Copy number variation (CNV) is defined as a segment of DNA that is 1kb or larger and is present at a variable copy number in comparison with the reference genome and they are present abundantly in the human genome. Because of their homology and tandem arrangement on chromosome 19q13.4, KIR genes are susceptible to non-allelic homologous recombination, which causes deletions or duplications of genes. We are currently screening for KIR3DL1/3DS1 CNV and thus far we have accumulated data from 2798 subjects. In collaboration with Dr. Dan Geraghty (Fred Hutchinson Cancer Research Center), we have characterized KIR haplotypes in individuals with KIR3DL1/S1 CNV. Our preliminary results reveal 10 distinct deletion KIR haplotypes and 10 distinct duplication haplotypes. Interestingly, the deletion haplotypes consistently show deletion of KIR2DL4 and KIR3DL1/3DS1, while in duplication haplotypes, KIR2DL4 and KIR3DL1/3DS1 are both duplicated, suggesting that this region is a hotspot for unequal crossing over and that KIR2DL4 and KIR3DL1/3DS1 travel as a cassette. We have also identified hybrid KIR genes in some deletion haplotypes. In collaboration with Drs. Geraghty and Stephen Anderson, we aim to further characterize duplication/deletion haplotypes, define the break point of the unequal crossover events, identify hybrid genes and determine the expression pattern and function of hybrid genes. Post-transcriptional regulation of HLA may be an important factor in differential surface expression, which might be important for immune surveillance of infectious agents and tumors. We have identified a polymorphic microRNA (miRNA) target site in the 3 untranslated region (3UTR) of HLA-C that appears to differentially alter levels of expression of HLA-C allotypes on cell surfaces. The common SNPs in the miRNA-binding site regulate binding of miRNA and subsequent post-transcriptional processing resulting in high or low HLA-C expression. Preliminary evidence suggests that the HLA-B gene is subjected to post-transcriptional regulation through miRNA, as well. Currently, there are no reports in the literature that point to a role of miRNA in regulation of expression of the classical HLA class I or class II molecules. We will screen the 3UTR of each of these genes for miRNA binding sites, determine whether or not they are polymorphic, and if so, measure any potential effect of the variants on HLA expression. Characterization of this regulation pathway could lead to identification of drug targets that modulate HLA expression, thereby modulating the host response in human disease. Inferences regarding selection may provide relevant information about the functional importance of the regions analyzed. There is strong evidence that genes involved in the immune response are under natural selection. The evolutionarily recent geographic expansion of humans, and the even more recent development of large, relatively dense human settlements, has exposed our species to challenging new environments. Potentially lethal pathogens are likely to have exerted important selective pressures on the human genome, so immunity genes can be expected to show molecular signatures of the adaptation of human populations to these recent environments. Native American populations are of particular interest for studies of natural selection in genes of the immune system, since they are exposed to an extensive geographic territory with significant environmental heterogeneity. Studies of Native American populations can help in building human evolutionary history and with Dr. Diogo Meyer (University of Sao Paulo) we have begun to characterize allelic variability of the 3DL1/S1 and 2DL4 genes in Native American populations as well as the regulatory regions of these genes. Population genetic studies of cis-regulatory variation have now revealed rich and varied histories of adaptation for a number of loci, and have been able to link selective and phenotypic effects for a subset of these. HLA-G is a class Ib HLA gene expressed at the maternal-fetal interface. Since it was first described, this molecule has attracted attention due to its immunotolerogenic properties. Recent studies also suggest the relevance of HLA-G in physiological and pathological contexts, such as transplantation, autoimmunity, cancer and hematological malignancies. Evidence of balancing selection at the HLA-G promoter region has previously been found in the African American, European American and Chinese populations, suggesting that its expression may be under strong selective pressures and tight regulation. In this regard, we are investigating sequence variation and haplotype structure of the HLA-G promoter region in Native American populations. We have sequenced the 1320 bp region immediately upstream of the HLA-G translation start site in 24 Native American individuals distributed throughout North and South America. The preliminary results show high levels of nucleotide variation. Nucleotide diversity was 0.00641, which is about eight times as high as the human genome average. Indeed, a significant departure from the expectation of evolutionary neutrality was observed (TajimasD = 1.97) which is consistent with balancing selection at this locus. The fact that we found retention of a substantial amount of diversity, even in populations that have undergone loss of heterozygosity, as is the case in Native Americans, reinforces the importance of the promoter region of this gene in human evolutionary history. To confirm the hypothesis postulated with our preliminary results, we will extend our analyses to Native American populations of the Amazon region. Finally, we plan to continue to use population based approaches to identify patterns of association between the unlinked KIR and HLA loci that may have evolved due to selection pressures using newly acquired populations from several collaborators including Drs. Ken and Judy Kidd (Yale University) and Dr. Sara Tishkoff (University of Pennsylvania).
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