Received: 6 April 2022 Revised: 17 June 2022 Accepted: 5 July 2022 DOI: 10.1111/tan.14734 ORIGINAL ARTICLE Diversity of NKG2C genotypes in a European population: Conserved and recombinant haplotypes in the coding, promoter, and 30-untranslated regions Judit Asenjo1 | Manuela Moraru1 | Karima Al-Akioui-Sanz1 | Mireia Altadill2 pez-Botet2,3 | Carlos Vilches1 | Aura Muntasell3,4 | Miguel Lo 1 Immunogenetics and Histocompatibility Lab, Instituto de Investigaci on Sanitaria NK cells monitor altered molecular patterns in tumors and infected cells through an Puerta de Hierro - Segovia de Arana, ample array of receptors. Two families of evolutionarily distant receptors have con- Madrid, Spain 2 verged to enable human NK cells to sense levels of HLA class I ligands, frequently Medicine and Life Sciences, University Pompeu Fabra, Barcelona, Spain abnormal in altered cells. Whilst different forms of polymorphism are a hallmark of 3 Immunology, Department of Pathology, killer-cell immunoglobulin-like receptors and their classic HLA-A, B, and C ligands, Hospital del Mar Medical Research genetic diversity of killer-cell lectin-like receptors for the non-classical HLA-E Institute (IMIM), Barcelona, Spain (CD94/NKG2 heterodimers) is less conspicuous and has attracted less attention. A 4 Department of Cell Biology, Physiology and Immunology, Universitat Autonòma common pattern of diversification in both receptor families is evolution of pairs of de Barcelona, Bellaterra, Catalonia, Spain inhibitory and activating homologs for a common ligand, the genes encoding activat- ing receptors being more frequently affected by copy number variation (CNV). This Correspondence Carlos Vilches, Servicio de Inmunología, is exemplified by the gene encoding the activating NKG2C subunit (KLRC2 or Hospital Universitario Puerta de Hierro, NKG2C), which marks an NK-cell subpopulation that differentiates or expands in Manuel de Falla 1 28222 Majadahonda, response to cytomegalovirus. We have studied NKG2C diversity in 240 South Spain. Email: carlos.vilches@yahoo.com European individuals, using polymerase chain reaction and sequencing methods to assess both gene CNV and single-nucleotide polymorphisms (SNPs) affecting its pro- Funding information moter, coding and 30 -untranslated (30 UT) regions. Sequence analysis revealed eight AEI/FEDER, Grant/Award Number: PID2019-110609RBC22/ common SNPs—one in the promoter, two in the coding sequence, and five in the AEI/10.13039/501100011033; Consejería 30 UT region. These SNPs associate strongly with each other, forming three conserved de Educacion, Juventud y Deporte, Comunidad de Madrid, Grant/Award extended haplotypes (frequencies: 0.456, 0.221, and 0.117). Homo- and heterozygous Number: SAF2016-80363-C2- 2-R; EU combination of these, together with complete gene deletion (0.175) and additional Youth Employment Initiative, European haplotypes with frequencies lower than 0.015, generate a diversity of NKG2C geno- Social Fund, Grant/Award Number: PEJ-2017-AI/BMD-7377; Fundaci on types of potential immunological importance. Científica Asociaci on Española Contra el Cancer, Grant/Award Number: KEYWORDS GCB15152947MELE; Consejería de alleles, genetic polymorphism, HLA-E, human genetics, natural killer cell lectin-like Educacion, Juventud y Deporte de la receptors, NKG2C receptor Comunidad de Madrid; European Social Fund Judit Asenjo and Manuela Moraru shared first authorship. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2022 The Authors. HLA: Immune Response Genetics published by John Wiley & Sons Ltd. HLA. 2022;100:469–478. wileyonlinelibrary.com/journal/tan 469 470 ASENJO ET AL. 1 | INTRODUCTION chromosome 12 by the KLRC2 gene (more commonly referred to as NKG2Ci),19,20 is characterized by a com- Natural killer cells monitor altered molecular patterns in plete gene deletion that affects in homo- or heterozygosis infected and tumor cells, including downregulation of ca. one third of individuals in different world MHC class I expression. The latter function is achieved populations.21–24 Furthermore, a few single nucleotide by means of inhibitory receptors, belonging to different polymorphisms (SNPs) are seen in its coding and non- protein superfamilies that have converged in using com- coding regions (Figure 1), but they have rarely been mon signaling motifs and pathways. Among them, investigated.19,21,25–27 To better characterize NKG2C and human killer-cell immunoglobulin-like receptors (KIR) its role in immunity, we have studied the distribution of and mouse lectin-like Ly49 molecules have indepen- its gene polymorphisms in healthy donors of South dently evolved as families of polymorphic receptors, each European origin. family member recognizing subsets of MHC class I allotypes.1–3 In contrast, CD94/NKG2 heterodimers rec- ognize in each species a non-classical MHC molecule of 2 | MATERIALS AND METHODS low polymorphism presenting the leader peptides encoded in other MHC class I loci.4–7 In humans, their 2.1 | Subjects and samples HLA-E ligand can also present a similar peptide encoded by cytomegalovirus (CMV) protein UL40.8–10 All these We studied a cohort of 240 unrelated healthy controls families of inhibitory receptors have evolved homologs recruited in our centers in Madrid and Barcelona, and a with NK-cell activating capacity. One of them, mouse collection of widely available cell lines, mainly ones Ly49H, enables NK cells to recognize the murine CMV- established in International Histocompatibility Work- encoded decoy MHC molecule m157, Ly49H genetic vari- shops. After informed consent, genomic DNA was iso- ation explaining resistance or susceptibility to the lated with standard methods from peripheral blood, virus.11,12 No human NK-cell receptor has been demon- mononuclear cell (PBMC) suspensions obtained by den- strated to play such a critical role in virus recognition or sity gradient centrifugation, or cultured cell lines. Total resistance. However, activating CD94/NKG2C heterodi- cytoplasmic RNA was isolated from PBMCs with the mers mark a human NK-cell subpopulation that differen- RNeasy Plus kit (Qiagen GmbH, D-40724), and cDNA tiates or expands in response to human CMV10,13,14; and was synthesized using a first-strand synthesis kit KIR2DS1 has been proposed to contribute to recognition (GE Healthcare-Fisher Scientific). of CMV-infected cells through ill-defined mechanisms.15 KIR, like their murine Ly49 analogues,16 exhibit extreme gene-copy number variation and allelic polymor- 2.2 | Sequence analysis phism, only inferior to that of their MHC ligands.3,17,18 In contrast, members of the CD94/NKG2 family are gener- The complete coding sequence (CDS) of NKG2C, and ally considered minimally polymorphic. However, parts of its flanking 50 and 30 sequences were amplified in human NKG2C, encoded in the NK complex on a single 5832-bp fragment from 28 genomic DNA samples Kbp 1 2 3 4 5 Exons: 1 2 3 4 5 6 c.-208 c.5G>A c.305C>T c.*20A>G T>G (Ser2Asn) (Ser102Phe) c.*105A>G F I G U R E 1 Schemes representing c.*285G>A c.*401A>G the NKG2C gene, its single-nucleotide c.*419T>C Cytoplasmic Transmembrane Stem Ligand-binding domain polymorphisms studied herein, and the tail region CD94/NKG2C heterodimer coupled to the signaling molecule DAP12 NKG2C NKG2C*01 NKG2C*02 NKG2C*03 (immunoreceptor tyrosine-based CD94 Ser102Phe Ser102 Phe102 Ser102 activation motifs marked in red). Polymorphic amino acids defining alleles NKG2C*01, *02 and *03 are Ser2Asn Ser2 Asn2 Asn2 represented on the NKG2C molecule. DAP12 Exons and introns are depicted approximately to scale. ASENJO ET AL. 471 using long-range polymerase chain reaction (PCR) with PCR). PCR conditions for both reactions were 95 C for primers KLRC2F–383 and KLRR+485b (promoter and 2 min; 30 cycles at 95 C for 10 s, 55 C for 30 s, 72 C for 30 -untranslated region [UTR], respectively, Table S1, and 1 min, and 72 C for 1 min. The final PCR product, of Figure S1). Approximately 100 ng of DNA were amplified 706 bp, spanned most of the CDS (exons 1–6). with Advantage 2 polymerase mix (BD-Clontech, Palo Sanger sequencing was performed using BigDye cycle Alto) in 20 μl of buffer containing 1 μM of each primer, sequencing kits, after eliminating excess PCR primers under the following conditions: initial denaturation at and nucleotides with ExoSAP-IT (Thermo Fisher Scien- 96 C for 2 min; 10 cycles of 20 s at 95 C, 30 s of primer tific). Sequencing reactions were purified with BigDye- annealing at 66 C and 7 min extension at 72 C; then XTerminator (Thermo Fisher Scientific) and analyzed in 30 cycles of 95 C for 20s, 60 C for 30 s 72 C for 7 min; an ABI Prism 3100-Avant Genetic analyzer (Applied and a final extension at 72 C for 2 min. The resulting Biosystems) in the central facility of IDIPHISA. amplicons were separated by electrophoresis in 2% aga- rose gels, visualized after ethidium bromide staining, and sequenced with the Sanger method and internal primers 2.3 | PCR genotyping of NKG2C (Table S1) that covered all six exons. Complete coding polymorphisms sequences were obtained from 19 donors, and partial ones spanning most exons, from an additional nine NKG2C copy-number variation was determined as in donors. Moraru et al.28 To assess the two common SNPs in the Additionally, we sequenced the polymorphic first and CDS (c.5G > A and c.305C > T), we developed a PCR- third exons of NKG2C in 75 donors, after amplifying SSP (sequence-specific primers) assay comprising four them in a single 2871-bp segment with the same forward reactions – two for detecting exon 1 polymorphisms primer and reverse KLRC2Ri4 + 197 (intron 4, Table S1, (c.5G > A) and another two for exon 3 (c.305C > T) and Figure S1). PCRs were performed in 20 μl of Advan- (Tables 1 and 1S). Each reaction contained 100 ng of tage 2 polymerase mix (BD-Clontech, Palo Alto, CA, genomic DNA and 1 U of BioTaq polymerase in 15 μl of USA) containing 125 μM deoxyribonucleotide triphos- buffer including primers mixes for each SNP and an phates, 100 ng of DNA and 1 μM of each primer, in the internal positive control (IPC) band (Table 1). PCR con- following conditions: 2 min at 96 C; 10 cycles of 20 s at ditions were 2 min at 96 C, 30 cycles of 20 s at 95 C, 95 C, 30 s at 64 C and 4 min at 72 C; 30 cycles of 20 s at 20 s at 61 C and 1 min at 72 C, then 4 min at 72 C, in 95 C, 30 s at 62 C and 4 min at 72 C; and 2 min at 72 C. ABI2720 thermocyclers (Applied Biosystems). Exon Amplified products were verified by agarose gel electro- 1 and 3 products were 400-bp and 1123-bp long, phoresis, and exons 1 and 3 were sequenced as above. respectively (Figure 2). Sequences covering 300 bp of the 50 -UT and promoter Another PCR-SSP assay, designed for rapid genotyp- regions upstream of exon 1 were obtained from 85 donors ing of the g.-208 T > G polymorphism, was carried out using the two aforementioned genomic fragments. combining, in two separate reactions, an NKG2C-pro- For 30 UTR analysis, an additional 587-bp fragment moter-specific forward primer, KLRC2F-358, and either was amplified in all 240 donors with KLRFg669 (exon 6) of two reverse allele-specific primers, KLRRt-208 and and KLRR+523 (30 UTR, Table S1, and Figure S1) in 20 μl KLRRg-208b (Tables 1 and S1). Reaction mixes contained of buffer (67 mM Tris–HCl, pH 8.8, 16 mM [NH4]2SO4, 100 ng genomic DNA and 1 U of BioTaq polymerase in 2 mM MgCl2, 0.01% Tween-20, 125 μM deoxyribonucleo- 15 μl of PCR buffer. Amplification conditions were 2 min tide triphosphates) containing 0.6 U of BioTaq polymer- at 96 C; 10 cycles of 20 s at 95 C, 20 s at 65 C and 30s at ase (Bioline), 0.5 μM of each primer and 30–300 ng of 72 C; 25 cycles of 20 s at 95 C, 20 s at 62 C and 30s at DNA. PCR conditions were 2 min at 96 C; 30 cycles of 72 C; then 4 min at 72 C. The method was validated on 10 s at 95 C, 20 s at 60 C and 20 s at 72 C; and 2 min at 42 sequenced individuals. 72 C. Amplified products were verified by electrophoresis in 2% agarose gel and sequenced with primer KLRFg669. Complementary DNA (cDNA) of NKG2C was ampli- 3 | RESULTS fied with a semi-nested PCR. The first PCR contained primers targeting the 50 - and 30 UTR (KLRC2F-26 and 3.1 | Sequence analysis of the NKG2C KLRC2R + 16, respectively, Table S1). For the second coding region PCR, the reverse primer was substituted by KLRRa544 (exon 6, Table S1). Both PCRs were performed in 20 μl of We explored NKG2C diversity in a South European popu- BioTaq buffer containing 0.5 U of polymerase, 1 μM of lation by first using a panel of 101 healthy unrelated each primer and 1 μl of cDNA (or product from the first donors to identify prevalent polymorphisms along with, 472 ASENJO ET AL. T A B L E 1 Primers and conditions for polymerase chain reaction-SSP of single-nucleotide polymorphisms in the NKG2C coding and promoter regions. Specific IPC Coding sequence primers primers Size Size Reaction Specificity Forward Reverse μM (bp) Forward Reverse μM (bp) 1 c.5G KLRC2F-274b KLRRg5 0.67 405 FDRA360 RDRA633 0.10 607 2 c.5A KLRC2F-274b KLRRa5b 0.67 403 FDRA360 RDRA633 0.10 607 3 c.305C KLRC2Fi1-186 KLRRc305 fwd 1.00, 1123 COCHFi8-86 COCHR11 + 95 0.33 1987 rev 0.33 4 c.305T KLRC2Fi1-186 KLRRt305 1.00 1123 COCHFi8-86 COCHR11 + 95 0.33 1987 Promoter region Specific primers IPC primers Reaction Specificity Forward Reverse μM Size (bp) Forward Reverse μM Size (bp) 1 c.-208 T KLRC2F-358 KLRRt-208 0.67 200 FDRA360 RDRA633 0.13 607 2 c.-208G KLRC2F-358 KLRRg-208b 0.67 195 FDRA360 RDRA633 0.13 607 Note: Primer sequences in Table S1. Abbreviations: Fwd, forward; IPC, internal positive control; rev, reverse. Reaction Genotype F I G U R E 2 Polymerase chain reaction-SSP genotyping of NKG2C 1 2 3 4 coding polymorphisms. Three DNA samples with different known genotypes were assayed with the four SSP reactions 2000- – IPC (~2.0 Kbp) DNA 1 for the single-nucleotide polymorphisms 1500- 1000- – c.305 (~1.1 Kbp) c.5G-c.305C of exons 1 (c.5G > A) and 750- 500- – IPC (~0.6 Kbp) NKG2C*01 – c.5 (~0.4 Kbp) 3 (c.305C > T), as indicated. This assay 250- (CNV: homozygous) does not inform of sample homo- or hemizygosity (CNV), which was determined as in Moraru et al.28 Exact 2000- – IPC (~2.0 Kbp) DNA 2 amplicon sizes are given in Table 1. 1500- 1000- – c.305 (~1.1 Kbp) c.5G/A-c.305C/T 750- – IPC (~0.6 Kbp) 500- – c.5 (~0.4 Kbp) NKG2C*01,*02 250- 2000- 1500- – IPC (~2.0 Kbp) DNA 3 1000- – c.305 (~1.1 Kbp) c.5A-c.305T 750- 500- – IPC (~0.6 Kbp) NKG2C*02 250- – c.5 (~0.4 Kbp) (CNV: homozygous) size c.5G c.5A c.305C c.305T marker Specificity possibly, new alleles, and their distribution. This was These analyses of the NKG2C CDS revealed only two accomplished through DNA sequencing of the CDS using already described SNPs: c.5G > A (exon 1, Ser2Asn, source templates including cDNA (12 donors), a genomic rs28403159) and c.305C > T (exon 3, Ser102Phe, DNA segment covering the whole NKG2C gene (28 rs1141715) (Figure 1). As previously reported in other donors), and a partial 2.9-kbp genomic amplicon that populations,21,26 and further analyzed in the following sec- contained its first and third exons, known to be polymor- tion, the two SNPs are tightly linked to each other in Span- phic (75 donors). A subset of the donors was sequenced iards, defining two alleles differing at two amino acids, with more than one method with consistent results. NKG2C*01 (Ser2-Ser102) and NKG2C*02 (Asn2-Phe102), ASENJO ET AL. 473 TABLE 2 NKG2C alleles of a panel of reference cell lines. NKG2C Cell lines alleles BM16, BM21, BOLETH, DEU, E4181324, IBW9, *01,*01 JESTHOM, KAS116, LUY, PAR, PITOUT, RML, SWEIG, NK3.3, NK92, YT, DU145 CB6B, DUCAF, HOM-2, HOR, KT12, LE023, *01,del MZ070782, PLH, RSH, WDV, WT24, HEK- 293T AMALA, BH, COX, DBB, OLGA, PE117, T7527, *01,*02 WT8, YAR, NKL, HeLa SPO010, WT51, HMy-2 C1R *02,del SK-BR3 del,del whose distribution is described below. However, we found that such linkage is incomplete—asparagine 2 is rarely found with serine 102, defining allele NKG2C*03, as we have described elsewhere29; whilst we observed no exam- ples of the reciprocal combination (Ser2-Phe102), which could define a fourth allele. Sequence analysis of cDNA synthesized from PBMCs RNA generally demonstrated no F I G U R E 3 Distribution of NKG2C CDS polymorphisms: allele trace of alternative splicing, except for one donor in whom (A) and genotype (B) frequencies. Colors: blue, allele NKG2C*01; intron 1 retention was detectable in part of the transcripts red, allele NKG2C*02; pink, NKG2C*03; white, gene deletion (Figure S2). whose CNV for NKG2C had been previously determined. 3.2 | A PCR-SSP typing assay to study the Direct sequencing and PCR-SSP analyses of 186 unambig- distribution of the coding dimorphisms uous individuals (hemizygotes and homozygotes for one NKG2C c.5G > A and c.305C > T or both SNPs) confirmed the nearly complete association of c.5G and c.305C (allele NKG2C*01), on one hand, and To facilitate higher-throughput study of NKG2C CDS c.5A and c.305T (allele NKG2C*02), on the other; with polymorphism and its putative association with disease only two c.5A-c.305C exceptions, corresponding to allele and immune responses, we designed a PCR-SSP typing NKG2C*03. method that, in combination with our method for asses- The distribution of coding NKG2C polymorphisms in sing CNV,28 allowed us to detect all possible genotypes of the whole sample, including allelic, carrier and genotypic the two SNPs in exons 1 and 3. The assay is based on four frequencies, is shown in Figure 3 and Table S2. reactions, each of which is specific for one of the alterna- NKG2C*01 was the major allele, carried by 90.4% of the tive bases of the two polymorphic sites (Table 1, individuals; followed by NKG2C*02, found in 25.0% of Figure 2). As is a common quality measure in PCR-SSP, the population. Two individuals, including the reference each reaction includes an internal positive control that donor in whom the allele was originally reported, carried alerts of technical failure when neither a specific nor an NKG2C*03 (c.5A and c.305C). In addition, as in earlier IPC band is present. For optimization and validation of reports of Spaniards and other populations,21–23,30 eight this technique, it was applied to 75 samples previously healthy donors (3.33%) lacked a NKG2C gene on both analyzed by direct sequencing. We also applied it to char- chromosomes. Eight of the nine possible CDS genotypes, acterize the NKG2C c.5G > A and c.305C > T SNPs in a considering CNV, were found in this sample; three geno- panel of worldwide available reference cell lines, which types (NKG2C*01,*01, NKG2C*01,del and NKG2C*01,*02) includes examples of all common genotypes (Table 2) had frequencies ranging 19%–47%, and, together, they and should help local adjustment and validation of the accounted for more than 90% of the population. The fre- method in other centers. quencies of the two observed SNPs are similar to those Finally, the PCR-SSP method was further used to observed previously in two East Asian populations21,25 genotype the NKG2C CDS in our cohort of 240 donors, (Table S2). 474 ASENJO ET AL. (A) 3’UTR haplotypes – IPC (607 bp) c.-208T Haplotype Carrier frequency Haplotype frequency – c.-208 (200 bp) N=240, n (%) N=480 aagat 166 (69.2%) 0.469 – IPC (607 bp) gaagt 91 (37.9%) 0.225 c.-208G – c.-208 (195 bp) ggggc 55 (22.9%) 0.125 ggagc 2 (0.8%) 0.004 Assigned DNA 1 DNA 2 DNA 3 DNA 4 DNA 5 H2O size type: G T T/G T T marker gaggt 1 (0.4%) 0.002 (CNV) (G/G) (T/del) (T/G) (T/T) (T/del) del 76 (31.7%) 0.175 F I G U R E 4 Polymerase chain reaction-SSP genotyping of (B) 3’UTR genotypes NKG2C promoter polymorphism. Five DNA samples with different gaagt,del 2x gaagt genotypes were assayed with two SSP reactions: one (top) for c.- 8.3% 7.1% 208T, and one (bottom) for c.-208 > G. This assay does not inform aagat,ggggc gaagt,ggggc ggggc,del (3.3%) of sample homo-or hemizygosity (CNV), which was determined as 11.3% 6.3% in Moraru et al.28 Others Others 10.0% 10.0% aagat,gaagt N=240 del,del (3.3%) TABLE 3 Frequencies of NKG2C non-coding polymorphism 16.3% 2x ggggc (2.1%) Frequencies aagat,del 2x aagat aagat,ggagc (0.8%) 16.3% 24.6% Major allele Minor allele gaggt,del (0.4%) SNP (carrier %) (carrier %) Promoter region F I G U R E 5 Distribution of NKG2C 30 untranslated region haplotype (A) and genotype (B) frequencies. Colors of common c.-208 T > G (rs76592132) 0.708 (91.2%) 0.117 (21.2%) haplotypes: dark and light blue, aagat and gaagt, respectively (both 0 3 -untranslated region associated with allele NKG2C*01); red, ggggc (associated with allele c.*20A > G (rs2981593) 0.469 (69.2%) 0.356 (55.8%) NKG2C*02); white, gene deletion c.*105A > G (rs3003) 0.696 (91.2%) 0.129 (23.7%) c.*285G > A (rs61917664) 0.596 (81.2%) 0.229 (37.9%) was seen in association with all CDS allotypes: 100% of c.*401A > G (rs2981594) 0.469 (69.2%) 0.356 (55.8%) NKG2C*01 and *03 donors/alleles, and a minority of c.*419 T > C (rs2981595) 0.696 (91.2%) 0.129 (23.7%) NKG2C*02 cases—9 of 60 donors (65 chromosomes) carrying this allele (global carrier frequency of c.-208 Note: Allele frequencies sum 0.825; the remaining 0.175 corresponds to gene deletion, determined as in Moraru et al.28 T-NKG2C*02, 3.75%). 3.3 | Analysis of the NKG2C promoter 3.4 | Analysis of the NKG2C 30 UTR region We also carried out an analysis of the NKG2C 30 UTR. By Considering the known variability in NKG2C surface means of DNA sequencing in all 240 individuals, we expression,14 we extended our genetic study to non- found five SNPs: c.*20A > G, c.*105A > G, c.*285G > A, coding regions that might participate in regulation. c.*401A > G, c.*419 T > C, whose frequencies are given Sequence analysis of 300 bp 50 of the start codon in in Table 3. No examples of the previously reported SNPs 85 donors detected a single SNP, c.-208 T > G. To facili- c.*8-9GC > AA21 were detected. Unambiguous phase tate the analysis of the complete sample, we set up an analysis of 68 hemizygotes showed that, of all potential assay in which each allele is identified by a PCR-SSP combinatorial associations, the five detected 30 UTR poly- reaction (Figure 4). After validation in 42 sequenced morphisms combine in, almost exclusively, three haplo- DNAs, we completed the analysis of c.-208 T > G in the types: in order of frequency, ‘aagat’, ‘gaagt’, and ‘ggggc’ whole cohort. Its frequencies, considering CNV (Figure 5A). As we analyze further in the following sec- (assessed as in Moraru et al.),28 are shown in Table 3. As tion, both ‘aagat’ and ‘gaagt’ associate with NKG2C*01, it will be analyzed in more detail in the last section, the whilst ‘ggggc’ associates with NKG2C*02. Those three minor allele c.-208G was seen exclusively in presence of haplotypes also explain virtually all 30 UTR genotypes c.5A, being found in 51 of 60 donors carrying the seen in donors with a full NKG2C-gene dose. Only two NKG2C*02 allele defined by the latter SNP (56 of variants of these nearly fixed 30 UTR haplotypes were 65 chromosomes). In contrast, the major allele c.-208 T detected: a ‘gaggt’ motif found in one hemizygote ASENJO ET AL. 475 (A) (B) F I G U R E 6 (A) NKG2C extended haplotypes and their frequencies (N = 480 haplotypes). Single-nucleotide polymorphisms and 30 untranslated region (UTR) haplotypes commonly associated with allele NKG2C*01 are depicted in blue, whereas those typically seen with NKG2C*02 are marked in pink; variant nucleotides in 30 UTR haplotypes are shaded in gray. (B) Frequencies of extended NKG2C genotypes (promoter-CDS-30 UTR). Haplotypes are ordered according to their frequencies. For the sake of visual clarity, bar colors are not based on those of (A), and they depend only on the first haplotype. Numbers are provided in Table S3 (the variant nucleotide is highlighted), and a ‘ggagc’ 143 individuals—68 lacking NKG2C on one chromosome, variant seen in two NKG2C*01,*02 heterozygotes (geno- and 75 being homozygous at all eight sites. An additional type: ‘aagat,ggagc’). The frequencies of all five 30 UTR 48 donors were homozygous for two of the three gene seg- haplotypes and their genotypic combinations are repre- ments, being heterozygous only at the 30 UTR (42 individ- sented in Figure 5. uals) or the CDS (six individuals), which, again, enabled us to phase their allotypes at all three regions. We thus defined unambiguously seven extended haplotypes (1a–d, 3.5 | Analysis of extended NKG2C 2a, 2d and 3 in Figure 6A), whose combinations explained haplotypes the genotypes of all but three individuals of the whole cohort. Of those eight-locus haplotypes, only three (1a, 1b The presented data on the distribution of eight NKG2C and 2a) had frequencies greater than 0.1, and, together polymorphisms (one in the promoter, two in the CDS, and with the null haplotype, had a cumulative frequency of five in the 30 UTR) revealed that, in at least certain gene 0.969. These three dominant haplotypes were formed by: regions, they tend to form conserved haplotypes, like those c.–208 T, allele NKG2C*01 in the CDS and either ‘aagat’ defining alleles NKG2C*01 and *02, and the three common or ‘gaagt’ in the 30 UTR (haplotypes 1a and 1b, respec- 30 UTR haplotypes. We therefore examined whether haplo- tively); and by NKG2C*02 preceded by c.–208G and fol- types extending across the three regions could also be lowed by the third 30 UTR motif, ‘ggggc’ (haplotype 2a). identified. To that end, we took advantage of the fact that As stated, only three individuals had genotypes not phasing ambiguities could be ruled out in at least explained by the seven defined extended haplotypes. 476 ASENJO ET AL. These remaining, unfitting genotypes were accounted for with CDS polymorphisms in conserved haplotypes. Taking by two variants of haplotype 2a – haplotype 2b, which advantage of availability of a high number of hemizygous differed from it only at the promoter (c.–208 T, one subjects, we have managed to establish unambiguously the donor); and haplotype 2c, having both c.–208 T and the composition of nine such extended haplotypes. In essence, ‘ggagc’ variant motif described in the previous the most prevalent allele in the coding region, NKG2C*01, is section (two individuals). The NKC of Europeans thus always seen with the same promoter allele, but combines harbors a minimum of nine NKG2C sequences. Random with either of two conserved 30 UTR haplotypes; whilst the combinations of CNV and those allotypes in the popula- other common CDS allele, NKG2C*02, associates strongly tion could theoretically produce 45 different genotypes, with the alternative promoter allele and the third common 18 of which were actually seen in the studied sample, 30 UTR motif; and all other combinations represent only with frequencies detailed in Figure 6B and Table S3. 3% of haplotypes. The homo- and heterozygous combina- tions of those haplotypes and NKG2C deletion generate a wide diversity of genotypes, the most common of which is 4 | DISCUSSION seen in only 22% of individuals, revealing a noticeable level of population diversity. The fact that these polymorphisms The CD94/NKG2 family of lectin-like NK-cell receptors are shared at detectable frequency by distant populations and their HLA-E ligand appear well conserved by contrast such as South Europeans and East Asians suggests existence with the extreme polymorphism of KIR and their classic of some evolutionary advantage in NKG2C diversity. The HLA-A, B, C ligands. However, both HLA-E and its recep- functional significance of NKG2C sequence polymorphism is tors display some degree of diversity. HLA-E is generally unknown. Analyses of the sequence surrounding c.- represented by two common alleles, E*01:01 and E*01:03, 208 T > G with programs Consite35 and sTRAP36,37ii suggest with different expression levels attributed to one amino that the SNP might modify its binding affinity for YY1 and acid substitution in the peptide-binding domain.31 On the other transcription factors (not shown). Furthermore, pre- receptors side, a common ancestor locus underwent dupli- computed predictions on NKG2C cDNA in the RNA22 tool38 cation and diversification to generate a family of genes (Computational Medicine Center at Thomas Jefferson Uni- that encode the different members of CD94/NKG2 hetero- versity) indicate, with p < 0.01, that nucleotides 260–308 of dimers. As in other receptors, including KIR, one extreme its 30 UTR (including c.*285G > A) could be targets for multi- example of divergent evolution in this family is a single ple microRNAs.iii However, potential significance of these ligand being recognized by pairs of receptors with opposed findings is uncertain, since no models have been proposed function – NK-cell inhibition (CD94/NKG2A) versus for regulation of NKG2C-gene expression. In addition, the activation (CD94/NKG2C). Other family members have non-conservative changes Ser2Asn (intracellular) and less-well characterized functions, and alternative splicing Ser102Phe (extracellular stem) might modulate receptor generates additional isoforms (NKG2A/B and NKG2E/H) expression or function but, unfortunately, predictions are which might confer an additional layer of functional diver- hindered by lack of crystal structures that include the sification.32,33 Finally, several coding and non-coding affected regions of NKG2C (or its NKG2A homolog).39–41 sequence polymorphisms appear to be present in all the Further investigation is therefore warranted to ascertain CD94 and NKG2 genes.19,21,25–27 In sharp contrast with whether and how genetic diversity modifies NKG2C func- KIR, only NKG2C among lectin-like receptors for HLA is tion and, possibly, immune responses and health conditions commonly affected by gene copy-number variation. None- conditioned by the receptor. We expect that the information theless, a common feature of both the KLRC- and the presented herein should facilitate such studies. KIR-gene complex is CNV affecting particularly the genes encoding activating receptors.3,21,34 AUTHOR CONTRIBUTIONS Here we have studied in detail the genotypic diversity of Judit Asenjo and Manuela Moraru designed and per- the NKG2C gene by analyzing the distribution of both CNV formed experiments, analyzed and interpreted data, and and a series of coding and non-coding SNPs. We have veri- wrote the manuscript. Karima Al-Akioui-Sanz performed fied that several previously reported polymorphisms of experiments. Aura Muntasell and Mireia Altadill contrib- uncertain distribution are represented in a South European uted samples. Miguel Lopez-Botet designed the study and population. Furthermore, we have confirmed that coding revised the manuscript. Carlos Vilches designed the polymorphisms define two conserved alleles (NKG2C*01 study, directed research, and wrote the manuscript. and *02), whilst the only detected hybrid of these (allele NKG2C*03) is represented at a low frequency. Our results ACKNOWLEDGMENTS also reveal the distribution of some SNPs in potential regula- This article is dedicated to the memory of Benny tory regions and demonstrate that these tend to be inherited P. Shum. We thank Dr. Elvira Ramil, from the DNA ASENJO ET AL. 477 sequencing core facility of Instituto de Investigacion Sanitaria 7. Carretero M, Cantoni C, Bellon T, et al. The CD94 and NKG2-a Puerta de Hierro – Segovia de Arana, for continued support. C-type lectins covalently assemble to form a natural killer cell This work was funded by grant PID2019-110609RB-C22/ inhibitory receptor for HLA class I molecules. Eur J Immunol. 1997;27(2):563-567. AEI/10.13039/501100011033 (AEI/FEDER, EU). MM and JA 8. Tomasec P, Braud VM, Rickards C, et al. Surface expression were hired by the latter grant and by GCB15152947MELE of HLA-E, an inhibitor of natural killer cells, enhanced by from the Asociacion Española contra el C ancer Foundation. human cytomegalovirus gpUL40. Science. 2000;287(5455): Karima Al-Akioui-Sanz was supported sequentially by grant 1031-1033. PEJ-2017-AI/BMD-7377, with co-financing by EU Youth 9. Ulbrecht M, Martinozzi S, Grzeschik M, et al. Cutting edge: the Employment Initiative, European Social Fund (91.89%), and human cytomegalovirus UL40 gene product contains a ligand Consejería de Educacion, Juventud y Deporte de la Comuni- for HLA-E and prevents NK cell-mediated lysis. J Immunol. 2000;164(10):5019-5022. dad de Madrid; and by grant SAF2016-80363-C2-2-R 10. Hammer Q, Ruckert T, Borst EM, et al. Peptide-specific recog- (AEI/FEDER, EU). nition of human cytomegalovirus strains controls adaptive nat- ural killer cells. Nat Immunol. 2018;19(5):453-463. CONFLICT OF INTEREST 11. Arase H, Mocarski ES, Campbell AE, Hill AB, Lanier LL. The authors declare no conflict of interest. Direct recognition of cytomegalovirus by activating and inhibi- tory NK cell receptors. Science. 2002;296(5571):1323-1326. DATA AVAILABILITY STATEMENT 12. Smith HR, Heusel JW, Mehta IK, et al. Recognition of a virus- The data that support the findings of this study are avail- encoded ligand by a natural killer cell activation receptor. Proc Natl Acad Sci USA. 2002;99(13):8826-8831. able from the corresponding author upon reasonable 13. Guma M, Angulo A, Vilches C, G omez-Lozano N, Malats N, request. Lopez-Botet M. Imprint of human cytomegalovirus infection on the NK ceH receptor repertoire. Blood. 2004;104(12):3664- ORCID 3671. Mireia Altadill https://orcid.org/0000-0002-3118-8654 14. Lopez-Botet M, Muntasell A, Vilches C. The CD94/NKG2C +- Carlos Vilches https://orcid.org/0000-0002-0300-9225 NK-cell subset on the edge of innate and adaptive immunity to human cytomegalovirus infection. 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