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

We characterized HLA and KIR combinatorial diversity in Malay and Malay Chinese, identified substantial allelic and structural diversity of the KIR locus in both populations and discovered novel variations at each analysis level. The Malay are more diverse than Malay Chinese, likely representing a unique history that includes admixture with immigrating populations spanning several thousand years. Characterizing the Malay are KIR haplotypes with large structural variants present in 10% individuals, and the Malaysian Chinese, a low frequency of interactions of KIR2DL1 with C2⁺HLA‐C.

Summary

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.

Abbreviations

Cyt

cytoplasmic domain

D0‐D1

Ig‐like domains

F_ST

F statistic

HBV

hepatitis B virus

HCV

hepatitis C virus

HLA

human leucocyte antigen

IPD

ImmunoPolymorphism database

ITAM

immune receptor tyrosine‐based activating motifs

ITIM

immune receptor tyrosine‐based inhibitory motifs

KIR

killer cell immunoglobulin‐like receptor

LD

linkage disequilibrium

MC

Malaysian Chinese

NK cell

natural killer cell

NPC

nasopharyngeal carcinoma

INTRODUCTION

Killer cell immunoglobulin‐like receptors (KIR) are expressed on the surface of natural killer (NK) cells and subsets of T cells. ¹ , ² KIR educate and regulate the function of these vital immune effector cells through interaction with polymorphic human leucocyte antigen (HLA) class I molecules expressed on tissue cells. ¹ These interactions have substantive roles in infection control, pregnancy, organ transplantation, cancer and autoimmune diseases. ³ , ⁴ , ⁵ Approximately 13 discrete KIR have been described, having inhibitory (KIR2DL1, KIR2DL2/3, KIR2DL5A/B, KIR3DL1, KIR3DL2 and KIR3DL3), activating (KIR2DS1‐2DS5 and KIR3DS1) or both (KIR2DL4) functions. Inhibitory KIR signal through immune receptor tyrosine‐based inhibitory motifs (ITIMs) in their intracellular domain, to block cellular activation signals. Activating KIR function through association with accessory molecules containing immune receptor tyrosine‐based activating motifs (ITAMs). ⁶ , ⁷ , ⁸ KIR interact differentially with subsets of HLA class I allotypes by binding specific outward‐facing amino acid motifs, which overlap with those that form TCR interfaces. ⁹ These motifs include the Bw4 epitope of residues 77–83 present on multiple HLA‐A and HLA‐B allotypes, and the mutually exclusive C1 or C2 epitope defined by residues 77–80 of HLA‐C. ¹⁰ , ¹¹ , ¹² , ¹³

The human KIR locus spans 150–350 kb of the leucocyte receptor complex on chromosome 19q13.4. ¹⁴ The locus varies by gene content, and the genes are organized into two haplotype classes, termed KIR‐A and KIR‐B. ¹⁵ KIR‐A haplotypes encode four inhibitory KIR (KIR2DL1, KIR2DL3, KIR3DL1 and KIR3DL2) and activating KIR2DS4 that are all specific for HLA class I, as well as KIR3DL3 and KIR2DL4, which are not known to bind the highly polymorphic HLA class I. ⁹ , ¹⁶ , ¹⁷ There are also two pseudogenes, KIR2DP1 and KIR3DP1. KIR‐B haplotypes have more variable gene content than KIR‐A and may also encode other activating KIR specific for HLA class I (KIR2DS1, KIR2DS2, KIR2DS5 and KIR3DS1). ⁴ , ⁹ Many of the KIR are highly polymorphic, with a total of 543 allotypes currently described (ImmunoPolymorphism Database Release 2.9.0, December 2019 ¹⁸ ). KIR allele and gene content diversity correlates directly with NK cell function through affecting receptor specificity, binding strength, signal transduction and NK cell development. ¹⁹ , ²⁰ Further copy‐number deviations caused by meiotic recombination enhance functional diversity of this complex system. ²¹ , ²² KIR variation thus distinguishes between individuals and populations in the repertoires of functional NK cells at their disposal. KIR variation is subject to natural selection and co‐evolves with HLA class I allotype variation, which also affects receptor–ligand interaction. ²³ , ²⁴ Accordingly, combinatorial diversity of receptor and ligand allotype impacts NK cell‐driven immune responses and affects resistance or susceptibility to immune‐mediated diseases. ²⁵ , ²⁶ , ²⁷ , ²⁸ , ²⁹ In these studies, KIR‐A haplotypes tend to associate with resistance to infectious disease, whereas KIR‐B haplotypes associate with protection from graft rejection and pregnancy syndromes. ¹⁹ , ²⁶ , ³⁰

Due to their importance for human health, as well as disparities in the incidence rates of immune‐mediated diseases across populations, a thorough understanding of worldwide KIR and HLA diversity is critical. Because Asians have not been adequately represented in previous analyses, here we study two populations from Malaysia. Malaysia spans 330,345 square kilometres north of the equator between the Pacific Ocean and the Indian Ocean. Mainland Peninsula Malaysians are comprised of three major population groups ³¹ : Malay (62%), Malaysian Chinese (21%) and Malaysian Indians (6%). The Malay group represents Deutero‐Malays, whose ancestors settled in Malaysia 3–5000 years ago ³² and subsequently admixed with immigrants from multiple origins including Arabs, Chinese and Indians. Previous studies of KIR diversity in Malaysians were limited to low‐resolution analyses. ³³ , ³⁴ , ³⁵ In the current study, we characterized the KIR and HLA genes of 92 Malay and 75 Malaysian Chinese individuals at high resolution using targeted high‐throughput sequencing technology, and then compared them with available data from other populations. The results will be important for understanding the nature of immune diversity in these groups, and for future studies of immune‐mediated disease or transplantation.

MATERIALS AND METHODS

Study population

Genetic diversity of KIR and HLA class I was determined from 92 Malay and 75 Malaysian Chinese individuals. Peripheral venous blood was collected into EDTA anticoagulant tubes from healthy unrelated blood donors and who were recruited at either Hospital Seberang Jaya in Pulau Pinang, Hospital Universiti Sains Malaysia in Kelantan, or Hospital Temerloh in Pahang. These locations represent the north‐east, north‐west and centre of the Malay Peninsula, respectively. Through questionnaire and analysis of family trees, the individuals were determined as unrelated (siblings, parents and grandparents were excluded) and to have no history of intermarriage with another ethnic group for the past three generations. Informed consent was obtained from all participants, and the samples were de‐identified. This study was approved by the Human Ethical Committee, University Sains Malaysia, Malaysia, and the Medical Research and Ethics Committee, Ministry of Health, Malaysia.

Library preparation, enrichment and sequencing

The KIR locus and individual HLA genes were targeted for DNA sequencing using a biotinylated DNA probe‐based capture method as described, ³⁶ and with modifications as follows. Genomic DNA (500 ng from each sample) was fragmented enzymatically using the NEBNext Ultra II FS Module (New England Biolabs, Boston, MA). We used 1 µl of the fragmentation enzyme with 5 µl of buffer, supplemented with 29 µL with 1× TE and incubated at 37° for 5 min and 65° for 30 min, then held at 4°. Individual samples were labelled uniquely using 3 µl of 15 µM custom dual‐index adapters (Integrated DNA Technologies, Coralville, IA) and the NEB ligation module. Post‐ligation cleanup was based on the Kapa Hyper Prep protocol (Kapa Biosystems, Wilmington, MA) and followed by dual size selection as previously described. ³⁷ The samples were pooled in two batches, then enriched using the capture probes and a modified version of the Nextera Rapid Capture enrichment protocol (Illumina) as previously described. ³⁶ The concentration of the library was determined by Qubit instrument, prepared to a final concentration of 12 pM and sequenced using a MiSeq instrument and v3 Reagent Kit (600‐cycle; Illumina Inc, San Diego, CA).

Sequence data analysis

KIR gene content, copy numbers and allele genotypes were determined using the Pushing Immunogenetics to the Next Generation (PING) pipeline as previously reported. ³⁶ The copy number was obtained according to the ratio of reads mapping to each KIR gene to those mapping to KIR3DL3, a reference gene present as one copy on each haplotype. ³⁸ Genes analysed were KIR3DL3, 2DS2, 2DL2/3, 2DL5A and 2DL5B, 2DS3/5, 2DP1, 2DL1, 2DL4, 3DL1/S1, 2DS1, 2DS4 and 3DL2. KIR2DL2 and KIR2DL3 are alleles of one gene (KIR2DL2/3), as are KIR2DS3 and KIR2DS5 (KIR2DS3/5) and KIR3DL1 and KIR3DS1 (KIR3DL1/S1). KIR3DL3 alleles were determined using PHASE 2.1. ³⁹ The following parameters for PHASE 2.1 were used: –f1, –x5 and –d1. The PING pipeline generates a high‐resolution KIR gene content and allele‐level genotype. ³⁶ It can also identify previously unreported single nucleotide polymorphisms (SNPs) and recombinant alleles. Novel allele sequences were analysed by visual inspection; reads specific to the relevant gene were isolated by bioinformatics filtering, aligned to the closest reference allele using MIRA 4.0.2 and inspected using Gap4 of the Staden package. ⁴⁰ , ⁴¹ All new alleles were confirmed by repeating the sequencing process, starting from the original gDNA sample. New allele sequences were deposited in GenBank and the ImmunoPolymorphism Database. ¹⁸ The HLA alleles were determined using NGSengine 2.10.0 (GenDX, Utrecht, the Netherlands) and HLA*LA, ⁴² with any discrepancies resolved using HLA Haplotype Validator. ⁴³

Frequencies of genotypes, alleles and haplotypes

Genotype names based on the presence or absence of KIR genes were designated according to the Allele Frequencies database (AlleleFrequencies.net ⁴⁴ ). Carrier frequencies of KIR genes and gene content genotypes were determined as their percentage of the total numbers of individuals for each population. Allele or haplotype frequencies were calculated by direct counting and the number observed divided by 2N (alleles duplicated on a single haplotype were not included, and gene absence was counted as a distinct allele). KIR haplotypes include a centromeric motif and a telomeric motif. The centromeric motif is defined as all genes from KIR3DL3 through KIR3DP1. The telomeric motif encompasses all genes present from KIR2DL4 to KIR3DL2 on the telomeric side of the KIR locus. The centromeric and telomeric haplotypes were designated based on linkage disequilibrium among KIR genes and the copy number of each KIR gene in an individual. The complete KIR haplotype is a combination of centromeric and telomeric haplotypes, and their frequencies were calculated by direct counting. HLA haplotype composition and frequencies were determined using ‘Arlequin 3.5’. ⁴⁵

HLA/KIR interactions

To determine the quantity of inhibitory receptor–ligand interactions per individual, the number of KIR/HLA allotype pairs that are known to interact was summed, and homozygous KIR or HLA allotypes were counted twice. The mean per population was then calculated. Broadly, HLA‐B or HLA‐C allotypes possessing asparagine at position 80 (C1⁺ HLA class I) are ligands for KIR2DL2/3; HLA‐C allotypes carrying lysine at this position (C2⁺ HLA class I) are ligands for KIR2DL1 – some are ligands for KIR2DL2/3; HLA‐A and HLA‐B allotypes that carry the Bw4 motif at residues 77‐83 are ligands for KIR3DL1 – HLA‐A*03 and HLA‐A*11 are ligands for KIR3DL2. ¹⁰ , ¹¹ , ¹² , ¹³ , ²⁴ The specific interactions that were counted are listed in Ref. 46.

Statistical analyses

Differences between populations were assessed by means of the chi‐square test for categorical variables. P values were calculated, with the Fisher exact test (pf) applied when appropriate. P values less than 0·05 were regarded as significant. The Bonferroni correction for multiple comparisons was applied. The statistical analyses were performed using GraphPad software. F _ST was calculated using the R package PopGenReport. ⁴⁷ The Fisher exact probability test was performed using the prop.test function in R. ⁴⁸

RESULTS

The frequencies and copy numbers of KIR genes in Malay and Malaysian Chinese

To examine the combinatorial diversity of KIR and HLA class I allotypes in mainland Peninsula Malaysia, we studied 92 Malay and 75 Malaysian Chinese individuals. We first analysed the variation of copy number in KIR genes. All individuals encoded at least one copy of the KIR2DL4, KIR2DL2/3, KIR3DL2 and KIR3DL3 genes and the KIR3DP1 pseudogene (Figure 1A). Of these, KIR2DL2/3, KIR3DL2 and KIR3DL3 were each observed at two copies (i.e. diploid) in every individual studied, and all other KIR genes and pseudogenes exhibited copy‐number variation (Figure S1). The most extreme in this regard are KIR2DL1 and KIR3DL1/S1, which were each observed from zero to four copies per individual (Figure 1A). Ten of the Malay individuals were observed having three copies of KIR2DL4 and KIR3DL1/S1, and one individual was observed having four copies of these genes (Figure 1A). In total, seven of the KIR genes were determined present at greater than diploid copy number in one or more of the Malay individuals analysed (Figure S1). Although excess copy number is less frequent in Malaysian Chinese, where only two individuals were observed having three copies each of KIR3DP1, KIR2DL4 and KIR3DL1/S1, five individuals have only one copy of all these genes (Figure 1A). These observations show that KIR haplotypes carrying gene duplications are likely more frequent in Malay than in Malaysian Chinese, whereas those having KIR gene deletions are more frequent in Malaysian Chinese.

Figure 1.

The KIR genotypes of Malay and Malaysian Chinese. (A) Shown are KIR gene content genotypes observed from 92 Malay and 75 Malaysian Chinese individuals. The colours of the boxes indicate the gene copy numbers, as given in the key at the bottom. At the left, the genotype IDs are assigned according to the Allelefrequencies.net database, ⁴⁴ and their observed frequencies are shown at the right. MC, Malaysian Chinese. (‐), not detected in that population. (B) Shown are the carrier frequencies of the four most frequent genotypes KIR‐AA, Bx2, Bx4 and Bx5 in nine representative populations. The populations are as follows: Japanese, ⁴⁹ Chinese Southern Han, ⁸⁸ Taiwan Han, ⁵¹ Eastern Chinese, ⁵¹ Europeans, ⁶⁰ sub‐Saharan Africans (Ghanaians) ⁵⁹ and South American Yucpa. ⁵⁶ (C) Shown is the distribution of centromeric and telomeric KIR‐A and B haplotypes in Malay and Malaysian Chinese.

Greater than 90% individuals in each population carried at least one copy of KIR2DL1, KIR2DP1, KIR3DL1/S1 and KIR2DS4. The high frequencies of these genes are consistent with high frequencies of the KIR‐A haplotype in both populations. Indeed, 37% of Malay and 38·7% Malaysian Chinese have the KIR‐AA genotype, which shows they are homozygous for the KIR‐A haplotype (Figure 1A). As observed previously, high frequencies of KIR‐AA genotypes are characteristic of East Asian populations (Figure 1B), likely though natural selection. ³⁵ , ⁴⁶ , ⁴⁹ , ⁵⁰ , ⁵¹ The Malaysians, however, have lower frequencies of KIR‐A haplotypes than other Asian populations analysed (Figure 1B). In the Malay, this difference is more evident in the telomeric part of the KIR locus (Figure 1C). That Malaysian Chinese have lower frequencies of KIR‐A than other East Asians therefore likely reflects a combination of founder effect and limited historical admixture with Malay.

The distribution of KIR alleles in Malay and Malaysian Chinese

We observed a total of 148 distinct KIR alleles across the two populations, 125 in Malay and 102 in Malaysian Chinese. Eleven of the KIR alleles we observed had not been described previously and were submitted to the ImmunoPolymorphism database ¹⁸ (Figure 2). All the novel KIR alleles encode distinct allotypes, two amino acid substitutions occur in the cytoplasmic domain, which may impact receptor signalling, ⁷ and nine of them are amino acid substitutions in the Ig‐like domains. Substitutions in the Ig‐domain loops of KIR can directly impact ligand binding; for example, the G‐R substitution at residue 43 of KIR2DL1 (Figure 2) occurs in the HLA binding site. ⁵² By contrast, those in the beta sheets may indirectly impact binding, for example KIR2DL2/3 residue 16, ¹⁰ which is observed here as polymorphic in KIR2DS4 (Figure 2). Thus, any of the nine Ig‐domain substitutions identified here could affect ligand binding strength or specificity. ⁷ These novel alleles were identified exclusively in Malay individuals, suggesting a greater diversity of NK cell receptor polymorphism in Malay than in Malaysian Chinese.

Figure 2.

KIR alleles discovered in the Malay population. Shown are the KIR alleles discovered in this study, which were all detected in Malay individuals. From left to right: the KIR gene, GenBank ID, new allele name, the closest match to previously identified alleles, nucleotide and corresponding amino acid substitutions compared with the closest match, and number of examples observed. D0–D1: Ig‐like domains, Cyt: cytoplasmic domain.

The most polymorphic KIR gene in both groups is KIR3DL3, where 32 alleles encoding 19 distinct allotypes were identified in Malay and 22 alleles encoding 15 allotypes were identified in Malaysian Chinese (Figure 3). Second and third most polymorphic were KIR3DL2, having 21 alleles in Malay and 15 in Malaysian Chinese, and KIR3DL1/S1, with 15 alleles in Malay and 12 in Malaysian Chinese (Figure 3). In total, there are 75 allotypes encoded by the six genes for inhibitory KIR present across the two populations. By contrast, the six genes for activating KIR encode a total of only ten allotypes, with each gene encoding one to three allotypes (Figure 3). Of the alleles of activating KIR present, only KIR2DS2 and KIR3DS1 express more than one allotype, and each is represented by one predominant and one low‐frequency allele (Figure 3). The alleles of KIR2DS1 (*00201 and *00202) represent synonymous mutation, and only one each of the KIR2DS3 and KIR2DS4 alleles is expressed (*002, and *00101, respectively ⁵³ , ⁵⁴ ). Thus, inhibitory KIR show substantially more functional polymorphism than activating KIR in both populations.

Figure 3.

KIR allele diversity of Malay and Malaysian Chinese. (A) Shown are the KIR alleles and their frequencies identified in 92 Malay and 75 Malaysian Chinese (MC). Red text indicates an allotype not expressed at the cell surface; Bold text, alleles showing marked frequency difference between the two populations; and (†), alleles discovered in this study. (−), not detected in that population.

Several KIR alleles show clear differences in frequency between the two populations studied, including KIR3DL3*00901 (21·2% vs 8%, P = 0·015, pc = ns), KIR3DL3*00902 (2·7% vs 13·3%, P = 0·017, pc = ns) and KIR3DL3*00301 (9·2% vs 1·3%, P = 0·019, pc = ns). KIR3DL3*00901 (21·2%) is the most frequently observed KIR3DL3 allele in Malay, whereas in Malaysian Chinese, this is KIR3DL3*01001 (17·3%). Despite differences in allele frequencies, the two KIR3DL3 allotypes are distributed evenly among the two groups, with KIR3DL3*009 observed at 23·9% in Malay and 21·3% in Malaysian Chinese; and KIR3DL3*010 at 27·2% in Malay and 32·7% in Malaysian Chinese, suggesting consistency of functional impact for these allotypes. Although the most frequent KIR3DL2 allele in both populations is KIR3DL2*00201, this also differs in frequency, at 39·1% in Malay and 57·3% in Malaysian Chinese (P = 0·016, pc = ns; Figure 3). Other frequent KIR3DL2 alleles common to both populations are KIR3DL2*00701 (19% Malay and 10% Malaysian Chinese) and KIR3DL2*01001 (9·8% Malay and 11·3% Malaysian Chinese). The most frequent KIR3DL1/S1 allele in both groups, the highly expressed KIR3DL1*01502, is also more frequent in the Malaysian Chinese individuals (34·8% vs 46%; Figure 3). Other common and frequent alleles are KIR3DL1*00501, which is expressed at low level, and KIR3DS1*01301, which encodes the activating allotype of KIR3DL1/S1. These observations show that all three ancient functional lineages of KIR3DL1/S1 ²¹ are maintained at high frequency in both of the populations studied. The Malay population has high frequencies of KIR2DL4*00501, KIR3DS1*01301, KIR2DL5A*00101, KIR2DS5*00201, KIR2DS1*00201 and KIR3DL2*007 (Figure 3), which are all characteristic of the telomeric KIR‐B haplotypes ⁴ that are more frequent in this population than in the Malaysian Chinese (Figure 1C). Finally, suggesting a requirement to maintain the expressed allotype of KIR2DS4, KIR2DS4*001, which is the only allele encoding a functional protein, was observed at a frequency of 39·6% in Malay and 49·3% in Malaysian Chinese.

Comparison of KIR allele distributions across representative populations showed that Asians are characterized by high frequencies of alleles that are rarer in other major population groups (Figure 4A–F). Specifically, KIR3DL1*01502, KIR3DL2*002 and KIR3DL3*010 are the predominant alleles of their respective genes (Figure 4A–C). In these genes, other populations studied are characterized by different sets of high‐frequency alleles. The distinction is most obvious for KIR3DL3, where Europeans are characterized by high frequency of allele *001, Amerindians by *003, West Africans by *005, Oceanians by *015 and Southern Africans by *038 (Figure 4C). The distributions are similar to those observed for HLA class I, where specific alleles can be characteristic of population groups, ⁵⁵ and illustrate the divergence of KIR and HLA class I polymorphism across human populations (Figure 4G). In these comparisons, however, the greatest mean population differentiation is observed for KIR2DL1 and KIR2DL2/3 (Figure 4D–E and Figure S2), which is consistent with the strong signatures of local adaptation observed for these KIR. ³⁷ , ⁴⁶ , ⁵⁶

Figure 4.

KIR allotype diversity across worldwide populations. (A–F) Shown are the frequencies of KIR3DL1/S1, KIR3DL2, KIR3DL3, KIR2DL1, KIR2DL2/3 and KIR2DS4 allotypes across representative human populations. Only allotypes with frequency greater than 5% in at least one population are shown, and they are ordered by highest to lowest frequency in Malay. Red text indicates allotype that is not expressed on the cell surface. Colour key at the right shows the populations studied. The populations are as follows: Chinese Southern Han, ⁴⁶ Japanese, ⁴⁹ Eastern Chinese, ⁵¹ sub‐Saharan Africans: Ga‐Adangbe from Ghana ⁵⁹ and KhoeSan from Southern Africa, ³⁷ Europeans ⁶⁰ and South American Yucpa. ⁵⁶ There is no KIR3DL3 allele information available from the Japanese population. (G) Mean pairwise F_ST measurements across the same genes and populations as panels A–F, shown also for HLA‐A, HLA‐B and HLA‐C.

High structural diversity of KIR haplotypes in Malay

When defined by their allele composition, we identified 78 distinct centromeric KIR haplotypes, with 52 of them observed in Malay and 49 in Malaysian Chinese (Figure S3A). Structurally, the majority in both populations are KIR‐A haplotypes (Figure 1), and similar to other Asian populations analysed, they encode allotypes of KIR2DL1 and KIR2DL3 that have relatively high affinity for HLA and strong signal transduction ability (*003 and *001, respectively). ⁴⁶ , ⁴⁹ The centromeric KIR‐A haplotypes are diversified substantially by their alleles of KIR3DL3. Indeed, the number of centromeric KIR haplotypes is reduced to 14 in Malay and 15 in Malaysian Chinese when KIR3DL3 alleles are excluded (Figure S3A). The impact of KIR3DL3 polymorphism is unknown, but it is rarely expressed by peripheral NK cells, and is unlikely to bind polymorphic HLA class I. ³⁸ Thus, although most allele‐level centromeric KIR‐A haplotypes differ in frequency between Malay and Malaysian Chinese (Figure 5A), they are functionally equivalent with respect to NK cell interaction with HLA class I. By contrast, the centromeric KIR‐B haplotypes present in Malaysia express the poorly functional KIR2DL1*004 ⁵⁷ or lack KIR2DL1 altogether (Figure 5A). Within the centromeric haplotypes, we identified two unusual configurations: the first having KIR2DL1*00302 linked to KIR2DL2 and KIR2DS2, which we term cB07; and the second that is identical to a centromeric KIR‐A haplotype but lacks KIR2DL1, which we term A_del (Figure S3A).

Figure 5.

KIR haplotype diversity in Malay and Malaysian Chinese. (A–B) Shown are the ten most frequent centromeric (panel A) and telomeric (panel B) KIR haplotypes and their frequencies identified from 92 Malay and 75 Malaysian Chinese individuals. Brackets indicate the frequency of that haplotype in the population if it was not in the top ten. KIR‐A haplotypes are shaded pink and KIR‐B haplotypes are shaded blue. (C–E) Shows three large structural variations observed. Grey shading indicates the additional segment in relation to more frequent haplotypes, as described above each with ‘insert’. (†) indicates allele or haplotype not previously observed. The corresponding centromeric KIR gene content motifs are indicated at the left.

In analysing the telomeric KIR genes, we identified 62 distinct haplotypes, 48 of them in Malay and 30 in Malaysian Chinese (Figure S3B). The large difference in number between the two groups is caused both by a greater allelic diversity of telomeric KIR‐A haplotypes (25 vs 16; Figure S3B) and by a greater structural diversity of the telomeric KIR‐B haplotypes in Malay (Figure 5B–E). KIR2DL4*00102–KIR3DL1*01502–KIR2DS4*00101–KIR3DL2*00201 is the most frequent telomeric KIR haplotype in both populations (27·7% in Malay and 40·7% in Malaysian Chinese). This haplotype is common to East Asians, Oceanians and Amerindians, ⁴⁶ , ⁵⁶ , ⁵⁸ but rare in Africans and Europeans. ³⁷ , ⁵⁹ , ⁶⁰ , ⁶¹ The most frequent telomeric KIR‐B haplotype observed in Malay (KIR2DL4*00501–KIR3DS1*01301–KIR2DL5A*00101–KIR2DS5*00201–KIR3DL2*00701) is common to most populations but rare or absent from Africans. ³⁷ , ⁵⁸ , ⁵⁹ , ⁶⁰ , ⁶¹ This distribution is consistent with the acquisition through admixture with ancient humans that is proposed for these haplotypes. ⁶²

KIR‐B haplotypes are characterized by structural diversity including deletion, duplication or insertion of one or more complete KIR genes. ²¹ , ²² , ⁶³ We identified six KIR haplotypes having deletions (Haplotypes 52‐56; Figure S3B) and 10 having duplications/insertions (Figure 5C–E), each case involving complete KIR genes. One of the duplication haplotypes (tB10; Figure 5C) was detected in four Malay individuals. The duplication/insertion haplotypes are defined by three major structural changes: addition of three genes, KIR3DP1, KIR2DL4 and KIR3DS1 (Figure 5C); addition of six genes KIR3DP1, KIR2DL4, KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DL1 (Figure 5D); or addition of seven genes, KIR3DP1, KIR2DL4, KIR3DS1, KIR2DL5, KIR2DS35, KIR2DP1 and KIR2DL1 (Figure 5E); the latter two structural variations being further distinguished by their alleles of 2DL1 (*00302 and *00201, respectively), 2DL3/5 (5*00201 and 3*00103, respectively) and 2DL5A (*00101 and *00501, respectively). In total, this high‐resolution analysis revealed there to be 19 distinct KIR gene content‐level haplotypes, 15 in Malay and 11 in Malaysian Chinese (Figure 6). Of these haplotypes, six are common to both populations and comprised of combinations of haplotype motifs that are frequent in most other populations, CenA01, CenB01, CenB02, TelA01 and TelB01. ⁴ The remaining 13 haplotypes are rare and characterized by the deletions and insertions described above.

Figure 6.

Structural diversity of KIR haplotypes in Malay and Malaysian Chinese. Shown are the KIR gene content haplotypes and their frequencies deduced from 92 Malay and 75 Malaysian Chinese individuals. Colour coding is used to indicate the ‘deletion’ or ‘insertion/duplication’ structural variants identified. MC, Malaysian Chinese.

Comparison of HLA ligand distributions in Malay and Malaysian Chinese

All HLA‐C allotypes and a subset of HLA‐A and HLA‐B allotypes are ligands for KIR. To evaluate and compare the combinatorial diversity of KIR and HLA class I allotypes in Malay and Malaysian Chinese, we next analysed high‐resolution genotypes of HLA‐A, HLA‐B and HLA‐C. We identified 26 alleles of HLA‐A (22 in Malay and 16 in Malaysian Chinese), 50 of HLA‐B (41 in Malay and 30 in Malaysian Chinese) and 28 of HLA‐C (26 in Malay and 15 in Malaysian Chinese; Figure 7A). The allele frequencies are given in Figure S4. Notable are HLA‐A*24:07, which was detected at 11% allele frequency in Malay but absent in Malaysian Chinese; and HLA‐B*40:01, present at 3% in Malay and 23% in Malaysian Chinese (P < 0·0001, pc = 0·005). Also notable are HLA‐B*15, which has ten distinct allotypes in Malay but only four in Malaysian Chinese; and HLA‐C*07 with seven allotypes in Malay but two in Malaysian Chinese. In contrast to these distinctions, HLA‐A*11:01 was detected at similarly high frequencies in both populations (21% and 26%; Figure S4). HLA‐A*11 is a ligand for inhibitory KIR3DL2 and activating KIR2DS4 ¹¹ , ⁶⁴ and is expressed by four of the ten most frequent HLA class I haplotypes in both populations (Figure 7B). HLA‐A*11:01 thus contributes substantially to the high proportion of HLA haplotypes encoding more than one KIR ligand that are present in both populations (Figure 7C).

Figure 7.

Combinatorial diversity of KIR and HLA class I allotypes in Malay and Malaysian Chinese. (A) Shown are the HLA‐A, HLA‐B and HLA‐C allele frequency spectra from 92 Malay and 75 Malaysian Chinese individuals. Each pie segment represents a distinct allele, coloured according to the expressed KIR binding motif: yellow – A3/11 (HLA‐A3 and HLA‐A11); green – Bw4 epitope (subsets of HLA‐A and HLA‐B); red – C1 (HLA‐C allotypes, plus HLA‐B*46); blue – C2 (HLA‐C allotypes that do not carry C1); and grey – allotypes that are not KIR ligands. Figure S4 lists all the HLA‐A, HLA‐B and HLA‐C allotypes present in the study population and shows which KIR ligand motifs they carry. (B) Shows the ten most frequent HLA class I haplotypes detected in each population. The frequencies are shown at the right, brackets indicate the frequency of that haplotype in the population if it was not in the top ten. Bold text indicates three haplotypes that are in the top ten in both populations. Coloured shading indicates HLA class I alleles that encode KIR ligands, as described in panel A. (C) Pie charts show the combined frequencies of HLA class I haplotypes encoding one (blue), two (gold) or three (green) KIR ligands in each population. (D) Shows the number of HLA‐A, HLA‐B, or HLA‐C allotypes present within each category of KIR ligand and their combined frequencies in each population. (E) Shown is a plot of the total number of KIR‐HLA allotype pairs per individual in Malay (mean 7·88) and Malaysian Chinese (mean 7·33). (F) Shown is the mean number of interactions between KIR and HLA allotypes per individual in Malay and Malaysian Chinese. The interacting KIR‐HLA pairs are shown at the left. (G) Shows the frequency of HLA‐B alleles encoding methionine (orange) or threonine (grey) at leader peptide residue 2. Methionine favours NK cell education through NKG2A/HLA‐E interaction. ⁶⁵ (H) Shows the phenotype frequency of HLA‐DPA1/‐DPB1 heterodimers that can bind NKp44 (blue).

In the Malay population, 47 of 89 HLA class I allotypes (53%), and in the Malaysian Chinese, 32 of 61 allotypes (50%) are KIR ligands (Figure 7D). Thus, all four major KIR ligands are present and the numbers and proportions of KIR ligands across the two populations are broadly similar (Figure 7A,C). The exceptions are a higher proportion of C2⁺ HLA‐C (29·9% vs 14·4%, P < 0·01, pc = 0·04) and Bw4⁺ HLA‐A (33·7% vs 15·1%, P < 0·01, pc = 0·03) in Malay, due in large part to the presence of alleles HLA‐C*04:03 and HLA‐A*24:07, respectively. These two alleles segregate in the population independently (Figure S3C). Similarly, when we examined the mean number of viable interactions per individual for each inhibitory receptor ligand pair, the values were comparable across the two populations (7·9 Malay and 7·3 Malaysian Chinese; Figure 7E). The exceptions are KIR2DL1 with C2⁺ HLA (1·1 per individual in Malay and 0·5 in Malaysian Chinese, P < 0·01) and KIR3DL1 with Bw4⁺HLA (1·9 Malay and 1·5 Malaysian Chinese, ns; Figure 7F). Contrasting the high number of KIR ligands in both groups is the low frequency of HLA‐B ‐21M, which favours development of NK cells that are educated by CD94/NKG2A instead of KIR. ⁶⁵ Only 10 of the 50 observed HLA‐B alleles encode a ‐21 M variant (combined frequency = 9·6% Malay and 12·3% Malaysian Chinese; Figure 7G). The most frequent HLA‐B allotype having ‐21 M is HLA‐B*38 (3% in Malay and 7·5% in Malaysian Chinese; Figure S4), which is also a KIR ligand. Representing a similar scenario to KIR interaction with HLA class I, a subset of HLA‐DP heterodimer allotypes was recently identified to bind the NK cell activating receptor NKp44. ⁶⁶ Because the constituent HLA‐DPA and HLA‐DPB alleles vary in their population distribution, ⁶⁷ we analysed the HLA class II allele frequencies of the two study populations. The allele frequencies of HLA‐DPA1, HLA‐DPB1, HLA‐DQA1, HLA‐DQB1, HLA‐DRB1 and HLA‐DRB3‐5 are given in Figure S5. In the Malay population, 60·5% of individuals possess at least one HLA‐DP heterodimer that can bind NKp44, whereas only 20·5% of Malaysian Chinese do so (the Fisher exact proportions test, P = 4·8 × 10⁻⁷; Figure 7H). There is thus a striking difference between the two Malaysian populations in the number of individuals who possess a ligand for NKp44. In summary, the proportions of HLA allotypes that can mediate viable interaction with NK cells are similar in the two populations analysed. The exceptions are significantly lower frequencies in the Malaysian Chinese of HLA‐C allotypes that bind inhibitory KIR2DL1 and HLA‐DP allotypes that bind activating NKp44.

DISCUSSION

We analysed the combinatorial diversity of KIR and HLA class I allotypes in Peninsular Malaysia, focusing on the two most populous groups, the Malay and Malaysian Chinese. The study uncovered substantial allelic and structural diversity of the KIR locus in both groups and characterized novel variations at each analysis level. We compared the KIR allele frequencies of Malaysians with those representing African, Asian, European, Oceanian and South Americans and identified specific KIR alleles that characterize these major population groups. Characteristic alleles are also observed for HLA, and together, they are likely to result in differences in immune function that underlie cross‐population differences in susceptibility to specific immune‐mediated diseases.

Our high‐resolution analysis of KIR copy number and allele diversity revealed considerable structural diversity of the KIR locus particularly in the Malay population, where 10% of individuals possess at least one haplotype carrying duplication of one or more KIR genes. Although previous analyses are limited, they suggest haplotypes carrying similar duplications are rare or absent from Africans, and present in 2%–5% of Europeans and East Asians, 2%–10% of South and South‐East Asians and up to 15% of Oceanians. ²¹ , ²² , ³⁷ , ⁵⁹ , ⁶⁰ , ⁶¹ , ⁶⁸ The frequency we observe in Malay is therefore consistent with ancestral population distributions. The mechanism forming these haplotypes is likely to be homologous recombination during meiosis. ⁶³ , ⁶⁹ In every example observed here, the duplicated segment encodes the alleles KIR2DL4*005 and KIR3DS1*013, raising the alternative possibility that this represents an ancestral form of KIR haplotype, and these segments are deleted in most modern haplotypes. These two alleles represent an expressed allotype of KIR2DL4 (2DL4*005 ⁷⁰ ) and the activating allotype of KIR3DL1/S1 (3DS1*013 ⁷¹ ). Importantly, expression of both activating and inhibitory KIR3DL1/S1 allotypes by a single haplotype broadens the repertoire of mature NK cells in the periphery above haplotypes expressing only one. ²¹ , ⁷² Extreme in this regard, one Malay individual was observed having four copies of KIR2DL1, KIR2DL4 and KIR3DL1/S1 and three copies of KIR2DL5A and KIR2DS5 (Figure 1A), indicating they carry two duplication haplotypes. Some of the structurally distinct haplotypes we describe have been reported previously, ²¹ , ²² , ⁶⁰ , ⁶³ , ⁶⁹ implying the deletion and duplication events are common, and some were identified for the first time, which may be because they are rare or that these large structural variations continue to arise.

When compared to published data from multiple representative populations, we found that Malaysian Chinese were relatively similar to Chinese Han, which is consistent with historical records showing that the Malaysian Chinese are mainly descendants of Chinese migrating from the Ming and Qing Dynasty of China. Although limited, the differences we observe with the ancestral populations are likely due to a combination of founder effects, and historical admixture of immigrating and resident populations. Similarly, in the Malay we observed several alleles and haplotypes that have only been observed previously in Remote Oceanic populations. These alleles, KIR2DL4*028, KIR3DL1*086, KIR3DL1*114 and KIR3DL2*101, ⁵⁸ , ⁶⁸ were identified in four Malaysian individuals. Each of the haplotypes carrying these alleles (haplotypes 11, 23 and 29; Figure S3B) was observed previously in Māori, Polynesian or Papua New Guineans. ⁵⁸ , ⁶⁸ These findings likely reflect the migration throughout South‐East Asia of Austronesian‐speaking populations, which originated from Taiwan, and subsequent admixture of the Deutero‐Malay with other indigenous groups in Peninsular Malaysia. ³¹ , ⁷³ Similarly, HLA‐A*34:01 and HLA‐B*15:21, which are frequent alleles in Remote Oceanic populations, ⁴⁴ , ⁷⁴ were identified here in Malay but not Malaysian Chinese. Indeed, the Malay population exhibits wide diversity of HLA‐B*15 allotypes. Among these allotypes is HLA‐B*15:13, which acts as a KIR ligand virtue of possessing the Bw4 motif, and is common to many South‐East Asian populations. ⁴⁴ , ⁷⁵ , ⁷⁶ Excluding the Bw4 motif, HLA‐B*15:13 is identical to the most frequent HLA‐B allotype in Malay, B*15:02, and therefore likely formed during a double‐crossover meiotic recombination event. A similar situation is observed in West Africa, where the common allotypes HLA‐B*35:01 and B*53:01 differ solely by the presence of a Bw4 motif in the latter. Both these allotypes can present peptides derived from Plasmodium falciparum and offer differing levels of protection from severe malaria that is likely dependent on polymorphic epitopes of the parasite. ⁷⁷ Thus, the only two distinctions between B*35:01 and B*53:01 are the peptide residues that can bind the F pocket and that B*53 can interact with KIR3DL1 in addition to TCR. Malarial disease also has a high prevalence in South‐East Asia, and the concurrent frequencies of HLA‐B*15:02 and HLA‐B*15:13 support the view that they may have the same roles in protection from malaria as B*35 and B*53 do in West Africa. ⁷⁵

Both alone and in combination, KIR and HLA allotypes are associated with variety of diseases, including autoimmunity and malignancies, and they influence the course of infections, transplantation and placentation. Although we observed the frequencies of allele‐level centromeric KIR‐A haplotypes differ between Malay and Malaysian Chinese, the most frequent one observed here (KIR3DL3*00901, KIR2DL3*001 and KIR2DL1*00302) has been detected in all populations studied to date. ³⁷ , ⁴⁶ , ⁵⁶ , ⁵⁸ , ⁵⁹ , ⁶⁰ However, the strong‐acting allotypes of KIR2DL1 and KIR2DL3 that are encoded by this haplotype have likely been subject to positive natural selection only in East Asians, ⁴⁶ , ⁷⁸ likely contributing to the increased interpopulation divergence we observed for these KIR genes (Figure 3). Malaysian Chinese exhibit a low frequency of C2⁺HLA‐C interactions with KIR2DL1, which are only half as frequent as those in Malay. KIR2DL1‐C2⁺HLA‐C interaction is a risk factor for preeclampsia, ⁷⁹ a hypertensive disorder caused by poor placentation, which has higher incidence in Malay than in Malaysian Chinese. ⁸⁰ , ⁸¹ Thus, the lower frequency of C2⁺HLA‐C interactions with KIR2DL1 in the Malaysian Chinese may offer protection from preeclampsia. Viral infection is another major selection pressure, including viral hepatitis, which is a growing problem in Malaysia. Here, 2% of the population were reported to carry HCV, with the Malay population showing a relatively higher prevalence than the Malaysian Chinese. ⁸² , ⁸³ In contrast to the strong KIR2DL1‐C2⁺HLA‐C interaction, the weaker KIR2DL3‐C1⁺HLA‐C interaction is correlated with better prognosis for HCV infection. ⁸⁴ Another C1⁺ allotype is HLA‐B*46:01, which is prominent in Malaysian Chinese and specific to East Asia, suggesting it underwent localized selection for disease resistance. ⁴⁶ , ⁸⁵ Interestingly, HLA‐A*02:07 is a risk factor for nasopharyngeal carcinoma (NPC) caused by Epstein–Barr virus, ⁸⁶ , ⁸⁷ and occurs in strong LD with HLA‐B*46:01 in Malaysian Chinese where NPC is more prevalent than Malay. By contrast, A*11:01, which interacts with KIR2DS4 and KIR3DL2, and protects from NPC, is found at equivalent frequencies in these populations. A further distinction between the two populations we studied is the lower frequency of HLA‐DP allotypes that bind NKp44 was observed in Malaysian Chinese. The simultaneous expression of HLA‐DP and NKp44 is a risk factor for the development of chronic HBV infection. ⁶⁶ This observation suggests again there will be distinct susceptibilities to chronic HBV across these groups.

In summary, we described the KIR and HLA ligand diversity in high‐resolution level in Malay and Malaysian Chinese. The description of KIR genes, allelic polymorphism, haplotypes and KIR‐HLA interactions will provide useful information for the studies of the potential interaction of associations of KIR‐HLA with disease and mechanistic insight into NK cell functions in disease in Malaysian populations.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

Supporting information

Figure S1. KIR gene copy number frequencies in Malaysia. A. Shown are the carrier frequencies of each copy number genotype observed for each KIR gene in MALAY (N = 92) and Malaysian Chinese (N = 75).

Figure S2. Pairwise F _ST values for KIR and HLA class I genes. Shows pairwise F _ST values obtained from comparisons of Malaysian Chinese (upper) or Malays (lower) allele distributions with those of other representative populations.

Figure S3. Haplotype frequencies. Shows the frequencies in Malay (2N = 184) and (MC) Malaysian Chinese (2N = 150) of: A. Centromeric KIR haplotypes; B. Telomeric KIR haplotypes. C. HLA class I haplotypes.

Figure S4. HLA‐A, ‐B and ‐C allele frequencies in Malay and Malaysian Chinese. Shows the HLA‐A, HLA‐B, and HLA‐C alleles and frequencies in Malay (2N = 184) and (MC) Malaysian Chinese (2N = 150). Coloured shading represents the expressed KIR binding motif: Yellow ‐ A3/11 (HLA‐A3 and ‐A11); Green ‐ Bw4 epitope (subsets of HLA‐A and ‐B); Red ‐ C1 (HLA‐C allotypes, plus HLA‐B*46); Blue ‐ C2 (HLA‐C allotypes that do not carry C1). (‐) not detected in that population. (#) indicates alleles showing significant difference across the two populations following correction for multiple testing.

Figure S5. HLA class II allele frequencies in Malay and Malaysian Chinese. Shows the HLA class II alleles and frequencies in Malay (2N = 184) and (MC) Malaysian Chinese (2N = 150). (#) indicates alleles showing significant difference across the two populations following correction for multiple testing.

 

 

DATA AVAILABILITY STATEMENT

The data necessary for the findings of this paper are all included in the Figures S1–S5 and Supplementary Material.