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

我们现在有初步数据显示HLA-A等位基因在mRNA表达水平上表现出等位基因特异性变异。不幸的是，没有特异性抗体可以完全证实这种多样性反映在蛋白质水平上。然而，我们已经确定了两种单克隆抗体，使我们能够在具有特定HLA-A基因型的个体亚群中显示mRNA和细胞表面蛋白表达水平之间的相关性。一些研究已经确定了表观遗传机制，特别是DNA甲基化，作为MHC区域内不同表达水平的一个促成因素，但等位基因水平的DNA甲基化模式尚未得到检验。我们假设DNA甲基化可以解释，至少部分，HLA-A mRNA的差异表达水平。我们现在已经显示了HLA-A等位基因之间的差异DNA甲基化，这可能解释了等位基因特异性mRNA表达水平的变化。在低表达水平的HLA-A等位基因中观察到较高的甲基化水平，这与较高的甲基化水平抑制转录并随后导致较低的基因表达水平的观点一致。到目前为止，我们的研究仅限于欧洲人后裔，与非洲人后裔相比，欧洲人后裔具有明显的HLA等位基因/等位基因频率。在与博士合作。夸祖鲁-纳塔尔大学的Thumbi Ndungu和Salim Karim，我们现在已经获得了280名南非黑人的队列，并计划确定这些人群中的等位基因表达水平。这将使我们能够将表达系数分配给由非洲背景组成的疾病队列。我们也有数据表明存在选择压力来维持HLA-C和潜在的HLA-A的差异表达水平。有趣的是，我们的初步数据显示，HLA-A mRNA的表达水平与HLA-C的表达水平呈正相关，但仅在携带miR-148a抑制的HLA-C等位基因（I/I）的受试者中，而在逃避miR-148a抑制的受试者中则没有（D/D）。我们还分析了来自不同种族背景的27个全球人群的基因型数据，使用来自高加索人的表达水平数据，我们观察到类似的相关性。我们也开始分析HLA-B表达水平。与HLA-C和HLA-A不同，HLA-B等位基因的mRNA表达水平没有差异。目前还没有一种抗体可以识别所有具有相同亲和力的HLA-B同种异体，但不能识别HLA-A或-C同种异体。我们使用了两种抗体，每一种抗体都能够检测两个或更多具有相同亲和力的HLA-B等位基因，使用LABScreen磁珠进行评估。根据我们目前的筛选，HLA-B的细胞表面表达似乎几乎没有变化，但我们的努力仍在继续。HLA I类或II类基因座同种异型的差异表达水平可以解释在HLA基因座变异和疾病结局之间观察到的许多强关联。在这方面，精确了解在大多数细胞上表达的HLA- a、HLA- b、HLA- c和HLA- e四种HLA- I类位点的相对蛋白表达水平似乎是基础。这对HIV感染的细胞也是有意义的，因为不知道哪个HLA位点在HIV感染的细胞上占主导地位，从而可能介导最大的保护性T细胞反应。与抗体介导的方法相比，质谱法能够区分高度同源的蛋白质，并且能够以相同的效率定量每个位点。到目前为止，我们已经证明该方法可以在ebv转化的B细胞系上工作。沉淀的HLA用胰蛋白酶消化，产生的肽片段电离，质谱分析。检测到这些个体中存在的四种HLA蛋白中每一种独有的多个肽片段，并对16个肽中的每一个（每个位点特异性的3-5个肽）进行了丰度定量。因此，我们已经证明，我们可以通过光谱法区分和量化这些供体中存在的所有4种HLA分子。我们现在的目标是使用从正常pbmc和体外HIV感染的原代CD4细胞中沉淀的HLA进行检测。

We now have preliminary data showing that HLA-A alleles exhibit allele-specific variation in mRNA expression levels. Unfortunately, no specific antibodies are available to fully confirm that this diversity is reflected at the protein level. However, we have identified two monoclonal antibodies that allowed us to show a correlation between mRNA and cell surface protein expression levels in a subset of individuals with specific HLA-A genotypes. Several studies have identified epigenetic mechanisms, specifically DNA methylation, as a contributing factor for varied expression levels within the MHC region, but DNA methylation patterns at the allelic level have not been examined. We hypothesized that DNA methylation may explain, at least in part, the differential expression levels of HLA-A mRNA. We have now shown differential DNA methylation across HLA-A alleles, which may explain allele-specific mRNA expression level variation. Higher methylation levels were observed for HLA-A alleles with low expression levels, consistent with the notion that higher methylation levels inhibit transcription and subsequently results in lower gene expression levels. Thus far, our studies have been limited to individuals of European descent, a population with distinct HLA alleles/allele frequencies compared to those of African descent. In collaboration with Drs. Thumbi Ndungu and Salim Karim at the University of Kwa Zulu Natal, we have now acquired a cohort of 280 black South Africans and plan to determine allelic expression levels in this population. This will allow us to assign expression coefficients to disease cohorts composed of African background. We also have data suggesting that there has been selection pressure to maintain differential expression levels of HLA-C and potentially HLA-A. Interestingly, our preliminary data show that HLA-A mRNA expression levels correlate positively with those of HLA-C, but only among subjects carrying miR-148a inhibited HLA-C alleles (I/I) and not among those that escape miR-148a inhibition (D/D). We have also analyzed genotype data in 27 worldwide populations from several ethnic backgrounds using expression level data derived from Caucasians and we observed a similar correlation. We have also begun to analyze HLA-B expression levels. Unlike HLA-C and HLA-A, no difference in mRNA expression levels was seen across HLA-B alleles. There is no single antibody available that recognizes all HLA-B allotypes with equal affinity, but does not recognize HLA-A or -C allotypes. We have used two antibodies, each of which was able to detect two or more HLA-B alleles with equal affinity as evaluated using LABScreen magnetic beads. Based on our screening thus far, there appears to be very little variation in cell surface expression of HLA-B, but our efforts are ongoing. Differential expression levels for allotypes of the HLA class I or II loci may explain some of the many strong associations observed between variation at the HLA loci and disease outcomes. Precise knowledge of the relative protein expression levels of the four HLA class I loci expressed on most cells, HLA-A, HLA-B, HLA-C and HLA-E, seems fundamental in this regard. This is also of interest on HIV-infected cells, as it is not known which HLA locus dominates on HIV infected cells, thereby potentially mediating the greatest protective T cell responses. Mass spectrometry has the ability to discriminate highly homologous proteins and is able to quantitate each locus with equal efficiency, in contrast to antibody-mediated approaches. Thus far we have demonstrated that the procedure can work using an EBV-transformed B cell line. Precipitated HLA was digested with trypsin and the peptide fragments produced were ionized and analyzed by mass spectrometry. Multiple peptide fragments unique to each of the four HLA proteins present in these individuals were detected and for each of 16 peptides (3-5 peptides specific to each locus), their abundance was quantified. Thus, we have shown that we can discriminate and quantify by spectrometry all 4 HLA molecules present in these donors. We now aim to perform the assay using HLA precipitated from normal PBMCs and from in vitro HIV infections of primary CD4 cells.