Differences in viral and host genetic risk factors for development of human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis between Iranian and Japanese HTLV-1-infected individuals

Amir H. Sabouri1, Mineki Saito1, Koichiro Usuku2, Sepideh Naghibzadeh Bajestan1, Mahmoud Mahmoudi3, Mohsen Forughipour4, Zahra Sabouri3, Zahra Abbaspour3, Mohammad E. Goharjoo4, Esmaeil Khayami5, Ali Hasani5, Shuji Izumo6, Kimiyoshi Arimura1, Reza Farid3 and Mitsuhiro Osame1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a neurological disease observed only in 1–2 % of infected individuals. HTLV-1 provirus load, certain HLA alleles and HTLV-1 tax subgroups are reported to be associated with different levels of risk for HAM/TSP in Kagoshima, Japan. Here, it was determined whether these risk factors were also valid for HTLV-1-infected individuals in Mashhad in northeastern Iran, another region of endemic HTLV-1 infection. In Iranian HTLV-1-infected individuals (n=132, 58 HAM/TSP patients and 74 seropositive asymptomatic carriers), although HLA-DRB1*0101 was associated with disease susceptibility in the absence of HLA-A*02 (P=0·038; odds ratio=2·71) as observed in Kagoshima, HLA-A*02 and HLA-Cw*08 had no effect on either the risk of developing HAM/TSP or HTLV-1 provirus load. All Iranian subjects possessed tax subgroup A sequences, and the protective effects of HLA-A*02 were observed only in Kagoshima subjects with tax subgroup B but not in those with tax subgroup A. Both the prevalence of HTLV-1 subgroups and the host genetic background may explain the different risks levels for HAM/TSP development in these two populations.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Human T-cell lymphotropic virus type 1 (HTLV-1) (Poiesz et al., 1980; Yoshida et al., 1982) is a causative agent of adult T-cell leukaemia (Hinuma et al., 1981; Yoshida et al., 1984) and the chronic neurodegenerative disorder HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) (Gessain et al., 1985; Osame et al., 1986). Only a minority of HTLV-1-infected individuals develop HAM/TSP, and most infected individuals remain healthy throughout their lives. A previous seroepidemiological survey in Kyushu Island, in southwestern Japan, where Kagoshima prefecture is located, estimated the incidence of HAM/TSP among HTLV-1-infected persons at 3·1x10–5 cases per year; assuming a lifespan of 75 years, the lifetime incidence is therefore approximately 0·25 % (Kaplan et al., 1990). In HAM/TSP patients from Kagoshima, the median provirus load in peripheral blood mononuclear cells (PBMCs) is more than ten times higher than HTLV-1-seropositive asymptomatic carriers (HCs) and high provirus load is also associated with an increased risk of progression to disease (Nagai et al., 1998). HTLV-1 provirus load has been correlated with progression of motor disability (Takenouchi et al., 2003) and the risk of sexual transmission of HTLV-1 (Kaplan et al., 1996). Thus, HTLV-1 provirus load is an important correlate of virus transmission as well as disease progression. A previous study indicated that the provirus load in PBMCs from HCs in genetic relatives of patients with HAM/TSP in Kagoshima was significantly higher than that of non-HAM/TSP-related HCs, suggesting the importance of genetic background for developing HAM/TSP (Nagai et al., 1998). In the Kagoshima population, an association between HLA-DRB1*0101, HLA-B*5401, HLA-A*02 and HLA-Cw*08 and the outcome of HTLV-1 infection has been reported, where HLA-A*02 and HLA-Cw*08 genes were each independently associated with a lower HTLV-1 provirus load and with protection from HAM/TSP, whereas HLA-DRB1*0101 and HLA-B*5401 were associated with an increased susceptibility to HAM/TSP (Jeffery et al., 1999, 2000). The association of HLA-DRB1*0101 with disease susceptibility was only evident in the absence of the protective effect of HLA-A*02 (Jeffery et al., 1999). These results are consistent with the hypothesis that a strong class I-restricted T-cell response is beneficial (Bangham, 2000). In another study, an association between HTLV-1 tax gene sequence variation and the risk of HAM/TSP was reported (Furukawa et al., 2000). The tax subgroup A was more frequently observed in HAM/TSP patients than in HCs and this effect was independent of HLA-A*02. These reports suggested that both host genetic factors and HTLV-1 subgroup independently play a part in determining the risk of developing HAM/TSP.

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.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Study populations.
Peripheral blood samples were studied from 58 Iranian patients with HAM/TSP and 74 HCs from blood donors of the Blood Transfusion Center in the city of Mashhad and Neyshabour, both located in HTLV-1-endemic northeastern Iran. The study population from Kagoshima consisted of 222 patients with HAM/TSP and 184 HCs, all of whom were enrolled in the previous Kagoshima studies (Nagai et al., 1998; Jeffery et al., 1999, 2000; Furukawa et al., 2000). The diagnosis of HAM/TSP was made according to the World Health Organization diagnostic criteria (Osame, 1990). Informed consent was obtained from all patients. This research was approved by the institutional review boards of the authors' institutions.

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 {beta}-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 {beta}-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 {beta}-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 7295–8356, 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 7257–7276, and PXO2+, 5'-TGAGCTTATGATTTGTCTTCA-3', nt 8447–8467). 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 7274–7293; PXI2+, 5'-GGCCATGCGCAAATACTCCC-3', nt 7618–7637; PXI3+, 5'-TTCCGTTCCACTCAACCCTC-3', nt 8001–8020; PXI1, 5'-GGGTTCCATGTATCCATTTC-3', nt 7644–7663, PXI2, 5'-GTCCAAATAAGGCCTGGAGT-3', nt 8024–8043; and PXI3, 5'-AGACGTCAGAGCCTTAGTCT-3', nt 8374–8393] 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 {chi}2 test, the Mann–Whitney U test and the odds ratio (OR) were used for statistical analysis. Values of P<0·05 were considered statistically significant.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Differences in HTLV-1 provirus load between HAM/TSP patients and asymptomatic carriers is significantly lower in Iranian HTLV-1-infected individuals than in Japanese
We used the previously analysed provirus load data of Kagoshima samples from our database (Nagai et al., 1998); all Iranian samples were newly analysed. The median age of HAM/TSP patients in both Kagoshima (57·3 years, range 15–80 years, 70·4 % female) and Iran (49·7 years, range 24–80 years, 72·1 % female) was greater than that of HCs in Kagoshima (39·4 years, range 16–64 years, 52·7 % female) and Iran (41·4 years, range 22–73 years, 38·3 % female), respectively. There was no significant difference in age between the control groups (HCs) of the two populations. All HCs in each group originated from unrelated blood donors. Since we extracted Japanese genomic DNA samples from PBMCs but Iranian samples from whole blood, direct comparison of HTLV-1 provirus load between the two populations was inappropriate. Since the main target of HTLV-1 infection is human T cells, whole blood-derived DNA contains more uninfected nucleated cells than PBMCs, and therefore the provirus load data in Iranians was likely to be underestimated if we used {beta}-actin as an internal control. Thus, we compared the HTLV-1 provirus load between HAM/TSP patients and asymptomatic carriers within each population. As shown in Fig. 1, although the HTLV-1 provirus load of Iranian HAM/TSP patients was significantly higher than that of Iranian HCs (P=0·009, Mann–Whitney U test), as reported in Japanese patients (Nagai et al., 1998), the differences in median provirus load between Iranian HAM/TSP patients and HCs (twofold greater in the HAM/TSP patients than in the HCs) was much smaller than that of Japanese subjects (13-fold). Interestingly, although provirus load data were probably underestimated in Iranian samples compared with Japanese samples, the HTLV-1 provirus load in Iranian HCs was still significantly higher than that of Japanese HCs (P=0·004, Mann–Whitney U test).



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Fig. 1. HTLV-1 provirus load of Japanese and Iranian HTLV-1-infected individuals. Mean HTLV-1 copy numbers per 104 PBMCs for Japanese samples and per 104 nucleated cells for Iranian samples determined by quantitative PCR are shown. The HTLV-1 provirus load of Iranian HAM/TSP patients was significantly higher than that of Iranian HCs (P=0·009, Mann–Whitney U test). The difference in median provirus load between Iranian HAM/TSP patients and HCs was much smaller than that of Japanese (Kagoshima) subjects, since HTLV-I provirus load in Iranian HCs is significantly higher than in Japanese HCs (P=0·004). Error bars indicate SEM.

 
HLA-A*02 and HLA-Cw*08 are not associated with a lower risk of HAM/TSP and a lower provirus load in Iranian HTLV-1-infected individuals
To examine whether the previously reported associations between class I and class II HLA alleles and HAM/TSP prevalence in Kagoshima was also valid for HAM/TSP development in the Iranian population, we genotyped HLA-DRB1*0101 and HLA-A*02, HLA-B*5401 and HLA-Cw*08 by PCR-based DNA typing in 132 Iranian HTLV-1-infected individuals (58 HAM/TSP and 74 HCs). All Japanese HLA data had been previously analysed and were extracted from our database (Jeffery et al., 1999, 2000). As shown in Table 1, the genotype frequency of HLA-A*02 and HLA-Cw*08 in Kagoshima subjects was significantly lower among the cases of HAM/TSP compared with HCs (P=0·0006 and 0·0196, respectively). In contrast, the genotype frequency of HLA-A*02 and HLA-Cw*08 was not significantly different between Iranian HAM/TSP and HCs (P=0·346 and 0·940, respectively). Also, whereas HLA-A*02 and HLA-Cw*08 were associated with a lower median provirus load in Kagoshima subjects (P=0·0003 for A*02 and P=0·009 for HLA-Cw*08; Mann–Whitney U test), this effect was not observed in Iranian subjects (P=0·071 for A*02 and P=0·75 for HLA-Cw*08; Mann–Whitney U test; Table 2), indicating that a protective effect of HLA-A*02 and HLA-Cw*08 was not observed in Iranian HTLV-1-infected individuals. As expected, HLA-B*5401, which is known to be almost exclusively found in East Asian populations, was not found in the Iranian subjects analysed.


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Table 1. HLA-A*02 and HLA-Cw*08 are not associated with a lower risk of HAM/TSP in Iranian HTLV-1-infected individuals

 

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Table 2. HLA-A*02 and HLA-Cw*08 are not associated with a lower provirus load in Iranian HTLV-1-infected individuals

 
HLA-DRB1*0101 increases the odds of HAM/TSP development in both Japanese and Iranian HLA-A*02-negative, but not in HLA-A*02-positive, HTLV-1-infected individuals
In contrast to HLA-A*02, HLA-DRB1*0101 was associated with susceptibility to HAM/TSP in both Japanese (P=0·049) and Iranian (P=0·035) populations (Table 3). This effect was observed only in the HLA-A*02-negative subjects but not in the HLA-A*02-positive subjects in both populations (Table 3). Although possession of HLA-DRB1*0101 was associated with a significantly lower provirus load in the Japanese HAM/TSP patients (Table 4, P=0·024) but not in HCs, HLA-DRB1*0101 was not associated with a difference in the provirus load in the Iranian HTLV-1-infected HAM/TSP patients and HCs (Table 4).


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Table 3. HLA-DRB1*0101 increases the odds of HAM/TSP development in Japanese and Iranian HLA-A*02-negative, but not in HLA-A*02-positive, HTLV-1-infected individuals

 

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Table 4. HLA-DRB1*0101 associated with lower HTLV-1 provirus load in Japanese but not in Iranian HAM/TSP patients

The DRB1-positive Japanese HAM/TSP patients developed HAM/TSP with a significantly lower provirus load than DRB1-negative HAM/TSP patients, but this effect was not observed in Iranian HAM/TSP patients.

 
All Iranian HTLV-1 isolates have 10 nt substitutions in the tax region including all the tax subgroup A substitutions
Based on the LTR gene sequence, HTLV-1 can be classified into three types: Melanesian, Central African and cosmopolitan types, while cosmopolitan types can be further classified into subtypes A, B and C (Miura et al., 1994). There are two distinct subtypes in Japan; the most frequently observed (nearly 80 %) Japanese subtype belongs to cosmopolitan subtype B, while a minor subtype (20 %), which seems to cluster in the southern islands of Kyushu and the Ryukyu Islands, belongs to cosmopolitan subtype A. A previous report suggested that, although Mashhadi HTLV-1 isolates belonged to cosmopolitan subtype A, this strain formed a tight cluster that was distinct from the other isolates of cosmopolitan subtype A from Japan, India, the Caribbean Basin and South America (Yamashita et al., 1995). A previous report indicated that the tax subgroup A was more frequently observed in HAM/TSP patients in the Kagoshima cohort and that this effect was independent of HLA-A*02 (Furukawa et al., 2000). The higher HAM/TSP risk tax subgroup A corresponds to the cosmopolitan subtype A, and the lower HAM/TSP risk tax subgroup B corresponds to the cosmopolitan subtype B according to the LTR sequence (Furukawa et al., 2000). We sequenced almost the entire tax region of HTLV-1 provirus (nt 7295–8356) from 20 different Iranian subjects (10 HAM/TSP and 10 HCs) by direct sequencing in both directions. As shown in Table 5, all Iranian HTLV-1 sequences (EMBL/GenBank/DDBJ accession no. AB181224) differed at 10 nt compared with the Japanese prototypic ATK-1 strain (Seiki et al., 1983). Among these, nt 7897, 7959, 8208 and 8344 were exactly the same as those in tax subgroup A. In addition to these four residues, the Iranian tax sequences had 6 nt differences, which encoded four additional amino acid differences from Japanese tax subgroup A. We further performed PCR-RFLP analysis to determine the HTLV-1 tax subgroup (tax A or B) of all of the remaining Iranian samples and found that all Iranian HTLV-1 isolates had tax subgroup A substitutions.


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Table 5. Nucleotide variations specific to Iranian HTLV-1

Amino acid changes in tax A resulting from the nucleotide substitution are shown. Nucleotide numbers correspond to those of the prototypic strain, ATK-1 (Seiki et al., 1983). N, No change.

 
HLA-A*02 is associated with a lower risk of HAM/TSP and a lower provirus load only in HTLV-1-infected individuals with tax subgroup B in Kagoshima subjects
As the majority of HTLV-1 isolates observed in the Kagoshima population were tax subgroup B, we examined further whether the effect of HLA-A*02 on the risk of HAM/TSP and HTLV-1 provirus load was observed only in HTLV-1 tax subgroup B-infected individuals in Kagoshima subjects. Japanese tax subgroup data were extracted from our existing database (Furukawa et al., 2000). As shown in Table 6, the effects of HLA-A*02 on the risk of HAM/TSP and provirus load were not observed in HTLV-1 tax subgroup A-infected subjects in Kagoshima. We next sought a possible interaction between HLA-A*02 and HTLV-1 provirus load among HTLV-1 tax subgroup A-infected subjects in Kagoshima (Table 7). HLA-A*02 was associated with a lower provirus load only in the tax subgroup B subjects in Kagoshima, but not in the tax subgroup A subjects in either Japan or Iran.


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Table 6. HLA-A*02 is associated with a lower risk of HAM/TSP development only in tax subgroup B subjects in Kagoshima

Japanese data were extracted from a database of previous analyses (Jeffery et al., 1999; Furukawa et al., 2000).

 

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Table 7. HLA-A*02 is associated with a lower provirus load only in tax subgroup B subjects in Kagoshima

Japanese data were extracted from a database of previous analyses (Nagai et al., 1998; Jeffery et al., 1999; Furukawa et al., 2000).

 

   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Currently, several different approaches including family-based linkage and population-based case–control studies have been used to identify genetic susceptibility to numerous infectious pathogens such as malaria, mycobacteria, hepatitis viruses and human immunodeficiency virus (Hill, 1998). The candidate gene approach (case–control studies) can only utilize known genes and will not identify unknown genes, but genome-wide linkage studies have less power than candidate gene studies to pick up genes that have only a small or moderate effect on disease risk; therefore the two approaches are complementary. Although our Kagoshima cohort of HAM/TSP is the world's largest, only 300 HAM/TSP patients were available for analysis. Also, extensive studies in one ethnic population may not disclose the marker-disease distance or exclude a possible spurious association due to admixture. Studies in different ethnic populations may thus provide useful information about marker-disease distance, as well as confirming the reliability of results from our previous association studies. In this study, we compared the risk factors for developing HAM/TSP in two ethnic groups living in quite different environments, namely, Kagoshima in southwest Japan and Mashhad in northeast Iran. It is almost certain a priori that there will be significant differences between populations in the genetic contribution to susceptibility to HAM/TSP, since HLA-B*5401 is prevalent in Japan and elsewhere in East Asian populations, but is virtually absent from many other populations. Since HLA-B*5401 has an important influence on the risk of disease in Kagoshima (Jeffery et al., 2000), its presence in the population is certain to influence the risk associated with other HLA alleles, and the absence of HLA-B*5401 in other populations with endemic HTLV-1 infection will alter the relative importance of other genes to the risk of developing HAM/TSP.

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, Mann–Whitney 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*1501–DQB1*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 Tax11–19 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.


   ACKNOWLEDGEMENTS
 
We thank the staff of the Blood Transfusion Center in Mashhad and Neyshabour, the personnel of the Bu-Ali Research Institute and the Faculty of Pharmacology in Mashhad University, and Dr Mahbubeh Naghibzadeh Bajestan for their cooperation, Professor Charles R. M. Bangham of Imperial College, London, for critical reading and comments on the manuscript, and Ms Tomoko Muramoto and Yoko Nishino of Kagoshima University for their excellent technical assistance. This work was supported by the Grant in Aid for Research on Brain Science of the Ministry of Health, Labor and Welfare, Japan.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 9 August 2004; accepted 2 December 2004.



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