Vitamin D receptor gene polymorphisms and susceptibility M. tuberculosis in Native Paraguayans
Introduction
Tuberculosis (TB) is a leading cause of death worldwide,1 and differences in susceptibility to it have been noted for decades.1, 2, 3 Because susceptibility to TB is multifactorial, polygenic, and displays population heterogeneity,4, 5, 6, 7, 8, 9, 10, 11 a broad array of human populations must be examined to elucidate commonalities and differences that may ultimately help communities and governments develop effective prevention and treatment strategies.
Few susceptibility studies have focused on Native American populations, despite extremely high TB prevalence. The Aché of Paraguay, for example, experience prevalence of infection greater than 30%, annual incidence of active pulmonary TB of 7%, and lifetime prevalence of active TB greater than 30%.12 A complex interplay between host genetics, and environmental and cultural factors likely contribute to these high rates. The Aché present a unique opportunity to analyze the effects of these factors separately, because the entire extant population shares similar socio-economic, nutrition, and exposure conditions, and further, genealogical information has been extensively documented.13 Here, vitamin D receptor (VDR) polymorphisms are examined in conjunction with the presence of pulmonary TB disease outcomes in the Aché and a neighboring group, the Avá.
The VDR is involved in a wide range of biological functions, including mediation of vitamin D3 interactions with the immune system.14, 15, 16, 17 Among Gujarati Asians living in the UK, a dose-response effect of vitamin D3 serum levels and risk of TB is seen,18 and two VDR gene polymorphisms,19 in combination with low serum vitamin D3, were strongly associated with pulmonary TB. The first, a TaqI RFLP in exon 9 (allele t), increases stability of the mRNA, and was also associated with pulmonary TB in native Gambian populations.8 There, the tt genotype was underrepresented in pulmonary TB patients. In India,20 the tt genotype was associated with the less severe tuberculoid form of leprosy (another mycobacterial disease), while the TT genotype was associated with lepromatous leprosy. These data imply21 an influence over the host's T-helper immune response, with tt assisting a shift to Th1.
The second polymorphism, a FokI restriction site in exon 2, increases the length of the protein.19 The F isoform has moderately higher transcriptional activity than the f variant.22, 23 Among UK Gujarati Asians, the ff genotype was associated with increased TB susceptibility, particularly extrapulmonary TB.18 In a predominantly Native Peruvian community, individuals homozygous for F were significantly more likely to have positive treatment outcome than Ff or ff individuals.1 Individuals of the Tt genotype were also more likely to have positive, and more rapid, treatment outcome than TT patients.
In south Indians, the TaqI, and FokI variants, as well as two others, a BsmI and an ApaI RFLP both in intron 8, were examined in a case-control study involving patients with spinal TB.24 The BsmI Bb genotype was found to be elevated in patients, as was the FF. Further, individuals with the BbAATtFF genotype were 7.2 times more likely to have spinal TB, and the batF haplotype was increased in spinal TB patients, while the BAtf haplotype was significantly increased in controls. In a companion study, the FF genotype was associated with active pulmonary TB among males,25 while in females,26, 27 the tt genotype was associated with spinal TB. Although VDR data appear to conflict across populations, this may be explained by the multiple roles of vitamin D3. Selvaraj et al.24 suggest that higher utilization of vitamin D3 for those with the FF genotype may result in lowered serum vitamin D3, increasing risk for spinal TB.
Given the associations of the VDR TaqI and FokI variants with TB in other populations, as well as the high prevalence of TB among Native South Americans,12 we examined the ApaI, TaqI and FokI polymorphisms in the Aché and Avá of Paraguay in relation to TB status. Among the Aché, we used a family-based analysis to test for allelic assocaition between the variants and three different TB outcomes. Among the Avá, we were able to examine a small number of individuals in conjunction with exposure to TB only.
Section snippets
Population
The Aché are an indigenous population from eastern Paraguay. Until the 1970s, when they came into permanent contact with outsiders, Aché lived in small mobile hunting and gathering bands of 15–70 individuals. Currently there are approximately 1000 Aché living in several agricultural settlements. Extensive historical, demographic, social, and medical data have been compiled for the Aché, including a complete genealogy for nearly the entire population.13
The Avá are also an indigenous population
Genotypes
Forty-one Aché and six Avá samples were sequenced at the exon 2 fragment containing the FokI polymorphism and at the intron 8/exon 9 fragment containing the TaqI and ApaI polymorphisms. No further variation was found in the exon 2 fragment in either population, and all samples were then typed for presence/absence of the restriction site using the FokI enzyme. In all cases for which there were both sequencing and restriction digest results, these were identical. Table 1 presents the distribution
Discussion
No associations were found between VDR polymorphisms and Avá TB status. At the TaqI site, no significant differences were noted by any measure of PCR positivity, but the T allele was more likely to have been transmitted to Aché offspring who had positive active disease. Individuals with the TT genotype, however, are significantly less likely to have a positive PPD than those with a copy of t. The PCR results indicate that Aché of all TaqI genotypes were equally likely to be exposed to M.
Acknowledgments
This work was supported by several programs: the National Science Foundation Dissertation Improvement (♯0334849) and Cultural Anthropology programs, and the Wenner Gren Individual Research, and National Institutes of Health Fogarty International programs (R01-TW005627-02S1). Funds were provided by the Diebold Foundation for collection of a portion of the samples. We thank the UNM School of Medicine for donated DNA from M. tuberculosis, used as a positive control. Joseph Feurstein, Jamie Smith
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