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The Lancet 2000; 355:618-621
DOI:10.1016/S0140-6736(99)02301-6
Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study
Robert J WilkinsonPhD a b, Martin LlewelynMRCP b, Zahra ToossiMD a, Punita PatelMSc b, Geoffrey PasvolDPhil b, Ajit LalvaniMRCP c, Dennis WrightPhD b, Mohammed LatifMRCP b and Dr Robert N DavidsonMD
b 
See Commentary
Summary
Introduction
Methods
Results
Discussion
References
Summary
Background Susceptibility to disease after infection by Mycobacterium tuberculosis is influenced by environmental and host genetic factors. Vitamin D metabolism leads to activation of macrophages and restricts the intracellular growth of M tuberculosis. This effect may be influenced by polymorphisms at three sites in the vitamin D receptor (VDR) gene. We investigated the interaction between serum vitamin D (25-hydroxycholecalciferol) concentrations and VDR genotype on susceptibility to tuberculosis.
Methods This study was a hospital-based case-control analysis of Asians of Gujarati origin, a mainly vegetarian immigrant population with a high rate of tuberculosis. We typed three VDR polymorphisms (defined by the presence of restriction endonuclease sites for Taq1, Bsm1, and Fok1) in 91 of 126 untreated patients with tuberculosis and 116 healthy contacts who had been sensitised to tuberculosis. Serum 25-hydroxycholecalciferol was recorded in 42 contacts and 103 patients.
Findings 25-hydroxycholecalciferol deficiency was associated with active tuberculosis (odds ratio 2?9 [95% Cl 1?3?6?5], p=0?008), and undetectable serum 25-hydroxycholecalciferol (<7 nmol/L) carried a higher risk of tuberculosis (9?9 [1?3?76?2], p=0?009). Although there was no significant independent association between VDR genotype and tuberculosis, the combination of genotype TT/Tt and 25-hydroxycholecalciferol deficiency was associated with disease (2?8 [1?2?6?5]) and the presence of genotype ff or undetectable serum 25-hydroxycholecalciferol was strongly associated with disease (5?1 [1?4?18?4]).
Interpretation 25-hydroxycholecalciferol deficiency may contribute to the high occurrence of tuberculosis in this population. Polymorphisms in the VDR gene also contribute to susceptibility when considered in combination with 25-hydroxycholecalciferol deficiency.
Back to top
Introduction
Susceptibility to disease after infection with Mycobacterium tuberculosis is influenced by environmental and host genetic factors.1,2 This gene-environment interaction is important because tuberculosis is predicted to be the largest single infectious cause of death between 1990 and 2020.3
Susceptibility to tuberculosis may be increased by deficiency of vitamin D (25-hydroxycholecalciferol).4,5 The active metabolite of 25-hydroxycholecalciferol, 1,25-dihydroxyvitamin D, helps mononuclear phagocytes to suppress the intracellular growth of M tuberculosis.6,7 Serum concentrations of 25-hydroxycholecalciferol in tuberculosis patients have been reported to be lower than in healthy controls in some populations,8 but do not differ substantially in others.9 There is an early summer peak in notifications of tuberculosis in the UK, suggesting that low post-winter trough concentrations of 25-hydroxycholecalciferol may contribute to the reactivation of latent infection.10 In addition, vegetarian diet may be a risk factor for tuberculosis in immigrant Asians living in south London.11,12
Genetic association studies of human tuberculosis have defined a role for HLA-DR2,13 haptoglobin 2-2,14 and variants of the human NRAMP1 gene15 in susceptibility to severe pulmonary tuberculosis. In addition, homozygotes (tt genotype) for a variant of the vitamin D receptor (VDR), which is associated with decreased bone-mineral density,16 are at decreased risk of tuberculosis.17 This VDR polymorphism, detected by the presence or absence of a restriction site for endonuclease Taq1, does not change the sequence of VDR but has been associated with increased VDR mRNA stability.16 A second VDR polymorphism, also associated with decreased bone-mineral density,18,19 results from a C-to-T transition that creates an alternative initiation codon (ATG) three codons from the downstream start site.20 This polymorphism, which has not been previously investigated in tuberculosis patients, is detected by the presence of a site for the restriction endonuclease Fok1. The VDR encoded by the f allele is increased in length by three aminoacids, and transcription of this allele is 1?7 times less efficient than the F allele.19
There is a very high rate of post-primary tuberculosis among Gujarati Asians living in Harrow, UK. The notification rate is as high as 809 per 100
000 population among those who arrived in the UK within the last 5 years.21 There is an unusual excess of extrapulmonary disease in these individuals and the overall rate is considerably higher than that recorded in their country of origin. However, poverty is not a factor because this immigrant community is generally prosperous. Within this population many marriages are arranged, marriage to non-Gujarati Hindus is rare, and marriage to non-Hindus is very rare. Most members of this community are strict vegetarians, and infants younger than 2 years have lower serum 25-hydroxycholecalciferol concentrations than white infants.22 This community is sufficiently homogenous for genetic study and of special interest to investigate the interaction between VDR polymorphism and acquired 25-hydroxycholecalciferol deficiency on reactivation of M tuberculosis infection.
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Methods
Participants
126 untreated patients with tuberculosis and 116 healthy tuberculosis contacts who were Hindu, resident in London, and of Gujarati origin were recruited from Northwick Park Hospital, Harrow, UK. 150 (62?0%) participants had been inoculated with BCG vaccine (including 78 untreated patients).
All 126 patients (mean age 45?5 years; 83 women, 43 men) were recruited during 3 years on the basis of their willingness to participate, and tuberculosis infection was confirmed by biopsy or culture. Culture confirmation of tuberculosis can take 8 weeks, so some patients were later excluded from analysis because the diagnosis could not be confirmed pathologically. Patients were stratified according to whether they had localised disease (tuberculosis confined to one anatomical site) or severe disease (pulmonary or miliary tuberculosis). We used this classification because the 5-year survival rate of untreated pulmonary tuberculosis is 37% and miliary disease is invariably fatal if untreated. By contrast, localised disease, although it may lead to deformation, does not have high mortality. Patients known to be immunosuppressed (eg, by HIV-1 infection, pregnancy, or corticosteroid therapy) were excluded.
116 tuberculosis contacts were recruited from our tuberculosis contact clinic (mean age 42?7 years; 53 women, 62 men). All contacts had household contact with tuberculosis patients (often multiple), all were positive to tuberculin by purified protein derivative skin test (Heaf grade 2 or above), all were symptom-free, all had a normal chest radiograph, and ten (8?6%) had received chemoprophylaxis for tuberculosis. The contacts were selected on the basis of genetic unrelatedness to any person included in the study as a patient. We ascertained this factor at interview and by simultaneously cross checking the database, which was available at the time of interview, as was a Gujarati speaking nurse in cases of ambiguity. All participants gave informed consent for inclusion, and permission to carry out the study was obtained from the Harrow local research ethics committee.
25-hydroxycholecalciferol concentrations
Samples for 25-hydroxycholecalciferol were taken from all patients at or very shortly after diagnosis. We also measured 25-hydroxycholecalciferol concentrations in 44 non-Gujarati patients who were diagnosed with tuberculosis during the study. 25-hydroxycholecalciferol was assayed by a competitive protein binding assay.23 Our routine laboratory criteria defines 25-hydroxycholecalciferol deficiency to be present at a concentration of 10 nmol/L or less. Others define clinical vitamin D deficiency as a serum concentration of less than 20 nmol/L.24
We also did a paired analysis of 25-hydroxycholecalciferol concentrations in 23 household patient-contact pairs who were concordant for dietary habit. Two blood samples were taken from each participant. An anticoagulated sample was used for DNA extraction, and a second clotted sample was used to obtain serum. Because 25-hydroxycholecalciferol concentrations can vary by season, the 42 contacts included in this part of the study were tested during the spring (March to May).
VDR polymorphisms
DNA was isolated by standard methods, and 5 ng DNA used in the PCR amplification of sequences containing previously described VDR restriction-fragment-length polymorphisms16,20 defined by the restriction endonucleases Fok1, Taq1, and. Bsm1. The primer sequences were as follows: 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ and 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′ for Fok1; 5′-GGGACGATGAGGGATGGACAGAGC-3′ and 5′-GGAAAGGGGTTAGGTTGGACAGGA-3′ for Taq1; and 5′-AACTTGCATGAGGAGGAGCATGTC-3′ and 5′-GGAGAGGAGCCTCTGTCCCATTTG-3′ for Bsm1.
Cycling conditions were 96?C for 1 min in all reactions, followed by 30 cycles at 94?C for 45 s, 60?C for 45 s, 72?C for 45 s (Fok1); 30 cycles at 94?C for 1 min, 55?C for 1 min, 72?C for 1 min (Taq1); or 30 cycles at 94?C for 1 min, 60?C for 1 min, 70?C for 1 min (Bsm1). PCR products were digested overnight with an excess of restriction endonuclease so that the reaction could proceed to completion, in accordance with the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA). Digested products were visualised on agarose gels stained with 1?5-2?0% ethidium bromide. The presence of a given restriction site was assigned by lower case (ie, f, t, b, for Fok1, Taq1, and Bsm1) and absence by upper case (ie, F, T, B). Typing was assigned without knowledge of diagnosis.
Statistical analysis
We analysed unpaired non-parametric variables by Mann-Whitney U test, and paired variables by Wilcoxon signed-rank test. Contingency analysis was done by the χ2 test with contingency correction when the lowest value in the 2?2 table was less than 10.
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Results
25-hydroxycholecalciferol concentrations
103 patients and 42 contacts had data available on 25-hydroxycholecalciferol concentration. Median 25-hydroxycholecalciferol concentrations were low in all participants, although significantly lower in tuberculosis patients (table 1). Over half the individuals in both groups had 25-hydroxycholecalciferol concentrations less than 20 nmol/L. 25-hydroxycholecalciferol deficiency (≤10 nmol/L) and undetectable 25-hydroxycholecalciferol concentrations were significantly associated with tuberculosis (table 1). However, there was no difference in the risk of severe versus localised tuberculosis.
Table 1. 25-hydroxycholecalciferol concentrations in untreated tuberculosis patients and tuberculosis contacts
The median concentration of 25-hydroxycholecalciferol in 32 strictly vegetarian contacts (18 nmol/L) was slightly higher than in ten non-vegetarian contacts (13 nmol/L). In a subanalysis of 23 household patient-contact pairs who were concordant for dietary habit, patients (five [22%] of 23) were more likely than contacts (none of 23) to have undetectable 25-hydroxycholecalciferol (p=0?025). However, the median difference in 25-hydroxycholecalciferol between these paired patients and contacts (8 nmol/L [IQR −6?5 to 11]) was not significant (p=0?13). The proportion of individuals with deficiency or undetectable concentrations was not influenced by sex (odds ratio 1?23 [95% CI 0?46?3?29] for an undetectable concentration; 1?17 [0?58?2?36] for deficiency).
The mean difference (after versus before treatment) in serum 25-hydroxycholecalciferol in 12 Gujarati patients. whose concentrations were measured after 77 days (IQR 38-157) of antituberculous chemotherapy was −5?1 nmol/L (95% CI −14?2 to 4?1). The median 25-hydroxycholecalciferol concentration in 44 non-Gujarati patients with newly-diagnosed tuberculosis (28 nmol/L) was also low, although significantly higher than in Gujarati patients (median difference 15?5 nmol/L [6?3-24?7], p<0?0001).
VDR genotype
91 patients and 116 controls had DNA available for comparison. There was no significant association between the overall risk of disease and positivity for any allele of the three VDR loci tested (allele T, odds ratio 1?17 [0?67-2?07]; allele b, 1?48 [0?70?3?03]; allele f, 1?34 [0?76?2?37]). When patients were stratified according to localised or severe disease there was a significant excess of patients with anatomically localised (extrapulmonary) tuberculosis bearing the f allele (table 2).
Table 2. VDR genotype in tuberculosis patients and tuberculosis contacts
VDR genotype and 25-hydroxycholecalciferol concentration data were available for 113 individuals. There was no association between genotype and 25-hydroxycholecalciferol concentration (median difference between Tt or tt vs TT, 3 nmol/L [95% CI −2?2 to 8?2]; Ff or ff vs FF, 3?5 nmol/L [−1?9 to 8?9]). When combinations of genotypes and 25-hydroxycholecalciferol concentrations were analysed, several interrelations became apparent (table 3).
Table 3. Association between tuberculosis and combinations of VDR genotypes and 25-hydroxycholecalciferol status
Alleles at all three VDR loci conformed to the Hardy-Weinberg equilibrium. T and b alleles were in strong linkage disequilibrium (p<0?0001). The alleles at the Fok1 locus segregated independently of both the B/b and T/t alleles. There was no evidence of increased risk of disease conferred by combinations of T or f alleles with or without 25-hydroxycholecalciferol deficiency.
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Discussion
There is a high prevalence of 25-hydroxycholecalciferol deficiency in Gujarati Asians living in London, a population in whom the incidence of tuberculosis is also high. The lowest serum 25-hydroxycholecalciferol concentrations were found in patients with active disease, and the greatest risk of tuberculosis (nearly ten-fold higher) was associated with an undetectable 25-hydroxycholecalciferol concentration. Although patients with severe disease showed a trend towards a lower frequency of the tt genotype, consistent with the association with severe pulmonary tuberculosis in the Gambia,17 our study was of insufficient power to confirm a moderate independent effect. However, the coexistence of a susceptibility genotype in combination with 25-hydroxycholecalciferol deficiency was strongly associated with disease.
Our analysis of 25-hydroxycholecalciferol concentrations during therapy suggests that tuberculosis itself does not lower 25-hydroxycholecalciferol concentrations. In addition, the association between 25-hydroxycholecalciferol concentrations and active tuberculosis showed dose dependency. However, antituberculosis chemotherapy can lower 25-hydroxycholecalciferol concentrations25 and the lack of change we saw during therapy may be due to the net effect of depression of 25-hydroxycholecalciferol by chemotherapy and the reversal of an M tuberculosis-induced depression.
The combination of 1,25-hydroxycholecalciferol and interferon-γ, a cytokine required to control the intracellular growth of mycobacteria,26 is more effective than either agent alone in restricting the growth of M tuberculosis in monocytes.7 However, the regulation of this circuit seems complex because the interferon-γ gene is directly downregulated by 1,25-hydroxycholecalciferol.27 In addition, several studies have not found an association between Taq1 VDR restriction-fragment-length polymorphism and bone-mineral density.28 Moreover, not all studies have found an effect of either the Taq1 or Fok1 alleles on VDR function.29,30 Clearly, the interaction between vitamin D, VDR, and effector molecules such as interferon-γ is complex and further research is required to understand metabolic and immunological factors in the pathogenesis of tuberculosis.
In our study, 23% of healthy contacts were of the genotype non-tt and 25-hydroxycholecalciferol deficient compared with 46% of tuberculosis patients, increasing to 52% of those with severe disease, suggesting a synergistic association between the T allele and 25-hydroxycholecalciferol deficiency. An interesting feature of our analysis is that the f allele was associated with extrapulmonary disease and has been associated with decreased transcriptional activation of the VDR gene, implying that this polymorphism may influence disease pattern, as well as susceptibility to tuberculosis. Other studies have also suggested an influence of genetic polymorphism on disease form.31,32
Although dietary deficiency contributes to the high proportion of 25-hydroxycholecalciferol deficiency in the Gujarati Asian population, our analysis of household pairs indicates that other factors (probably sunlight exposure) are important. Thus, 25-hydroxycholecalciferol deficiency may also be present in immigrants who are not usually vegetarian, but in whom there is a high incidence of postprimary tuberculosis. The tuberculosis notification rate for largely non-vegetarian Muslim immigrants to the UK is also high,21 and we have begun to routinely assay 25-hydroxycholecalciferol in such patients. Because dietary habit was of limited predictive value, and because well over half the patients in the study had substantial deficiency, the most rational intervention may be to recommend 25-hydroxycholecalciferol supplements to all tuberculosis contacts of Gujarati origin. The greatest risk of tuberculosis was associated with an undetectable 25-hydroxycholecalciferol concentration, so even moderate supplementation may be useful.
25-hydroxycholecalciferol deficiency may account for a proportion of the acquired susceptibility of Gujarati immigrants to tuberculosis. Polymorphisms in the VDR gene also contributed to the risk of tuberculosis especially when assessed in combination with 25-hydroxycholecalciferol deficiency. 25-hydroxycholecalciferol supplementation deserves further investigation in contacts and recent immigrants for the prevention of tuberculosis.
Contributors
Robert Wilkinson and Robert Davidson had the idea for the study and supervised its design, execution, and analysis. Martin Llewelyn and Robert Wilkinson provided the clinical data and samples with the assistance of Geoffrey Pasvol, Ajit Lalvani, and Mohammed Latif. Punita Patel, Robert Wilkinson, and Zahra Toossi did the VDR genotyping. Dennis Wright measured the 25-hydroxycholecalciferol concentrations. Robert Wilkinson wrote the paper with input from all investigators, especially Zahra Toossi, Ajit Lalvani, and Robert Davidson.
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Acknowledgments
We thank T Ross Eccleshall of Stanford University for advice on typing Taq1 alleles; R A Wall and I Cropley for clinical assistance; Dina Shah for secretarial assistance; Georges Snounou for technical assistance; the nursing staff of the Infectious Diseases Unit and tuberculosis contact clinic at Northwick Park Hospital; and Adrian Hill of the Wellcome Centre for Human Genetics, Oxford, for helpful discussion.
This study was funded by a Wellcome Trust Fellowship in Clinical Tropical Medicine to Robert Wilkinson, and by an MSc studentship from Imperial College of Science, Technology and Medicine to Punita Patel.
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<!--start tail=-->References
1. Spence DPS, Hotchkiss J, Williams CSD, Davies PDO. Tuberculosis and poverty. BMJ 1993; 307: 759-761. MEDLINE
2. Comstock GW. Tuberculosis in twins: a re-analysis of the Prophit survey. Am Rev Respir Dis 1978; 117: 621-624. MEDLINE
3. Murray CJL, Lopez AD. Alternative projections of mortality and disability
DOI:10.1016/S0140-6736(99)02301-6
Influence of vitamin D deficiency and vitamin D receptor polymorphisms on tuberculosis among Gujarati Asians in west London: a case-control study
Robert J WilkinsonPhD a b, Martin LlewelynMRCP b, Zahra ToossiMD a, Punita PatelMSc b, Geoffrey PasvolDPhil b, Ajit LalvaniMRCP c, Dennis WrightPhD b, Mohammed LatifMRCP b and Dr Robert N DavidsonMD
b See Commentary
Summary
Introduction
Methods
Results
Discussion
References
Summary
Background Susceptibility to disease after infection by Mycobacterium tuberculosis is influenced by environmental and host genetic factors. Vitamin D metabolism leads to activation of macrophages and restricts the intracellular growth of M tuberculosis. This effect may be influenced by polymorphisms at three sites in the vitamin D receptor (VDR) gene. We investigated the interaction between serum vitamin D (25-hydroxycholecalciferol) concentrations and VDR genotype on susceptibility to tuberculosis.
Methods This study was a hospital-based case-control analysis of Asians of Gujarati origin, a mainly vegetarian immigrant population with a high rate of tuberculosis. We typed three VDR polymorphisms (defined by the presence of restriction endonuclease sites for Taq1, Bsm1, and Fok1) in 91 of 126 untreated patients with tuberculosis and 116 healthy contacts who had been sensitised to tuberculosis. Serum 25-hydroxycholecalciferol was recorded in 42 contacts and 103 patients.
Findings 25-hydroxycholecalciferol deficiency was associated with active tuberculosis (odds ratio 2?9 [95% Cl 1?3?6?5], p=0?008), and undetectable serum 25-hydroxycholecalciferol (<7 nmol/L) carried a higher risk of tuberculosis (9?9 [1?3?76?2], p=0?009). Although there was no significant independent association between VDR genotype and tuberculosis, the combination of genotype TT/Tt and 25-hydroxycholecalciferol deficiency was associated with disease (2?8 [1?2?6?5]) and the presence of genotype ff or undetectable serum 25-hydroxycholecalciferol was strongly associated with disease (5?1 [1?4?18?4]).
Interpretation 25-hydroxycholecalciferol deficiency may contribute to the high occurrence of tuberculosis in this population. Polymorphisms in the VDR gene also contribute to susceptibility when considered in combination with 25-hydroxycholecalciferol deficiency.
Back to top
Introduction
Susceptibility to disease after infection with Mycobacterium tuberculosis is influenced by environmental and host genetic factors.1,2 This gene-environment interaction is important because tuberculosis is predicted to be the largest single infectious cause of death between 1990 and 2020.3
Susceptibility to tuberculosis may be increased by deficiency of vitamin D (25-hydroxycholecalciferol).4,5 The active metabolite of 25-hydroxycholecalciferol, 1,25-dihydroxyvitamin D, helps mononuclear phagocytes to suppress the intracellular growth of M tuberculosis.6,7 Serum concentrations of 25-hydroxycholecalciferol in tuberculosis patients have been reported to be lower than in healthy controls in some populations,8 but do not differ substantially in others.9 There is an early summer peak in notifications of tuberculosis in the UK, suggesting that low post-winter trough concentrations of 25-hydroxycholecalciferol may contribute to the reactivation of latent infection.10 In addition, vegetarian diet may be a risk factor for tuberculosis in immigrant Asians living in south London.11,12
Genetic association studies of human tuberculosis have defined a role for HLA-DR2,13 haptoglobin 2-2,14 and variants of the human NRAMP1 gene15 in susceptibility to severe pulmonary tuberculosis. In addition, homozygotes (tt genotype) for a variant of the vitamin D receptor (VDR), which is associated with decreased bone-mineral density,16 are at decreased risk of tuberculosis.17 This VDR polymorphism, detected by the presence or absence of a restriction site for endonuclease Taq1, does not change the sequence of VDR but has been associated with increased VDR mRNA stability.16 A second VDR polymorphism, also associated with decreased bone-mineral density,18,19 results from a C-to-T transition that creates an alternative initiation codon (ATG) three codons from the downstream start site.20 This polymorphism, which has not been previously investigated in tuberculosis patients, is detected by the presence of a site for the restriction endonuclease Fok1. The VDR encoded by the f allele is increased in length by three aminoacids, and transcription of this allele is 1?7 times less efficient than the F allele.19
There is a very high rate of post-primary tuberculosis among Gujarati Asians living in Harrow, UK. The notification rate is as high as 809 per 100
000 population among those who arrived in the UK within the last 5 years.21 There is an unusual excess of extrapulmonary disease in these individuals and the overall rate is considerably higher than that recorded in their country of origin. However, poverty is not a factor because this immigrant community is generally prosperous. Within this population many marriages are arranged, marriage to non-Gujarati Hindus is rare, and marriage to non-Hindus is very rare. Most members of this community are strict vegetarians, and infants younger than 2 years have lower serum 25-hydroxycholecalciferol concentrations than white infants.22 This community is sufficiently homogenous for genetic study and of special interest to investigate the interaction between VDR polymorphism and acquired 25-hydroxycholecalciferol deficiency on reactivation of M tuberculosis infection.Back to top
Methods
Participants
126 untreated patients with tuberculosis and 116 healthy tuberculosis contacts who were Hindu, resident in London, and of Gujarati origin were recruited from Northwick Park Hospital, Harrow, UK. 150 (62?0%) participants had been inoculated with BCG vaccine (including 78 untreated patients).
All 126 patients (mean age 45?5 years; 83 women, 43 men) were recruited during 3 years on the basis of their willingness to participate, and tuberculosis infection was confirmed by biopsy or culture. Culture confirmation of tuberculosis can take 8 weeks, so some patients were later excluded from analysis because the diagnosis could not be confirmed pathologically. Patients were stratified according to whether they had localised disease (tuberculosis confined to one anatomical site) or severe disease (pulmonary or miliary tuberculosis). We used this classification because the 5-year survival rate of untreated pulmonary tuberculosis is 37% and miliary disease is invariably fatal if untreated. By contrast, localised disease, although it may lead to deformation, does not have high mortality. Patients known to be immunosuppressed (eg, by HIV-1 infection, pregnancy, or corticosteroid therapy) were excluded.
116 tuberculosis contacts were recruited from our tuberculosis contact clinic (mean age 42?7 years; 53 women, 62 men). All contacts had household contact with tuberculosis patients (often multiple), all were positive to tuberculin by purified protein derivative skin test (Heaf grade 2 or above), all were symptom-free, all had a normal chest radiograph, and ten (8?6%) had received chemoprophylaxis for tuberculosis. The contacts were selected on the basis of genetic unrelatedness to any person included in the study as a patient. We ascertained this factor at interview and by simultaneously cross checking the database, which was available at the time of interview, as was a Gujarati speaking nurse in cases of ambiguity. All participants gave informed consent for inclusion, and permission to carry out the study was obtained from the Harrow local research ethics committee.
25-hydroxycholecalciferol concentrations
Samples for 25-hydroxycholecalciferol were taken from all patients at or very shortly after diagnosis. We also measured 25-hydroxycholecalciferol concentrations in 44 non-Gujarati patients who were diagnosed with tuberculosis during the study. 25-hydroxycholecalciferol was assayed by a competitive protein binding assay.23 Our routine laboratory criteria defines 25-hydroxycholecalciferol deficiency to be present at a concentration of 10 nmol/L or less. Others define clinical vitamin D deficiency as a serum concentration of less than 20 nmol/L.24
We also did a paired analysis of 25-hydroxycholecalciferol concentrations in 23 household patient-contact pairs who were concordant for dietary habit. Two blood samples were taken from each participant. An anticoagulated sample was used for DNA extraction, and a second clotted sample was used to obtain serum. Because 25-hydroxycholecalciferol concentrations can vary by season, the 42 contacts included in this part of the study were tested during the spring (March to May).
VDR polymorphisms
DNA was isolated by standard methods, and 5 ng DNA used in the PCR amplification of sequences containing previously described VDR restriction-fragment-length polymorphisms16,20 defined by the restriction endonucleases Fok1, Taq1, and. Bsm1. The primer sequences were as follows: 5′-AGCTGGCCCTGGCACTGACTCTGCTCT-3′ and 5′-ATGGAAACACCTTGCTTCTTCTCCCTC-3′ for Fok1; 5′-GGGACGATGAGGGATGGACAGAGC-3′ and 5′-GGAAAGGGGTTAGGTTGGACAGGA-3′ for Taq1; and 5′-AACTTGCATGAGGAGGAGCATGTC-3′ and 5′-GGAGAGGAGCCTCTGTCCCATTTG-3′ for Bsm1.
Cycling conditions were 96?C for 1 min in all reactions, followed by 30 cycles at 94?C for 45 s, 60?C for 45 s, 72?C for 45 s (Fok1); 30 cycles at 94?C for 1 min, 55?C for 1 min, 72?C for 1 min (Taq1); or 30 cycles at 94?C for 1 min, 60?C for 1 min, 70?C for 1 min (Bsm1). PCR products were digested overnight with an excess of restriction endonuclease so that the reaction could proceed to completion, in accordance with the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA). Digested products were visualised on agarose gels stained with 1?5-2?0% ethidium bromide. The presence of a given restriction site was assigned by lower case (ie, f, t, b, for Fok1, Taq1, and Bsm1) and absence by upper case (ie, F, T, B). Typing was assigned without knowledge of diagnosis.
Statistical analysis
We analysed unpaired non-parametric variables by Mann-Whitney U test, and paired variables by Wilcoxon signed-rank test. Contingency analysis was done by the χ2 test with contingency correction when the lowest value in the 2?2 table was less than 10.
Back to top
Results
25-hydroxycholecalciferol concentrations
103 patients and 42 contacts had data available on 25-hydroxycholecalciferol concentration. Median 25-hydroxycholecalciferol concentrations were low in all participants, although significantly lower in tuberculosis patients (table 1). Over half the individuals in both groups had 25-hydroxycholecalciferol concentrations less than 20 nmol/L. 25-hydroxycholecalciferol deficiency (≤10 nmol/L) and undetectable 25-hydroxycholecalciferol concentrations were significantly associated with tuberculosis (table 1). However, there was no difference in the risk of severe versus localised tuberculosis.
Click to view table
Table 1. 25-hydroxycholecalciferol concentrations in untreated tuberculosis patients and tuberculosis contacts
The median concentration of 25-hydroxycholecalciferol in 32 strictly vegetarian contacts (18 nmol/L) was slightly higher than in ten non-vegetarian contacts (13 nmol/L). In a subanalysis of 23 household patient-contact pairs who were concordant for dietary habit, patients (five [22%] of 23) were more likely than contacts (none of 23) to have undetectable 25-hydroxycholecalciferol (p=0?025). However, the median difference in 25-hydroxycholecalciferol between these paired patients and contacts (8 nmol/L [IQR −6?5 to 11]) was not significant (p=0?13). The proportion of individuals with deficiency or undetectable concentrations was not influenced by sex (odds ratio 1?23 [95% CI 0?46?3?29] for an undetectable concentration; 1?17 [0?58?2?36] for deficiency).
The mean difference (after versus before treatment) in serum 25-hydroxycholecalciferol in 12 Gujarati patients. whose concentrations were measured after 77 days (IQR 38-157) of antituberculous chemotherapy was −5?1 nmol/L (95% CI −14?2 to 4?1). The median 25-hydroxycholecalciferol concentration in 44 non-Gujarati patients with newly-diagnosed tuberculosis (28 nmol/L) was also low, although significantly higher than in Gujarati patients (median difference 15?5 nmol/L [6?3-24?7], p<0?0001).
VDR genotype
91 patients and 116 controls had DNA available for comparison. There was no significant association between the overall risk of disease and positivity for any allele of the three VDR loci tested (allele T, odds ratio 1?17 [0?67-2?07]; allele b, 1?48 [0?70?3?03]; allele f, 1?34 [0?76?2?37]). When patients were stratified according to localised or severe disease there was a significant excess of patients with anatomically localised (extrapulmonary) tuberculosis bearing the f allele (table 2).
Click to view table
Table 2. VDR genotype in tuberculosis patients and tuberculosis contacts
VDR genotype and 25-hydroxycholecalciferol concentration data were available for 113 individuals. There was no association between genotype and 25-hydroxycholecalciferol concentration (median difference between Tt or tt vs TT, 3 nmol/L [95% CI −2?2 to 8?2]; Ff or ff vs FF, 3?5 nmol/L [−1?9 to 8?9]). When combinations of genotypes and 25-hydroxycholecalciferol concentrations were analysed, several interrelations became apparent (table 3).
Click to view table
Table 3. Association between tuberculosis and combinations of VDR genotypes and 25-hydroxycholecalciferol status
Alleles at all three VDR loci conformed to the Hardy-Weinberg equilibrium. T and b alleles were in strong linkage disequilibrium (p<0?0001). The alleles at the Fok1 locus segregated independently of both the B/b and T/t alleles. There was no evidence of increased risk of disease conferred by combinations of T or f alleles with or without 25-hydroxycholecalciferol deficiency.
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Discussion
There is a high prevalence of 25-hydroxycholecalciferol deficiency in Gujarati Asians living in London, a population in whom the incidence of tuberculosis is also high. The lowest serum 25-hydroxycholecalciferol concentrations were found in patients with active disease, and the greatest risk of tuberculosis (nearly ten-fold higher) was associated with an undetectable 25-hydroxycholecalciferol concentration. Although patients with severe disease showed a trend towards a lower frequency of the tt genotype, consistent with the association with severe pulmonary tuberculosis in the Gambia,17 our study was of insufficient power to confirm a moderate independent effect. However, the coexistence of a susceptibility genotype in combination with 25-hydroxycholecalciferol deficiency was strongly associated with disease.
Our analysis of 25-hydroxycholecalciferol concentrations during therapy suggests that tuberculosis itself does not lower 25-hydroxycholecalciferol concentrations. In addition, the association between 25-hydroxycholecalciferol concentrations and active tuberculosis showed dose dependency. However, antituberculosis chemotherapy can lower 25-hydroxycholecalciferol concentrations25 and the lack of change we saw during therapy may be due to the net effect of depression of 25-hydroxycholecalciferol by chemotherapy and the reversal of an M tuberculosis-induced depression.
The combination of 1,25-hydroxycholecalciferol and interferon-γ, a cytokine required to control the intracellular growth of mycobacteria,26 is more effective than either agent alone in restricting the growth of M tuberculosis in monocytes.7 However, the regulation of this circuit seems complex because the interferon-γ gene is directly downregulated by 1,25-hydroxycholecalciferol.27 In addition, several studies have not found an association between Taq1 VDR restriction-fragment-length polymorphism and bone-mineral density.28 Moreover, not all studies have found an effect of either the Taq1 or Fok1 alleles on VDR function.29,30 Clearly, the interaction between vitamin D, VDR, and effector molecules such as interferon-γ is complex and further research is required to understand metabolic and immunological factors in the pathogenesis of tuberculosis.
In our study, 23% of healthy contacts were of the genotype non-tt and 25-hydroxycholecalciferol deficient compared with 46% of tuberculosis patients, increasing to 52% of those with severe disease, suggesting a synergistic association between the T allele and 25-hydroxycholecalciferol deficiency. An interesting feature of our analysis is that the f allele was associated with extrapulmonary disease and has been associated with decreased transcriptional activation of the VDR gene, implying that this polymorphism may influence disease pattern, as well as susceptibility to tuberculosis. Other studies have also suggested an influence of genetic polymorphism on disease form.31,32
Although dietary deficiency contributes to the high proportion of 25-hydroxycholecalciferol deficiency in the Gujarati Asian population, our analysis of household pairs indicates that other factors (probably sunlight exposure) are important. Thus, 25-hydroxycholecalciferol deficiency may also be present in immigrants who are not usually vegetarian, but in whom there is a high incidence of postprimary tuberculosis. The tuberculosis notification rate for largely non-vegetarian Muslim immigrants to the UK is also high,21 and we have begun to routinely assay 25-hydroxycholecalciferol in such patients. Because dietary habit was of limited predictive value, and because well over half the patients in the study had substantial deficiency, the most rational intervention may be to recommend 25-hydroxycholecalciferol supplements to all tuberculosis contacts of Gujarati origin. The greatest risk of tuberculosis was associated with an undetectable 25-hydroxycholecalciferol concentration, so even moderate supplementation may be useful.
25-hydroxycholecalciferol deficiency may account for a proportion of the acquired susceptibility of Gujarati immigrants to tuberculosis. Polymorphisms in the VDR gene also contributed to the risk of tuberculosis especially when assessed in combination with 25-hydroxycholecalciferol deficiency. 25-hydroxycholecalciferol supplementation deserves further investigation in contacts and recent immigrants for the prevention of tuberculosis.
Contributors
Robert Wilkinson and Robert Davidson had the idea for the study and supervised its design, execution, and analysis. Martin Llewelyn and Robert Wilkinson provided the clinical data and samples with the assistance of Geoffrey Pasvol, Ajit Lalvani, and Mohammed Latif. Punita Patel, Robert Wilkinson, and Zahra Toossi did the VDR genotyping. Dennis Wright measured the 25-hydroxycholecalciferol concentrations. Robert Wilkinson wrote the paper with input from all investigators, especially Zahra Toossi, Ajit Lalvani, and Robert Davidson.
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Acknowledgments
We thank T Ross Eccleshall of Stanford University for advice on typing Taq1 alleles; R A Wall and I Cropley for clinical assistance; Dina Shah for secretarial assistance; Georges Snounou for technical assistance; the nursing staff of the Infectious Diseases Unit and tuberculosis contact clinic at Northwick Park Hospital; and Adrian Hill of the Wellcome Centre for Human Genetics, Oxford, for helpful discussion.
This study was funded by a Wellcome Trust Fellowship in Clinical Tropical Medicine to Robert Wilkinson, and by an MSc studentship from Imperial College of Science, Technology and Medicine to Punita Patel.
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1. Spence DPS, Hotchkiss J, Williams CSD, Davies PDO. Tuberculosis and poverty. BMJ 1993; 307: 759-761. MEDLINE
2. Comstock GW. Tuberculosis in twins: a re-analysis of the Prophit survey. Am Rev Respir Dis 1978; 117: 621-624. MEDLINE
3. Murray CJL, Lopez AD. Alternative projections of mortality and disability
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