Allelic diversity of KIR3DL1/3DS1 in a southern Chinese population

The inhibitory KIR3DL1 and the activating KIR3DS1 segregate as alleles of the same locus. KIR3DL1 is highly diversified at the allele level and KIR3DL1 alleles exhibit varied levels of expression and ligand binding affinity resulting in varied degrees of NK cell inhibition. Previous studies have shown that the KIR3DL1/3DS1 polymorphism associated with viral infection, cancer and transplantation. However, little is known about the population distribution of KIR3DL1/3DS1 alleles in Chinese. The present study examined allelic diversity of KIR3DL1/3DS1 in a southern Chinese population (N = 306) using PCR-SSP and sequencing based typing. The presence of KIR3DL1 and KIR3DS1 were detected in 97.1% and 34.0% of the tested individuals respectively. A total of 10 KIR3DL1 alleles (including 2 novel ones) and 6 KIR3DS1 alleles (including 5 novel ones) were identified. Common KIR3DL1 alleles (>10%) were KIR3DL1*01502 (74.8%), KIR3DL1*00501 (23.9%) and KIR3DL1*00701 (15.7%). KIR3DS1*01301 was the predominant KIR3DS1 allele with other KIR3DS1 alleles only sporadically observed. The knowledge of the allelic polymorphism of KIR3DL1/3DS1 may help to better understand the role played by KIR3DL1/3DS1 in associated diseases and clinical transplantation in southern Chinese.     

浙江汉族人群KIR3DL2等位基因的多态性分析

目的探讨浙江汉族人群KIR3DL2等位基因的多态性分布情况。方法KIR3DL2等位基因分型采用聚合酶链反应寡核苷酸探针杂交方法，利用引物特异性扩增KIR3DL2基因的两个片段，合成21条探针检测KIR3DL2基因的多态性位点，根据探针的格局判断出样本的KIR3DL2等位基因情况。结果样本中共发现17种探针格局，检测到7种等位基因，其中KIR3DL2＊002等位基因占57％。1例样本无法用现有等位基因探针格局进行分析。结论聚合酶链反应寡核苷酸探针杂交方法可有效检测KIR3DL2等位基因，浙江汉族人群KIR3DL2等位基因的分布有其特点。     

Investigation of Killer Cell Immunoglobulin‐Like Receptors KIR2DL2 and KIR2DL3 Diversity and Identification of Ten Novel KIR2DL3 Alleles in the Chinese Han Population

KIR2DL2 and KIR2DL3 are important inhibitory receptors that recognize a subset of HLA‐C allelic products carrying Ser77 and Asn80. In this study, we have determined KIR2DL2 and KIR2DL3 diversity in the Chinese Han population by a PCR sequence‐based typing. Based on sequencing, the coding regions of 166 Chinese Han individuals, seven new polymorphic sites (238G>A, 405G>A, 476A>G, 550G>A, 608G>A, 789T>C, 947T>C) were found. KIR2DL2*00301, *00101, KIR2DL3*00101,*00201,*013, *015 and ten new KIR2DL3 variants (KIR2DL3*00105, 00106, 00107, 00108, 019, 020, 021, 022, 023 and 024) were identified, of which KIR2DL3*00101 was the most frequent allele. Compared with the sequences of KIR2DL3*00101, all sequences of 2DL3*00105, 2DL3*00106, 2DL3*00107 and 2DL3*00108 had one nucleotide substitution(789T>C, 261C>T, 489G>A and 405G>A),but none resulted in amino acid change. An A>G substitution was observed in nucleotide position 476 in 2DL3*019, 608 G>A in 2DL3*020, 824T>C in 2DL3*021 and 238 G>A in 2DL3*023. In addition, 2DL3*022 probably arose from 2DL3*00201 with a nucleotide substitution G>A at 550. There were more HLA‐C1 positive individuals than HLA‐C2. In conclusion, the data of allelic polymorphism for KIR2DL2 and KIR2DL3 were obtained in the Chinese Han population and ten novel KIR2DL3 alleles were identified.     

The elucidation of KIR2DL4 gene polymorphism

The killer cell immunoglobulin-like receptors (KIRs) on NK cells recognize defined groups of HLA class I alleles. By this mechanism the NK cells fulfil a significant role in the first line of defense against infectious agents and cancer. For the treatment of leukaemia this NK cell allorecognition is of great importance. Still, an appropriate effect against the leukaemic cells requires sufficient expression of both KIR and HLA proteins. KIR gene polymorphism influence membrane expression of the KIR protein. We addressed KIR2DL4 gene polymorphism by a newly developed DNA and cDNA based direct sequencing based typing (SBT) and cloning approach. A panel of 44 individuals revealed a variety of KIR2DL4 alleles. Three new alleles have been identified, among those one allele showed alternatively spliced products. In conclusion, this approach is applicable for routine KIR2DL4 allele typing and enables the characterisation of new KIR2DL4 alleles.     

Investigation of killer cell immunoglobulin-like receptors gene KIR3DL2 diversity and confirmation of KIR3DL2*015 in a Chinese population

Human killer cell immunoglobulin-like receptors (KIRs) are a subfamily of the immunoglobulin superfamily. Here, we established polymerase chain reaction-sequence-based typing (PCR-SBT) procedure to identify alleles of KIR3DL2 gene. The method was designed around the specific amplification of exon 3 to exon 4 and exon 8 to exon 9 of the KIR3DL2 gene. Genomic DNA from 104 healthy, unrelated Chinese Han individuals was typed for KIR3DL2 alleles. Each sample was assigned to the putative allele combination according to the sequences of all KIR3DL2 alleles. We observed 18 different genotypes and eight KIR3DL2 alleles in the population, with KIR3DL2*002 having the highest frequency of 0.558, and confirmed the new KIR3DL2*015 allele. Our data showed that the established PCR-SBT methods for KIR3DL2 allele typing were reliable, and Chinese Han population is distinct in KIR3DL2 allele frequencies.     

Allelic expression patterns of KIR3DS1 and 3DL1 using the Z27 and DX9 antibodies

KIR3DL1 is one of the best-characterised inhibitory NK cell receptors. Unusually, one common allele at the 3DL1 locus encodes an activating receptor known as 3DS1. There is genetic evidence for a protective role of 3DS1 in certain viral diseases, but there has been uncertainty about expression of the 3DS1 protein. Using transfection, we show that surface expression of 3DS1 is reliant on the adaptor protein DNAX-activating protein 12 (DAP12). KIR3DS1 was recognised by the antibody Z27, a reagent that also detects KIR3DL1 but no other killer immunoglobulin-like receptor (KIR) molecule. Z27 stained 3DS1 on the surface of fresh circulating NK cells from 3DS1/3DS1 homozygotes. By double-staining with Z27 and DX9, an antibody specific for 3DL1, we obtained evidence that in 3DS1/3DL1 heterozygous donors significant numbers of NK cells express 3DS1 without co-expressing 3DL1 and that NK cells expressing both alleles are difficult to detect.     

Association of KIR2DS4 and its variant KIR1D with syphilis in a Chinese Han population

Syphilis is a sexually transmitted infection caused by the Treponema pallidum subspecies pallidum spirochaete bacterium. The killer immunoglobulin-like receptor (KIR) gene family encodes cell surface receptors that are found on natural killer (NK) cells and certain T-cell subsets. NK cells are fast-acting effector lymphocytes of innate immunity that respond to infection. The activity of NK cells depends on the dynamic balance of activating and inhibitory signals that are transmitted through respective receptors including KIRs. KIR2DS4 is the only activating KIR gene in KIR haplotype A. KIR1D is a partial deletion KIR2DS4 variant encoding protein devoid of transmembrane region. Up to now, there is no knowledge of association of KIR2DS4 and its variant KIR1D with syphilis in a population that belongs to KIR gene haplotype A. Polymerase chain reaction with sequence-specific primers (PCR-SSP) method was used to genotype KIR genes in 190 patients with syphilis and 192 healthy controls. The gene frequencies of KIR2DS4 and KIR1D were analysed for the association with syphilis in patients and healthy controls who belong to KIR gene haplotype A. The gene frequency of KIR1D/KIR1D in patients with syphilis classified as haplotype A was 16.85% and was significantly higher than that in the control group (6.59%) (P = 0.032). However, there was no significant difference for the gene frequencies of KIR2DS4/KIR2DS4 and KIR2DS4/KIR1D between the two groups (P > 0.05). KIR1D/KIR1D was found in association with susceptibility to syphilis in the Chinese Han population that belongs to KIR gene haplotype A.     

Isolation of a novel KIR2DL3-specific mAb: comparative analysis of the surface distribution and function of KIR2DL2, KIR2DL3 and KIR2DS2

In recent years an increasing number of sequences coding for new KIRs have been described. However, the limited availability of mAbs with unique KIR specificities has hindered an exhaustive assessment of their actual function, HLA-specificity, expression at the cell surface and distribution in different cell populations. In this study we report the generation of a novel mAb (ECM41) specific for KIR2DL3 molecules. By the use of cell transfectants expressing one or other KIR we show that this reagent allows discrimination of KIR2DL3 from other GL183 mAb-reactive molecules such as KIR2DL2 and KIR2DS2. Moreover we show that this novel mAb can be used to assess the surface expression and distribution of KIR2DL3 in different polyclonal NK populations and in NK cell clones. Along this line, we were able to analyze the HLA class I specificity of NK clones expressing either KIR2DL3 or KIR2DL2, two inhibitory receptors that were so far serologically undistinguishable. Finally, the combined use of GL183 and ECM41 mAbs in redirected killing assays allowed us to investigate the functional outcome of the simultaneous engagement of KIR2DL3 and KIR2DS2 in NK cell clones co-expressing KIRs that display opposite (inhibitory vs activating) function.     

Allelic diversity in KIR2DL4 in a bone marrow transplant population: description of three novel alleles

Genomic DNA sequencing was used to identify alleles of KIR2DL4 from 78 unrelated individuals involved in hematopoietic stem cell transplants. Eight known alleles were observed. Three new alleles, KIR2DL4*00203, *00502, *0080104, which differ from known alleles at the nucleotide but not at the protein sequence level, were also identified.     

Two New Cases of KIR3DP1, KIR2DL4-Negative Genotypes,One of which is also Lacking KIR3DL2

The killer immunoglobulin-like receptor (KIR) genes KIR2DL4, KIR3DL2, and KIR3DP1 are present in virtually all humans. KIR2DL4 encodes a receptor present on uterine and decidual natural killer (NK) cells and some peripheral blood NK cells. Its only known ligand is the human leukocyte antigen-G molecule expressed on extravillous trophoblasts, and on tissues in some diseases. KIR3DL2 binds HLA-A*03 and HLA-A*11 as well as HLA-B*27 dimers, and microbial CpG DNA. KIR3DP1 is a pseudogene. During our immunogenetic studies we found two individuals, one from Lower Silesia district in Poland, and another from Western Ukraine, who were reproducibly negative for KIR2DL4 and KIR3DP1 genes, using three different PCR systems. Both individuals displayed very similar genotypes, possessing only KIR3DL3, KIR2DL3, KIR2DP1, KIR2DS1, and probably a rare variant of KIR2DL1. The Pole had also KIR3DL2, which the Ukrainian was apparently lacking. The Lower Silesia has been populated after the Second World War by a remarkable percentage with displaced people from Western Ukraine, which might contribute to genetic similarity of the two individuals described here.     

Investigation of killer cell immunoglobulin-like receptor (KIR) gene diversity: KIR2DL2, KIR2DL5 and KIR2DS5

Human killer cell immunoglobulin-like receptor (KIR) genes are important for restraining natural killer cytotoxicity toward cells with autologous human leukocyte antigen (HLA) while targeting cells lacking or expressing low levels of self-HLA molecules. KIR gene content and alleles vary across individual genomes and populations, requiring specialized laboratory tools for their characterization. Here, we detail methods based on sequence-specific polymerase chain reaction amplification and oligonucleotide probe hybridization to identify alleles of KIR2DL2, KIR2DL5A, KIR2DL5B and KIR2DS5. Allele frequencies for a Northern Irish population of 354 individuals typed with this system are given, along with results from 132 cell lines from the International Histocompatibility Workshop that cover many world populations. This information complements published reports by our laboratory for allele-level typing of other KIR members, totaling 12 of the 17 known genes. These methods are allowing us to characterize KIR haplotypes in our population.     

Investigation of killer cell immunoglobulin-like receptor gene diversity, KIR2DL1 and KIR2DS1

Polymorphism in the alleles of the killer cell immunoglobulin-like receptor 2DL1 and 2DS1 genes has been investigated by the development of polymerase chain reaction-sequence-specific oligonucleotide probing systems. The methods have been applied to 77 Northern Irish families, establishing allele frequencies from the unrelated parents. Additionally, cell line DNA from individuals and CEPH families of the 13th International Histocompatibility Workshop panel were investigated. Eight of the reported KIR2DL1 alleles and only the KIR2DS1*002 allele were identified in the groups studied. Two individuals were positive for three alleles of KIR2DL1, and a putative variant of KIR2DL1*001 was observed. Results also indicated an inherited KIR2DL1/2DS1 splice variant present in a haplotype with several core loci absent, in two families.     

Genetic profile of KIR and HLA in southern Chinese Han population

KIR and their HLA ligands are encoded by two of the most diverse gene families in the human genome. The function of KIR on the NK cell is highly dependent on the normal expression of class I HLA on the target cell. Previous population studies in southern Chinese have been focused on the KIR framework genes and genotypes but little is known about the compound profiles of KIR/HLA. The present study examined 503 unrelated individuals from southern Chinese Han population for the polymorphism of KIR and class I HLA genes. All 16 KIR genes were detected in the study population and the four framework genes KIR3DL2, 3DL3, 3DP1, and 2DL4 were present in all individuals. Thirty unique KIR gene profiles were found reflecting a rather limited number of KIR haplotypes in this population. KIRAA1 was the most common profile observed in 54.7% of the samples. Among the AA1 individuals, 15.6% were homozygous for the deleted KIR2DS4. Haplotype A (74.8%) was more common than haplotype B (25.2%). HLA-C1 was a much more common ligand for 2D KIRs than C2. Bw4-80I, Bw4-80T, and the Bw4-bearing HLA-A alleles were detected at similar frequencies. The matched KIR+HLA pairs 2DL2/3+C1 (98.1%), 3DL1+Bw4 (73.3%), 3DL2+A3/11 (60.0%) were the most common ones whereas 3DS1+Bw4-80I was the least common (9.4%). A total of 193 unique compound profiles of KIR–HLA were identified in 480 informative individuals, 130 of the profiles being detected only once. The study provided a comprehensive analysis of the KIR/HLA profiles in southern Chinese in regards of the presence/absence of KIR genes, HLA ligands, matched KIR+HLA pairs, and KIR/HLA compound profiles. The results could help to better understand the role played by KIR/HLA interaction in associated diseases and clinical transplantation in southern Chinese.     

上海地区汉族人群KIR2DL5基因多态性及单元型分析

KIR2DL5基因是杀伤细胞免疫球蛋白样受体 (KIR )单元型B的特征性标志 ,至少有 4个变异体 ,其中 2DL5A 0 0 1和2DL5B 0 0 3是表达型 ,统称为KIR2DL5 1;2DL5B 0 0 2和 2DL5B 0 0 4是不表达型 ,统称为KIR2DL5 2。本文用序列特异性引物PCR法 (PCR SSP )分析了KIR2DL5表达型KIR2DL5 1和不表达型KIR2DL5 2在上海地区正常汉族人群的分布及其在KIR单元型的组成。结果发现 (1)在 87名健康汉族人和 14个家族中表达型KIR2DL5 1和不表达型KIR2DL5 2基因频率分别为0 1977和 0 0 4 11;(2 )KIR2DL5基因变异体在不同单元型中分布不同 ,有 5种不同组合 ,其中单元型 1、 2、 3、 4、 11、 12、15和 16不含KIR2DL5及其变异体 ;单元型 5和 14只含有KIR2DL5 1;单元型 13和 17只含有KIR2DL5 2 ;单元型 6含有单一KIR2DL5 1或二者兼有 ;单元型 8和 9同时含有KIR2DL5 1和KIR2DL5 2。     

High-throughput genotyping of KIR2DL2/L3, KIR3DL1/S1, and their HLA class I ligands using real-time PCR

Killer immunoglobulin-like receptors (KIRs) expressed on natural killer cells are critical components of innate immunity. Interactions between KIRs and their human leukocyte antigen (HLA) ligands have been shown to influence autoimmune and infectious disease course in defined populations. However, the low throughput and high cost of current methods impede confirmation of the universality of these findings. To support large epidemiology surveys, we developed a high-throughput real-time polymerase chain reaction-based assay to identify carriers of KIR3DL1, KIR3DS1, KIR2DL2, and KIR2DL3 and their HLA ligands. The platform performed with 100% sensitivity and specificity in detection of carrier and non-carrier on reference samples. The application of this platform will further clarify the nature and impact of the KIR–HLA epistatic interaction on disease course in large global population-based studies.     

Multiple copies of KIR 3DL/S1 and KIR 2DL4 genes identified in a number of individuals

Multiple copies of the killer immunoglobulin-like receptor gene, 3DL/S1, have been identified in certain individuals. Additionally, allele determination of the killer immunoglobulin-like receptor gene (KIR), 2DL4, has identified three alleles of this gene present in these same individuals. This event has been confirmed by isolating three distinct KIR2DL4 allele clones in each individual, which sequenced as the alleles identified by the allele identification technique. It is our assumption that an unequal crossover event has occurred between differing KIR haplotypes resulting in the duplication of the 2DL4, 3DS1/3DL1 genes on the newly formed haplotype(s).     

Lack of association of KIR2DL1-R245 and KIR2DL1-C245 with HIV-1 control in black South Africans with HLA-C2

Activating/inhibitory Killer-cell Immunoglobulin-like Receptors (KIRs) partly regulate Natural Killer (NK) cells. KIR2DL1 allotypes with cysteine at position-245 (KIR2DL1-C²⁴⁵) express at lower levels and demonstrate weaker inhibitory signaling compared to allotypes with arginine at position-245 (KIR2DL1-R²⁴⁵). The functional consequence of either allotype in infectious diseases is unknown. Since NK cells mediate antiviral immunity, we investigated KIR2DL1-R²⁴⁵ and KIR2DL1-C²⁴⁵ in association with HIV-1 virological control in untreated immunocompetent black South Africans. Allotype carriage, determined by KIR2DL1 sequencing, was similar between uninfected South Africans (n = 104) and other black African populations, but differed significantly from Europeans, while no significant differences were noted between uninfected and HIV-1-infected individuals (n = 52). KIR2DL1 expression, measured by flow cytometry, in uninfected individuals showed higher KIR2DL1-R²⁴⁵ expression compared to KIR2DL1-C²⁴⁵ in white donors (n = 27), while black donors (n = 21) generally expressed lower levels of both allotypes. KIR2DL1 expression was reduced in HLA-C2 carriers, most evident in black HLA-C2/C2 donors. KIR2DL1-R²⁴⁵ and KIR2DL1-C²⁴⁵ did not associate with viral load when HLA-C2 ligands were present, however in HLA-C1 homozygotes, individuals with only KIR2DL1-R²⁴⁵, showed lower viral loads compared to carriers of both allotypes. The lack of association of KIR2DL1-R²⁴⁵ or KIR2DL1-C²⁴⁵ with HIV-1 control in HLA-C2 carriers may relate to lower KIR2DL1 expression levels in a population with high HLA-C2 prevalence.     

Investigation of killer cell immunoglobulin-like receptor KIR2DL4 diversity by sequence-based typing in Chinese population

Human killer cell immunoglobulin-like receptors (KIRs) play an important role in controlling natural killer (NK) cell function. Here, polymerase chain reaction sequence-based typing (PCR-SBT) procedures identifying alleles of the KIR2DL4 gene have been established. The method was designed around the specific amplification of exon 3 to exon 5 and exon 7 to exon 9 of the KIR2DL4 gene and produce discrimination of KIR2DL4 alleles. Genomic DNAs from 83 healthy unrelated Chinese Han individuals were typed for KIR2DL4 alleles by this method. Each sample was assigned to the putative KIR2DL4 allele combination according to the nucleotide polymorphism profiles of all KIR2DL4 alleles. Twenty-one different genotypes and seven KIR2DL4 alleles were observed in the population, with KIR2DL4*00102 having the highest frequency, 0.5. Five individuals bear a recombinant allele KIR3DP*004 that associated with three putative KIR2DL4 alleles. Our data demonstrated that the established PCR-SBT method for KIR2DL4 allele typing was reliable, and Chinese Han population is distinct in KIR2DL4 allele frequencies in comparison to some other populations.     

HLA-B40组等位基因多态性和血清学分型不明确标本分析

目的：利用DNA分型技术调查上海汉族人群HLA－B40组等位基因多态性并比较配型标本中HLA－B40抗原血清学和DNA分型的结果：方法：采用反向PCR－SSOP技术进行DNA分型,可检出HLA－B＊4001－4011等11个等位基因,结果：所有标本DNA分型均获成功,无假阳性和假阴性结果出现,质控DNA分型结果与UCLA结果相符,上海地区汉族人群共检出B＊4001－4003,4005－4007,4011等等位基因,未检出B＊4004,4008－4010等等位基因,296名无关个体中HLA－B40＊组等位基因频率为0．1402,血清学方法检测HLA－B40组抗原错误率为12．82％（10／78）,结论：该技术用于HLA－B40分型分辨率高,分型结果较血清学方法更加精确,可确保HLA分型的准确。