计划扩大基因分型数据集，以提高统计功效。此外，我们将通过在721.220细胞中重建两种版本的tapasin（Thr/Arg 240）的表达来进一步表征tapasin依赖性。我们还将在M553细胞系中进行类似的实验，以观察不同的细胞如何影响tapasin依赖性。