Association of VDR Gene Polymorphism and Haplotype with Tuberculosis Susceptibility, Treatment Outcome, Bacillary Load and Extrapulmonary Site
Published On July 10, 2015
Association of VDR Gene Polymorphism and Haplotype with Tuberculosis Susceptibility, Treatment Outcome, Bacillary Load and Extrapulmonary Site
Singla N1, Gupta D2, Birbian N1, Joshi A3, Batra N4, Singh J1*
1Department of Biotechnology, Panjab University, Chandigarh-160014, India
2Department of Pulmonary Medicine, PGIMER, Chandigarh-160012, India
3Department of Biotechnology, SGGS College Sector-26, Chandigarh-160019, India
4Department of Biotechnology, GGDSD College Sector-32, Chandigarh-160032, India
*Corresponding author: Jagtar Singh, Department of Biotechnology, Panjab University, Chandigarh 160014, India, Tel: +91-172-253-4065; Fax: +91-172-254-1409; E-mail: firstname.lastname@example.org
Citation: Singla N, Gupta D, Birbian N, Singh J (2015) Association of VDR Gene Polymorphism and Haplotype with Tuberculosis Susceptibility, Treatment Outcome, Bacillary Load and Extrapulmonary Site. J Mole Biol Tech 1(1): 101.
Background: A case-control study was conducted to study three VDR gene polymorphism, FokI, BsmI and TaqI, with Tuberculosis (TB)-susceptibility, treatment outcome, bacillary load and site of extrapulmonary TB.
Methods: Three hundred and Fifty seven TB patients and 392 healthy unrelated controls with no clinical symptoms or family history of TB belonging to same ethnicity were enrolled.
Results: This study reported a significant association of ‘ff’ genotype of VDR FokI gene with susceptibility to TB. These results were highly significant in extrapulmonary cases (EP), pleural effusion patients carrying polymorphic variant ‘f’ were at significantly higher risk of developing the disease where as presence of ‘b’ allele of BsmI variant contributed to protection against disease. BsmI polymorphism analysis reported a protective role of ‘bb’ genotype against TB. Treatment outcome analysis revealed ‘Bb’ genotype conferring risk in patients with unsuccessful treatment when compared to those with successful treatment. To the best of our knowledge no other study has reported a similar association. Low bacilli load was reported to be significantly higher in ‘ff’ carriers when compared to control group whereas ‘bb’ genotype was reported to confer protection against the same. Data in this study showed a positive association of smoking status and TB risk. Forty nine percent of smoker males belong to < 35 age-group. The frequency of TBf and TBF haplotype was reported to be significantly higher in case group when compared to control group. Haplotype TbF was found to be more frequent in control group than cases.
Conclusion: A significant association of VDR gene polymorphisms and haplotypes with disease susceptibility, treatment outcome, bacillary load and site of extrapulmonary TB along with smoking status and TB susceptibility was found.
Keywords: VDR; Gene polymorphism; Haplotype tuberculosis susceptibility; Bacillary load; Extrapulmonary TB
India accounts for an estimated one quarter (24%) of all the global tuberculosis (TB) incident cases alone . It is estimated that annually around 2,40,000 Indians die due to TB . Many investigations have confirmed that host genetic factors are involved in the tuberculosis disease, and these include adoption studies , twin studies , genome wide linkage  and population-based case–control association studies [5-7]. Recently, a number of genes have been studied in various case-control studies with reference to tuberculosis, among which are NRAMP1 (natural resistance associated macrophage protein one), HLA-DQB1 (major histocompatibility complex, class II, DQ beta-1) and VDR (vitamin D receptor). The VDR gene is of particular interest as Vitamin D modulates monocyte-macrophage activity in the body and plays a role in human innate immunity to infectious agents like Mycobacterium tuberculosis. Vitamin D exerts its actions through vitamin D receptor (VDR), a nuclear hormone receptor. Liver metabolises Vitamin D into 25-hydroxyvitamin D3 (calcidiol/25(OH) 2D3), which is further converted to 1, 25 (OH) 2D3 by kidneys (active form of vitamin D). A meta-analysis recently showed that vitamin D deficiency might play a role as risk factor during the development of TB .
The VDR gene is located on the long arm of chromosome12 (12q12-14) and is composed of 10 exons, the first of which is not transcribed . Polymorphisms in the VDR gene, which may influence VDR activity and subsequent downstream vitamin D-mediated effects, are therefore studied as potential candidates of risk markers for various clinical outcomes . Three of these VDR polymorphisms can be distinguished by digestion with restriction enzymes. Two of them do not change the translated protein (Bsm I and Taq I). The polymorphisms recognized by BsmI (BB, Bb and bb) are located in intron 8 of the VDR gene and the one recognized by Taq I (TT, Tt and tt ) is located in exon 9, that leads to a silent codon change, with ATT and ATC, both coding for isoleucine and has been associated with increased VDR mRNA stability. FokI VDR polymorphism recognized by Fok I restriction enzyme (FF, Ff and ff), results from a C→T transition that creates an alternative initiation codon (ATG), and three codons from the downstream start site. The VDR encoded by the f allele from the Fok I restriction enzyme recognition is increased in length by three amino acids, and the transcription of this allele is 1-7 times less efficient than the F allele and can alter the amount of VDR produced .
There is strong evidence to suggest that VDR gene variants may be related to the development of TB [8-12]. However, these polymorphisms were seemed to play different roles in different populations. Gao et al  evaluated VDR gene polymorphism in different ethnic population studies published prior to April 2009 with regard to TB followed by a recent meta-analysis conducted by Cao et al.  in Chinese Han population. A significant association was observed for Asian subjects carrying different VDR gene variants .
Vegans and other vegetarians who limit their intake of animal products may be at greater risk of vitamin D deficiency than non-vegetarians, because foods providing the highest amount of vitamin D per gram naturally are all from animal sources . Asians from the Indian subcontinent having vegetarian diet were reported to have vitamin D deficiency, known to affect immunological competence which might result in increased mycobacterial reactivation . To check for the same we included the nutrition status of our subjects and analysed them with respect to VDR genotype. Recently Sharma et al.  reported an association of VDR polymorphism with bacillary load. Validation of this preliminary report in a different population is attempted in this study.
To the best of our knowledge, no studies have reported the influence of VDR genes on outcome to TB treatment in north Indian population. We therefore, conducted a well characterised case-control study and a number of other demographic and clinical factors to reproduce the reports of the role of VDR polymorphism with TB in our study population as well as association of the same with treatment outcome, bacillary load and site of extrapulmonary TB.
Three hundred and fifty seven TB patients were enrolled for this longitudinal study after taking a written consent from each subject, from DOTS centre Rampur Bushahr, District Shimla of Himachal Pradesh (H.P.). These tuberculosis patients were classified into five groups according to Revised National Tuberculosis Control Programme (RNTCP) standard classification: 1) Extrapulmonary (EP) 2) Pulmonary Negative (PN; sputum smear negative pulmonary patients) 3) Pulmonary Positive (PP; sputum smear positive pulmonary patients) 4) Pulmonary All (PTB, Pulmonary Positive + Pulmonary Negative) and 5) TB All (TB, Pulmonary All + Extrapulmonary). Following WHO protocol the sputum smear status of subjects was established after screening of slides for Tubercle bacilli and radiological (X-ray) investigations . Fifty patients (14%) out of 357 cases were relapse case belonging to pulmonary positive, negative as well as extrapulmonary category. According to WHO guidelines for disease classification of extrapulmonary TB on the basis of disease severity the patients could be divided into two categories: Severe (meningitis, miliary, pericarditis etc.) and Less-severe (lymph node, pleural effusion, abdominal etc.) . EP cases here included 25 abdominal, 1 breast, 16 lymph node, 27 pleural effusion, 1 spine and 1 urinary tract case. For this study the study population was stratified on the basis of sputum bacillary load, EP site and treatment outcome. For analysis of treatment outcome and VDR gene polymorphism, the outcome was stratified into percentages of successful and unsuccessful outcomes, among all patients who underwent standard Directly Observed Treatment, Short-course (DOTS) therapy. For this part only pulmonary positive patients (PP) were included as their follow-up sputum smear reports were available thus, making assess of disease outcome convenient and unbiased. Integrated Counselling and Testing Centres (ICTCs) for HIV counselling and testing services was started in March 2009 in our hospital. None of the study patients taken after the date of start of this facility was found to be HIV positive.
Three hundred and eighty healthy unrelated controls with no clinical symptoms or family history of TB belonging to same ethnicity were taken. A strict exclusion criterion was applied for preventing biasing of the population by selecting only those patients who were settled in this area from minimum of three generations. Marriage within the local community is a common trend observed in this population. The study was approved by Postgraduate Institute of Medical Education and Research (PGIMER), Chandigarh and Indira Gandhi Medical College (IGMC), Shimla (H.P.).
One millilitre peripheral blood was taken and genomic DNA was then isolated by phenol-chloroform method  for the purpose of genotyping. Primers were used as in Wilkinson et al. . PCR conditions for VDR TaqI polymorphism included initial 95 oC for 4 minutes followed by 35 cycles at 95 oC for 30 seconds, annealing at 60 oC for 30 seconds and extension at 72 oC for 90 seconds and a final extension at 72 oC for 7 minutes. For FokI polymorphism conditions included initial 96oC for 1 minutes followed by 30 cycles at 94 oC for 45 seconds, annealing at 61.5 oC for 45 seconds and extension at 72 oC for 60 seconds and a final extension at 72 oC for 5 minutes. BsmI product was amplified by initial 96 oC for 1 minutes followed by 30 cycles at 94 oC for 60 seconds, annealing at 60 oC for 60 seconds and extension at 72 oC for 60 seconds and a final extension at 72 oC for 1 minutes.
The PCR product was digested with excess of restriction enzyme 6-8 hrs (TaqI and BsmI at 65 oC and FokI at 37 oC). The digested product was run on 2% agarose gel pre stained with ethidium bromide and visualised using a UV transilluminator. The presence of a restriction site was assigned a lowercase letter and its absence an uppercase letter, according to convention (i.e., b and B for BsmI, f and F for FokI, and t and T for TaqI) (Fig 1, 2 and 3, respectively).
Figure 1: Genotyping of vitamin D receptor (VDR) BsmI polymorphism was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Lane 1, 2 and 4: polymorphic variant, Lane 3: wild, Lane 5-8: heterozygote and, Lane 9: 100bp marker.
Figure 2: Genotyping of vitamin D receptor (VDR) FokI polymorphism was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Lane 1,3,4,6 and 7: wild type, Lane 2: heterozygote, Lane 5: polymorphic variant and Lane 8: 100bp marker
Figure 3: Genotyping of vitamin D receptor (VDR) TaqI polymorphism was done using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Lane 1 and 5: wild, Lane 2 and 4: heterozygote, Lane 6: mutant and Lane 3: 100bp marker
SPSS Ver. 17 and Epi Info (TM) 3.4.3 were used for statistical analysis. Odds ratio (OR) and 95% confidence intervals (95% CI) were calculated. The overall genotypic frequency was compared between the groups using 3x2 χ2 test and the allelic frequency was compared by 2x2 χ2 test. All data were considered significant when p < 0.05. Whenever an expected cell value was less than 5, Fisher’s exact test was used. Construction of the VDR haplotypes was done with the help of Plink ver. 1.07-dos , pairwise linkage disequilibrium estimation and D’ calculation was performed using the Haploview ver. 4.2 .
Demographic characteristics of case and controls
A total of 737 subjects, including 357 tuberculosis patients (37% females) and 380 healthy controls (46% females) belonging to same ethnicity were analysed for VDR gene polymorphism and its role in TB susceptibility. All the three polymorphism studied were found in Hardy Weinberg Equilibrium. The details of the baseline characteristics of the studied population are given in Table 1. The mean age of TB patients and controls was 33.14 ± 15.42 and 36.33 ± 14.87, respectively. 61.60% of the cases and 50% of the controls belonged to age-group < 35 years. Ever-smoker’s were reported to be at higher risk of developing TB (p < 0.001, OR 2.61 95% CI 1.90-3.59) as compared to controls. Majority of cases and controls lived in Semi-pucca houses (63.30% cases and 70% controls). Dietary regime analysis, as a possible indicator of Vitamin D deficiency in vegetarians as compared to non-vegetarians, showed a majority of study subjects following non-vegetarian diet-style (75% cases and 69% controls) (p = 0.11, OR 0.77 95% CI 0.55-1.08). 12% of the cases were exposed to the disease whereas the exclusion criteria for controls were no exposure to TB and thus none of the healthy controls was exposed to disease. Indian education system has been divided into primary (up to 14 years of age), secondary (14-18 years of age) followed by higher education including college and professional courses. Stratification on basis of educational status, a risk factor for disease as well as an indicator of socio-economic status, majority of case pursued higher education (41%) followed by primary (29%) whereas in controls majority received primary (37.80%) followed by secondary education(30.60%) (Table 1).
Table 1: Baseline characteristics of all tuberculosis cases and controls
VDR genotype in cases and controls
Genotypic distribution of VDR BsmI polymorphism was first analysed using 3x2 χ2 test (Table 2) reporting an overall genotypic difference between the case groups and controls. A closer look at the same showed a significantly higher prevalence of ‘bb’ genotype in the control group as compared to the case groups; EP(p < 0.01, OR 0.33, 95% CI 0.14-0.79), PN (p = 0.01, OR 0.33, 95% CI 0.12- 0.86), PP (p < 0.001, OR 0.42 95% CI 0.24-0.71) and PTB (p < 0.001, OR 0.40 95% CI 0.24-0.65) and TB (p < 0.001 OR 0.38 95% CI 0.24-0.60) groups. The allele frequency distribution showed a significantly higher frequency of ‘b’ allele in the control group as compared to all case groups (Table 2).
Table 2: Frequency distribution of the BsmI polymorphism of VDR in study population. (aCI=confidence interval, OR=odds ratio)
Genotypic distribution of VDR FokI polymorphism showed a significant difference between case groups and controls (Table 3). Closer look at the same showed a significantly higher prevalence of ‘ff’ genotype in the case groups; EP (p <0.001, OR 5.86 95% CI 2.07-16.50), PP (p = 0.02, OR 2.36 95% CI 1.07-5.26), PTB (p = 0.02, OR 2.25 95% CI 1.05-4.85) and TB (p < 0.01, OR 2.72 95% CI 1.33-5.64). In case of EP patients, presence of a polymorphic variant (‘f ’) was observed to be significantly associated with disease (p < 0.001, OR 2.08 95% CI 1.40-3.09) (Table 3).
Table 3: Frequency distribution of the FokI polymorphism of VDR in study population (OR=odds ratio, CI=confidence interval)
For TaqI variant of VDR gene no overall significant association between the allele and genotype distribution of case groups and control group was observed (Table 4).
Table 4: Frequency distribution of the TaqI polymorphism of VDR in study population (OR=odds ratio, CI=confidence interval)
VDR genotype and disease outcome
As per WHO definition of cases, successful cases included cured and patients who completed their treatment (174) and unsuccessful included failures and patients who died during treatment (22) . Thirty two patients were excluded as they were either transfer-out or defaulters (the patient who did not take the medication for two consecutive months).
Tendency was reported for carriers of VDR BsmI heterozygote (Bb) and disease outcome (p = 0.5, OR 3.38 95% CI 0.91-18.66) (Table 5). No significant association was reported between overall risk of unsuccessful treatment and genotype and allele distribution for any other VDR variant studied (Table 5).
Table 5: Association between Treatment Outcome and VDR Gene Polymorphism (OR=odds ratio, CI=confidence interval, * Successful cases included cured and patients who completed their treatment, ** Unsuccessful cases included failures and patients who died during treatment)
VDR genotype and Bacillary load
Microscopically diagnosed sputum smear positive patients were further subcategorized into two clinical groups according to their bacilli load 1+ as low bacillary load and 2+ and 3+ as high bacillary load (Table 6).
Of the 228 pulmonary patients included for this analysis 147(64.50%) were high bacillary load carriers (2+ and 3+) and 81(35.50%) were low bacillary load carriers (1+). Each of these categories was separately compared with healthy controls for each genotype. Variant genotype (ff) of VDR FokI variant was seen to be associated with risk of having low bacillary load as compared to controls (p < 0.01, OR 3.68 95%CI 1.35-9.67) (Table 6). However, the ‘bb’ variant of VDR BsmI gene was observed to be significantly higher in control group when compared to low bacilli load carriers (p < 0.001, OR 0.23 95% CI 0.09-0.55) (Table 6). Tendency was also observed for VDR Taq I mutant genotype ‘tt’ and low bacillary load on comparison with the control group (p = 0.05, OR 2.24 CI 0.91-5.42). No such association was observed for high bacillary load carriers and control group (Table 6).
Table 6: Association between Bacilli Load and VDR Gene Polymorphism( OR=odds ratio, CI=confidence interval, * High load constitutes 2+ and 3+ bacilli load patients, ** Low load constitutes 1+bacilli load patients)
VDR genotype and Extrapulmonary Site
EP site and VDR polymorphism analysis consisted of 71 patients. 68 patients belonging to abdominal, lymph node and pleural effusion EP TB were selected (Table 7). With respect to pleural effusion site, presence of polymorphic variant ‘f’ for VDR FokI gene was reported to be highly associated with risk of developing the disease (p < 0.001, OR 3.39 95% CI 1.87-6.15). However, polymorphic variant for VDR BsmI gene (b) was observed to confer protection against Pleural TB (p < 0.01, OR 0.47 95% CI 0.25-0.87). No association was reported for any other EP site and VDR gene polymorphism (Table 7).
Table 7: Association of Extrapulmonary Site and VDR Gene Polymorphism (OR=odds ratio, CI=confidence interval)
Linkage disequilibrium analysis and Haplotype analysis
Pair-wise linkage disequilibrium (D’) was analysed between all three studied polymorphisms. TaqI was not linked to either BsmI or FokI (D’ = 0.005 and D’ = 0.03). No linkage disequilibrium (LD) was observed between BsmI and FokI either (D’ = 0.04). On haplotype analysis, the frequency of TBf and TBF haplotype was reported to be significantly higher in case group when compared to control group (p = 0.03 and p = 0.03, respectively). Haplotype TbF was found to be more frequent in control group than cases (p < 0.001) (Table 8).
Table 8: Three locus haplotypes for VDR TaqI, BsmI and FokI gene polymorphism for Tuberculosis cases and controls.
A non-silent VDR gene polymorphism is the FokI polymorphism, found in exon 2, which is at a translation initiation start site and is predicted to change the structure of the coded protein. Although the difference between the two proteins is only three amino acids, it has been suggested that the more commonly observed shorter VDR protein is functionally more active . This study reported a significant association of ‘ff’ genotype and ‘f’ allele with susceptibility to TB, as reported by Sharma et al . The role of ‘ff’ genotype has been found to confer a significantly increased risk to TB among Asians but no evident association was observed among African . These results were highly significant in extrapulmonary cases (‘f’ allele frequency 0.38 versus 0.23 in controls). On further stratification of EP cases in based on site of disease, pleural effusion patients carrying of polymorphic variant ‘f’ were at significantly higher risk of developing the disease where as presence of ‘b’ allele of BsmI variant may contribute to protection against disease. Results of this study were in concordance to those observed in a Gujarati Indian population  living in London having several forms of extrapulmonary tuberculosis as well as blacks with extrapulmonary TB, studied by Motsinger-Reif et al.  and spinal TB study in Chinese Han population .
Two of the most widely studied VDR polymorphisms are located in 3′ untranslated region- TaqI and BsmI, which have been shown to be related to VDR activity or expression . For BsmI polymorphism we found a significantly higher prevalence of ‘bb’ genotype in the control group when compared to the case groups inferring the protective role of this genotype against TB. These results are consistent with those concluded by a meta-analysis reporting a significant decreased risk for ‘bb’ carriers against TB [8,9]. Ates et al.  reported an overrepresentation of ‘B’ allele in case-group (48% compared to 30% in controls) similar to our results (59% in cases and 48% in controls)
With regard to TaqI polymorphism, no association was reported in this study similar to those reported in East Indian , Tanzanian , South African  and a recent analysis done on Chinese Han population . However, a South Indian reported an association of ‘tt’ genotype with PTB in females . This difference could be contributed to Indo-Dravidian origin of the later compared to Indo-Aryan population in this study.
WHO’s strategy and framework for effective TB control includes evaluation of treatment outcome of TB patients undergoing standard therapy . Treatment success is defined as sum of patients cured and those who completed their treatment . Analysis of VDR gene polymorphism and treatment outcome revealed a higher prevalence of ‘Bb’ genotype in patients with unsuccessful treatment when compared to those with successful treatment. To the best of our knowledge no other study has reported a similar association between treatment outcome and VDR gene polymorphism.
Low bacilli load was reported to be significantly higher in ‘ff’ carriers when compared to control group whereas ‘bb’ genotype was reported to confer protection against the same. A recent study on Indian population, however, reported an association of ‘tt’ genotype with high bacillary load not found in this study population . The later might be contributed to difference in ethnicity between two populations as well as difference in study design.
Demographic analysis of studied population revealed that 61.60% of the cases belonged to < 35 age-group which includes not only the socio-economically more important generation but also woman of reproductive age. Data in this study showed a positive association of smoking status and TB risk. 90% of the ever-smoker cases were males as smoking has been widespread among Indian males since many decades but to our surprise 49% of these males belonged < 35 age-group. A study by Gajalaxami et al.  underlined that mortality from TB is 4 times as greater among smokers than non-smokers. It has been suggested that the link between VDR polymorphisms and disease susceptibility might be modulated by vitamin D status. Stratchan et al. reported that vitamin D deficiency, associated with a vegetarian diet, is known to affect immunological competence and increased mycobacterial reactivation among Asians from the Indian subcontinent. To validate this aspect in our population, we included the nutrition status of cases and controls. But to our surprise 75% of the cases were non-vegetarian compared to 69% controls. Therefore, we might conclude that vegetarian diet is not one of the risk factors for TB susceptibility in our population.
In conclusion, this study reported VDR gene variant to play an important role in TB susceptibility as well as treatment outcome and bacillary load. The study population belongs to a small town in Himachal Pradesh with subjects living in that area from a minimum of three generations and marriage within the community is a common trend providing an unbiased population for the study. Therefore, we believe that similar studies in different populations within India as well as outside India will definitely prove beneficial with regard to better understanding of immunogenetics of TB. The era of personalised medicine and genetic markers have the potential to be used for early targeting of a high burden population or population at higher risk to TB. We strongly recommend that the variant homozygous genotype of FokI variant of VDR (‘ff’) should be further explored with reference to extrapulmonary tuberculosis in different ethnic groups.
J. Singh is thankful to the Council of Scientific & Industrial Research, New Delhi, India, for providing research grant [F. No. 27(280)/12-EMR-II].
We acknowledge the help of DOTS unit Rampur Bushahr for providing the samples. We thank Dr. Surinder Kashyap, Principal, IGMC, Shimla, Dr. Uppal, Dr. Bhatia, Dr. Hemant, Mr. Kaundal and Mr. Singh for their technical assistance.
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