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

白细胞免疫球蛋白样受体(LILR)B3和LILRA6是一对胞外结构域相同、配体未知的抑制/激活受体。LILRB3可以通过其胞浆尾部的基于免疫受体酪氨酸的抑制基序(ITIMs)来介导抑制信号，而LILRA6可以通过与激活的接头分子FcRGamma结合来传递信号，FcRGamma带有基于免疫受体酪氨酸的激活基序(ITAM)的细胞质尾巴。这些受体是由人类19号染色体上白细胞受体复合体(LRC)内两个高度多态的相邻基因编码的。我们对LILRB3/A6基因座进行了全面的遗传分析，并研究了基因特异性表达。这些数据证实了这两个基因存在高度的非同义变异，大多数多态位点是相同的。此外，LILRA6基因表现出拷贝数变异(CNV)，而LILRB3没有。对健康的高加索人的筛查表明，32%的受试者拥有两个以上的LILRA6拷贝，而4%的受试者每个二倍体基因组只有一个拷贝的基因。因此，很明显，随着时间的推移，该基因已经经历了非等位基因同源重组(Nahr)，导致激活的LILRA6基因的拷贝数可变，但抑制LILRB3基因的单一固定拷贝保持不变。对PBMCs主要组分的mRNA表达分析表明，LILRA6主要在单核细胞中表达，与LILRB3相似，其表达水平与基因的拷贝数有关。我们认为LILRA6CNV可能影响细胞表面激活受体的水平，从而潜在地影响LILRB3/A6连接的信号转导。人类KIR基因至少有六种主要的基因内容单倍型，它们都是四个着丝粒基序和两个端粒基序的组合。还存在几种频率较低或较小的单倍型，包括主要单倍型的KIR基因的插入、缺失和杂交。这些KIR基因的单倍型结构及其伴随的连锁不平衡表明，通过对KIR区单倍型的总体测量，可以从KIR遗传学和复杂疾病的研究中获得更有意义的相关数据。以前对KIR区域的研究已经获得了关于KIR单倍型结构的详细信息，这些单倍型结构独特地来源于完整的阶段性基因组序列，揭示了已知单倍型的亚结构，并进一步确定了KIR基因之间的连锁。考虑到许多已报道的KIR基因多态性与疾病之间的相关性，有必要将KIR单倍型结构，包括潜在的区域内上位性，纳入关联研究，以便识别KIR的致病变异。此外，KIR区重复的基因内容结构可能通过异常重组机制促进快速进化，这可能是由KIR的免疫功能驱动的，并为理解整个基因组区域的变异提供了进一步的推动力。为此，使用了我们的合作者开发的KIR单倍型方法，该方法报告了KIR基因内容单倍型的明确组合，包括每个KIR的阶段和拷贝数。在4512个个体中总共检测到37种不同的基因含量单倍型，并从以前没有详细结构的单倍型中获得了新的序列数据。在单拷贝中检测到另外10种单倍型，但未经测序证实。37个KIR单倍型被分为10种结构改变类型，包括基因缺失、插入和杂交，这些类型共同表明在KIR进化过程中可能发生了许多重组事件。这些新结构暗示了KIR进化过程中的一些特定重组事件，并增加了来自基因复制、缺失和杂交的潜在新KIR单倍型的多样性。这些数据提供了有关可能影响疾病结果的KIR遗传因素的重要信息。

Leukocyte immunoglobulin-like receptor (LILR) B3 and LILRA6 represent a pair of inhibitory/activating receptors with identical extracellular domains and unknown ligands. LILRB3 can mediate inhibitory signaling via immunoreceptor tyrosine-based inhibition motifs (ITIMs) in its cytoplasmic tail whereas LILRA6 can signal through association with an activating adaptor molecule, FcRgamma, which bears a cytoplasmic tail with an immunoreceptor tyrosine-based activation motif (ITAM). The receptors are encoded by two highly polymorphic neighboring genes within the Leukocyte Receptor Complex (LRC) on human chromosome 19. We undertook a comprehensive genetic analysis of the LILRB3/A6 locus and investigated gene-specific expression. The data confirm the presence of high levels of non-synonymous variation in both genes with the majority of polymorphic sites being identical. In addition, the LILRA6 gene exhibits copy number variation (CNV) whereas LILRB3 does not. A screen of healthy Caucasians indicated that 32% of the subjects possessed more than 2 copies of LILRA6, whereas 4% have only one copy of the gene per diploid genome. Thus it is apparent that this locus has been subjected to non-allelic homologous recombination (NAHR) over time, resulting in variable copy numbers of the activating LILRA6 gene, but maintenance of a single fixed copy of the inhibitory LILRB3 gene.. Analysis of mRNA expression in the major fractions of PBMCs showed that LILRA6 is primarily expressed in monocytes, similarly to LILRB3, and its expression level correlates with copy number of the gene. We suggest that the LILRA6 CNV may influence the level of the activating receptor on the cell surface, potentially affecting signaling upon LILRB3/A6 ligation. The human KIR genes are arranged in at least six major gene-content haplotypes, all of which are combinations of four centromeric and two telomeric motifs. Several less frequent or minor haplotypes also exist, including insertions, deletions, and hybridization of KIR genes derived from the major haplotypes. These haplotype structures and their concomitant linkage disequilibrium among KIR genes suggest that more meaningful correlative data from studies of KIR genetics and complex disease may be achieved by measuring haplotypes of the KIR region in total. Previous studies of the KIR region have yielded detailed information about KIR haplotype structures derived uniquely from complete phased genomic sequences, revealing substructures of known haplotypes and further defined linkage among the KIR genes. Taking into account the many reported correlations between KIR polymorphism and disease, there is some imperative to incorporate KIR haplotype structures, including potential intraregion epistasis, into the association studies so that causative variation at KIR can be identified. In addition, the repetitive gene content structure of the KIR region may contribute to rapid evolution through aberrant recombination mechanisms, possibly driven by the immune function of KIR and providing further impetus towards understanding overall genomic region variation. Towards that end, a KIR haplotyping method developed by our collaborators that reports unambiguous combinations of KIR gene-content haplotypes, including both phase and copy number for each KIR was utilized. A total of 37 different gene content haplotypes were detected from 4,512 individuals and new sequence data was derived from haplotypes where the detailed structure was not previously available. An additional 10 haplotypes were detected in single copy but were not confirmed by sequencing. The 37 KIR haplotypes were sorted into 10 types of structural alterations, including gene deletions, insertions, and hybridizations, which together suggest a number of recombination events that might have occurred during KIR evolution. These new structures suggest a number of specific recombinant events during the course of KIR evolution, and add to an expanding diversity ofpotential new KIR haplotypes derived from gene duplication, deletion, and hybridization. The data provide important information regarding KIR genetic factors that may contribute to disease outcome.