High-resolution KIR and HLA genotyping in three Chinese ethnic minorities reveals distinct origins.

Polymorphism of killer-cell immunoglobulin-like receptors (KIRs) and their HLA class I ligands impacts the effector activity of cytotoxic NK cell and T cell subsets. Therefore, understanding the extent and implications of KIR and HLA class I genetic polymorphism across various populations is important for immunological and medical research. In this study, we conducted a high-resolution investigation of KIR and HLA class I diversity in three distinct Chinese ethnic minority populations. We studied the She, Yugur, and Tajik, and compared them with the Zhejiang Han population (Zhe), which represents the majority Southern Han ethnicity. Our findings revealed that the Tajik population exhibited the most diverse KIR copy number, allele, and haplotype diversity among the four populations. This diversity aligns with their proposed ancestral origin, closely resembling that of Iranian populations, with a relatively higher presence of KIR-B genes, alleles, and haplotypes compared with the other Chinese populations. The Yugur population displayed KIR distributions similar to those of the Tibetans and Southeast Asians, whereas the She population resembled the Zhe and other East Asians, as confirmed by genetic distance analysis of KIR. Additionally, we identified 12.9% of individuals across the three minority populations as having KIR haplotypes characterized by specific gene block insertions or deletions. Genetic analysis based on HLA alleles yielded consistent results, even though there were extensive variations in HLA alleles. The observed variations in KIR interactions, such as higher numbers of 2DL1-C2 interactions in Tajik and Yugur populations and of 2DL3-C1 interactions in the She population, are likely shaped by demographic and evolutionary mechanisms specific to their local environments. Overall, our findings offer valuable insights into the distribution of KIR and HLA diversity among three distinct Chinese ethnic minority populations, which can inform future clinical and population studies.

Determination for KIR genotype and allele copy number via real-time quantitative PCR method.

Killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) play crucial roles in regulating NK cell activity. Here, we report a real-time quantitative PCR (qPCR) to genotype all KIR genes and their copy numbers simultaneously. With 18 pairs of locus-specific primers, we identified KIR genes by Ct values and determined KIR copy number using the 2-∆Ct method. Haplotypes were assigned based on KIR gene copy numbers. The real-time qPCR results were consistent with the NGS method, except for one sample with KIR2DL5 discrepancy. qPCR is a multiplex method that can identify KIR copy number, which helps obtain a relatively accurate haplotype structure, facilitating increased KIR research in laboratories where NGS or other high-resolution methods are not available.

The novel allele, HLA-B*15:627 was identified by next-generation sequencing in a Chinese cord blood donor.

HLA-B*15:627 shows one nucleotide substitution G to A at position 601 compared with HLA-B*15:02:01:01.

Identification of the novel KIR3DP1*00604 allele in a Chinese individual.

KIR3DP1*00604 differs from KIR3DP1*0060101 by one single nucleotide substitution G > C at position 252.

Characterization of the novel HLA-C*01:225 allele in a Chinese individual.

HLA-C*01:225 has one nucleotide change compared with HLA-C*01:02:01:01 in codon 110 of exon 3.

Allelic variation of KIR and HLA tunes the cytolytic payload and determines functional hierarchy of NK cell repertoires.

The functionality of natural killer (NK) cells is tuned during education and is associated with remodeling of the lysosomal compartment. We hypothesized that genetic variation in killer cell immunoglobulin-like receptor (KIR) and HLA, which is known to influence the functional strength of NK cells, fine-tunes the payload of effector molecules stored in secretory lysosomes. To address this possibility, we performed a high-resolution analysis of KIR and HLA class I genes in 365 blood donors and linked genotypes to granzyme B loading and functional phenotypes. We found that granzyme B levels varied across individuals but were stable over time in each individual and genetically determined by allelic variation in HLA class I genes. A broad mapping of surface receptors and lysosomal effector molecules revealed that DNAM-1 and granzyme B levels served as robust metric of the functional state in NK cells. Variation in granzyme B levels at rest was tightly linked to the lytic hit and downstream killing of major histocompatibility complex-deficient target cells. Together, these data provide insights into how variation in genetically hardwired receptor pairs tunes the releasable granzyme B pool in NK cells, resulting in predictable hierarchies in global NK cell function.

Simultaneous genotyping for human platelet antigen systems and HLA-A and HLA-B loci by targeted next-generation sequencing.

In order to treat the alloimmunization platelet transfusion refractoriness (PTR), human leukocyte antigen (HLA)-type and/or human platelet antigen (HPA)-type matched platelets between donors and patients are usually used. Therefore, genotyping of HLA-A and HLA-B loci, as well as HPA systems, for donors and patients, is of great significance. However, there is a rare report of genotyping for HLA-A and HLA-B loci as well as HPA systems at the same time. In this study, a high-throughput method for simultaneous genotyping of HLA-A and HLA-B loci, as well as HPA genotyping, was developed. A RNA capture probe panel was designed covering all exon sequences of the GP1BA, GP1BB, ITGA2, CD109, ITGB3, and ITGA2B genes and HLA-A and HLA-B loci. The HLA-A, HLA-B, and 34 HPA systems were genotyped using a targeted next-generation sequencing (NGS) method. The genotypes of the HLA-A and HLA-B loci, as well as the HPA, were assigned based on the nucleotides in the polymorphism sites. Using the NGS method, 204 unrelated blood specimens were successfully genotyped for all 34 HPA systems as well as HLA-A and HLA-B loci. The accuracy of the NGS method was 100%. Only HPA-2, HPA-3, HPA-5, HPA-6w, HPA-15, and HPA-21w showed polymorphism with frequencies of 0.9412, 0.6863, 0.9853, 0.9779, 0.4314, and 0.9951 for a allele, respectively. Thirty-two single nucleotide variants (SNVs) were detected. Of them, 12 SNVs can lead to amino acid change. HLA-A*11:01 and HLA-B*46:01 are the most common alleles for HLA-A and HLA-B loci. A targeted next-generation sequencing method for simultaneously genotyping HPA systems and HLA-A and HLA-B loci was first established, which could be used to create a database of HLA-typed and/or HPA-typed unrelated donors.

Description of two novel HLA alleles: HLA-C*01:02:73 and HLA-C*01:02:75.

Compared with HLA-C*01:02:01:01, the alleles HLA-C*01:02:73 and HLA-C*01:02:75 each show one single nucleotide substitution respectively.

HLA-DRB1*15:01:43 and HLA-DRB1*15:01:44 alleles were identified by next-generation sequencing.

Compared with HLA-DRB1*15:01:01:01, the alleles HLA-DRB1*15:01:43 and HLA-DRB1*15:01:44 each show one nucleotide substitution respectively.

The novel HLA-DRB1*14:222N allele was identified by next-generation sequencing.

HLA-DRB1*14:222N differs from HLA-DRB1*14:03:01 by one single nucleotide substitution at position 262.

The novel HLA-DRB1*15:01:42 allele was identified by next-generation sequencing.

HLA-DRB1*15:01:42 differs from HLA-DRB1*15:01:01:01 by one single nucleotide substitution at position 732 C>T.

Identification of the novel HLA-DRB1*11:271 allele by next-generation sequencing.

HLA-DRB1*11:271 differs from HLA-DRB1*11:01:01:01 by a single nucleotide substitution at position 610G > A.

[Study of the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang].

OBJECTIVE: To study the distribution of KIR3DL2 alleles among ethnic Han Chinese from Zhejiang. METHODS: Genomic DNA was extracted by using a magnetic bead method. The full sequence of the KIR3DL2 gene was amplified with four pairs by PCR primers. The coding regions of 208 unrelated ethnic Han Chinese blood donors were analyzed using a BigDye Terminator v3.1 Sequencing Kit. The genotypes were assigned based on the nucleotide polymorphism of the KIR3DL2 gene. RESULTS: Among the 208 samples, 133 were KIR3DL2 heterozygotes and 75 were homozygotes. Forty six KIR3DL2 genotypes were detected. Respectively, 70, 33 and 23 individuals were found to have a KIR3DL2*00201/KIR3DL2*00201, KIR3DL2*00201/KIR3DL2*00701, and KIR3DL2*00201/KIR3DL2*01001 genotype. Twenty-two KIR3DL2 alleles were discovered, and the frequencies of KIR3DL2*00201, KIR3DL2*00701 and KIR3DL2*01001 were 57.45%, 13.46% and 9.13%, respectively. CONCLUSION: The distribution of KIR3DL2 alleles among ethnic Han Chinese in Zhejiang has been determined and fits the criteria for genetic polymorphism.

High-Resolution Analysis Identifies High Frequency of KIR-A Haplotypes and Inhibitory Interactions of KIR With HLA Class I in Zhejiang Han.

Killer cell immunoglobulin-like receptors (KIR) interact with human leukocyte antigen (HLA) class I molecules, modulating critical NK cell functions in the maintenance of human health. Characterizing the distribution and characteristics of KIR and HLA allotype diversity across defined human populations is thus essential for understanding the multiple associations with disease, and for directing therapies. In this study of 176 Zhejiang Han individuals from Southeastern China, we describe diversity of the highly polymorphic KIR and HLA class I genes at high resolution. KIR-A haplotypes, which carry four inhibitory receptors specific for HLA-A, B or C, are known to associate with protection from infection and some cancers. We show the Chinese Southern Han from Zhejiang are characterized by a high frequency of KIR-A haplotypes and a high frequency of C1 KIR ligands. Accordingly, interactions of inhibitory KIR2DL3 with C1+HLA are more frequent in Zhejiang Han than populations outside East Asia. Zhejiang Han exhibit greater diversity of inhibitory than activating KIR, with three-domain inhibitory KIR exhibiting the greatest degree of polymorphism. As distinguished by gene copy number and allele content, 54 centromeric and 37 telomeric haplotypes were observed. We observed 6% of the population to have KIR haplotypes containing large-scale duplications or deletions that include complete genes. A unique truncated haplotype containing only KIR2DL4 in the telomeric region was also identified. An additional feature is the high frequency of HLA-B*46:01, which may have arisen due to selection pressure from infectious disease. This study will provide further insight into the role of KIR and HLA polymorphism in disease susceptibility of Zhejiang Chinese.

The novel HLA-A*26:174 allele was identified in a Chinese individual.

HLA-A*26:174 shows a single nucleotide substitution at position 148 G>T when compared to HLA-A*26:01:01:01.

Identification of the novel HLA-B*40:125:03 allele in a Chinese bone marrow donor.

HLA-B*40:125:03 shows a single nucleotide substitution at position 378 C > T when compared with HLA-B*40:125:02.

Identification of the novel KIR3DL2*00711 allele by sequencing-based typing in a Chinese individual.

KIR3DL2*00711 differs from KIR3DL2*0070101 by a single nucleotide substitution at position 1344G>A.

Identification of the novel HLA-A*02:837 and -A*02:888 alleles by next-generation sequencing in two Chinese individuals.

Compared with HLA-A*02:06:01:01, HLA-A*02:837 and HLA-A*02:888 show one single nucleotide substitution respectively.

The combinatorial diversity of KIR and HLA class I allotypes in Peninsular Malaysia.

Killer cell immunoglobulin-like receptors (KIRs) interact with polymorphic human leucocyte antigen (HLA) class I molecules, modulating natural killer (NK) cell functions and affecting both the susceptibility and outcome of immune-mediated diseases. The KIR locus is highly diverse in gene content, copy number and allelic polymorphism within individuals and across geographical populations. To analyse currently under-represented Asian and Pacific populations, we investigated the combinatorial diversity of KIR and HLA class I in 92 unrelated Malay and 75 Malaysian Chinese individuals from the Malay Peninsula. We identified substantial allelic and structural diversity of the KIR locus in both populations and characterized novel variations at each analysis level. The Malay population is more diverse than Malay Chinese, likely representing a unique history including admixture with immigrating populations spanning several thousand years. Characterizing the Malay population are KIR haplotypes with large structural variants present in 10% individuals, and KIR and HLA alleles previously identified in Austronesian populations. Despite the differences in ancestries, the proportion of HLA allotypes that serve as KIR ligands is similar in each population. The exception is a significantly reduced frequency of interactions of KIR2DL1 with C2+ HLA-C in the Malaysian Chinese group, caused by the low frequency of C2+ HLA. One likely implication is a greater protection from preeclampsia, a pregnancy disorder associated with KIR2DL1, which shows higher incidence in the Malay than in the Malaysian Chinese. This first complete, high-resolution, characterization of combinatorial diversity of KIR and HLA in Malaysians will form a valuable reference for future clinical and population studies.

HLA-DRB1*14:54:09 and -DRB1*14:54:10, were identified by next-generation sequencing in Chinese cord blood donors.

The sequences of HLA-DRB1*14:54:09 and HLA-DRB1*14:54:10 differ from HLA-DRB1*14:54:01:01 by one single nucleotide substitution, respectively.