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

在HIV队列中进行的第一次GWAS鉴定了位于HLA-C基因上游35 Kb处的单核苷酸多态性（SNP），称为-35，其与HIV病毒载量的控制相关。-35 SNP也与HLA-C的表面表达水平相关。我们随后在HLA-C的3个非翻译区（3UTR）中鉴定了一个microRNA（miRNA）结合位点，该位点具有多态性，并解释了HLA-C在各种同种异型中的差异细胞表面表达。 该结合位点的变异与-35 SNP处于接近完美的连锁不平衡。 我们通过多种方法明确地表明，miR-148 a与低表达HLA-C等位基因的HLA-C 3UTR中存在的特定序列强烈结合，这导致细胞表面低表达，但该miRNA与高表达等位基因的3UTR中存在的该序列的替代形式结合较差，从而使相应的mRNA保持完整。 我们还发现miR-148 a结合位点的变化与HIV病毒载量控制密切相关。我们进一步证明了所有现存HLA-C等位基因的共同祖先被miR-148 a抑制。阻止miR-148 a结合的取代是由3- 5 MYA发生的HLA-C等位基因和HLA-B等位基因之间的序列交换事件引起的，导致HLA-C变体逃避miR-148 a下调。此外，我们发现选择在HLA-C逃逸等位基因的成功传播中发挥了作用。miR-148 a基因区域（MIR 148 A）也是多态性的，并已被证明在世界各地的各种人群中具有显着的选择特征。miR-148 a参与调节与细胞发育、分化、增殖、凋亡、神经元发育、基因表达的表观遗传控制和免疫系统相关的分子途径。越来越多的数据表明，miR-148 a表达的失调与许多人类疾病密切相关。我们的初步数据表明，在MIR 148 A基因区域的变体与miR-148 a的表达，并与HLA-C 3UTR的miR-148 a结合区的变化上位相互作用，以控制HIV病毒复制。我们认为MIR 148 A区域的多态性调节其表达，进而影响细胞和免疫途径，影响宿主对病原体和肿瘤的防御。我们正在进一步研究MIR 148 A区域的多态性，这些多态性调节MIR 148 A区域的表达，进而影响宿主防御病原体和肿瘤的下游细胞和免疫途径。HLA-C 07：01：01 G等位基因组由三个非同义等位基因组成，C 07：01：01，C 07：06和C 07：18，加上C 07：01：02，它与C 07：01：01同义。所有这些等位基因具有相同的外显子2、3和4，但外显子5或6不同。因此，外显子2和3的常规基于序列的分型（SBT）无法分辨这些亚型，导致人群和疾病队列研究中的分型结果不明确。我们充分表征了欧洲人和非洲裔美国人中的C 07：01：01 G亚型，并检查了它们的相对频率分布。在欧洲裔美国人中，C 07：01：01 G主要由C 07：01：01（94.4%）代表，而C 07：01：02（1.1%）和C 07：18（4.5%）相对较少检测到。在非裔美国人中，C 07：18（42.4%）显示出与C 07：01：01（44.7%）相似的高频率，而C 07：06的检测频率较低（4.7%）。C 07：06在两个民族中均只存在于B44：03上，但C 07：18与HLA-B存在多重连锁关系。这些结果表明，C 07：01：01 G是一个异质性的等位基因组，特别是在非洲起源的个体中。如果C 07：01：01 G亚型确实具有功能差异，则它们的表征可能为临床移植的HLA配型效果以及HLA与人类疾病的关联提供进一步的见解。

The first GWAS performed in an HIV cohort identified a single nucleotide polymorphism (SNP) located 35Kb upstream of the HLA-C gene termed -35 that associated with control of HIV viral load. The -35 SNP also associated with surface expression levels of HLA-C. We subsequently identified a microRNA (miRNA) binding site in the 3untranslated region (3UTR) of HLA-C, which is polymorphic and explains the differential cell surface expression of HLA-C across the various allotypes. Variation in this binding site is in near perfect linkage disequilibrium with the -35 SNP. We showed definitively through multiple approaches that miR-148a binds strongly to a specific sequence that is present in the HLA-C 3UTR of low expression HLA-C alleles, which causes the low cell surface expression, but this miRNA binds poorly to the alternative form of that sequence which is present in the 3UTR of high expression alleles thus leaving the corresponding mRNA intact. We also showed that the variation in the miR-148a binding site associates strongly with HIV viral load control. We further demonstrated that the common ancestor of all extant HLA-C alleles was suppressed by miR-148a. Substitutions that prevent miR-148a binding arose by a sequence exchange event between an HLA-C allele and an HLA-B allele that occurred 3-5MYA, resulting in an HLA-C variant that escaped from miR-148a downregulation. In addition, we showed that selection played a role in the successful spread of the HLA-C escape alleles.The miR-148a gene region (MIR148A) is also polymorphic and has been shown to have remarkable selection signature in various populations across the world. miR-148a is involved in the regulation of molecular pathways related to cellular development, differentiation, proliferation, apoptosis, neuronal development, epigenetic control of gene expression and immune system. Data is accumulating to suggest deregulation of miR-148a expression is closely associated with a multitude of human diseases. Our preliminary data suggest that a variant in the MIR148A gene region associates with expression of miR-148a and also epistatically interacts with the variation in the miR-148a binding region of HLA-C 3UTR to control HIV viral replication. We propose that the polymorphisms in the MIR148A region modulate its expression and in turn affect cellular and immune pathways affecting host defense against pathogens and tumors. We are further characterizing the polymorphism(s) in the MIR148A region that modulate its expression and in turn affect downstream cellular and immune pathways involved in host defense against pathogens and tumors.The HLA-C07:01:01G allele group consists of three nonsynonymous alleles, C07:01:01, C07:06 and C07:18, plus C07:01:02, which is synonymous to C07:01:01. All of these alleles have identical exons 2, 3 and 4, but differ in exons 5 or 6. Therefore routine sequence-based typing (SBT) of exons 2 and 3 is unable to resolve these subtypes, resulting in ambiguous typing results in population and disease cohort studies. We fully characterized C07:01:01G subtypes in European and African Americans and examined their relative frequency distributions. In European Americans C07:01:01G is predominantly represented by C07:01:01 (94.4%), whereas C07:01:02 (1.1%) and C07:18 (4.5%) were detected relatively infrequently. In African Americans C07:18 (42.4%) showed a high frequency similar to that of C07:01:01 (44.7%) whereas C07:06 was detected at a low frequency (4.7%). C07:06 was found exclusively on B44:03 carrying haplotypes in both ethnic groups, but C07:18 showed multiple linkage relationships with HLA-B. These results demonstrate that C07:01:01G as defined by routine SBT is a heterogeneous group of alleles, especially among individuals of African origin. If indeed C07:01:01G subtypes confer functional differences, their characterization may provide further insights into the effect of HLA matching for clinical transplantation and HLA association with human disease.