Genes and Immunity (2001) 2, 156–158  2001 Nature Publishing Group All rights reserved 1466-4879/01 $15.00 www.nature.com/gene BRIEF COMMUNICATION Novel polymorphisms in human inflammatory protein-1 alpha (MIP-1␣) gene

X Xin1, K Nakamura2, H Liu3, EE Nakayama3, M Goto1, Y Nagai4, Y Kitamura1, T Shioda3 and A Iwamoto1 1Division of Infectious Diseases, Advanced Clinical Research Center, Institute of Medical Science, and 2Department of Dermatology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; 3Department of Immune and Living Body Defense, Institute of Microbal Disease, Osaka University, Japan; 4National Institute of Infectious Disease, Tokyo, Japan

Human macrophage inflammatory protein-1 alpha (MIP-1␣) is a chemotactic , which binds to , T cells, and B cells affecting their activation. We found novel polymorphisms at four sites within MIP-1␣ gene in Japanese population: C to T in exon 2; A to G in intron 2; C to G and A to G in exon 3. They occurred on the same allele. Although MIP-1␣ effectively suppresses the replication of HIV-1 in vitro, we observed no statistically significant difference in the allele frequency of this polymorphism between HIV-1-infected and uninfected individuals in Japanese population. Since an increased transcription level of MIP-1␣ has been reported to be associated with inflammatory diseases such as atopic dermatitis, we also investigated the frequency of these polymorphisms among patients with atopic dermatitis, HIV-1- infected individuals (with a normal IgE level), and healthy donors. A small increase in ratio of homozygotes to other genotypes was observed in patients with atopic dermatitis (P = 0.04). Genes and Immunity (2001) 2, 156–158.

Keywords: MIP-1␣; polymorphism; HIV-1; atopic dermatitis; Japanese

Macrophage inflammatory protein-1 alpha (MIP-1␣), also novel polymorphic sites in both the coding and 3Ј-nonco- referred to as LD78␣,1,2 has already been demonstrated ding regions in the MIP-1␣ gene in Japanese population, to have phagocyte stimulating and proinflammatory and demonstrate that the mutation homozygosity may be properties.3 MIP-1␣ belongs to the C-C family involved in the pathogenesis of atopic dermatitis. and is mainly produced by CD8-positive lymphocytes. It Human genomic DNA was obtained from peripheral acts as one of the natural ligands of blood mononuclear cells (PBMC) from healthy donors, CCR5 and moderately suppresses in vitro replication of patients with atopic dermatitis, and HIV-1-infected indi- macrophage tropic strains of HIV-1.4 viduals with normal IgE level. Genomic sequence of 1889 Recently, polymorphisms in chemokine receptor and nucleotides spanning the entire two introns and three their natural ligand genes have been shown to modify exons of MIP-1␣ gene were determined. We identified HIV-1 transmission and disease progression, and allergic polymorphisms at four positions: a C to T substitution at diseases.5–9 MIP-1␣ gene has been mapped to chromo- position +954 (exon 2), A to G at +1245 (intron 2), C to some 17q11-q1210 and consists of three exons and two Gat+1728 (exon 3), and A to G at position +1771 (exon introns (Figure 1). Evidence of increased transcription 3), respectively (Figure 1). The mutations in exons are level of MIP-1␣ in peripheral blood mononuclear cells in silent, that is, no amino acid change was observed. All patients with atopic dermatitis (AD) has been reported.11 the SNPs were found neither to create nor to destroy a Recently, Al-Sharif et al reported a biallelic dinucleotide restriction endonuclease site. Table 1 summarizes the microsatellite repeat within the promoter region in the genotypes of all four polymorphisms haplotypes, their healthy UK Caucasoid population.12 These prompted us combination and frequencies in 65 healthy donors, 57 to search for polymorphisms in the other regions of MIP- HIV-1-infected individuals, and 39 AD patients. All the 1␣, and to evaluate a possible role of polymorphisms in four nucleotide changes were completely associated with HIV-1 infection and allergic disease such as atopic der- each other: we concluded that they occurred on the same matitis. In this paper, we report the presence of four chromosome. Thus, we found two alleles of MIP-1␣ present in the Japanese population in our study. We designated the wild-type allele (C at 954, A at 1245, C at Correspondence: Yoshihiro Kitamura, Division of Infectious Diseases, 1728, and A at 1771) as C-A-C-A allele (wt); and the Advanced Clinical Research Center, Institute of Medical Science, Univer- mutant allele (T at 954, G at 1245, G at 1728, and G at sity of Tokyo, 4-6-1 Shiroganedai, Minato-ku, Tokyo 108-8639, Japan. E- Ȱ 1771) as T-G-G-G allele (mt) (Table 1). mail: yochan nih.go.jp In HIV-1-infected individuals with a normal IgE level, This work was supported by grants from Ministry of Education, Science, Sports, and Culture; the Ministry of Health and Welfare; the frequency of T-G-G-G allele was 22.8%, showing no and Japan Society for the Promotion of Science. difference from that in ether patients with AD or healthy Received 5 January 2001; revised and accepted 9 March 2001 donors (26.9%, 28.5%, P = 0.31). Patients with AD showed Novel polymorphism in human MIP-1␣ gene X Xin et al 157

Figure 1 Schematic representation of genomic structure of MIP-1␣. Boxes indicate exons. the transcription start site marked by +1. The numbers −906 indicates the dinucleotide repeat position within the promoter region.11 The number 954, 1245, 1728, and 1771 indicate the novel polymorphic positions. DNA fragment corresponding to genomic region of the entire two introns and three exons of the MIP-1␣ gene was PCR amplified by using primer pair M␣5–1 (−11 5Ј-CAGAAGGACACGGGCAGCAGACAGTGG-3Ј +12), and M␣2003 (+2003 5Ј- CCTCCCCATCTCTCCCAAATTTCC-3Ј +1981). PCR was carried out at 95°C, 5 min followed by 40 cycles of denaturation at 95°C for 30 sec, annealing and extension at 64°C for 3 min. Direct fluorescence-based automated cycle sequencing of the PCR products was then performed by an ABI 377 sequencer by using primer M␣2(+1314 5Ј-CCACCATTCTGCTCTCTGT-3Ј +1296) and LD6 (+1423 5Ј-AGTGAG- GAGTGGGTCCAGAA-3Ј +1442). Primer positions and direction used for PCR and sequencing are indicated by arrows. Genomic and cDNA sequences are derived from D90144. ATG: translation initiation; Term: translation termination.

Table 1 Frequencies of MIP-1␣ genotypes and alleles among patients with atopic dermatitis, HIV-1-infected individuals, and healthy donors

Genotype/Haplotype Healthy donor HIV-1-infected Atopic dermatitis (frequency %) (frequency %) (frequency %) 954 1245 1728 1771

Genotypes 1 C/C A/A C/C A/A 35 (53.8) 33 (57.9) 24 (61.5) 2 C/T A/G C/G A/G 23 (35.4) 22 (38.6) 9 (23.1) 3 T/T G/G G/G G/G 7 (10.8) 2 (3.5) 6 (15.4) Total 65 (100) 57 (100) 39 (100) Alleles C-A-C-A C A C A 93 (71.5) 88 (77.2) 57 (73.1) T-G-G-G T G G G 37 (28.5) 26 (22.8) 21 (26.9) Total 130 (100) 114 (100) 78 (100)

P value (HWEa) 0.52 0.66 0.04 P value (t test) 0.31 0.52 aHWE, Hardy-Weinberg euqilibrium. more T-G-G-G homozygotes, showing a statistically sig- Japanese population. Therefore, further studies would be nificant deviation from Hardy–Weinberg expectation (P = required to determine whether or not this allele affects 0.04). This deviation was not observed in HIV-1-infected levels of MIP-1␣ production in vivo. Since evidences for individuals or healthy donors. But this result does not the involvement of the production of RANTES in the soundly support the suggestion that T-G-G-G allele pathogenesis of AD been reported,13 our data that homozygosity may be related to AD susceptibility. For patients with AD showed more mutation homozygotes example, this finding may be explained by the assump- would make it interesting to determine the possible effect tion that the population in the AD group is under selec- of a polymorphism in IL-8 or RANTES on AD. tive pressure of AD disease itself. We also investigated MIP-1␣ gene promoter polymor- phism in Japanese population by using the primers Acknowledgements described previously.12 Our results confirmed the pres- We thank Drs Ai Tachikawa, Tomohiko Koibuchi, and ence of the biallelic dinucleotide repeat (TATA repeat at Mioo Satoh for clinical samples and data, and Dr Deshan position −906), and showed a similar frequency (data not Yu for discussion. shown) of heterozygotes to that in UK Caucasoid.12 Fur- thermore, this microsatellite was also found to be associa- ted with the new alleles reported above. References ␣ Although MIP-1 effectively suppresses the replication 1 Obaru K, Fukuda M, Maeda S, Shimada K. A cDNA clone used of HIV-1 in vitro, we observed no statistically significant to study mRNA inducible in human tonsillar lymphocytes by a difference in allele frequencies of newly-identified allele tumor promoter. J Biochem (Tokyo) 1986; 99: 885–894. between HIV-1-infected and uninfected individuals in 2 Davatelis G, Tekamp-Olson P, Wolpe SD et al. Cloning and

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