A Study On The High-resolution Level Polymorphism Of 14 Functional KIR Genes In The Southern Chinese Han Population

BackgroundThe killer cell immunoglobulin-like receptors(KIRs)gene family,which plays an important role in innate immunity,contains 14 functional genes and 2 pseudogenes.KIR genes not only exhibit extensive allelic polymorphism,but also present extra diversity on haplotype content.Identification of KIR alleles has functional and clinical significance.Up to now,there is only low-resolution commercial kit for identification the presence or absence of KIR genes in the worldwide,however high-resolution KIR sequence-based typing(SBT)commercial kit is not available.As for the published documents related to KIR SBT method,some of them only tested partial exons of KIR genes;although several documents had reported KIR SBT method covering all the exons of each functional KIR genes,simultaneously genotyping of 14 functional KIR genes by SBT has not been reported owing to the varied annealing temperature or the varied fragment size of PCR amplicons and PCR extension time.The technical bottleneck resulted in little comprehensive data of KIR allelic polymorphism.The high-resolution level polymorphism of 14 functional KIR genes having been reported was only seen in sub-Saharan Africans,Caucasians,Japanese and population of Maori,the high-resolution level polymorphism of 14 functional KIR genes in the southern Chinese Han population has not been investigated and reported yet.ObjectBlood samples were randomly collected from 306 unrelated healthy southern Chinese Han voluntary blood donors with informed consent.MethodsIn the present paper,a total of 306 cases of peripheral blood samples from unrelated healthy individuals of southern Chinese Han population were subjected to sequence-based typing for all 14 functional KIR genes using the previously-established KIR SBT patent assay supported by the National Natural Science Foundation of China(81373158).The experimental work of this paper including:(1)PCR Amplification for the entire coding sequence of each functional KIR gene was performed by using 3-4 pairs KIR gene-specific PCR primers,PCR product was run electrophoresis on the agarose gel and the presence or absence of KIR genes identified by our KIR-SBT PCR primers were compared with the results of KIR SSP commercial kit.The positive specific PCR products were purified and then sequenced by ABI3730 sequencing analyzer.(2)As for the ambiguous allele combinations emerged in KIR SBT test,we designed the group-specific PCR primers and performed group-specific PCR amplification and SBT re-test to identify the exact KIR genotypes.(3)For samples with no conclusive SBT results,we collected the fresh peripheral blood and identified the potential novel alleles by means of molecular cloning and haplotype sequencing,the coding sequences of demonstrated novel alleles were then submitted to GenBank and IPD-KIR database to obtain the official name assigned by the WHO Nomenclature Committee for factors of the HLA system.(4)Having obtained the KIR SBT results of all the study samples,we analyzed the high-resolution level diversity of KIR alleles,allelic KIR profiles,single nucleotide polymorphisms(SNPs)and phylogenetic tree analysis using the DNASP4 and MEGA6 software.Results(1)We had acquired more than 44500 sequences in KIR SBT at 14 functional KIR genes.A total of 116 alleles were identified in this paper,in which 46 KIR novel alleles were officially-named by the WHO Nomenclature Committee for factors of the HLA system.The three structural KIR genes,namely 2DL4,3DL2 and 3DL3 showed high polymorphism as 11,18 and 19 different alleles were determined in 2DL4,3DL2 and 3DL3 genes,respectively.However,the diversity of activated KIR genes seemed to be rather conserved,for example only one allele was identified in 2DS5,two alleles were identified in both 2DS2 and 2DS3,three alleles were identified in 2DS1.(2)A total of 258 allelic KIR profiles were detected in 306 study samples,124 different allelic KIR profiles were identified in 167 individuals with the common AA1 haplotype group,119 different allelic KIR profiles were identified in 124 individuals with the AB haplotype group and 15 different allelic KIR profiles were identified in 15 individuals with the BB haplotype group.(3)The average nucleotide mutation ratios(π)of exons 3-5 encoding extracellular domains and exons 7-9 encoding the cytoplasmic tail are much higher than that of the exons 1-2 encoding leader peptide and exon 6 encoding transmembrane region in all functional KIR genes.(4)The gamma(dn/ds)values of coding sequence region were varied among of different KIR gene,indicating the different role of natural selection in evolution.Conclusions and significanceIn this paper,we have established the group-specific PCR amplification and SBT retest assay to identify the ambiguous KIR allele combinations.A total of 46 novel KIR alleles were officially-named by the WHO Nomenclature Committee for factors of the HLA system.The coding sequences of these 46 novel KIR alleles have been submitted to IPD-KIR Database,which can be downloaded and utilized in the worldwide.The identification of these novel alleles will greatly improve the accuracy of KIR genotyping in Chinese Han population.For the first time,we have acquired the comprehensive data of high-resolution level KIR polymorphism in southern Chinese Han population by sequence-based typing of 14 functional KIR genes,which can be widely used in transplantation,KIR-associated disease studies,developing more accurate KIR genotyping commercial kit and human evolution.