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

MHC是人类基因组中多态性最多的位点，也是与疾病相关最丰富的基因组片段，包括细菌、病毒、炎症和自身免疫性疾病、癌症、神经系统疾病和药物过敏。广泛的多态性、密集的基因含量、基因对之间强烈的连锁不平衡以及MHC内基因编码的分子的功能相似性，使这些疾病关联的机制难以确定。HLA I类表达的调控是由一系列核心启动子基序介导的，这些核心启动子基序位于起始位点上游500bp以内，相对保守。从核心启动子上游更远的调控蛋白的结合位点也已被确定，并且可能存在其他的结合位点，其中一些可能是多态的。事实上，位于3和5个非翻译区（utr）以及编码区内的遗传变异和表观遗传修饰已被证明可以改变HLA等位基因的表达水平，但到目前为止，这些机制仅占各自基因中发现的表达变异的一小部分。HLA表达水平的差异最近被证明与许多疾病有关。我们确定了来自57个经典HLA I类基因主要谱系的68个等位基因在转录起始位点上游延伸近2 kb的序列。该启动子片段内的核苷酸多样性与编码区内的核苷酸多样性大致相同，HLA B的多样性最高（约1.9%），其次是HLA A（约1.8%），HLA C的多样性最低（约1.8%）。0.9%)。尽管存在更大的多样性，178名欧洲裔美国人的HLA B mRNA表达水平并没有等位基因或谱系特异性的变化，这与之前报道的HLA A或HLA C的差异表达水平不同。大多数HLA A和C基因座的表达模式相似大致反映了系统发育树中启动子序列的接近性。虽然启动子序列的差异可能会影响启动子的活性，但我们观察到启动子区域的成对核苷酸差异所代表的系统发育结构与经典I类位点mRNA表达水平的估计差异之间没有明确的联系。此外，没有一对I类基因位点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。这些数据为更深入地分析经典HLA I类位点启动子区域变异的功能后果奠定了基础。HLA I类肽装载涉及包括tapasin在内的几种蛋白，其功能是编辑HLA I类所呈现的肽库。实验数据表明，HLA I类同种异体对tapasin的依赖性存在差异，以形成稳定的肽复合物。在tapasin缺陷细胞系中，一些HLA I类异体可以在细胞表面以正常水平表达，而其他的几乎无法检测到。值得注意的是，HIV疾病中代表保护性和易感等位基因的两个等位基因表现出相反的表型：tapasin依赖性B*57:01和tapasin非依赖性B*35:01。鉴于tapasin在某些HLA同种异体组装中的重要性及其对肽库的潜在影响，我们假设tapasin/HLA相互作用的遗传定义变异可能影响HIV疾病的预后。最近，在密歇根大学Malini Raghavan博士的实验室中，一组27个HLA B等位基因在tapasin缺陷的M553细胞系中进行了测试，显示出广泛的表达水平。这些表达水平可以作为HLA I类等位基因tapasin依赖性的特征，并作为统计分析的变量。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人或黑人人群中频率超过0.5%的所有HLA B等位基因。用慢病毒构建体在721.220 tapasin阴性B细胞株中表达这些等位基因，用流式细胞仪检测其表达水平。每个HLA I类等位基因基于这些表达水平被赋予tapasin依赖值（表达越高对应的等位基因越独立）。编码tapasin的基因变异包括一个非同义变异（rs2071888），对应于成熟肽240位的精氨酸和苏氨酸两个版本的蛋白质。这些变异在白人中几乎相同，而Arg在黑人中更常见（72%的等位基因频率）。分子动力学模拟表明，pos 240的变异可能影响与ERp57（肽装载复合物的另一组分）和HLA i类的相互作用。我们在HIV队列中对rs2071888进行了基因分型，我们的初步分析表明，在具有tapasin依赖等位基因的个体中，Thr240与较低的病毒载量相关。我们正计划扩大基因分型数据集，以增加统计能力。此外，我们将通过重组721.220个细胞中两种版本的tapasin （Thr/Arg240）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，看看不同的细胞如何影响tapasin依赖性。

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.