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: 8937846
• 负责人: Mary N. Carrington
• 金额: $ 28.66万
• 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/8937846
• 关键词: 14q32.33; Accounting; Affinity; African; Alleles; Antibodies; B-Lymphocytes; Biological Assay; Biology; CD4 Positive T Lymphocytes; Caucasians; Caucasoid Race; Cell Line; Cell Surface Proteins; Cell surface; Cells; Characteristics; Collaborations; Complex; DNA; DNA Methylation; DNA Sequence Rearrangement; Data; Disease; Disease Outcome; Epigenetic Process; European; Exhibits; Gene Cluster; Gene Expression; Gene Frequency; Genes; Genetic; Genetic Polymorphism; Genetic Population Study; Genetic Transcription; Genome; Genotype; Goals; HIV; HIV Infections; HLA Antigens; Haplotypes; Homologous Gene; Homologous Protein; Human; Human Chromosomes; Human Herpesvirus 4; Immune Response Genes; Immune response; Immunoglobulin Fragments; Immunoglobulins; In Vitro; Individual; Killer Cells; Knowledge; Laboratories; Leukocytes; Linkage Disequilibrium; Major Histocompatibility Complex; Mass Spectrum Analysis; Mediating; Meiotic Recombination; Messenger RNA; Methylation; Molecular Genetics; Monoclonal Antibodies; Pattern; Peptide Fragments; Peptides; Population; Predisposition; Procedures; Process; Property; Proteins; Receptor Gene; Relative (related person); Resistance; Sampling; Spectrometry; T cell response; Trypsin; Universities; Variant; Work; base; cohort; genome wide association study; human disease; human leukocyte antigen gene; interest; mRNA Expression; magnetic beads; population based; pressure; protein expression; receptor; screening

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.

我们现在有初步数据表明，人类白细胞抗原-A等位基因在基因表达水平上表现出等位基因特异性的差异。不幸的是，没有特定的抗体可以完全证实这种多样性反映在蛋白质水平上。然而，我们已经鉴定出两种单抗，这两种抗体使我们能够在具有特定的人类白细胞抗原-A基因型别的个体的子集中显示出信使核糖核酸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促成因素，但尚未检查等位基因水平上的DNA甲基化模式。我们推测，DNA甲基化可能至少部分解释了人类白细胞抗原-A基因的差异表达水平。我们现在已经显示了不同的人类白细胞抗原-A等位基因之间的DNA甲基化，这可能解释了等位基因特异性的mRNA表达水平的差异。低表达水平的HLA-A等位基因的甲基化水平较高，这与甲基化水平较高抑制转录并随后导致基因表达水平较低的概念一致。到目前为止，我们的研究仅限于欧洲血统的个人，与非洲血统相比，欧洲血统的人群具有不同的HLA等位基因/等位基因频率。在夸祖鲁纳塔尔大学的Thumbi Ndungu博士和Salim Karim博士的合作下，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具有非洲背景的疾病队列。我们也有数据表明，一直存在维持人类白细胞抗原-C和潜在的人类白细胞抗原-A的差异表达水平的选择压力。有趣的是，我们的初步数据显示，人类白细胞抗原-A基因的表达水平与人类白细胞抗原-C的表达水平呈正相关，但仅在携带miR-148A抑制的等位基因(I/I)的受试者中，而在那些逃脱miR-148A抑制的受试者(D/D)中则不相关。我们还使用来自高加索人的表达水平数据分析了来自几个种族背景的27个世界人口的基因型数据，我们观察到了类似的相关性。我们也开始分析人类白细胞抗原-B的表达水平。与人类白细胞抗原-C和人类白细胞抗原-A不同，人类白细胞抗原-B等位基因之间的基因表达水平没有差异。目前还没有一种抗体能以相同的亲和力识别所有的人类白细胞抗原B同种异型，但不能识别人类白细胞抗原-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到两个或更多具有相同亲和力的HLA-B等位基因，这一点通过LABScreen磁珠进行了评估。根据我们到目前为止的筛查，细胞表面的HLA-B表达似乎变化很小，但我们的努力仍在继续。人类白细胞抗原I类或II类基因座异型的不同表达水平可能解释了所观察到的人类白细胞抗原基因座变异与疾病结局之间的许多强烈关联。在这方面，准确了解在大多数细胞上表达的四个HLAI类基因座的相对蛋白表达水平似乎是至关重要的。这对HIV感染的细胞也很感兴趣，因为目前还不知道HIV感染的细胞上哪个HLA基因座占主导地位，从而潜在地介导了最大的保护性T细胞反应。与抗体介导的方法相比，质谱学具有区分高度同源蛋白质的能力，并能够以同样的效率定量每个基因座。到目前为止，我们已经证明了这个过程可以使用EBV转化的B细胞株来工作。用胰酶消化沉淀的人类白细胞抗原，并对产生的多肽片段进行电离和质谱分析。对存在于这些个体中的四种HLA蛋白中的每一种都检测到了多个独特的多肽片段，并对16种多肽(每个座位特有的3-5种多肽)的丰度进行了定量。因此，我们已经证明，我们可以通过光谱分析来区分和量化这些捐赠者中存在的所有4个HLA分子。我们现在的目标是使用从正常PBMCs和体外HIV感染的原代CD4细胞中析出的HLA来进行检测。