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 Woude syndrome, type 2, and neoplastic diseases such as rhabdomyosarcoma and uterine leiomyomata.5,11,12 Chemokine receptors belong to the G-protein coupled On the other hand, CXCR3 was identified as a receptor receptor (GPCR) family, which have seven transmem- for T-lymphocyte-specific chemokines. It is strongly brane regions and signal through heterotrimeric G-pro- expressed in IL-2-activated T lymphocytes, but not in 1 teins. Among them, CXCR1 (IL8RA) and CXCR2 (IL8RB) freshly isolated peripheral blood leukocytes or related were identified as receptors for interleukin-8 (IL-8), are cell lines.13 However, its intense expression was shown both expressed in neutrophils, and have 77% amino acid in T cells isolated from synovial fluid from patients with 2,3 identity with each other. CXCR1 binds only two ELR- RA, from tissues of chronically inflamed vaginal and CXC chemokines: IL-8 and granulocyte chemotactic pro- colonic mucosa, or from cerebrospinal fluid from patients tein (GCP)-2, while CXCR2 binds other ELR-CXC chemo- with multiple sclerosis.14,15 Activated Th1/Tc1 subsets 1 kines as well. CXCR1, CXCR2 and IL8RBP, the pseudo- have been shown to express CXCR3 and CCR5.14,16,17 gene bearing greater similarity to CXCR2, are clustered Genetic contribution of chemokine receptor polymor- 4,5 at chromosome 2q35. Physical distances among them phisms to several human disorders has been reported, have not yet been clear. These genes are considered as such as CCR5-⌬32 allele associated with protective effect candidate genes for several human disorders. The from human immunodeficiency virus (HIV)-1 infection.18 mapped susceptibility loci for rheumatoid arthritis (RA), In autoimmune diseases, a significant association was systemic lupus erythematosus (SLE), insulin-dependent shown between CCR5-⌬32 allele and the clinical and diabetes mellitus (IDDM) and juvenile amyotrophic lat- immunological characteristics of RA, or between CCR2– 6–10 eral sclerosis include 2q35. Moreover, structural 64I allele and the susceptibility to IDDM.19,20 To assess abnormalities of this chromosomal region have been the genetic contribution of other chemokine receptors to reported in Waardenburg syndrome, type 1, van der human diseases, polymorphisms in these genes need to be screened and registered as the essential information. In our previous study, we hypothesized that genetic Correspondence: Dr Naoyuki Tsuchiya, Department of Human Genetics, Graduate School of Medicine, University of Tokyo, 7–3-1 Hongo, Bunkyo- variations of chemokine receptors CCR3 and CCR4, pref- 16,17 ku, Tokyo, Japan 113–0033. E-mail: tsuchiya-tkyȰumin.ac.jp erentially expressed in the Th2 subset of lymphocytes, Grant support: This study was supported by the Grant-in-Aid for might be associated with the susceptibility to rheumatic Scientific Research (B11470505) from the Ministry of Education, diseases.21 Since the imbalance of Th1/Th2 cells had been Science, Sports and Culture. implicated in autoimmune rheumatic diseases, it was Sequence Accession numbers: The nucleotide sequence data possible that genetic variations of CCR3 or CCR4 might reported in this paper have been submitted to the DDBJ/EMBL/GenBank nucleotide sequence databases and have cause the imbalance and might influence the suscepti- 22 been assigned the Accession number AB032728 – AB032738. bility to the diseases. However, although several new Received 18 January 2000; revised and accepted 1 March 2000 variations were detected, no association was observed cSNPs in human CXCRs H Kato et al 331 between the susceptibility to rheumatic diseases and any CXCR2 in Japanese, since they were present in 97.9% and of the variations. On the other hand, as for Th1 subset in 89.3% of healthy population, respectively. Other markers, no attempts have been reported on polymor- sequences were regarded as variants. As shown in phisms in CXCR3, while a number of reports are present Table 1, all of them were single nucleotide substitutions. for CCR5.23 In CXCR1, two non-synonymous and two synonymous Thus far, several genomic DNA or cDNA sequences of substitutions were detected. The non-synonymous substi- CXCR1 and CXCR2 have been published.2–4,24–28 How- tution, 827 G → C (S276T), was present in 19.8% of heal- ever, they have a difference at one nucleotide in CXCR1, thy population, while the two synonymous substitutions, 827 G/C (counting from ATG initiation codon) coding 741 C → T (V247V) and 915 C → T (Y305Y) were rare for amino acid 276 S/T. Similarly, in CXCR2, two variations. In CXCR2, one non-synonymous and two syn- sequences have been published possessing 786C or T, onymous substitutions were detected. 786 C → T (L262L) leading to synonymous substitution encoding amino acid was the most frequent variant observed in 52.5% of heal- 262L. In addition, restriction fragment length polymor- thy individuals. In CXCR3, two non-synonymous substi- phism (RFLP) in the region encompassing CXCR2 gene tutions were detected. 875 G → A (R292Q) was detected was reported, and a haplotype containing the RFLP and in one patient with SLE and one healthy individual, and GT dinucleotide repeat number polymorphism in the 1087 G → A (A363T) was detected in only one patient natural resistance associated macrophage protein-1 gene with scleroderma (SSc). Both patients were female, and (NRAMP1) promoter region was demonstrated to be were heterozygous for the common allele and the substi- linked with the susceptibility to RA.7,29 However, it has tution allele. Hence, CXCR3 is considered to be highly not been shown whether the CXCR2 RFLP site is pos- conserved, at least in the Japanese population. itioned within or outside of CXCR2 gene. Systematic In addition, in the process of primer design for CXCR3 screening of the entire coding sequences has not been exon 1, we confirmed that CXCR3 gene has one intron reported for CXCR1, CXCR2 and CXCR3. between exon 1 and exon 2, the size of which is approxi- In this study, we attempted to detect variations in the mately 1 kb (data not shown). One polymorphic site was entire coding regions of human CXCR1, CXCR2, CXCR3, incidentally detected within intron 1 [+235 (G/A)] (The estimated each allele frequency in Japanese, and exam- nucleotide sequence data of the 5′ portion of intron 1 ined the possible association between their variations and have been submitted DDBJ/EMBL/GenBank database; the susceptibility to rheumatic diseases. accession numbers: AB032737–AB032738). Results Analysis of association between detected variations and rheumatic diseases Identification of new variations of CXCRs The detected variation sites were subsequently examined Several new nucleotide sequence variations, in addition for the possible association with rheumatic diseases using to the previously reported ones, were detected using case-control analysis. As summarized in Table 1, no sig- polymerase chain reaction (PCR)-single-strand confor- nificant association between the positivity of these vari- mation polymorphism (SSCP) method. The representa- ations and any of the tested rheumatic diseases was tive SSCP patterns are shown in Figure 1. From the observed. Among the variations, CXCR1-827G/C and observed frequency of each sequence, we considered CXCR2-786C/T were frequently observed and were con- CXCR1-827G (coding for 276S) and CXCR2-786C (262L) sidered as single nucleotide polymorphisms within the as the common standard sequences of CXCR1 and coding sequence (cSNPs). For these cSNPs, genotype fre- 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, CXCR2- 238C/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).