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

我们现在有初步的数据表明，HLA-A等位基因表现出等位基因特异性的mRNA表达水平的变化。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，使我们能够显示特定HLA-A基因型个体亚组中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，但在等位基因水平的DNA甲基化模式尚未被检查。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，这可以解释等位基因特异性mRNA表达水平的变化。对于具有低表达水平的HLA-A等位基因，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血统的个体，与非洲血统的个体相比，欧洲血统的个体具有不同的HLA等位基因/等位基因频率。在与夸祖鲁纳塔尔大学的Thumbi Ndungu和Salim Karim博士的合作下，我们现在已经获得了一组280名南非黑人，并计划确定这一人群中的等位基因表达水平。这将使我们能够分配表达系数的疾病队列组成的非洲背景。我们也有数据表明，有选择压力，以保持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表达水平与HLA-C的表达水平呈正相关，但仅在携带miR-148 a抑制的HLA-C等位基因的受试者中（I/I），而在逃避miR-148 a抑制的受试者中（D/D）。我们还分析了来自几个种族背景的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。我们还开始分析HLA-B表达水平。与HLA-C和HLA-A不同，在HLA-B等位基因之间没有观察到mRNA表达水平的差异。没有一种抗体能以相同的亲和力识别所有的HLA-B同种异型，但不识别HLA-A或-C同种异型。我们使用了两种抗体，每种抗体都能够检测两种或更多种HLA-B等位基因，其亲和力与使用LABScreen磁珠评估的亲和力相同。根据我们迄今为止的筛选，HLA-B的细胞表面表达似乎变化很小，但我们的努力仍在进行中。HLA I类或II类基因座的同种异型的差异表达水平可以解释在HLA基因座的变异和疾病结果之间观察到的许多强关联中的一些。在大多数细胞上表达的四个HLA I类基因座（HLA-A、HLA-B、HLA-C和HLA-E）的相对蛋白质表达水平的精确知识在这方面似乎是基本的。这在HIV感染的细胞上也是令人感兴趣的，因为不知道哪个HLA基因座在HIV感染的细胞上占主导地位，从而潜在地介导最大的保护性T细胞应答。与抗体介导的方法相比，质谱法具有区分高度同源蛋白质的能力，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，该程序可以使用EBV转化的B细胞系。用胰蛋白酶消化纯化的HLA，将产生的肽片段电离并通过质谱法分析。检测这些个体中存在的四种HLA蛋白中的每一种所特有的多个肽片段，并且对于16种肽中的每一种（每个基因座特异性的3-5种肽），定量其丰度。因此，我们已经表明，我们可以通过光谱法区分和定量存在于这些供体中的所有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CD 4细胞中沉淀的HLA进行测定。

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.