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

在HIV队列中进行的第一次GWAS鉴定出位于HLA-C基因上游35Kb的单核苷酸多态性（SNP），称为-35，与HIV病毒载量的控制有关。-35 SNP也与HLA-C的表面表达水平相关。随后，我们在HLA-C的3非翻译区（3UTR）发现了一个microRNA （miRNA）结合位点，该位点具有多态性，解释了HLA-C在不同同种异型细胞表面表达的差异。该结合位点的变异与-35 SNP的连锁不平衡接近完美。我们通过多种方法明确地表明，miR-148a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，从而导致低细胞表面表达，但该miRNA与高表达等位基因3UTR中存在的该序列的替代形式结合较差，从而使相应的mRNA完整。我们还发现miR-148a结合位点的变异与HIV病毒载量控制密切相关。我们进一步证明，所有现存HLA-C等位基因的共同祖先都被miR-148a抑制。阻止miR-148a结合的替换是由HLA-C等位基因和发生在3-5MYA的HLA-B等位基因之间的序列交换事件引起的，导致HLA-C变体逃脱了miR-148a的下调。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-148a基因区域（MIR148A）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著的选择特征。miR-148a参与调控细胞发育、分化、增殖、凋亡、神经元发育、基因表达的表观遗传调控和免疫系统等相关分子通路。越来越多的数据表明，miR-148a表达的失调与多种人类疾病密切相关。我们的初步数据表明，MIR148A基因区域的变异与miR-148a的表达相关，并且还与HLA-C 3UTR的miR-148a结合区域的变异相互作用，以控制HIV病毒的复制。我们提出MIR148A区域的多态性调节其表达，进而影响影响宿主防御病原体和肿瘤的细胞和免疫途径。我们正在进一步表征MIR148A区域的多态性，该多态性调节其表达，进而影响下游细胞和免疫途径，参与宿主对病原体和肿瘤的防御。HLA-C07:01:01G等位基因组由三个非同义等位基因C07:01:01、C07:06和C07:18，以及与C07:01:01同义的C07:01:02组成。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，常规的基于序列的外显子2和3分型（SBT）无法解决这些亚型，导致人群和疾病队列研究中的分型结果不明确。我们对欧洲人和非裔美国人的C07:01:01G亚型进行了全面表征，并检查了它们的相对频率分布。在欧洲裔美国人中，C07:01:01 g以C07:01:01（94.4%）为主，而C07:01:02（1.1%）和C07:18（4.5%）的检出相对较少。在非裔美国人中，C07:18（42.4%）的频率与C07:01:01（44.7%）相似，而C07:06的频率较低（4.7%）。C07:06仅在B44:03上发现携带单倍型，但C07:18与HLA-B存在多重连锁关系。这些结果表明，常规SBT定义的C07:01:01G是一个异质等位基因群体，特别是在非洲裔个体中。如果C07:01:01G亚型确实具有功能差异，那么它们的特征可能会进一步揭示HLA配型对临床移植的影响以及HLA与人类疾病的关系。

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.