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1 Laboratory of Molecular Microbiology, St Petersburg Pasteur Institute, St Petersburg 197101, Russia
2 Research Institute of Phthisiopulmonology, St Petersburg 193063, Russia
Correspondence
Igor Mokrousov
imokrousov{at}mail.ru
or
igormokrousov{at}yahoo.com
Received 18 June 2007
Accepted 11 January 2008
2=3.2, 1 d.f., P=0.2). The –762C allele was highly and almost equally represented in both groups in this study (68.2 % in patients and 69.3 % in controls). This result differs strikingly from a Gambian study where this allele was found in only 7 and 12 % of pulmonary TB patients and controls, respectively [Li, C. M., Campbell, S. J., Kumararatne, D. S., Bellamy, R., Ruwende, C., McAdam, K. P. W. J., Hill, A. V. S. & Lammas, D. A. (2002). J Infect Dis 186, 1458–1462]. In contrast, the frequency of the C allele at position 1513 in exon 13, resulting in a loss of P2X7 function, was significantly higher among pulmonary TB patients in this study (P=0.02). Thus, analysis of the P2X7 receptor gene in the Russian Slavic population showed that the 1513C allele, acting dominantly, is a possible risk factor for clinical TB, whereas the –762 P2RX7 polymorphism did not appear to be associated with human susceptibility to TB.
Abbreviations: CI, confidence interval; HIV, human immunodeficiency virus; HWE, Hardy–Weinberg equilibrium; LD, linkage disequilibrium; OR, odds ratio; SNP, single-nucleotide polymorphism; TB, tuberculosis.
A table showing the results of stratification analysis for the –762 and 1513 genotypes and alleles is available as supplementary material with the online version of this paper.
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INTRODUCTION |
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 TOP INTRODUCTION METHODSRESULTSDISCUSSIONREFERENCES |
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M. tuberculosis is a facultative intracellular pathogen, and macrophages act both as the principal reservoir of infection and as the primary effector cells regulating bacterial growth and viability. ATP ligation of P2X7 receptors expressed on human macrophages that are infected with mycobacteria induces cell death and subsequent loss of intracellular bacterial viability. The ATP-mediated killing of mycobacteria by macrophages is independent of other mechanisms, linked to SCL11A1 (formerly NRAMP1), VDR, HLA, IL, MBL and some other human genes (reviewed by Bellamy, 1998, 2005; Marquet & Schurr, 2001). The P2X7 response to ATP has been shown to involve a calcium-dependent and phospholipase D-dependent effector pathway, which induces mycobacterial death via the promotion of phagosome–lysosome fusion (Fernando et al., 2007; Li et al., 2002).
The human P2RX7 gene encoding the P2X7 receptor comprises 13 exons localized on chromosome 12q24 (Buell et al., 1998). A homozygous A
C substitution in exon 13 at position 1513 is associated with loss of P2X7 function, whereas the heterozygous state results in cells with half the function of cells with the germline P2X7 protein (Gu et al., 2001). Additionally, a protective association against TB in the Gambian population in West Africa has been found for a single-nucleotide polymorphism (SNP) at nucleotide position –762 in the putative P2RX7 promoter (Li et al., 2002).
This study is the first evaluation of the allelic distribution and possible link to susceptibility to TB of these two P2RX7 polymorphisms in the Russian Slavic population. We also compared our results with available data on these SNPs in other human populations.
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METHODS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Genotyping. DNA was extracted from whole blood with a DNA Sorb B kit (AmpliSens). A PCR-RFLP assay was used to detect the A/C polymorphism at position 1513 in exon 13 in P2RX7, as described previously (Li et al., 2002).
An allele-specific PCR assay was used to detect the C/T polymorphism at position –762 in P2RX7 (position 1014 in GenBank accession no. Y12851) as follows. Two outer primers, P2X73 (5'-GAAACAGGGCCCTGGGTCCTC-3', forward) and P2X74 (5'-TGGTGGGGGTGGAGGGGC-3', reverse) flanked the entire region under study and amplified a 373 bp fragment in all strains. Two allele-specific inner primers, P2X75 (5'-GGTGTCCCTCACTGAATAGGTCAAT-3', forward), and P2X76 (5'-GGCAGTCCAACAAAGTTAGGTTTG-3', reverse), were also used. For the –762C allele, a 235 bp fragment was amplified using the outer forward (P2X73) and inner reverse (P2X76) primers. For the –762T allele, a 186 bp fragment was amplified using the inner forward (P2X75) and outer reverse (P2X74) primers. Purified DNA (1 µl) was added to the PCR mixture (final volume of 20 µl) containing 1.5 mM MgCl2, 1 U Taq DNA polymerase (Sileks), 100 µM each dNTPs, and primers P2X73 and P2X76 (5 pmol each) and P2X74 and P2X75 (20 pmol each). The reaction was performed in a PTC-100 thermal controller (MJ Research) under the following conditions: initial denaturation at 96 °C for 3 min; five cycles of 95 °C for 45 s, 68 °C for 1 min and 72 °C for 30 s; five cycles of 95 °C for 40 s, 67 °C for 50 s and 72 °C for 30 s; 22 cycles of 94 °C for 50 s, 66 °C for 40 s and 72 °C for 30 s; and a final elongation at 72 °C for 3 min. The amplified PCR fragments were subjected to electrophoresis in 1.5 % standard agarose gels.
Statistical analysis.
The existence of population subdivision among the case and control sets was tested using Wright's F statistics according to the non-biased method of Weir and Cockerham (
value; Weir & Cockerham, 1984) using GDA software (Lewis & Zaykin, 2001). Bootstrap tests (1000 replicates) were performed to determine results that were significantly different from 0.
Genotype frequencies in each group were tested for correspondence to Hardy–Weinberg equilibrium (HWE). Linkage disequilibrium between the two SNPs was evaluated by calculating the normalized D' measure of Lewontin (1964). The statistical significance (P value) of the pairwise disequilibrium was tested by excluding within-locus HWE. These analyses were carried out using GDA software (Lewis & Zaykin, 2001) using 1000 runs.
Overall genotype frequencies in each patient group were compared using a 3x2 2 test with 2 degrees of freedom (d.f.). A 2x2 2 test was also used: (i) to detect any significant difference between the two groups in allele frequencies [Yates-corrected 2 and P values were calculated with a 95 % confidence interval (CI)] and (ii) to test dominant and recessive models. This analysis was carried out using EpiCalc 2000 version 1.02 software (Gilman & Myatt, 1998).
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RESULTS |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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shows the results of genotyping of the two P2RX7 polymorphisms, 1513 A/C and –762 C/T.
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All genotypes in both loci were found to be in HWE (Table 1
), although a slight under-representation of the –762 heterozygotes was noted in the control goup (Hobs=0.365 compared with Hexp=0.429). The linkage disequilibrium (LD) analysis revealed the major alleles of the two loci to be in a weak LD (D'=–0.36, P=0.04) in the combined sample of the patient and control groups. Based on published recommendations (Ardlie et al., 2002; Tiret et al., 2002), we considered this LD not to be sufficient to influence a possible independent (non-)association of these two SNPs with TB.
Analysis of the –762 P2RX7 promoter SNP revealed no significant difference in the genotype distribution in the two groups in our study using a 3x2 2 test (P=0.2). The –762C allele was both highly prevalent and almost equally represented (68–69 %) in both groups in our study [2x2 2=0.05, odds ratio (OR)=0.95 (95 % CI 0.67–1.34), 1 d.f.; P=0.8].
For the 1513 polymorphism, an overall difference in genotype distribution between the two groups was at borderline significance level (3x2 2=3.2, 2 d.f.; P=0.06). A closer look at the genotype distribution revealed that the wild-type AA homozygotes were significantly more prevalent in healthy controls (P=0.02), whereas AC heterozygotes, in which half of the P2X7 function was lost, were already significantly associated with the TB patient group (P=0.05). Taken together, the frequency of the 1513C mutant allele was significantly higher in the TB patient group compared with the control subjects [Table 1; 2x2 2=5.2, OR 1.71 (95 % CI 1.10–2.66), 1 d.f.; P=0.02].
The effect of the P2RX7 gene variants was assessed using both recessive and dominant models. This revealed that the overall OR for the 1513C deleterious allele as a risk factor was 1.81 (95 % CI 1.09–3.01, P=0.03) under a dominant model and 2.06 (95 % CI 0.55–7.77, P=0.4) under a recessive model. Testing of alleles in the –762 position revealed no statistical significance of either dominant or recessive model (P=0.3 in both cases).
The frequency data of the two SNPs were further compared by stratification of the studied groups by sex or age, or, for patients, by diagnosis. This comparison revealed no significant difference in the –762 and 1513 genotypes and alleles (see Supplementary Table S1, available in JMM Online, for detailed information).
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DISCUSSION |
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TOPINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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We found that the P2RX7 1513C allele was associated with an increased risk of pulmonary TB in Russians (Table 1). This is not unexpected in view of the available knowledge of the role of this P2X7 mutation in clinical development of TB in West Africans (Li et al., 2002) and the Australian Vietnamese population (Fernando et al., 2007), although the latter study found an association with extrapulmonary rather than pulmonary TB. A role of selection in the population frequency differences of this allele is possible but is not clear at this stage. In the Russian setting, testing of dominant versus recessive models for the major allele 1513A revealed a statistical significance for the recessive model. In fact, this implies a dominant model for the minor, mutant allele 1513C, for which deleterious action is manifested even in the heterozygous state.
No data have been published yet on the –762 P2RX7 SNP from areas of the world other than West Africa. A case–control study of the Gambian population showed that the –762CC genotype was found more frequently in controls (P=0.03), whilst the –762C allele appeared to be associated with a protective effect against TB (P=0.003; Li et al., 2002). In the Gambia, the C allele was found in 32.9 vs 25.4 % and the CC genotype in only 12.7 vs 7.1 % of controls and TB patients, respectively. In contrast, the Russian Slavic population showed much higher frequencies of both the –762C ‘protective’ allele and CC homozygotes in both TB patients and controls groups (Table 1); the reason, albeit speculative, may lie in selection unrelated to TB and acting on this locus or another in LD with it. The sixfold difference in the frequency of the –762C allele in the Russian and Gambian populations is striking and goes far beyond a genome variation observed among human populations. Thus, it appears that variation in the –762 position is not linked to human susceptibility to TB, at least in Caucasians, although further studies in other settings are required.
In conclusion, analysis of these two SNPs in the P2RX7 gene in the Russian Slavic population showed that the P2RX7 1513 polymorphism is probably associated with host control of M. tuberculosis infection. The 1513C allele, acting dominantly, is a possible risk factor for clinical TB, whereas the –762 C/T P2RX7 polymorphism does not appear to be associated with human susceptibility to TB.
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ACKNOWLEDGEMENTS |
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