1 Department of Neurology and Geriatrics, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
2 Department of Medical Information Science, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
3 Department of Immunology and Immunology Research Center, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
4 Department of Neurology, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
5 Khorasan Blood Transfusion Center, Mashhad, Iran
6 Department of Molecular Pathology, Center for Chronic Viral Diseases, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8520, Japan
Correspondence
Mineki Saito
mineki{at}m3.kufm.kagoshima-u.ac.jp
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ABSTRACT |
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INTRODUCTION |
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HTLV-1 is also endemic in the Caribbean Basin (Blattner et al., 1982), Africa (Biggar et al., 1984
), South America (Zamora et al., 1990
; Cartier et al., 1993
; Zaninovic et al., 1994
) and the Melanesian islands (Yanagihara et al., 1990
). The city of Mashhad in northeastern Iran has also been reported as an endemic centre for HTLV-1 (Safai et al., 1996
). In a recent study, the prevalence of HTLV-I infection was reported to be 0·77 % among blood-bank donors of Mashhad (Abbaszadegan et al., 2003
), but the prevalence and incidence of HAM/TSP are unknown in this population. Since there has been no report to compare the genetic risk factors for HAM/TSP among different ethnic populations, it was interesting to study whether genetic risk factors found in Kagoshima, Japan, were also valid for HAM/TSP development in the Mashhadi Iranian population. We therefore analysed the HTLV-1 provirus load, HTLV-1 tax subgroup and the allele frequencies of HLA-A*02, HLA-B*5401, HLA-Cw*08 and HLA-DRB1*0101 in Iranian HTLV-1-infected individuals using the same methods and techniques that were used in the Kagoshima studies (Nagai et al., 1998
; Jeffery et al., 1999
, 2000
). The effect of host genetic factors and HTLV-1 tax subgroups on the risk of HAM/TSP development in different ethnic groups is discussed.
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METHODS |
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DNA preparation.
All Japanese and Iranian blood samples were taken by vacuum tube pre-filled with the anticoagulant EDTA. Genomic DNA extraction procedures were different for each population. In the case of Kagoshima samples, fresh PBMCs were isolated by Histopaque-1077 (Sigma) density-gradient centrifugation and genomic DNA was extracted using a QIAamp Blood kit (Qiagen). For Iranian samples, for economical and technical reasons, fresh blood specimens were frozen immediately after collection and frozen whole-blood samples were transported to Kagoshima University on dry ice. Genomic DNA of nucleated blood cells was isolated from whole blood in Kagoshima University using the PureGene DNA Purification kit (Gentra Systems).
Provirus load measurement.
To assay the HTLV-1 provirus load, we carried out a quantitative PCR using ABI Prism 7700 (PE Applied Biosystems) with 100 ng genomic DNA (equivalent to approx. 104 cells) from PBMCs (for Kagoshima samples) or nucleated blood cells (for Iranian samples) as reported previously (Nagai et al., 1998). Using
-actin as an internal control, the amount of HTLV-1 provirus DNA was calculated using the following formula: copy number of HTLV-1 tax per 104 PBMCs (for Japanese samples) or nucleated blood cells (for Iranian samples)=[(copy number of tax)/(copy number of
-actin/2)]x104. All samples were tested in triplicate. The lower limit of detection was one copy of HTLV-1 tax per 104 PBMCs. In this study, we used the previously analysed provirus load data of Kagoshima samples from our database (Nagai et al., 1998
). All Iranian samples and some randomly selected Kagoshima samples were analysed using the same kit (AmpliTaq Gold and TaqMan probe; PE Applied Biosystems) and machine (ABI Prism 7700) at the same time. The same standard DNA for tax and
-actin was used throughout the study and there was no discrepancy between old and new data (not shown).
Sequencing of the HTLV-1 tax gene.
Randomly selected Iranian samples from 10 HAM/TSP patients and 10 HCs were sequenced over almost the entire HTLV-1 tax gene (nt 72958356, nucleotide numbers correspond to those of the prototypic strain, ATK-1; Seiki et al., 1983). PCR was done on extracted DNA to amplify provirus DNA, and nucleotide sequences were determined by direct sequencing in both directions. We amplified 100 ng DNA in 35 cycles of PCR, using an expanded high-fidelity PCR system (Boehringer Mannheim) and 1 µM primers (PXO1+, 5'-TCGAAACAGCCCTGCAGATA-3', nt 72577276, and PXO2+, 5'-TGAGCTTATGATTTGTCTTCA-3', nt 84478467). Each PCR cycle consisted of denaturation at 94 °C for 60 s, annealing at 58 °C for 75 s, extension at 72 °C for 90 s and a final extension at 72 °C for 10 min. Amplified DNA products were purified using a purification kit (QIAquick; Qiagen) and 0·1 µg PCR product was sequenced with a dye terminator DNA sequencing kit (Applied Biosystems) with 3·2 pmol each primer [PXI1+, 5'-ATACAAAGTTAACCATGCTT-3', nt 72747293; PXI2+, 5'-GGCCATGCGCAAATACTCCC-3', nt 76187637; PXI3+, 5'-TTCCGTTCCACTCAACCCTC-3', nt 80018020; PXI1, 5'-GGGTTCCATGTATCCATTTC-3', nt 76447663, PXI2, 5'-GTCCAAATAAGGCCTGGAGT-3', nt 80248043; and PXI3, 5'-AGACGTCAGAGCCTTAGTCT-3', nt 83748393] in an automatic DNA sequencer (model 377; Applied Biosystems).
Restriction fragment length polymorphism (RFLP) analysis of the HTLV-1 tax gene.
To determine the HTLV-1 tax gene subgroup (tax A or B) in Iranian samples, we carried out a PCR-RFLP analysis as previously described (Furukawa et al., 2000). For RFLP analysis, 4 µl PCR product was digested with 5 U AccII (Takara) in 10 µl total volume at 37 °C for 1 h followed by electrophoresis on 2 % Nusieve agarose gel. The previously analysed tax subgroup data of Kagoshima samples (Furukawa et al., 2000
) were extracted from our database. Positive and negative controls of known Japanese samples of tax gene subgroups A and B, which were confirmed by direct sequence analysis, were included in all experiments.
HLA typing.
PCR sequence-specific primer reactions were performed to detect HLA-A*02, HLA-B*5401, HLA-Cw*08 and HLA-DRB1*0101 as previously described (Bunce et al., 1995; Olerup & Zetterquist, 1992
). We used previously analysed HLA data of Kagoshima samples from our database (Jeffery et al., 1999
, 2000
).
Statistical analysis.
Statistical analysis was performed using the SPSS for Windows release 7.0, run on an IBM-compatible computer (Analytical Software, version 7). The 2 test, the MannWhitney U test and the odds ratio (OR) were used for statistical analysis. Values of P<0·05 were considered statistically significant.
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RESULTS |
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DISCUSSION |
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We first examined the HTLV-1 provirus load in Iranian HAM/TSP patients and HCs, since one of the major risk factors for developing HAM/TSP is the provirus load (Nagai et al., 1998). The median HTLV-1 provirus load of Iranian HAM/TSP patients was twofold greater in HAM/TSP patients than in HCs, whereas that of Japanese HAM/TSP patients was 13-fold greater than in HCs. Interestingly, despite differences in the methods of DNA extraction between the two study groups (whole blood-derived DNA for Iranian samples vs PBMC-derived DNA for Japanese samples), the HTLV-1 provirus load in Iranian HCs was still significantly higher than Japanese HCs (P=0·004, MannWhitney U test). This may be the main cause of the smaller observed ratio of median provirus load between HAM/TSP patients and HCs in the Iranian study group. To investigate the reason for this difference between the two populations, we further analysed the frequencies of certain HLA alleles and the HTLV-1 tax subgroup in the Iranian population.
In the Kagoshima population, possession of either of the HLA class I genes HLA-A*02 or HLA-Cw*08 was associated with a statistically significant reduction in both HTLV-1 provirus load and the risk of HAM/TSP (Jeffery et al., 1999, 2000
). However, in Mashhadi Iranian subjects, both HLA-A*02 and HLA-Cw*08 had no effect on either the risk of HAM/TSP or provirus load. In contrast, HLA-DRB1*0101 was associated with increased susceptibility to HAM/TSP both in Kagoshima (P=0·049) and Iran (P=0·035). In HAM/TSP, CD4+ cells are the predominant cells present early in the active lesions (Umehara et al., 1993
) and are also the HTLV-1-infected cells in the inflammatory spinal cord lesions (Moritoyo et al., 1996
). Moreover, HLA-DRB1*0101 restricts CD4+ T-cell immunodominant epitopes of HTLV-1 env gp21 (Yamano et al., 1997
; Kitze et al., 1998
). Therefore, it is possible that HLA-DRB1*0101 is associated with susceptibility to HAM/TSP via an effect on CD4+ T-cell activation and subsequent bystander damage in the central nervous system (Ijichi et al., 1993
; Bangham, 2000
). However, since possession of HLA-DRB1*0101 was associated with a significantly lower provirus load in the Japanese HAM/TSP patients but not in the Iranian HAM/TSP patients, the underlying mechanism involving HLA-DRB1*0101 may not be the same between Iranian and Japanese HTLV-1-infected individuals. Differences in other genetic factors, including non-HLA genes, may also be important for explaining the observed differences between the populations.
Another possible explanation of the observed differences in the present study is that certain HLA genotypes are associated with different effects on different subtypes of the virus. In human papilloma virus (HPV) infection, the association of the DRB1*1501DQB1*0602 haplotype with HPV-related cervical carcinoma was reported to be specific for the viral type HPV-16, suggesting that specific HLA haplotypes may influence the immune response to specific virus-encoded epitopes and affect the risk of viral disease (Apple et al., 1994). To test this possibility, we sequenced almost the entire region of the tax gene in 20 Mashhad Iranian HTLV-1-infected individuals (10 HAM/TSP and 10 HCs) and compared the sequence with that of two Japanese strains, tax subgroups A and B. Although we could not identify any amino acid differences in the Tax1119 immunodominant epitope between the Iranian and Japanese tax subgroups A and B, we found that Iranian HTLV-1 possessed 10 different nucleotides in the tax region compared with Japanese tax subgroup B. Among these, nt 7897, 7959, 8208 and 8344 were identical to tax subgroup A. Therefore, Iranian tax sequences have four additional different amino acids compared with Japanese tax subgroup A and six additional different amino acids compared with Japanese tax subgroup B. These findings suggest that both the lack of consistency of host genetic influences and the smaller difference in median provirus load between HAM/TSP patients and HCs in Iran may be due in part to different strains of HTLV-1. Our present observation that HLA-A*02 was associated with a lower provirus load only in the tax subgroup B-infected subjects in Kagoshima, but not in tax subgroup A-infected subjects, is consistent with this hypothesis. Further studies to examine functional differences between Iranian and Japanese HTLV-1 Tax proteins will provide important information to clarify this point.
The interaction between different genes and/or environmental factors is also likely to contribute to the observed differences between the two populations. For example, co-infection with Strongyloides stercoralis (Gabet et al., 2000) can affect the HTLV-1 provirus load. In Japan, S. stercoralis infection is endemic in the southwestern islands Amami and Ryukyu, but is rarely reported on the mainland including Kagoshima (Arakaki et al., 1992
). However, there are no data on the prevalence of S. stercoralis infection in Mashhad, Iran, and therefore future epidemiological studies are necessary to clarify this possibility.
It seems likely that the same evolutionary selection pressures that induce polymorphisms in infection-resisting genes' have contributed to marked allele-frequency differences at the same loci. When geographical variation in pathogen polymorphism is superimposed on this host genetic heterogeneity, considerable variation in detectable allelic associations is likely to result in the different populations. In other words, genetic resistance to infectious diseases that is formed by complex host genetic effects is complicated further by pathogen diversity and environmental factors. Considering this background of complexity, the most practical approach to finding reliable results may be first to identify disease-associated genes in a single large population, and secondly to analyse subsequently whether a similar effect is found in other ethnic populations, as we have shown in this study.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Apple, R. J., Erlich, H. A., Klitz, W., Manos, M. M., Becker, T. M. & Wheeler, C. M. (1994). HLA DR-DQ associations with cervical carcinoma show papillomavirus-type specificity. Nat Genet 6, 157162.[Medline]
Arakaki, T., Kohakura, M., Asato, R., Ikeshiro, T., Nakamura, S. & Iwanaga, M. (1992). Epidemiological aspects of Strongyloides stercoralis infection in Okinawa, Japan. J Trop Med Hyg 95, 210213.[Medline]
Bangham, C. R. (2000). The immune response to HTLV-1. Curr Opin Immunol 12, 397402.[CrossRef][Medline]
Biggar, R. J., Saxinger, C., Gardiner, C., Collins, W. E., Levine, P. H., Clark, J. W., Nkrumah, F. K. & Blattner, W. A. (1984). Type-I HTLV antibody in urban and rural Ghana, West Africa. Int J Cancer 34, 215219.[Medline]
Blattner, W. A., Kalyanaraman, V. S., Robert-Guroff, M. & 7 other authors (1982). The human type-C retrovirus, HTLV, in Blacks from the Caribbean region, and relationship to adult T-cell leukemia/lymphoma. Int J Cancer 30, 257264.[Medline]
Bunce, M., O'Neill, C. M., Barnardo, M. C., Krausa, P., Browning, M. J., Morris, P. J. & Welsh, K. I. (1995). Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens 46, 355367.[Medline]
Cartier, L., Araya, F., Castillo, J. L. & 8 other authors (1993). Southernmost carriers of HTLV-I/II in the world. Jpn J Cancer Res 84, 13.[Medline]
Furukawa, Y., Yamashita, M., Usuku, K., Izumo, S., Nakagawa, M. & Osame, M. (2000). Phylogenetic subgroups of human T cell lymphotropic virus (HTLV) type I in the tax gene and their association with different risks for HTLV-1-associated myelopathy/tropical spastic paraparesis. J Infect Dis 182, 13431349.[CrossRef][Medline]
Gabet, A. S., Mortreux, F., Talarmin, A. & 7 other authors (2000). High circulating proviral load with oligoclonal expansion of HTLV-1 bearing T cells in HTLV-1 carriers with strongyloidiasis. Oncogene 19, 49544960.[CrossRef][Medline]
Gessain, A., Barin, F., Vernant, J. C., Gout, O., Maurs, L., Calender, A. & de The, G. (1985). Antibodies to human T-lymphotropic virus type-I in patients with tropical spastic paraparesis. Lancet ii, 407410.
Hill, A. V. (1998). The immunogenetics of human infectious diseases. Annu Rev Immunol 16, 593617.[CrossRef][Medline]
Hinuma, Y., Nagata, K., Hanaoka, M., Nakai, M., Matsumoto, T., Kinoshita, K. I., Shirakawa, S. & Miyoshi, I. (1981). Adult T-cell leukemia: antigen in an ATL cell line and detection of antibodies to the antigen in human sera. Proc Natl Acad Sci U S A 78, 64766480.[Abstract]
Ijichi, S., Izumo, S., Eiraku, N. & 8 other authors (1993). An autoaggressive process against bystander tissues in HTLV-1-infected individuals: a possible pathomechanism of HAM/TSP. Med Hypotheses 41, 542547.[CrossRef][Medline]
Jeffery, K. J., Usuku, K., Hall, S. E. & 14 other authors (1999). HLA alleles determine human T-lymphotropic virus-I (HTLV-I) proviral load and the risk of HTLV-1-associated myelopathy. Proc Natl Acad Sci U S A 96, 38483853.
Jeffery, K. J., Siddiqui, A. A., Bunce, M. & 8 other authors (2000). The influence of HLA class I alleles and heterozygosity on the outcome of human T cell lymphotropic virus type I infection. J Immunol 165, 72787284.
Kaplan, J. E., Osame, M., Kubota, H., Igata, A., Nishitani, H., Maeda, Y., Khabbaz, R. F. & Janssen, R. S. (1990). The risk of development of HTLV-1-associated myelopathy/tropical spastic paraparesis among persons infected with HTLV-1. J Acquir Immune Defic Syndr 3, 10961101.[Medline]
Kaplan, J. E., Khabbaz, R. F., Murphy, E. L. & 12 other authors (1996). Male-to-female transmission of human T-cell lymphotropic virus types I and II: association with viral load. The Retrovirus Epidemiology Donor Study Group. J Acquir Immune Defic Syndr Hum Retrovirol 12, 193201.[Medline]
Kitze, B., Usuku, K., Yamano, Y., Yashiki, S., Nakamura, M., Fujiyoshi, T., Izumo, S., Osame, M. & Sonoda, S. (1998). Human CD4+ T lymphocytes recognize a highly conserved epitope of human T lymphotropic virus type 1 (HTLV-1) env gp21 restricted by HLA DRB1*0101. Clin Exp Immunol 111, 278285.[CrossRef][Medline]
Miura, T., Fukunaga, T., Igarashi, T. & 7 other authors (1994). Phylogenetic subtypes of human T-lymphotropic virus type I and their relations to the anthropological background. Proc Natl Acad Sci U S A 91, 11241127.
Moritoyo, T., Reinhart, T. A., Moritoyo, H., Sato, E., Izumo, S., Osame, M. & Haase, A. T. (1996). Human T-lymphotropic virus type I-associated myelopathy and tax gene expression in CD4+ T lymphocytes. Ann Neurol 40, 8490.[Medline]
Nagai, M., Usuku, K., Matsumoto, W. & 8 other authors (1998). Analysis of HTLV-1 proviral load in 202 HAM/TSP patients and 243 asymptomatic HTLV-1 carriers: high proviral load strongly predisposes to HAM/TSP. J Neurovirol 4, 586593.[Medline]
Olerup, O. & Zetterquist, H. (1992). HLA-DR typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours: an alternative to serological DR typing in clinical practice including donorrecipient matching in cadaveric transplantation. Tissue Antigens 39, 225235.[Medline]
Osame, M. (1990). Review of WHO Kagoshima meeting and diagnostic guidelines for HAM/TSP. In Human Retrovirology: HTLV, pp. 191197. Edited by W. A. Blattner. New York: Raven Press.
Osame, M., Usuku, K., Izumo, S., Ijichi, N., Amitani, H., Igata, A., Matsumoto, M. & Tara, M. (1986). HTLV-1 associated myelopathy, a new clinical entity. Lancet i, 10311032.
Poiesz, B. J., Ruscetti, F. W., Gazdar, A. F., Bunn, P. A., Minna, J. D. & Gallo, R. C. (1980). Detection and isolation of type C retrovirus particles from fresh and cultured lymphocytes of a patient with cutaneous T-cell lymphoma. Proc Natl Acad Sci U S A 77, 74157419.[Abstract]
Safai, B., Huang, J. L., Boeri, E., Farid, R., Raafat, J., Schutzer, P., Ahkami, R. & Franchini, G. (1996). Prevalence of HTLV type I infection in Iran: a serological and genetic study. AIDS Res Hum Retroviruses 12, 11851190.[Medline]
Seiki, M., Hattori, S., Hirayama, Y. & Yoshida, M. (1983). Human adult T-cell leukemia virus: complete nucleotide sequence of the provirus genome integrated in leukemia cell DNA. Proc Natl Acad Sci U S A 80, 36183622.[Abstract]
Takenouchi, N., Yamano, Y., Usuku, K., Osame, M. & Izumo, S. (2003). Usefulness of proviral load measurement for monitoring of disease activity in individual patients with human T-lymphotropic virus type I-associated myelopathy/tropical spastic paraparesis. J Neurovirol 9, 2935.[Medline]
Umehara, F., Izumo, S., Nakagawa, M., Ronquillo, A. T., Takahashi, K., Matsumuro, K., Sato, E. & Osame, M. (1993). Immunocytochemical analysis of the cellular infiltrate in the spinal cord lesions in HTLV-1-associated myelopathy. J Neuropathol Exp Neurol 52, 424430.[Medline]
Woolf, B. (1955). On estimating the relationship between blood group and disease. Ann Hum Genet 19, 251253.[Medline]
Yamano, Y., Kitze, B., Yashiki, S. & 7 other authors (1997). Preferential recognition of synthetic peptides from HTLV-1 gp21 envelope protein by HLA-DRB1 alleles associated with HAM/TSP (HTLV-1-associated myelopathy/tropical spastic paraparesis). J Neuroimmunol 76, 5060.[CrossRef][Medline]
Yamashita, M., Achiron, A., Miura, T. & 7 other authors (1995). HTLV-I from Iranian Mashhadi Jews in Israel is phylogenetically related to that of Japan, India, and South America rather than to that of Africa and Melanesia. Virus Genes 10, 8590.[Medline]
Yanagihara, R., Jenkins, C. L., Alexander, S. S., Mora, C. A. & Garruto, R. M. (1990). Human T lymphotropic virus type I infection in Papua New Guinea: high prevalence among the Hagahai confirmed by western analysis. J Infect Dis 162, 649654.[Medline]
Yoshida, M., Miyoshi, I. & Hinuma, Y. (1982). Isolation and characterization of retrovirus from cell lines of human adult T-cell leukemia and its implication in the disease. Proc Natl Acad Sci U S A 79, 20312035.[Abstract]
Yoshida, M., Seiki, M., Yamaguchi, K. & Takatsuki, K. (1984). Monoclonal integration of human T-cell leukemia provirus in all primary tumors of adult T-cell leukemia suggests causative role of human T-cell leukemia virus in the disease. Proc Natl Acad Sci U S A 81, 25342537.[Abstract]
Zamora, T., Zaninovic, V., Kajiwara, M., Komoda, H., Hayami, M. & Tajima, K. (1990). Antibody to HTLV-1 in indigenous inhabitants of the Andes and Amazon regions in Colombia. Jpn J Cancer Res 81, 715719.[Medline]
Zaninovic, V., Sanzon, F., Lopez, F. & 9 other authors (1994). Geographic independence of HTLV-I and HTLV-II foci in the Andes highland, the Atlantic coast, and the Orinoco of Colombia. AIDS Res Hum Retroviruses 10, 97101.[Medline]
Received 9 August 2004;
accepted 2 December 2004.
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