and Immunity (2003) 4, 160–167 & 2003 Nature Publishing Group All rights reserved 1466-4879/03 $25.00 www.nature.com/gene Variations of human killer cell lectin-like receptors: common occurrence of NKG2-C deletion in the general population

K Hikami1, N Tsuchiya1, T Yabe2 and K Tokunaga1 1Department of Human Genetics, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; 2Department of Research, Tokyo Metropolitan Red Cross Blood Center, Tokyo, Japan

CD94 and NKG2 are members of the NK cell receptor families, and are encoded in the natural killer complex (NKC) on human 12p12–13, one of the candidate chromosomal regions for rheumatic diseases. To examine a possible association between variations in CD94 and NKG2 genes and genetic susceptibility to rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), we carried out a systematic polymorphism screening of NKG2-A (KLRC1), NKG2-C (KLRC2) and CD94 (KLRD1) genes on a population basis. In NKG2-A, previously considered to be highly conserved, 10 polymorphisms in the noncoding region and introns, as well as one rare variation leading to an amino acid substitution within the transmembrane region, c.238T4A (Cys80Ser), were detected. In NKG2-C, in addition to the previously described two nonsynonymous substitutions, c.5G4A (Ser2Asn) and c.305C4T (Ser102Phe), two polymorphisms were newly detected in the noncoding region. In CD94, only one single nucleotide substitution was identified in the 50 untranslated region. When the patients and healthy individuals were genotyped for these variations, no significant association was observed. However, although statistically not significant, NKG2-A c.238T4A (Cys80Ser) was observed in three patients with RA, but in none of the healthy individuals and the patients with SLE. Unexpectedly, in the process of polymorphism screening, we identified homozygous deletion of NKG2-C in approximately 4.3% of healthy donors; under the assumption of Hardy–Weinberg equilibrium, the allele frequency of NKG2-C deletion was estimated to be 20.7%. These results demonstrated that, although human NKG2-A, -C and CD94 are generally conserved with respect to amino acid sequences, NKG2-A is polymorphic in the noncoding region, and that the number of genes encoded in the human NKC is variable among individuals, as previously shown for the leukocyte receptor complex (LRC), HLA and Fcg receptor (FCGR) regions. Genes and Immunity (2003) 4, 160–167. doi:10.1038/sj.gene.6363940

Keywords: NKG2; CD94; polymorphism; deletion; systemic lupus erythematosus; rheumatoid arthritis

Introduction nonclassical MHC class I molecule, HLA-E, in humans.10–12 NKG2-A/CD94 and NKG2-C, -E/CD94 NK cells are lymphocytes that serve as components of transmit inhibitory and activation signals into NK the early, innate immune system.1 NK cells can kill cells, respectively.13 NKG2 (KLRC) (Human Gene cellular targets such as tumor cells and infected cells, and Nomenclature Database (http://www.gene.ucl.ac.uk/ also secrete that influence subsequent devel- nomenclature) approves the following symbols: KLRC1 opment of adaptive immunity.2 Such functions of NK (NKG2-A), KLRC2 (NKG2-C), KLRC3 (NKG2-E), KLRC4 cells are regulated by the balance of signals via a (NKG2-F), KLRD1 (CD94) and D12S2469E (NKG2-D)) multitude of activating and inhibitory NK cell recep- and CD94 (KLRD1) genes are encoded on human tors.3,4 The imbalance of the signals could lead to chromosome 12p12–13,14–16 which has been shown to unregulated activation of both cytotoxicity and be one of the candidate susceptibility regions of rheu- release by NK cells. Indeed, a role for NK cells has often matic diseases.17,18 been implicated in autoimmunity.5 Human chromosome 12p12–13 encodes many C-type NKG2 and CD94 are members of C-type lectin-like lectin preferentially expressed in NK cells, and NK cell receptors.6,7 NKG2-A, NKG2-C and NKG2-E is called ‘the natural killer complex (NKC)’.15,16,19 In the form heterodimers with CD948,9 and recognize the human NKC, five loci coding for NKG2 molecules NKG2-A (KLRC1), -C (KLRC2), -D (D12S2469E), -E (KLRC3) and NKG2-F (KLRC4) are present. NKG2-A, -C, Correspondence: Dr N Tsuchiya, Department of Human Genetics, -E and NKG2-F share substantial , Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, while NKG2-D is distantly related to the other NKG2 Bunkyo-ku, Tokyo 113-0033, Japan. E-mail: [email protected] family members.20–22 In contrast to killer cell Ig-like This study was supported by Grants-in-Aid for Scientific Research (B), and a Grant-in-Aid for Scientific Research on Priority Areas (C) receptor (KIR) genes, where the number of loci possessed ‘Medical Genome Science’ from the Ministry of Education, Science, by an individual is variable because of the structural Sports and Culture of Japan. polymorphism of the leukocyte receptor complex (LRC) Human NKG2 variations and NKG2-C deletion K Hikami et al 161 on chromosome 19q13, gross structural polymorphism of the NKG2 gene family has not been known, and all individuals are assumed to possess all of the NKG2 genes.23,24 Although recent reports demonstrated poly- morphisms of NKG2 and CD94 genes from the analysis of a relatively small number of healthy individuals,23,25 neither population-based variation screening, nor analy- sis on the association between the polymorphisms and rheumatic diseases, has been reported. This study was aimed at screening for polymorphisms of NKG2-A, -C and CD94 genes, and at testing their association with the susceptibility to rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Unex- pectedly, in the process of polymorphism screening, evidence for a structural polymorphism resulting in NKG2-C deletion was obtained.

Results New variations in NKG2-A, NKG2-C and CD94 genes To detect new variations in NKG2-A, NKG2-C and CD94, we performed a variation screening of the entire coding regions of these genes with PCR-single strand conforma- tion polymorphism (SSCP) using genomic DNA from 10 patients with RA, 10 patients with SLE and 12 healthy individuals (New variations detected in this study have been submitted to the DDBJ/EMBL/GenBank databases and assigned the accession numbers AB073867, AB073868, AB073869, AB073870, AB073871, AB073872, AB073873 and AB073874.) We detected 11 nucleotide sequence variations in NKG2-A (Figure 1a). Only one of Figure 1 Variations detected in NKG2-A, NKG2-C and CD94 genes. them, c.238T4A (the designations of the variations The designations of the variations are based on references (ref. 26; are based on references (ref. 26; http://www.dmd.nl/ http://www.dmd.nl/mutnomen.html. mutnomen.html)), was located in the coding region and resulted in a nonsynonymous substitution, Cys80Ser, but in none of the patients with SLE or healthy within the transmembrane domain of NKG2-A. The individuals. other 10 variations observed were located in introns or in untranslated regions (UTRs). Among them, c.284-67_- Detection of NKG2-C deficiency in the general 62delAAACTT had already been deposited in the JSNP population database (http://snp.ims.u-tokyo.ac.jp/). The other 10 In the process of variation screening of NKG2-C,we variations had not previously been reported. found that some genomic DNA samples could not be In NKG2-C, in addition to the two previously reported amplified by PCR using two sets of primers specific to nonsynonymous substitutions, c.5G4A (Ser2Asn) and two different segments of NKG2-C (see Materials and c.305C4T (Ser102Phe) (GenBank accession number methods). We postulated that the lack of amplification 23,25 AJ001684 and NM_002260, respectively), two new might be caused by homozygous deletion of NKG2-Cin variations, c.287-29T4A and c.704_705GC4AA, were such individuals. observed in the noncoding region (Figure 1b). Next we tried long PCR to amplify approximately Only one substitution, c.-134A4T, was detected in the 21,27 0 24 kb encompassing NKG2-C, by placing the primers 5 UTR of CD94. Variations were not detected in the at the 30 end of NKG2-A and 50 upstream region of NKG2- coding region of CD94 (Figure 1c). E, as shown in Figure 2a. Even though we used high-molecular-weight genomic DNA as templates, the Analysis of association of NKG2-A and CD94 expected 24 kb products were not amplified in any of the variations with susceptibility to RA or SLE samples, because the size of the fragment was too large. We subsequently examined the detected variation sites Instead, PCR product of approximately 8 kb was de- in NKG2-A and CD94 for possible association with RA tected in a proportion of samples (Figure 2b). The 8 kb or SLE. As summarized in Table 1, statistically significant fragment was always observed in samples in which no association was not observed between the detected amplification was obtained in the PCR to amplify NKG2- variations and RA or SLE. Association was not observed C-specific intragenic fragments (Figure 2c). These ob- with any of the clinical subsets of SLE defined by the age servations strongly suggested that the genomic region of of onset (r20), presence or absence of lupus nephritis, approximately 16 kd encompassing NKG2-C was homo- central nervous system lupus, anti-dsDNA antibody and zygously deleted in such samples. anti-Sm antibody (data not shown). However, of interest, To confirm the deletion, we performed Southern blot the nonsynonymous substitution, c.238T4A (Cys80Ser) analysis to detect structural polymorphism in NKG2 in NKG2-A, was detected only in three patients with RA, gene family region. Southern blot was done using three

Genes and Immunity Human NKG2 variations and NKG2-C deletion K Hikami et al 162 Table 1 Genotype frequencies of detected variations in NKG2-A and CD94

RA SLE Healthy individuals

NKG2A (n=201) (n=123) (n=215) c.-169G>A (50UTR) G/G 135 (67.2) 92 (74.8) 152 (70.7) G/A 58 (28.9) 28 (23.4) 59 (27.4) A/A 8 (4.0) 3 (2.4) 4 (1.9)

c.-31-4 A>G (intron1) A/A 136 (67.7) 91 (74.0) 152 (70.7) A/G 57 (28.4) 29 (24.2) 59 (27.4) G/G 8 (4.0) 3 (2.6) 4 (1.9)

c.238T>A (Cys 80 Ser) (exon3) T/T 198 (98.5) 123 (100) 215 (100) T/A 3 (1.5) 0 (0) 0 (0) A/A 0 (0) 0 (0) 0 (0)

c.284-67 -62 delAAACTT (intron3) n/n 158 (78.6) 101 (82.1) 178 (82.8) n/del 42 (20.9) 18 (14.6) 36 (16.7) del/del 1 (0.5) 4 (3.3) 1 (0.5)

c.338-90A>G (intron4) A/A 94 (46.8) 66 (53.7) 113 (52.6) A/G 84 (41.8) 47 (38.2) 88 (40.9) G/G 23 (11.4) 10 (8.1) 14 (6.5)

c.1015C>T (30UTR) C/C 137 (68.2) 91 (74.0) 153 (71.2) C/T 55 (27.3) 29 (24.2) 58 (27.0) T/T 9 (4.5) 3 (2.6) 4 (1.9)

c.1077C>T (30UTR) C/C 76 (37.8) 56 (45.5) 98 (45.6) C/T 96 (47.8) 55 (44.7) 86 (40.0) T/T 29 (14.4) 14 (11.4) 31 (14.4)

c.1146 1150 delGATTT (30UTR) n/n 131 (65.2) 82 (66.7) 144 (67.0) n/del 63 (31.3) 34 (27.6) 63 (29.3) del/del 7 (3.5) 7 (5.7) 8 (3.7)

CD94 (n=194) (n=114) (n=210) c.-134A>T A/A 72 (37.1) 52 (45.6) 80 (38.1) A/T 92 (47.4) 52 (45.6) 98 (46.7) T/T 30 (15.5) 10 (8.8) 32 (15.2)

The percentages are shown in parentheses. Statistically significant difference between patients and healthy individuals was not detected in any of the polymorphisms.

different restriction enzymes, EcoRI, BamHI and TaqI, ordinary genomic DNA preparation, only the frequency with the probe for 30 region of NKG2-C. As shown in of homozygous NKG2-C deletion was compared. To our Figure 3a, all enzymes revealed different band patterns surprise, 4.3% of healthy donors were homozygous for between the samples with or without putative NKG2-C the deletion of NKG2-C (Table 2). No significant associa- deletion. The DNA samples with the putative deletion of tion was observed between homozygous deletion and NKG2-C lacked one of the bands in every preparation, tested diseases. indicating the structural difference in the NKG2 gene We also tested possible associations of c.5G4A and family region. The other two bands detected in each c.305C4TinNKG2-C with RA and SLE. To distinguish preparation were considered to derive from cross- homozygotes and hemizygotes (ie variant allele on one hybridization of the probe to NKG2-A and NKG2-E, chromosome and deletion on the other) of the variant caused by substantial sequence homology among these allele, high-quality genomic DNA samples suitable for genes. the amplification of 8 kb in a long PCR shown in Figure To further confirm the deletion of NKG2-C,we 2b were required; however, such samples were not performed RT-PCR analysis. As shown in Figure 3b, available for most of the sample. Therefore, in this study, NKG2-A, -E and CD94 transcripts were detected in all of only allele carrier frequencies were compared between the samples tested. However, NKG2-C transcripts were the patients and controls. A statistically significant not detected in individuals with putative homozygous difference was not observed (Table 2). Association with deletion of NKG2-C. disease subset of SLE was not observed either (data not shown). Lack of association of NKG2-C polymorphisms with RA and SLE Finally, we examined whether NKG2-C deficiency or Discussion nonsynonymous substitutions are associated with sus- ceptibility to RA and SLE. Since heterozygous deletion The most remarkable finding in this study is that was not distinguishable from the common type using the individuals lacking NKG2-C are commonly present in

Genes and Immunity Human NKG2 variations and NKG2-C deletion K Hikami et al 163

Figure 2 Evidence for NKG2-C deletion. (a) Genomic organization of NKG2 genes and primers used for PCR analysis of NKG2-C. Primers 3 and 4 were used to amplify a segment of NKG2-C, and primers 1 and 2 were used to amplify the whole region encompassing NKG2-C.(b) PCR with primers 1 and 2. (c) PCR with primers 3 and 4. Same genomic samples (a, b and c) were used in each lane of (b) and (c). The results suggested that sample a has NKG2-C genes on both , sample b is heterozygous for NKG2-C deletion and sample c is homozygous for NKG2-C deletion. The sequences of the primers are listed in Materials and methods. the general population. Although our current genotyping system cannot detect heterozygous deficiency, under the assumption of Hardy–Weinberg equilibrium, the pre- sence of 4.3% of homozygous deficiency indicates that the frequencies of the deficient allele and heterozygous individuals amount to as much as 20.7 and 32.9%, Figure 3 Southern blot and RT-PCR analysis of NKG2-C. Samples respectively. (a, b and c) are the same as those shown in Figure 2. (a) Southern Our finding contrasts with earlier studies which blotting. Sample c lacks one band in each enzyme digest. (b)RT- suggested that human NKC is relatively conserved, PCR. In each panel, the upper band indicates b-actin (internal whereas NKCs are structurally polymorphic in chim- control) and the arrow indicates the specific band for each gene. No amplification of NKG2-C message was detected in sample c, while panzee, rhesus monkey and mouse: some chimpanzees NKG2-A, -E and CD94 messages were detected. have two NKG2-C genes and others lack NKG2-C or NKG2-D,23,25 the presence of multiple alleles in NKG2-A and NKG2-C was described in rhesus monkey,28 each It has already been known that polymorphisms strain of mice has been shown to have different numbers derived from the presence or absence of some loci at of Ly49 genes,29 and DBA/2J mice have recently been the genomic level are present in gene clusters such as shown to be deficient in CD94.30 The discrepancy HLA, KIR and FCGR.31–35 Like these loci, NKG2 genes are between this study and earlier studies with respect to highly homologous to one another, suggesting that the human NKC diversity may possibly derive from the NKC was evolved through multiple duplication and differences in the sample size and populations. Thus, in unequal crossover events. the previous studies, relatively limited numbers (68 and The functional consequence of NKG2-C deficiency is 11) of donors were examined,23,25 and it is not surprising at this stage unclear. Activating NKG2-C and inhibitory that individuals with homozygous deficiency were not NKG2-A are highly homologous in their extracellular present in the donors. In addition, in the previous studies domains, and both of them recognize HLA-E as a ligand. the donors were mainly Caucasians, and it is possible While the function of NKG2-A is to inhibit the cytotoxic that the frequency of deletion is lower in the Caucasians activity of NK cells, the role of NKG2-C remains unclear. than in the Japanese. It has been suggested that NKG2-C and NKG2-A are

Genes and Immunity Human NKG2 variations and NKG2-C deletion K Hikami et al 164 Table 2 Frequency of NKG2-C genotypes for the deletion of NKG2-C in a proportion of general Japanese population. Our present data indicated RA SLE Healthy that NKG2-A, NKG2-C and CD94 are unlikely to be (n=192) (n=114) individuals susceptibility genes to these diseases, although a role for (n=210) NKG2-A Cys80Ser substitution in a small proportion of RA cannot be excluded. Relatively common occurrence NKG2C del/del 12 (6.3) 7 (6.1) 9 (4.3) of NKG2-C deletion in general Japanese population is a c.5G>A new finding of particular interest. Its significance in G/G or G/del 123 (64.1) 76 (66.7) 136 (64.8) terms of susceptibility to viral and evolution- G/A 37 (19.3) 21 (18.4) 37 (17.6) ary pathway of NKC requires further investigation. A/A or A/del 20 (10.4) 10 (8.8) 28 (13.3)

c.305C>T C/C or C/del 121 (63.0) 76 (66.7) 135 (64.3) Patients and methods C/T 38 (19.8) 22 (19.3) 38 (18.1) Subjects T/T or T/del 21 (10.9) 9 (7.9) 28 (13.3) A total of 201 patients with RA (male 26, female 175, mean age 57.5 7 11.6), 123 with SLE (male 13, female 110, Percentages are shown in parentheses. Statistically significant mean age 40.5 7 13.3) and 215 healthy individuals difference was not observed. (male:female B1 : 1, age 20–60) were studied. RA and SLE were diagnosed according to the American College 43,44 expressed in different subsets of NK cells.29,36,37 In the of Rheumatology criteria. Healthy individuals con- absence of NKG2-A, NKG2-C may be able to transmit sisted of the researchers, laboratory workers and activation signals to kill target cells which lost expression students. All individuals were unrelated Japanese living of HLA-B or -C (thus eliminating the inhibitory signals in Tokyo area, the central part of Japan. This area has through KIR), but not that of HLA-E. been shown to be homogeneous with respect to genetic background, permitting the case–control approach em- Alternatively, it is possible that NKG2-C is in some 45 sense specific. NKG2-C may detect decoy ployed in this study. This study was reviewed and ligand that produce for the purpose of inhibiting approved by the research ethics committee of the NK cells, or may recognize viral antigens in the context Graduate School of Medicine, The University of Tokyo. of HLA-E.38 In the case of mouse, Ly49h, a C-type lectin- like receptor encoded in the mouse NKC, has been Preparation of genomic DNA shown to recognize specifically murine cytomegalovirus Genomic DNA for variation screening and genotyping (MCMV), and Ly49h deficiency causes susceptibility to was purified from the peripheral blood leukocytes using MCMV.39–41 Similarly, rmp1 locus, which is associated a QIAmp blood (Qiagen, Hilden, Germany). High- with susceptibility to ectromelia (mousepox) infec- molecular-weight genomic DNA for Southern blotting tion, is located in the mouse NKC.42 No matter what the was isolated from peripheral blood using a PUREGENE exact role of NKG2-C may be, our data indicating that a DNA Isolation Kit (Gentra systems, Minneapolis, MN, proportion of apparently healthy individuals lack NKG2- USA). C suggest that either NKG2-C is not essential for survival and reproduction, and/or NKG2-C and NKG2-E have Variation screening of NKG2-A, NKG2-C a redundant role in humans. More frequent occurrence and CD94 genes of polymorphisms associated with nonsynonymous Variation screening was performed using PCR-SSCP substitutions in NKG2-C as compared with NKG2-A analysis.46 Genomic DNA from 12 healthy individuals, may also support this idea. Although our present data do 10 RA patients and 10 SLE patients was used for not support association of NKG2-C deletion with RA or screening. Each coding region of NKG2-A, NKG2-C and SLE in humans, association with other conditions, CD94 was amplified using the specific primer sets especially with resistance to certain viral infections, designed within flanking introns or UTRs, based on the needs to be investigated in the future. nucleotide sequences AF023840 for NKG2-A, AJ001684 We also detected a number of new polymorphisms in for NKG2-C, and AB010084, Y14287 and Y14288 for NKG2-A, and one in CD94. Among them, Cys80Ser in the CD94. After the PCR reaction, 1 ml each of the PCR transmembrane region of NKG2-A was found only in product was mixed with 7 ml of denaturing solution (95% three patients with RA, but not in SLE or in healthy formamide, 20 mM EDTA, 0.05% bromophenol blue, individuals. Although a statistically significant differ- 0.05% xylene cyanol FF). The mixtures were denatured ence was not observed, it is possible that this rare at 961C for 5 min and immediately cooled on ice. One variation might be contributing to the susceptibility in microliter of the mixtures was applied to polyacrylamide a small proportion of RA. Of interest, one of the three gel (acrylamide: bis ¼ 49 : 1). Electrophoresis was carried patients with this substitution had a sister who was also out in 0.5 Â TBE under a constant electric current of suffering from RA. Unfortunately, this sister was not 20 mA/gel for 90 min, using a minigel electrophoresis available for the study. Functional consequence of this apparatus with a constant temperature control system substitution within the transmembrane region requires (ATTO, Tokyo, Japan). The electrophoresis was per- further investigation. In general, the paucity of poly- formed in eight conditions with four different buffer morphism in the coding regions of NKG2-A and CD94 temperatures with or without the addition of 5% glycerol agrees with previous studies.23,25 into gels. After electrophoresis, single-strand DNA In conclusion, we detected a number of new poly- fragments in the gel were visualized by silver staining morphisms in NKG2-A and CD94, and also evidence (Daiichi Pure Chemicals, Tokyo, Japan).

Genes and Immunity Human NKG2 variations and NKG2-C deletion K Hikami et al 165 Direct sequencing c.1015C4T was done using the PCR-SSCP method.46 The nucleotide sequences of the samples showing Primers for genotyping are shown in Table 3. PCR different SSCP patterns were determined by direct was carried out using commercially available reagents sequencing. PCR products were purified using the (GeneAmp and AmpliTaq Gold, Perkin-Elmer, cartridge filter Suprec-02 (Takara, Otsu, Japan) and both Norwalk, CT, USA). After preheating for 10 min at sense and antisense strands were directly sequenced 961C, 35 cycles of PCR reaction that consists of using the same primers as those used for the SSCP denaturation for 30 s at 961C, annealing for 30 s at the analysis. Fluorescence-based automated cycle sequen- temperature shown in Table 3, and extension for 30 s at cing of PCR products was performed with an automated 721C were performed using thermal cyclers. Subse- sequencer (Applied Biosystems, Foster City, CA, USA) quently, SSCP electrophoresis was carried out under using a dRhodamine Terminator Cycle Sequencing- the conditions shown in Table 3. For NKG2-A c.1015C4T Ready Reaction Kit (Applied Biosystems), according to genotyping, 7.5% instead of 10% acrylamide gel was the manufacturer’s instructions. used and electrophoresis was carried out for 120 min instead of 90 min. Genotyping For NKG2-A c.1077C4T, c.1146_1150delGATTT and Genotyping of NKG2-A c.-169G4A, c.-31-4A4G, NKG2-C c.5G4A and c.305G4T, nested PCR was c.238T4A, c.284-67_-62delAAACTT, c.388-90A4G and employed to overcome extensive homology between

Table 3 Primers and SSCP conditions of variation screening and genotyping

Variation Sequence (50–30) Size (bp) ATa ETb Glyc

NKG2-A c.-169G>A (50UTR) CTG ATT TCA CAA TAG CCC ATG T 307 58 10 + CAC CCA CTA AAT GAG GTA GGC c.-31-4A>G (intron 1) GTT CTC CAC CTC ACC CTT TT 347 56 10 À GCT GCA CAT CCT AGA CTG TT c.238T>A (exon 3) TGT ATC CAC CTC TCC TTT CG 239 56 7 + CAA TGA GAA CTC TAT TCC CTG

c.284-67 -62delAAACTT (intron 3) GAG ATG GTG AAA TTT GGT TCT 323 52 25 À TTT GTA CAG CCT AAG ATC AAG c.338-90A>G (intron 4) AGC CCA TGA AGA TGT ATA GAT 329 46 20 À ATA TTA TCG ACC GAA AGA AGC

c.1015C>T (30UTR) AGC ATA AGC TTT AGA GGT AAA GCG 369 53 10 + CAG TTA GAG GTC ATC TCC TAT TTC

c.1077C>T (30UTR) First PCR AGC ATA AGC TTT AGA GGT AAA GCG 651 60 CAC AGT ACA TTG AAG GAA ACA CT Second PCR TAG AAT AGT GGT TGC CAA TGT CTG 142 58 15 À GAT GTT TAG TAT ATT CGC AGA GTT A

c.1146 1150delGATTT (30UTR) First PCR AGC ATA AGC TTT AGA GGT AAA GCG 651 54 CAC AGT ACA TTG AAG GAA ACA CT Second PCR TAG AAT AGT GGT TGC CAA TGT CTG 339 54 SSLP CAC AGT ACA TTG AAG GAA ACA CT NKG2-C c.5G>A (exon 1) First PCR GAG CAC AGT CCC TCA CAT CA 2914 68 AAC AGT GCA CCG TAA ACC AC Second PCR GAG CAC AGT CCC TCA CAT CA 276 68 7 À TGC ATT CAA CTG CAC ATC CT

c.305C>T (exon 3) First PCR GAG CAC AGT CCC TCA CAT CA 2914 68 AAC AGT GCA CCG TAA ACC AC Second PCR GAA ACT AAA CTC GTT ATG GTT CAT C 210 54 FRET TTT TCT GCA AAA ATG CCA CT

CD94 c.-134A>T (50UTR) Forward (-134A) ATC ATT TAA ATA CAC AAT TTT TCA TTC TCT A (-134C) ATC ATT TAA ATA CAC AAT TTT TCA TTC TCT C Reverse CCA AAA TCA GCC AAT CCA AG 240 60 SSP

AT: annealing temperature of PCR; ET: buffer temperature of SSCP electrophoresis; SSLP: simple sequence length polymorphism; FRET: fluorescence resonance electron transfer; SSP: sequence-specific primer; Gly (À) and Gly (+): the gel condition of SSCP; gels without glycerol and with 5% glycerol were used respectively.

Genes and Immunity Human NKG2 variations and NKG2-C deletion K Hikami et al 166 NKG2-A, NKG2-C and NKG2-E genes. The first PCR Pharmacia Biotech, Buckinghamshire, UK). The probe was carried out using a Takara LA Taq kit (Takara). for the NKG2-C was made by PCR using the primers The PCR conditions were initial denaturation at 50-AAAGAGGACCCTAGGTGGTTTAC-30 and 50- 961C for 10 min, followed by 35 (for NKG2-A) CTGATGCACTGTAAACGCAAAT-30 (2053 bp), and or 20 (for NKG2-C) cycles of denaturation at 961C was labeled using DIG-High Prime (Roche Diagnostics, for 30 s, annealing at the temperature shown in Table 3 Basel, Switzerland). This probe hybridizes with NKG2-C for 30 s, and extension at 721C for 1 (for NKG2-A) or 3 (for intron 4 through 30 UTR in exon 6. Hybridization and NKG2-C) min. Subsequently, NKG2-A c.1077C4T was detection were performed according to the manufac- genotyped using SSCP, c.1146_1150delGATTT by PCR- turer’s instructions of a DIG Nucleic Acid Detection Kit simple sequence length polymorphism SSLP, while (Roche Diagnostics). genotyping of NKG2-C c.305C4T was performed by PCR-fluorescence resonance energy transfer (FRET) RNA extraction and reverse transcription (RT)-PCR method using LightCycler (Roche Diagnostics, Man- Total cellular RNA was prepared from peripheral blood nheim, Germany), according to the manufacturer’s leukocytes using RNeasy Total RNA System (Qiagen). instructions. For the latter, first PCR products were First-strand cDNA was synthesized from extracted RNA used as templates, and second PCR amplification using oligo(dT) primer (Promega, Madison, WI, USA) was performed using the reaction mixture containing and MMLV reverse transcriptase (Toyobo, Osaka, Japan) 0.4 mM MgCl2,10mM primers and 4 mM of hybridization at 421C for 45 min. RT-PCR was performed as previously 0 probes (5 -LCRed-ATTCGGGGAAGAATTGTT-phos- described,23 except for NKG2-C. RT-PCR for NKG2-C was phate-30 and 50-GTACTAATATCAGAACATTGAAAA- done using the primers 50-TTTCTGGCCAGCATTT- 0 TAAAAATGTACCTTTCTGCGTTCTTG- fluorescein-3 ) TACCT-30 and 50-CTGATGCACTGTAAACGCAAAT-30, for 50 cycles of denaturation for 5 s at 951C, anneal- because of the difficulty in amplification of NKG2-C ing for 10 s at 541C, and extension for 15 s at 721C. using the primer set described in Ref. 23. Genotypes were determined from the melting curve analysis. Statistical analysis CD94 c. -134A4T was genotyped by PCR-sequence- The genotype frequency of the detected variations and specific primer (SSP) (Table 3). NKG2-A c.591-86C4A, the frequency of homozygous deletion of NKG2-C were c.591-22T4A and c.591-9C4T, and NKG2-C c.287- compared between patients and healthy controls using w2 29T4A and c.704_705GC4AA were not examined test or Fisher’s exact test. Fisher’s exact test was used because of the difficulty in genotyping, and also because 0 when one or more variables within 2 Â 2 tables were less such polymorphisms within introns and 3 UTR were than five. P-values o0.05 were regarded as statistically considered less likely to be associated with functional significant. significance.

Detection of NKG2-C deletion To detect NKG2-C deletion, PCR amplification was Acknowledgements performed with two sets of intragenic primers: a previously described set placed within exon 5 and exon The authors are indebted to Dr Hirohiko Hohjoh 6(50-TTTCTGGCCAGCATTTTACCT-30 and 50-CTGATG- (Department of Human Genetics, The University of CACTGTAAACGCAAAT-30, primers 3 and 4 in Figure Tokyo) for valuable suggestions, and to Tomoko Suzuki 2), which amplifies 1.1 kb fragment,23 and the primers for help in the manuscript preparation. used for the first PCR for c.5G4A genotyping listed in Table 3. In addition, a long PCR was also designed to amplify a References segment of approximately 24 kb encompassing NKG2-C locus. The primers were placed within exon 7 of NKG2-A 1 Trinchieri G. Biology of natural killer cells. Adv Immunol 1989; and the promoter region of NKG2-E (50-GCATTTG- 47: 187–376. CAGTGCATCAGATAAATTG-30 and 50-GGTTCCTTCT- 2 Biron CA, Nguyen KB, Pien GC, Cousens LP, Salazar-Mather CTAATCATGACTTACTGTCTG-30, primers 1 and 2 in TP. Natural killer cells in antiviral defense: function and Figure 2). The PCR was performed using a Takara LA Taq regulation by innate cytokines. Annu Rev Immunol 1999; 17: kit (Takara). The PCR condition consisted of initial 189–220. 1 3 Lanier LL. NK cell receptors. Annu Rev Immunol 1998; 16: denaturation at 96 C for 10 min, followed by 35 cycles 359–393. 1 1 of denaturation at 96 C for 30 s, annealing at 68 C for 4 Raulet DH, Vance RE, McMahon CW. Regulation of the 8 min 30 s and extension at 721C for 10 min. receptor repertoire. Annu Rev Immunol 2001; 19: 291–330. Southern blotting 5 Seaman WE. Natural killer cells and natural killer T cells. Southern blots were prepared by alkaline transfer of Arthritis Rheum 2000; 43: 1204–1217. DNAs. A total of 10 mg each of high-molecular-weight 6 Chang C, Rodriguez A, Carretero M, Lopez-Botet M, Phillips DNA was digested with EcoRI, BamHI or TaqI. Digestion JH, Lanier LL. Molecular characterization of human CD94: a was performed in 100 ml reaction mixtures containing type II membrane glycoprotein related to the C-type lectin superfamily. Eur J Immunol 1995; 25: 2433–2437. 10 mg genomic DNA, 100 U of enzyme in the manufac- 1 1 7 Yabe T, McSherry C, Bach FH et al. A multigene family on turer’s buffer at 37 C for EcoRI and BamHI, or at 65 C for human encodes natural killer-cell lectins. TaqI, overnight. Digests were separated by electrophor- Immunogenetics 1993; 37: 455–460. esis on 0.8% agarose gels at 25 V for 15 h and blotted 8 Lazetic S, Chang C, Houchins P, Lanier LL, Phillips JH. overnight onto Hybond-N+ membranes (Amersham Human natural killer cell receptors involved in MHC class I

Genes and Immunity Human NKG2 variations and NKG2-C deletion K Hikami et al 167 recognition are disulfide-linked heterodimers of CD94 and 29 Takei F, McQueen KL, Maeda M et al. Ly49 and CD94/NKG2: NKG2 subunits. J Immunol 1996; 157: 4741–4745. developmentally regulated expression and evolution. Immunol 9 Carretero M, Cantoni C, Bellon T et al. The CD94 and NKG2-A Rev 2001; 181: 90–103. C-type lectins covalently assemble to form a natural killer cell 30 Vance RE, Jamieson AM, Cado D, Raulet DH. Implications inhibitory receptor for HLA class I molecules. Eur J Immunol of CD94 deficiency and monoallelic NKG2A expression for 1997; 27: 563–567. natural killer cell development and repertoire formation. Proc 10 Borrego F, Ulbrecht M, Weiss EH, Coligan JE, Brooks AG. Natl Acad Sci USA 2002; 99: 868–873. Recognition of human histocompatibility leukocyte antigen 31 Bohme J, Andersson M, Andersson G, Moller E, Peterson PA, (HLA)-E complexed with HLA class I signal sequence-derived Rask L. HLA-DR beta genes vary in number between different peptides by CD94/NKG2 confers protection from natural DR specificities, whereas the number of DQ beta genes is killer cell-mediated lysis. J Exp Med 1998; 187: 813–818. constant. J Immunol 1985; 135: 2149–2155. 11 Lee N, Llano M, Carretero M et al. HLA-E is a major ligand for 32 Tokunaga K, Saueracker G, Kay PH, Christiansen FT, Anand the natural killer inhibitory receptor CD94/NKG2A. Proc Natl R, Dawkins RL. Extensive deletions and insertions in different Acad Sci USA 1998; 95: 5199–5204. MHC supratypes detected by pulsed field gel electrophoresis. 12 Braud VM, Allan DS, O’Callaghan CA et al. HLA-E binds to J Exp Med 1988; 168: 933–940. natural killer cell receptors CD94/NKG2A, B and C. Nature 33 Wagtmann N, Rojo S, Eichler E, Mohrenweiser H, Long EO. A 1998; 391: 795–799. new human gene complex encoding the killer cell inhibitory 13 Houchins JP, Lanier LL, Niemi EC, Phillips JH, Ryan JC. receptors and related monocyte/macrophage receptors. Curr Natural killer cell cytolytic activity is inhibited by NKG2-A Biol 1997; 7: 615–618. and activated by NKG2-C. J Immunol 1997; 158: 3603–3609. 34 Wende H, Colonna M, Ziegler A, Volz A. Organization of the 14 Plougastel B, Jones T, Trowsdale J. Genomic structure, leukocyte receptor cluster (LRC) on human chromosome chromosome location, and alternative splicing of the human 19q13.4. Mamm Genome 1999; 10: 154–160. NKG2A gene. Immunogenetics 1996; 44: 286–291. 35 de Haas M, Kleijer M, van Zwieten R, Roos D, von dem Borne 15 Suto Y, Yabe T, Maenaka K, Tokunaga K, Tadokoro K, Juji T. AE. Neutrophil Fcg RIIIb deficiency, nature, and clinical The human natural killer gene complex (NKC) is located on consequences: a study of 21 individuals from 14 families. Blood chromosome 12p13.1–p13.2. Immunogenetics 1997; 46: 159–162. 199; 86: 2403–2413. 16 Renedo M, Arce I, Rodriguez A et al. The human natural killer 36 Valiante NM, Uhrberg M, Shilling HG et al. Functionally and gene complex is located on chromosome 12p12–p13. Immuno- structurally distinct NK cell receptor repertoires in the genetics 1997; 46: 307–311. peripheral blood of two human donors. Immunity 1997; 7: 17 Corne´lis F, Faure S, Martinez M et al. New susceptibility locus 739–751. for rheumatoid arthritis suggested by a genome-wide linkage 37 Smith HR, Chuang HH, Wang LL, Salcedo M, Heusel JW, study. Proc Natl Acad Sci USA 1998; 95: 10 746–10 750. Yokoyama WM. Nonstochastic coexpression of activation 18 Moser KL, Neas BR, Salmon JE et al. Genome scan of human receptors on murine natural killer cells. J Exp Med 2000; 191: systemic lupus erythematosus: evidence for linkage on 1341–1354. chromosome 1q in African-American pedigrees. Proc Natl 38 Tomasec P, Braud VM, Rickards C et al. Surface expression of Acad Sci USA 1998; 95: 14 869–14 874. HLA-E, an inhibitor of natural killer cells, enhanced by human 19 Plougastel B, Trowsdale J. Sequence analysis of a 62-kb region cytomegalovirus gpUL40. Science 2000; 287: 1031. overlapping the human KLRC cluster of genes. Genomics 1998; 39 Daniels KA, Devora G, Lai WC, O’Donnell CL, Bennett M, 49: 193–199. Welsh RM. Murine cytomegalovirus is regulated by a discrete 20 Renedo M, Arce I, Montgomery K et al. A sequence-ready subset of natural killer cells reactive with monoclonal anti- physical map of the region containing the human natural body to Ly49H. J Exp Med 2001; 194: 29–44. killer gene complex on chromosome 12p12.3–p13.2. Genomics 40 Lee SH, Girard S, Macina D et al. Susceptibility to mouse 2000; 65: 129–136. cytomegalovirus is associated with deletion of an activating 21 Glienke J, Sobanov Y, Brostjan C et al. The genomic natural killer cell receptor of the C-type lectin superfamily. Nat organization of NKG2C, E, F, and D receptor genes in the Genet 2001; 28: 42–45. human natural killer gene complex. Immunogenetics 1998; 48: 41 Brown MG, Dokun AO, Heusel JW et al. Vital involvement of a 163–173. natural killer cell activation receptor in resistance to viral 22 Brostjan C, Sobanov Y, Glienke J et al. The NKG2 natural killer . Science 2001; 292: 934–937. cell receptor family: comparative analysis of promoter 42 Delano ML, Brownstein DG. Innate resistance to lethal sequences. Genes Immun 2000; 1: 504–508. mousepox is genetically linked to the NK gene complex on 23 Uhrberg M, Valiante NM, Shum BP et al. Human diversity in chromosome 6 and correlates with early restriction of virus killer cell inhibitory receptor genes. Immunity 1997; 7: replication by cells with an NK phenotype. J Virol 1995; 69: 753–763. 5875–5877. 24 Trowsdale J. Genetic and functional relationships between 43 Arnett FC, Edworthy SM, Bloch DA et al. The American MHC and NK receptor genes. Immunity 2001; 15: 363–374. Rheumatism Association 1987 revised criteria for the classifica- 25 Shum BP, Flodin LR, Muir DG et al. Conservation and tion of rheumatoid arthritis. Arthritis Rheum 1988; 31: variation in human and common chimpanzee CD94 and 315–324. NKG2 genes. J Immunol 2002; 168: 240–252. 44 Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for 26 Dunnen JT, Antonarakis SE. Mutation nomenclature exten- the classification of systemic lupus erythematosus. Arthritis sions and suggestions to describe complex mutations: a Rheum 1982; 25: 1271–1277. discussion. Hum Mutat 2000; 15: 7–12. 45 Tokunaga K, Imanishi T, Takahashi K, Juji T. On the origin and 27 Sobanov Y, Glienke J, Brostjan C, Lehrach H, Francis F, Hofer dispersal of East Asian populations as viewed from HLA E. Linkage of the NKG2 and CD94 receptor genes to D12S77 in haplotypes. In: Szathmary EJ (ed). Prehistoric Mongoloid the human natural killer gene complex. Immuogenetics 1999; Dispersals. Oxford University Press: Oxford, 1996, pp 187–197. 49: 99–105. 46 Hikami K, Tsuchiya N, Tokunaga K. New variations in 28 LaBonte ML, Hershberger KL, Korber B, Letvin NL. The KIR human OX40 ligand (CD134L) gene. Genes Immun 2000; 1: and CD94/NKG2 families of molecules in the rhesus monkey. 521–522. Immunol Rev 2001; 183: 25–40.

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