Single nucleotide polymorphisms in the coding regions of ... - Nature

Genes and Immunity (2000) 1, 330–337  2000 Macmillan Publishers Ltd All rights reserved 1466-4879/00 $15.00 www.nature.com/gene

Single nucleotide polymorphisms in the coding regions of human CXC-chemokine receptors CXCR1, CXCR2 and CXCR3 H Kato, N Tsuchiya and K Tokunaga Department of Human Genetics, Graduate School of Medicine, University of Tokyo, Japan

Chemokines and their receptors have critical roles in inflammatory and immunological responses, and thus their genetic contribution to various human disorders needs investigation. In this study, systematic variation screening of the entire coding regions of CXCR1 (IL8RA), CXCR2 (IL8RB) and CXCR3 was carried out, using genomic DNA from a large number of Japanese healthy individuals and patients with rheumatic diseases. In addition to the previously reported variations in CXCR1 and in CXCR2, two non-synonymous, two synonymous substitutions and one nonsense mutation of CXCR1, one non-synonymous and two synonymous substitutions of CXCR2, two non-synonymous substitutions of CXCR3 were newly identified. The common single nucleotide polymorphisms (SNPs) at CXCR1 codon 827 and CXCR2 codon 786 were in strong linkage disequilibrium. In addition, familial analysis indicated that human CXCR3 is located on chromosome X. No significant association was observed between the variations and the tested rheumatic diseases. However, CXCR variations identified in this study will provide valuable information for the future studies in medical sciences as well as in human genetics. Genes and Immunity (2000) 1, 330–337. Keywords: CXCR1; CXCR2; CXCR3; cSNP; haplotype; rheumatic diseases

Introduction Chemokine receptors belong to the G-protein coupled receptor (GPCR) family, which have seven transmembrane regions and signal through heterotrimeric G-proteins.1 Among them, CXCR1 (IL8RA) and CXCR2 (IL8RB) were identified as receptors for interleukin-8 (IL-8), are both expressed in neutrophils, and have 77% amino acid identity with each other.2,3 CXCR1 binds only two ELRCXC chemokines: IL-8 and granulocyte chemotactic protein (GCP)-2, while CXCR2 binds other ELR-CXC chemokines as well.1 CXCR1, CXCR2 and IL8RBP, the pseudogene bearing greater similarity to CXCR2, are clustered at chromosome 2q35.4,5 Physical distances among them have not yet been clear. These genes are considered as candidate genes for several human disorders. The mapped susceptibility loci for rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), insulin-dependent diabetes mellitus (IDDM) and juvenile amyotrophic lateral sclerosis include 2q35.6–10 Moreover, structural abnormalities of this chromosomal region have been reported in Waardenburg syndrome, type 1, van der Correspondence: Dr Naoyuki Tsuchiya, Department of Human Genetics, Graduate School of Medicine, University of Tokyo, 7–3-1 Hongo, Bunkyoku, Tokyo, Japan 113–0033. E-mail: tsuchiya-tky얀umin.ac.jp Grant support: This study was supported by the Grant-in-Aid for Scientific Research (B11470505) from the Ministry of Education, Science, Sports and Culture. Sequence Accession numbers: The nucleotide sequence data reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases and have been assigned the Accession number AB032728 – AB032738. Received 18 January 2000; revised and accepted 1 March 2000

Woude syndrome, type 2, and neoplastic diseases such as rhabdomyosarcoma and uterine leiomyomata.5,11,12 On the other hand, CXCR3 was identified as a receptor for T-lymphocyte-specific chemokines. It is strongly expressed in IL-2-activated T lymphocytes, but not in freshly isolated peripheral blood leukocytes or related cell lines.13 However, its intense expression was shown in T cells isolated from synovial fluid from patients with RA, from tissues of chronically inflamed vaginal and colonic mucosa, or from cerebrospinal fluid from patients with multiple sclerosis.14,15 Activated Th1/Tc1 subsets have been shown to express CXCR3 and CCR5.14,16,17 Genetic contribution of chemokine receptor polymorphisms to several human disorders has been reported, such as CCR5-⌬32 allele associated with protective effect from human immunodeficiency virus (HIV)-1 infection.18 In autoimmune diseases, a significant association was shown between CCR5-⌬32 allele and the clinical and immunological characteristics of RA, or between CCR2– 64I allele and the susceptibility to IDDM.19,20 To assess the genetic contribution of other chemokine receptors to human diseases, polymorphisms in these genes need to be screened and registered as the essential information. In our previous study, we hypothesized that genetic variations of chemokine receptors CCR3 and CCR4, preferentially expressed in the Th2 subset of lymphocytes,16,17 might be associated with the susceptibility to rheumatic diseases.21 Since the imbalance of Th1/Th2 cells had been implicated in autoimmune rheumatic diseases, it was possible that genetic variations of CCR3 or CCR4 might cause the imbalance and might influence the susceptibility to the diseases.22 However, although several new variations were detected, no association was observed

cSNPs in human CXCRs H Kato et al

between the susceptibility to rheumatic diseases and any of the variations. On the other hand, as for Th1 subset markers, no attempts have been reported on polymorphisms in CXCR3, while a number of reports are present for CCR5.23 Thus far, several genomic DNA or cDNA sequences of CXCR1 and CXCR2 have been published.2–4,24–28 However, they have a difference at one nucleotide in CXCR1, 827 G/C (counting from ATG initiation codon) coding for amino acid 276 S/T. Similarly, in CXCR2, two sequences have been published possessing 786C or T, leading to synonymous substitution encoding amino acid 262L. In addition, restriction fragment length polymorphism (RFLP) in the region encompassing CXCR2 gene was reported, and a haplotype containing the RFLP and GT dinucleotide repeat number polymorphism in the natural resistance associated macrophage protein-1 gene (NRAMP1) promoter region was demonstrated to be linked with the susceptibility to RA.7,29 However, it has not been shown whether the CXCR2 RFLP site is positioned within or outside of CXCR2 gene. Systematic screening of the entire coding sequences has not been reported for CXCR1, CXCR2 and CXCR3. In this study, we attempted to detect variations in the entire coding regions of human CXCR1, CXCR2, CXCR3, estimated each allele frequency in Japanese, and examined the possible association between their variations and the susceptibility to rheumatic diseases.

CXCR2 in Japanese, since they were present in 97.9% and in 89.3% of healthy population, respectively. Other sequences were regarded as variants. As shown in Table 1, all of them were single nucleotide substitutions. In CXCR1, two non-synonymous and two synonymous substitutions were detected. The non-synonymous substitution, 827 G → C (S276T), was present in 19.8% of healthy population, while the two synonymous substitutions, 741 C → T (V247V) and 915 C → T (Y305Y) were rare variations. In CXCR2, one non-synonymous and two synonymous substitutions were detected. 786 C → T (L262L) was the most frequent variant observed in 52.5% of healthy individuals. In CXCR3, two non-synonymous substitutions were detected. 875 G → A (R292Q) was detected in one patient with SLE and one healthy individual, and 1087 G → A (A363T) was detected in only one patient with scleroderma (SSc). Both patients were female, and were heterozygous for the common allele and the substitution allele. Hence, CXCR3 is considered to be highly conserved, at least in the Japanese population. In addition, in the process of primer design for CXCR3 exon 1, we confirmed that CXCR3 gene has one intron between exon 1 and exon 2, the size of which is approximately 1 kb (data not shown). One polymorphic site was incidentally detected within intron 1 [+235 (G/A)] (The nucleotide sequence data of the 5′ portion of intron 1 have been submitted DDBJ/EMBL/GenBank database; accession numbers: AB032737–AB032738).

Results

Analysis of association between detected variations and rheumatic diseases The detected variation sites were subsequently examined for the possible association with rheumatic diseases using case-control analysis. As summarized in Table 1, no significant association between the positivity of these variations and any of the tested rheumatic diseases was observed. Among the variations, CXCR1-827G/C and CXCR2-786C/T were frequently observed and were considered as single nucleotide polymorphisms within the coding sequence (cSNPs). For these cSNPs, genotype fre-

Identification of new variations of CXCRs Several new nucleotide sequence variations, in addition to the previously reported ones, were detected using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) method. The representative SSCP patterns are shown in Figure 1. From the observed frequency of each sequence, we considered CXCR1-827G (coding for 276S) and CXCR2-786C (262L) as the common standard sequences of CXCR1 and

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Figure 1 SSCP patterns of amplified fragments of CXCR1 (a, b), CXCR2 (c, d), and CXCR3 (e). Each PCR-SSCP condition was shown in Table 5. (a) CXCR1–5: Lane 1, CXCR1-741C/T, 827G/G, Lane 2, 741C/C, 827C/C, Lane 3, 741C/C, 827G/C, Lane 4, 741C/C, 827G/G. (b) CXCR1–6: Lane 1, 4, CXCR1-915C/C, 1003C/C, Lane 2, 915C/C, 1003C/T, Lane 3, 915C/T, 1003C/C. (c) CXCR2–2: Lanes 1, 2, 4, CXCR2238C/C, Lane 3, 238C/T. (d) CXCR2–4: Lane 1, CXCR2-768C/C, 786C/C, Lane 2, 768C/T, 786C/C, Lane 3, 768C/C, 786T/T, Lane 4, 768C/C, 786C/T, Lane 5, 768C/T, 786C/T. (e) CXCR3-exon2–5: Lane 1, CXCR3-875G/A, 1087G/G (heterozygote, female), Lane 2, 875G/G, 1087G/A (heterozygote, female), Lane 3, 875G, 1087G (hemizygote, the father), Lane 4, 875A, 1087G (hemizygote, the son [the proband]), Lane 5, 875G/A, 1087G/G (heterozygote, the mother). These SSCP patterns (Lanes 3–5) indicated that CXCR3 gene was located on chromosome X, not on autosome. 875G/G, 1087G/G (homozygote, female) showed identical pattern to Lane 3. Genes and Immunity

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Table 1 Detected nucleotide substitutions and positivities among the patients with rheumatic diseases and healthy individuals (% in parentheses) Substitutions nucleotidea

Control

RA

SLE

SSc

BD

Sjs

n = 242

n = 146

n = 80

n = 14

n = 12

n = 12

AAa

position

CXCR1 741(C→T) 827(G→C)b 915(C→T) 1003(C→T)

V247V S276T Y305Y R335C

TM6 EC3 TM7 C-tail

CXCR2 238(C→T) 768(C→T) 786(C→T)b

R80C V256V L262L

IC1 TM6 TM6

2 (0.8) 6 (2.5) 127 (52.5)

CXCR3 875(G→A) 1087(G→A)

R292Q A363T

EC3 C-tail

1 (0.4) 0 (0)

0 48 0 2

(0) (19.8) (0) (0.8)

0 30 1 4

(0) (20.5) (0.7) (2.7)

1 (0.7) 5 (3.4) 85 (58.2) 0 (0) 0 (0)

1 16 0 0

(1.3) (20.0) (0) (0)

0 1 0 0

(0) (7.1) (0) (0)

0 2 0 0

(0) (16.7) (0) (0)

0 2 0 0

(0) (16.7) (0) (0)

0 (0) 1 (1.3) 44 (55.0)

1 (7.1) 0 (0) 6 (42.9)

0 (0) 0 (0) 6 (50.0)

1 (8.3) 0 (0) 6 (50.0)

1 (1.3) 0 (0)

0 (0) 1 (7.1)

0 (0) 0 (0)

0 (0) 0 (0)

AA, amino acid; TM, transmembrane region (eg, TM6 stands for the sixth transmembrane region); EC, extracellular loop region; IC, intracellular loop region; C-tail, the intracellular carboxy-terminal tail region; RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; SSc, systemic sclerosis (scleroderma); BD, Behc¸et disease; Sjs, Sjo¨gren’s syndrome. aNumbering starts at the ATG translation initiation. bThese sequence variations were previously reported.2,25

quencies were compared between patients and controls. Based on the similarity of the pathogenesis, patients with SLE and Sjo¨gren’s syndrome (Sjs) were grouped together for this analysis. Although CXCR1-827C/C was not present in RA, significant difference in the genotype frequency was observed neither between RA and controls, nor between SLE + Sjs and controls (Table 2). In addition, association with clinical features or disease severity was not observed (data not shown). CXCR1-1003T (335C) allele was present with slightly higher frequency in the patients with RA (4/146, 2.7%) than that in healthy individuals (2/242, 0.8%). In addition, two of the four patients possessing 1003T allele had affected sib with RA, who also possessed 1003T allele. At this point, it remained possible that 1003T allele is weakly associated with the susceptibility to RA, especially in the patients who have family members affected with RA. Thus, a large number of non-familial patients as well as affected sib-pairs with RA were additionally screened for only CXCR1–6 segment, encompassing 1003C/T site. In a total of 575 unrelated patients with RA, 10 patients possessed 1003T allele Table 2 Genotype frequencies of CXCR1-827 G/C and CXCR2-786 C/T SNPs in patients and healthy individuals (% in parentheses) RA n = 146

SLE + Sjsa n = 91

Controls n = 242

CXCR1-827 G/G G/C C/C

116 (79.5) 30 (20.5) 0 (0)

73 (80.2) 17 (18.7) 1 (1.1)

194 (80.1) 43 (17.8) 5 (2.1)

CXCR2-786 C/C C/T T/T

61 (41.8) 65 (44.5) 20 (13.7)

40 (44.0) 44 (48.4) 7 (7.7)

114 (47.1) 103 (42.6) 25 (10.3)

Significant difference was observed neither between RA and controls, nor between SLE, Sjo¨gren’s syndrome (Sjs) and controls (␹2 analysis from 2 × 3 contingency tables). aPatients with SLE or Sjs. One patient fulfilled classification criteria for both SLE and Sjs. Genes and Immunity

(10/575, 1.7%), which was not significantly different from the positivity among the controls, excluding the possibility of weak association. In affected sibs, no individuals except for the previously mentioned two sib-pairs had 1003T allele. Incidentally, two new variations were detected during the screening of the additional samples: 915 C → A (Y305-stop) and 916 G → A (A306T) both positioned in the seventh transmembrane region (TM7). Both were rare mutations detected in only one sample as a heterozygote. Assignment of human CXCR3 gene on chromosome X During the analysis of CXCR3, one healthy male individual was noted to possess only 875A (292Q) allele, and no common allele (875G) (Table 1). Two conflicting chromosomal localizations have been reported for human CXCR3, 8p12-p11.2 and Xq13.30,31 Because of the low allele frequency of 875A, it was considered highly unlikely that this individual was homozygous for the 875A allele of CXCR3 located on 8p. In order to confirm that CXCR3 is located on X chromosome, the genotypes of his parents were examined. As shown in Figure 1e (Lanes 3–5), his father had only the common allele, and his mother were heterozygous for the common allele and 875A allele. If the proband was homozygous for 875A allele on the autosome, his father should possess 875A allele. Thus, our data indicated that human CXCR3 gene is located on chromosome X. Allele frequencies of each IL8Rs (CXCR1 and CXCR2) in Japanese In this study, we detected two polymorphic sites in CXCR1 and three in CXCR2 in the healthy population (Table 1). Since no subjects possessed two or more CXCR1 variations, three CXCR1 alleles are considered to be present in the healthy individuals. Allele frequencies calculated by direct counting in Japanese are shown in Table 3A. In the case of CXCR2, two polymorphic sites, 768 C/T and 786 C/T, were present in the same PCR fragment. Only three of the four possible combinations

cSNPs in human CXCRs H Kato et al

Table 3 Frequencies of CXCR1 and CXCR2 alleles in 242 Japanese healthy individuals (A)

CXCR1 allele frequencies 827

1003

Allele Frequency

G C G

C C T

0.886 0.110 0.004

(B)

CXCR2 allele frequencies 238

768

786

Allele Frequency

C C C T

C C T C

C T C T

0.674 0.310 0.012 0.004

were observed; 768C–786C, 768C–786T, 768T–786C (Figure 1d). Among all tested samples, five individuals (two controls and three patients) were 238 C/T: heterozygous for R80C substitution. Four of them were 786 T/T, and the other one was 786 C/T. Based on these genotypes, it was deduced that four alleles are present for CXCR2. The estimated allele frequencies in Japanese healthy individuals are shown in Table 3B. Linkage disequilibrium between cSNPs of CXCR1 and CXCR2 Because CXCR1, CXCR2 and the homologous pseudogene (IL8RBP) are clustered at human chromosome 2q35,4,5 it was considered likely that cSNPs of these genes are in linkage disequilibrium. To test this possibility, linkage disequilibrium was examined using the genotypes of the 242 healthy population. Using the data at the most polymorphic CXCR1-827 and CXCR2-786 cSNPs, it was demonstrated that these two cSNPs are in strong linkage disequilibrium (␹2 = 104.6, P ⬍ 0.0001, Table 4A). The Table 4 Frequency of estimated haplotypes formed by CXCR1 codon 827 and CXCR2 codon 786 SNPs HFa

LDb

RLDc

(A) Healthy individuals (n = 242) G C 0.678 G T 0.212 C C 0.008 C T 0.102

0.067 −0.067 −0.067 0.067

0.897 −0.897 −0.897 0.897

(B) RA (n = 146) G G C C

C T C T

0.632 0.265 0.009 0.094

0.057 −0.057 −0.057 0.057

0.869 −0.869 −0.869 0.869

(C) SLE or Sjo¨gren G G C C

syndrome (n = 91) C 0.674 T 0.221 C 0.007 T 0.097

0.064 −0.064 −0.064 0.064

0.901 −0.901 −0.901 0.901

Haplotype CXCR1-827

a

CXCR2-786

HF, haplotype frequency; bLD, linkage disequilibrium parameter; RLD, relative linkage disequilibrium value.

c

estimated haplotype frequencies were not significantly different among controls, patients with RA and patients with SLE or Sjs (Table 4B, 4C).

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Discussion In this study, we carried out a variation screening of the entire coding regions of human CXCR1, CXCR2, CXCR3, and detected several new variations. GPCRs containing chemokine receptors are predominantly intronless within coding regions and often they are clustered in human genome, and therefore, they are suggested to have been duplicated by retroposition and by recombination events.32,33 When compared with previously published sequences of various chemokine receptor family genes, we found that the identical amino acid substitutions are present at the same or similar positions of the different receptors: CXCR1-827G/C (276S/T) and CCR3-827G828C/827C-828G (276S/T); CXCR3-875G/A (292R/Q) and CCR3-824G/A (275R/Q); CXCR1-741C/T (247V/V) and CXCR2-768C/T (256V/V). Considering that these variations are not so common, it seems unlikely that the variations had already existed before the gene family emerged and branched off by duplication events from ancestral gene(s). Therefore, the presence of these variations may indicate a mechanism of convergent evolution from the viewpoint that identical substitutions independently took place at similar positions. Such a hypothesis remains speculative and should be tested by further analyses, such as studies on other populations or even other species, and screening for the variations of other GPCRs. Conflicting observations have been reported concerning the chromosomal localization of human CXCR3 gene. CXCR3 was initially reported to localize at 8p12-p11.2 by fluorescence in situ hybridization (FISH) using the genomic DNA clone GPR9 as the hybridization probe.30 However, it was later reported to be located on chromosome X by PCR analysis using hamster/human hybrid lines,34 and subsequently localized at Xq13 by FISH.31 Our data of SSCP analysis strongly supported the latter, although the possibility that the father (shown in Figure 1e, Lane 3) has a deletion in one autosome (null allele), and that the null allele was transmitted to the proband, cannot be completely excluded. Because human chemokine receptor (HCR) clone was identified at Xq13 as well,35 the localization of CXCR3 at Xq13 suggests the presence of a novel mini-cluster region of chemokine receptor genes at this chromosomal position. Recently, mouse CC chemokine, secondary lymphoid tissue chemokine (SLC), was shown to serve as a functional ligand for mouse CXCR3, though human SLC does not have such a function.34,36 In addition, certain CC chemokines, especially CCR3 ligands such as eotaxin, show higher affinity to CXCR3 than CXC chemokines other than CXCR3 agonists, and act as antagonists in human.37 Thus, based on the chromosomal localization, the presence of an intron between the separated coding regions, and other several unique functions, CXCR3 is considered to hold a unique position among the chemokine receptors. It is interesting to examine the possible association between the susceptibility to the diseases such as asthma and the CXCR3 variations, which might influence Th1/Th2 balance or antagonists’ binding to CXCR3, Genes and Immunity

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because the antagonists serve as the CCR3 agonists to Th2 function. Human IL8R genes (CXCR1, CXCR2, IL8RBP) are clustered at chromosome 2q35, approximately 130 kb telomeric to NRAMP1 gene and 10 苲 30 kb centromeric to the villin1 gene (VIL1), which encodes a Ca2+-regulated actinbinding protein.29,38 The mouse homologue of NRAMP1, Ity/Lsh/Bcg, was found to control susceptibility to the intracellular pathogens Salmonella typhimurium, Leishmania donovani, and Mycobacterium bovis BCG.39 Besides, NRAMP1 was shown to upregulate mRNA of ELR-CXC chemokines.40 Shaw et al7 reported positive association between NRAMP1-IL8RB(CXCR2)-VIL1 haplotype and susceptibility to RA by the identity by descent analysis using affected sib pairs. Subsequently, John et al41 suggested a role for NRAMP1 polymorphism in a subset of patients who do not possess HLA susceptibility alleles. In the present case-control study, no association of IL8Rs variations and RA was observed in Japanese, suggesting that IL8Rs may not be responsible for the reported association with RA. It should be noted that none of the CXCR2 variations detected in this study corresponded to the reported CXCR2 RFLP site,29 possibly because the RFLP site was positioned out of the coding region of CXCR2, or such variation was too rare to be detected in Japanese. Although we focused on rheumatic diseases in this study, it will be interesting to examine the variations for the susceptibility to the intracellular pathogens such as tuberculosis, because of the close relationship between polymorphisms of NRAMP1 and those of IL8Rs. Although the frequency is rare, some of the variations identified in this study seem to be potentially important in terms of receptor functions. For example, CXCR1R335C and CXCR2-R80C may confer potentiality of homo- or heterodimerization through disulfide bonds,42,43 while CXCR3-A363T increases the number of potential phosphorylation sites in the C terminal tail. Functional analysis as well as larger scale association studies might reveal important role for such rare variations in the future. In addition, the linkage disequilibrium between the haplotype of NRAMP1-IL8RB-VIL1 and the nucleotide sequence variations detected in this study needs to be analyzed both in Japanese and in other populations. In conclusion, we detected new variations of CXCR1, CXCR2, and CXCR3 in Japanese, and some of which were considered to form specific haplotypes. Such information will become important in the genomic approaches to the wide spectrum of immunological diseases from autoimmunity to infections, and also, in the analysis of the evolutionary pathway of a family of chemokine receptor genes.

Materials and methods Subjects A total of 242 healthy individuals and 264 patients with rheumatic diseases were examined. The healthy individuals consisted of the researchers, laboratory workers and students at the institutions where this study was carried out. The patient group consisted of 146 patients with RA, 80 patients with SLE, 14 patients with SSc, 12 patients with Behc¸et disease (BD) and 12 patients with Sjo¨gren’s syndrome (Sjs). These diseases were diagnosed according Genes and Immunity

to the generally accepted criteria.44–48 All examined individuals were unrelated Japanese living in Tokyo area, the central part of Japan. The area has been shown to be homogeneous with respect to genetic background,49 permitting the case-control approach employed in this study. Informed consent was obtained from the subjects. Furthermore, for the screening of a certain polymorphic site, additional patients with RA were recruited for the case-control association analysis or familial analysis; in total, the genotypes at the particular site were determined in 575 unrelated RA samples and 20 affected sib-pair families with RA (19 families with two RA sibs, one family with three RA sibs). Genomic DNA isolation Genomic DNA from each individual was purified from peripheral blood leukocytes using a commercially available DNA extraction kit (QIAamp blood kit, Qiagen, Hilden, Germany) following the manufacturer’s protocol. PCR-SSCP analysis The variation screening was performed using PCR-SSCP method. The primers used for PCR and the PCR-SSCP conditions are summarized in Table 5. The specific primer pairs were designed according to the published genomic DNA sequences: CXCR1 (GenBank accession number: L19592),24 CXCR2 (M99412),28 GPR9 (U32674).30 Both CXCR1 and CXCR2 have no intron within their coding regions of 1053 bp and 1083 bp, respectively. GPR9 corresponds to the putative CXCR3 exon 2 and consists of partial CXCR3 coding region [CXCR3 (5–368)] of 1095 bp and the 3′ untranslated region (3′UTR).13 Since the optimal size of PCR product for SSCP was determined to be 200–400 bp in the previous studies,50 specific primer sets were designed to amplify six overlapping segments of the entire coding region of CXCR1. CXCR2 was divided into five fragments and CXCR3 exon 2 into five as well. Because no flanking sequence data were available to design the primer set for exon 1, the sense primer was designed within 5′ untranslated region (5′UTR) based on the published cDNA sequence of CXCR3 (X95876)13 and the antisense primer within intron 1 based on the results of direct sequencing (data not shown). PCR was carried out using commercially available reagents (GeneAmp and AmpliTaq Gold, Perkin-Elmer, Norwalk, CT, USA). After preheating for 10 min at 96°C, 35 cycles of denaturation for 30 s at 96°C, annealing for 30 s at the temperature shown in Table 4, and extension for 30 s at 72°C were performed, using thermal cyclers (GeneAmp PCR system 9700 and 9600, Perkin–Elmer Applied Biosystems, Foster City, CA, USA, and Thermal cycler MP, Takara, Kyoto, Japan). The amplified DNA was analyzed by SSCP method. SSCP analysis was performed essentially according to the method described previously.21 One microliter of solution containing the PCR product was mixed with 7 ␮l of denaturing solution (95% formamide, 20 mm EDTA, 0.05% bromphenol blue, 0.05% xylene cyanol FF). The mixtures were thermally denatured at 96°C for 5 min and immediately cooled on ice. One microliter of the mixtures was applied to 10% polyacrylamide gel (acrylamide : bisacrylamide = 49:1). Electrophoresis was carried out in 0.5 × TBE (45 mm Tris-borate [pH 8.0], 1 mm EDTA) under constant electric current of

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Table 5 Primers used for the detection of variations and PCR-SSCP conditionsa Name

Primer sequence

CXCR1–5 CXCR1-F5 CXCR1-R5 CXCR1–6 CXCR1-F6 CXCR1-R6 CXCR2–2 CXCR2-F2 CXCR2-R2 CXCR2–4 CXCR2-F4 CXCR2-R4 CXCR3-exon2–5 CXCR3-ex2-F5 CXCR3-ex2-R5

Fragment size

Annealing temperature

Electrophoresis temperaturec

positionb

5′-ATGGATTCACCCTGCGTACA-3′ 5′-GTTGAGGCAGCTATGGAGAA-3′

IC3 TM7

239 bp

55°C

20°C

5′-AGCGCCGCAACAACATCGG-3′ 5′-CCTGTCCAGAGCCAGATCAC-3′

EC3 3′UTR

312 bp

58°C

10°C

5′-GGTCATTATCTATGCCCTGG-3′ 5′-TTGACCAAGTAGCGCTTCTG-3′

TM1 IC2

336 bp

55°C

8°C

5′-CAATGTTAGCCCAGCCTGCT-3′ 5′-GAATCTCGGTGGCATCCAGA-3′

EC2 TM7

335 bp

59°C

15°C

5′-TGGACATCCTCATGGACCTG-3′ 5′-GAAGTCAGACTGTGGGCGAA-3′

EC3 3′UTR

319 bp

62°C

15°C

a

Only the primer sequences and experimental conditions for the fragments containing variations are listed. Others are available upon request. bPrimer position: IC, intracellular loop region (eg, IC3 stands for the third intracellular loop region); TM, transmembrane region; EC, extracellular loop region; 3′UTR, 3′ untranslated region. cSSCP conditions: Electrophoresis was carried out in 10% polyacrylamide gels without glycerol at the shown temperature for 90 min duration under constant current 20 mA/gel. These conditions were applicable in only the fragments listed in this table, not always in others.

20 mA/gel, using a minigel electrophoresis apparatus with a constant temperature control system (90 × 80 × 1 mm, AE-6410 and AE-6370; ATTO, Tokyo, Japan). The optimal conditions of electrophoresis were determined by preliminary experiments. To detect variations sensitively, each fragment was analyzed with and without 5% glycerol in the gel. Single-strand DNA fragments in the gel were visualized by silver staining (Daiichi Pure Chemicals, Tokyo, Japan) following the manufacturer’s protocol. Direct sequencing The nucleotide sequences of the samples showing different SSCP patterns were determined by direct sequencing. PCR products were amplified, and both sense and antisense strands were directly sequenced using the same primers as those for the SSCP analysis. Fluorescencebased automated cycle sequencing of PCR products was performed with an automated sequencer (DNA Sequencing System ABI PRISM 377 and ABI PRISM 310, Perkin–Elmer Applied Biosystems), using dye-terminator method according to the manufacturer’s instructions (ABI PRISM dRhodamine Terminator Cycle Sequencing-Ready Reaction Kit). Statistical analysis Allele positivity was defined as the proportion of the individuals possessing the allele. Fisher’s exact probability test was used to analyze the distribution of each variation in the patients and in healthy individuals. Odds ratio (OR) was also calculated to evaluate the strength of associations between rheumatic diseases and variations. P ⬍ 0.05 was considered significant. Haplotype frequencies and linkage disequilibrium parameters were estimated from the typing results using the EH algorithm.51 The linkage disequilibrium (LD) value of a twolocus haplotype is the difference between the estimated haplotype frequency of AiBj, f(AiBj) and the product of the allele frequencies of Ai and Bj, f(Ai)f(Bj). In the case of

a multi-locus haplotype, LD is defined as the difference between the estimated haplotype frequency and the product of each allele frequency. The relative linkage disequilibrium (RLD) value is defined as the ratio of LD to the absolute value of possible maximum (when LD ⭓ 0) or possible minimum (when LD ⬍ 0) of LD.52

Acknowledgements The authors are indebted to Drs Kunio Matsuta (Matsuta Clinic), Yasuoki Moroi, Akira Watanabe (National Ito Spa Hospital), Kenji Ikebe (Nanasato Hospital), Shoji Uchida, Yoshiaki Kuga (Tokyo Metropolitan Bokuto Hospital), Yuichi Nishioka (Yamanashi Central Hospital), Akira Hashimoto, Fumiaki Fukuhara, Sumako Fukuhara (Fukuhara Hospital) for the recruitment of the patients, to Dr Jun Ohashi (Department of Human Genetics, University of Tokyo) for statistical analysis, and to Michiko Shiota (Department of Human Genetics, University of Tokyo) for technical assistance.

References 1 Baggiolini M. Chemokines and leukocyte traffic. Nature 1998; 392: 565–568. 2 Holmes WE, Lee J, Kuang WJ, Rice GC, Wood WI. Structure and functional expression of a human interleukin-8 receptor. Science 1991; 253: 1278–1280. 3 Murphy PM, Tiffany HL. Cloning of complementary DNA encoding a functional human interleukin-8 receptor. Science 1991; 253: 1280–1283. 4 Mollereau C, Muscatelli F, Mattei MG, Vassart G, Parmentier M. The high-affinity interleukin 8 receptor gene (IL8RA) maps to the 2q33-q36 region of the human genome: cloning of a pseudogene (IL8RBP) for the low-affinity receptor. Genomics 1993; 16: 248–251. 5 Morris SW, Nelson N, Valentine MB et al. Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35. Genomics 1992; 14: 685–691. Genes and Immunity

cSNPs in human CXCRs H Kato et al

336

6 Corne´lis F, Faure S, Martinez M et al. New susceptibility locus for rheumatoid arthritis suggested by a genome-wide linkage study. Proc Natl Acad Sci USA 1998; 95: 10746–10750. 7 Shaw MA, Clayton D, Atkinson SE et al. Linkage of rheumatoid arthritis to the candidate gene NRAMP1 on 2q35. J Med Genet 1996; 33: 672–677. 8 Gaffney PM, Kearns GM, Shark KB et al. A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families. Proc Natl Acad Sci USA 1998; 95: 14875–14879. 9 Copeman JB, Cucca F, Hearne CM et al. Linkage disequilibrium mapping of a type 1 diabetes susceptibility gene (IDDM7) to chromosome 2q31-q33. Nat Genet 1995; 9: 80–85. 10 Hadano S, Nichol K, Brinkman RR et al. A yeast artificial chromosome-based physical map of the juvenile amyotrophic lateral sclerosis (ALS2) critical region on human chromosome 2q33-q34. Genomics 1999; 55: 106–112. 11 Mitelman F, Kaneko Y, Trent J. Report of the committee on chromosome changes in neoplasia. Cytogenet Cell Genet 1991; 58: 1053–1079. 12 Ishikiriyama S. Gene for Waardenburg syndrome type I is located at 2q35, not at 2q37.3. Am J Med Genet 1993; 46: 608. 13 Loetscher M, Gerber B, Loetscher P et al. Chemokine receptor specific for IP10 and Mig: structure, function, and expression in activated T-lymphocytes. J Exp Med 1996; 184: 963–969. 14 Qin S, Rottman JB, Myers P et al. The chemokine receptors CXCR3 and CCR5 mark subsets of T cells associated with certain inflammatory reactions. J Clin Invest 1998; 101: 746–754. 15 Sorensen TL, Tani M, Jensen J et al. Expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients. J Clin Invest 1999; 103: 807– 815. 16 Bonecchi R, Bianchi G, Bordignon PP et al. Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med 1998; 187: 129–134. 17 Sallusto F, Lenig D, Mackay CR, Lanzavecchia A. Flexible programs of chemokine receptor expression on human polarized T helper 1 and 2 lymphocytes. J Exp Med 1998; 187: 875–883. 18 Samson M, Libert F, Doranz BJ et al. Resistance to HIV-1 infection in caucasian individuals bearing mutant alleles of the CCR5 chemokine receptor gene. Nature 1996; 382: 722–725. 19 Garred P, Madsen HO, Petersen J et al. CC chemokine receptor 5 polymorphism in rheumatoid arthritis. J Rheumatol 1998; 25: 1462–1465. 20 Szalai C, Csaszar A, Czinner A et al. Chemokine receptor CCR2 and CCR5 polymorphisms in children with insulin-dependent diabetes mellitus. Pediatr Res 1999; 46: 82–84. 21 Kato H, Tsuchiya N, Izumi S et al. New variations of human CC-chemokine receptors CCR3 and CCR4. Genes Immun 1999; 1: 97–104. 22 Charlton B, Lafferty KJ. The Th1/Th2 balance in autoimmunity. Curr Opin Immunol 1995; 7: 793–798. 23 Mummidi S, Ahuja SS, Gonzalez E et al. Genealogy of the CCR5 locus and chemokine system gene variants associated with altered rates of HIV-1 disease progression. Nat Med 1998; 4: 786– 793. 24 Sprenger H, Lloyd AR, Meyer RG, Johnston JA, Kelvin DJ. Genomic structure, characterization, and identification of the promoter of the human IL-8 receptor A gene. J Immunol 1994; 153: 2524–2532. 25 Ahuja SK, Shetty A, Tiffany HL, Murphy PM. Comparison of the genomic organization and promoter function for human interleukin-8 receptor A and B. J Biol Chem 1994; 269: 26381– 26389. 26 Ahuja SK, Ozcelik T, Milatovitch A, Francke U, Murphy PM. Molecular evolution of the human interleukin-8 receptor gene cluster. Nat Genet 1992; 2: 31–36. 27 Lee J, Horuk R, Rice GC, Bennett GL, Camerato T, Wood WI. Characterization of two high affinity human interleukin-8 receptors. J Biol Chem 1992; 267: 16283–16287. 28 Sprenger H, Lloyd AR, Lautens LL, Bonner TI, Kelvin DJ. Struc-

Genes and Immunity

29

30

31

32 33 34

35

36

37

38

39

40

41

42 43

44

45

46

47 48

49

ture, genomic organization, and expression of the human interleukin-8 receptor B gene. J Biol Chem 1994; 269: 11065–11072. White JK, Shaw MA, Barton CH et al. Genetic and physical mapping of 2q35 in the region of the NRAMP and IL8R genes: Identification of a polymorphic repeat in exon2 of NRAMP. Genomics 1994; 24: 295–302. Marchese A, Heiber M, Nguyen T et al. Cloning and chromosomal mapping of three novel genes, GPR9, GPR10, and GPR14, encoding receptors related to interleukin 8, neuropeptide Y, and somatostatin receptors. Genomics 1995; 29: 335–344. Loetscher M, Loetscher P, Brass N, Meese E, Moser B. Lymphocyte-specific chemokine receptor CXCR3: regulation, chemokine binding and gene localization. Eur J Immunol 1998; 28: 3696– 3705. Gentles AJ, Karlin S. Why are human G-protein-coupled receptors predominantly intronless? Trends Genet 1999; 15: 47–49. Brosius J. Many G-protein-coupled receptors are encoded by retrogenes. Trends Genet 1999; 15: 304–305. Soto H, Wang W, Strieter RM et al. The CC chemokine 6Ckine binds the CXC chemokine receptor CXCR3. Proc Natl Acad Sci USA 1998; 95: 8205–8210. Fan P, Kyaw H, Su K et al. Cloning and characterization of a novel human chemokine receptor. Biochem Biophys Res Commun 1998; 243: 264–268. Jenh CH, Cox MA, Kaminski H et al. Species specificity of the CC chemokine 6Ckine signaling through the CXC chemokine receptor CXCR3: human 6Ckine is not a ligand for the human or mouse CXCR3 receptors. J Immunol 1999; 162: 3765–3769. Weng Y, Siciliano SJ, Waldburger KE et al. Binding and functional properties of recombinant and endogenous CXCR3 chemokine receptors. J Biol Chem 1998; 273: 18288–18291. Pringault E, Arpin M, Garcia A, Finidori J, Louvard D. A human villin cDNA clone to investigate the differentiation of intestinal and kidney cells in vivo and in culture. EMBO J 1986; 5: 3119– 3124. Blackwell JM, Barton CH, White JK et al. Genetic regulation of leishmanial and mycobacterial infections: the Lsh/Ity/Bcg gene story continues. Immunol Lett 1994; 43: 99–107. Roach TI, Chatterjee D, Blackwell JM. Induction of earlyresponse genes KC and JE by mycobacterial lipoarabinomannans: regulation of KC expression in murine macrophages by Lsh/Ity/Bcg (candidate Nramp). Infect Immun 1994; 62: 1176– 1184. John S, Marlow A, Hajeer A, Ollier W, Silman A, Worthington J. Linkage and association studies of the natural resistance associated macrophage protein 1 (NRAMP1) locus in rheumatoid arthritis. J Rheumatol 1997; 24: 452–457. Bockaert J, Pin JP. Molecular tinkering of G protein-coupled receptors: an evolutionary success. EMBO J 1999; 18: 1723–1729. Mellado M, Rodriguez-Frade JM, Vila-Coro AJ, de Ana AM, Martinez AC. Chemokine control of HIV-1 infection. Nature 1999; 400: 723–724. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 1988; 31: 315–324. Tan EM, Cohen AS, Fries JF et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982; 25: 1271–1277. Subcommittee for scleroderma criteria of the American Rheumatism Association Diagnostic and Therapeutic Criteria Committee: Preliminary criteria for the classification of systemic sclerosis (scleroderma). Arthritis Rheum 1980; 23: 581–590. International Study Group for Behc¸et’s Disease. Criteria for diagnosis of Behc¸et’s disease. Lancet 1990; 335: 1078–1080. Vitali C, Bombardieri S, Moutsopoulos HM et al. Preliminary criteria for the classification of Sjo¨gren’s syndrome. Results of a prospective concerted action supported by the European Community. Arthritis Rheum 1993; 36: 340–347. Tokunaga K, Imanishi T, Takahashi K, Juji T. On the origin and dispersal of East Asian populations as viewed from HLA haplo-

cSNPs in human CXCRs H Kato et al

types. In: Akazawa T, Szathmary EJ (ed). Prehistoric Mongoloid Dispersals. Oxford University Press: Oxford, 1996, pp 187–197. 50 Bannai M, Tokunaga K, Lin L et al. Discrimination of human HLA-DRB1 alleles by PCR-SSCP (single-strand conformation polymorphism) method. Eur J Immunogenet 1994; 21: 1–9.

51 Terwilliger JD, Ott J. Handbook of human linkage analysis. Johns Hopkins University Press: Baltimore, 1999, pp 188–193. 52 Lewontin RC. The interaction of selection and linkage. I. General considerations, heterotic models. Genetics 1964; 49: 49–67.

337

Genes and Immunity