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

MHC是人类基因组中最具多态性的基因座，也是基因组中与疾病相关的最丰富的片段，包括细菌、病毒、炎症和自身免疫性疾病、癌症、神经疾病和药物过敏。这些疾病关联的机制被广泛的多态、密集的基因内容和一对基因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所混淆。人类白细胞抗原I类基因表达的调控是由一系列核心启动子基序介导的，这些启动子基序位于起始点上游500bp内，相对保守。位于核心启动子上游的调控蛋白的结合位点也已确定，很可能还存在其他调控蛋白，其中一些可能是多态的。事实上，位于第3和第5非翻译区(UTRs)以及编码区的遗传变异和表观遗传修饰已经被证明可以改变HLA等位基因的表达水平，但到目前为止，这些机制只占各自基因内发现的表达变异的一小部分。最近研究表明，不同的人类白细胞抗原表达水平与许多疾病有关。我们确定了来自57个经典的人类白细胞抗原I类基因主要谱系的68个等位基因的转录起始点上游近2kb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，其中HLAB的多样性最高(约1.9%)，其次是HLAA(约1.8%)，HLAC的多样性最低(约1.8%)。0.9%)。尽管具有更大的多样性，但178名欧洲裔美国人的HLABmRNA表达水平并没有以等位基因或谱系特异性的方式发生变化，这与先前报道的HLAA或HLAC的差异表达水平不同。大多数HLAA和C基因座表达模式的相似性大致反映了系统发育树中启动子序列的接近程度。尽管启动子序列的差异可能会影响启动子的活性，但我们没有观察到由启动子区域的成对核苷酸差异所代表的系统发育结构之间的明显联系，而经典的I类基因座的mRNA表达水平估计存在差异。此外，没有一对I类基因座显示出协调的表达水平，这表明在非刺激条件下，不同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的人类白细胞抗原I类基因座内启动子区变异的功能后果奠定了基础。人类白细胞抗原I类多肽负载涉及包括Tapasin在内的多种蛋白质，其功能是编辑人类白细胞抗原I类提出的多肽谱系。实验数据表明，人类白细胞抗原I类异型对Tapasin形成稳定的多肽复合体有不同的依赖性。在Tapasin缺乏的细胞系中，一些HLAI类同种异型可以在细胞表面正常水平表达，而另一些几乎检测不到。值得注意的是，代表HIV疾病保护和易感等位基因的两个等位基因表现出相反的表型：分别依赖于Tapasin的B*57：01和不依赖Tapasin的B*35：01。考虑到Tapasin在某些HLA同种异型组装中的重要性及其对多肽库的潜在影响，我们假设Tapasin/HLA相互作用中的基因定义变异可能会影响HIV疾病的结果。最近，密歇根大学Malini Raghavan博士的实验室在缺乏Tapasin的M553细胞系中测试了一组27个HLAB等位基因，显示了广泛的表达水平。这些表达水平可作为人类白细胞抗原I类等位基因对Tapasin依赖性的特征，并可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HLAB等位基因，这些等位基因代表了在白人或黑人群体中频率超过0.5%的所有HLAB等位基因。用慢病毒载体在721.220个Tapasin阴性B细胞系中表达这些等位基因，并用流式细胞仪检测其表达水平。根据这些表达水平为每个HLAI类等位基因分配Tapasin依赖值(表达水平越高，相应等位基因的独立性越强)。Tapasin编码基因的变异包括一个非同义变异(Rs2071888)，对应于成熟多肽的240位带有精氨酸和苏氨酸的两个版本的蛋白质。这些变异在白人中几乎相同，而Arg在黑人中更常见(等位基因频率为72%)。分子动力学模拟表明，PoS240的变异可能影响与肽负载复合体的另一组分ERp57和人类白细胞抗原I类的相互作用。我们在我们的HIV队列中发现了rs2071888基因型别，初步分析表明，在携带Tapasin依赖等位基因的个体中，Thr240与较低的病毒载量有关。我们计划扩大基因分型数据集，以增加统计能力。此外，我们将通过在721.220细胞中重组Tapasin的两个版本(Thr/Arg240)的表达来进一步表征Tapasin的依赖性。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。

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.