Diversity Of Killer Cell Immunoglobulin-like Receptor KIR3DL1,KIR2DL1,KIR2DL2/3 Genes

Background and ObjectiveDonor killer cell immunoglobulin-like receptors (KIR) cluster and recipient human leukocyte antigen (HLA) mismatching induce NK cell alloreactivity in allo-hematopoietic stem cell transplantation. NK cells alloreactivity kill recipient leukemia cells, T cells and dendritic cells, also improve HSCT outcome by reducing the relapse rate, increasing graft survival, reducing acute graft-versus-host disease incidence and severity. Donor selection by testing KIR gene cluster can effectively improve HSCT outcome. KIR3DL1, KIR2DL1 and KIR2DL2/3 mismatching improved HSCT outcome has been confirmed. However, domestic labs only are capable of typing KIR genotype, can't typing KIR cluster in allele level. There are few laboratories can investigate KIR gene DNA full-length sequence.So we designed this research to develop KIR3DL1, KIR2DL1 and KIR2DL2/3 high resolution typing methods, systematically study these genes in the genotype level, allele level and ligand-receptor system in China and Georgetown University, United States.MethodsPart I 90 whole blood samples of healthy Mongolian donors were collected from Horqin prairie Tongliao City, Inner Mongolian. Genomic DNA was extracted following manufacture protocol. Each sample was amplified by two published KIR gene sequence specific primer sets, while amplified positive control, negative control and blank control,GH orβ-actin was amplified as the internal control. PCR products were checked by agarose gel electrophoresis. A few samples have discrepancy results by primer set A and set B. The third set of primers was used to confirm amplified results. KIR gene frequency and genetic linkage disequilibrium have been calculated according to the results of amplification. Individual genotype was searched from a website. Mongolian KIR frequency and 24 published population data were analyzed by SAS8.0. Scatter gram was constructed by Principal component analysis. Genetics tree was drew according to Nei genetic distance by Phylogenetic Software.Part II 100 African-American samples'B cell lines were transfected by EB virus. Genetic DNA was extracted. KIR3DL1 and KIR2DL1 were amplified by 2~3 overlapped pieces to cover whole DNA sequence. KIR2DL2/3 should be prepared by haploprep beads and/or restriction enzyme digestion to remove similar gene before amplified. Each amplicon was typing by bigdye and sequence primers.Part III HLA-A,HLA-B or HLA-C was amplified from genomic DNA. After purified by magnetic beads, amplicons were sequence by sequence reaction. According to available NMDP allele code, Samples were checked in the website to acquaint whether it's HLA-A,B loci carried HLA-Bw4 epitope and HLA-C classificationPart IV KIR3DL1, KIR2DL1 and KIR2DL2/3 high-resolution results showed some samples had suspicious new alleles. Each new allele should be separated to confirm. Alleles were separated by allele-specific primer PCR, haploprep beads and PCR product cloning, then amplified and sequenced as same as allele typing. New alleles were calculated amino code alteration and submitted to Genebank and WHO.Results1 All KIR genes were found in Mongolian population. The framework genes KIR3DL2, KIR3DL3 frequency was 100%. A sample lack KIR2DL4 gene. The frequencies of KIR2DL1, KIR2DL3 and KIR3DL1 were 93.3%, 93.3% and 95.6%, respectively. The frequency of KIR2DL2 was lower as 30.0%, between Mongoloid and Caucasoid. The frequency of haplotype AA in Mongolian population was 37.78%, also between Mongoloid and the Caucasoid. KIR2DL2-KIR2DS2 and KIR2DL5-KIR2DS1-KIR2DS3-KIR2DS5 showed linkage disequilibrium.2 Principal component analysis for Mongolian populations and other 24 populations, constructed scatter diagram. Caucasoid populations: Europeans, European Americans, Argentinean, Lebanese and Chinese Uighurs gathered in the central, Mongoloid populations: Chinese Han in Shanghai, Yunnan Han, Yunnan Yi, Japanese, Korean and Singapore Han in the left of the diagram, while the Mongolian population was located between Caucasoid and Mongoloid. Genetic tree constructed by Physical6.22 Package also showed a similar result. 3 KIR3DL1, KIR2DL1 and KIR2DL2/3 were amplified by large, overlapped pieces and sequenced and compared to European Americans typing results. The frequency of KIR in African-American population was 99%. KIR3DL1*01501 and KIR3DL1*01502 is the most common alleles in African-American population. KIR3DL1*002, *00402, *008, *019 and *029 existed in the African-American population, but wasn't found in African population proved that African-American has gradually mixed with other races KIR3DL1 alleles after their ancestors emigrated to American. Three samples carrying heterozygous KIR3DL1*059, an allele derived from KIR3DL1 and KIR3DL2 fusion gene. There were two alleles similar to KIR3DL1*059 should be separated and analyzed.4 3% African American population didn't carry any KIR2DL1 allele. 42 samples carried an allele or homozygous alleles. 55 samples carried heterozygous KIR2DL1 alleles. KIR2DL1*00302 and KIR2DL1*00401 were the most popular alleles in African population, their frequencies were 68% and 22%.The frequency of KIR2DL1*006, KIR2DL1*007 in African-Americans were 11% and 7%.5 African Americans carried more KIR2DL2*001 than European Americans, and KIR2DL2*003 carried less. KIR2DL2*00602 was specific allele of African Americans. KIR2DL3*001 in African Americans and in European Americans were counted over 60%, but African American carried less KIR2DL3*002.6 19 samples contained KIR3DL1 genes, but not be combined with HLA Bw4 epitope. A sample contained homozygous KIR3DL1*004, which can't expressed in the surface of NK cells, and can't induced KIR3DL1+ functional NK cells. 25% of the samples did not contain HLA-C2 group alleles, with 32% of the samples did not contain HLA-C1 group alleles. 43% of the people have both HLA-C1 group and HLA-C2 group alleles.7 7 KIR3DL1 novel alleles and 11 KIR2DL1 novel alleles were successfully isolated by different methods. The whole DNA sequences were typing and submitted to WHO.Conclusions1 To Study diversity of KIR cluster in Mongolian population on the genotype level. Mongolian population carries more activating KIR genes than Mongoloid, but less than Caucasoid. The frequency of haplotype AA in Mongolian population was lower than Mongoloid, higher than Caucasoid. Principal component analysis and genetic tree without roots showed Mongolian population between Caucasoid and Mongoloid. Mongolian population had both genetic characteristics from Caucasoid or Mongoloid.2 To study polymorphism of KIR inhibitory genes in African Americans on the allele level. The allele distribution of inhibitory gene KIR3DL1 showed that African-Americans and African people have a closer relationship.3 3% donors in African Americans did not carry KIR2DL1. KIR2DL1*00302 and KIR2DL1*00401 is the most common allele in African American population Hou L, Chen M, Jiang B4 The frequency of KIR2DL2*001 was the highest in African Americans. KIR2DL2*00602 is a specific allele in African Americans. KIR2DL3 * 001 is the most common in African-Americans.5 Analyzing KIR inhibitory genes and their HLA ligands was found that 19% of African-Americans lack HLA Bw4 epitope. 25% of African Americans have homozygous HLA-C1 group alleles, 32% have homozygous HLA-C2 group alleles. Only 43% people carried heterozygous HLA-C1 group and HLA-C2 group alleles.6 7 KIR3DL1 novel alleles and 11 KIR2DL1 alleles were successfully isolated and submitted to Genebank and WHO. WHO have given official names to these new alleles.