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Clinical Science (2000) 98, 245–250 (Printed in Great Britain)
G L A X O / M R S
P A P E R
Identifying genetic susceptibility factors for
tuberculosis in Africans: a combined
approach using a candidate gene
study and a genome-wide screen*
Richard BELLAMY
Department of Infectious Diseases, University Hospital of Wales, Cardiff CF14 4XW, Wales, U.K.
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There is convincing evidence that host genes affect the outcome of infection in human
tuberculosis. Two complementary strategies were used to identify the genes involved. A linkagebased genome-wide screen was carried out to locate the positions of genes exerting a major
population-wide effect on tuberculosis susceptibility. A candidate-gene-based case–control
study was used to examine the effects of genes that may exert a more moderate effect on risk of
clinical tuberculosis. The genome screen was conducted in two stages. In the first stage 299
microsatellite markers, spanning all 23 chromosomes, were typed in 92 independent sib-pairs,
and seven regions showed some evidence of co-segregation with the disease. These seven regions
were examined in a second set of 81 sib-pairs, and markers on chromosomes 15q and Xq showed
evidence of linkage to tuberculosis. An X chromosome susceptibility gene may contribute to the
excess of males with tuberculosis observed in many populations. The candidate gene approach
compared the frequency of polymorphisms in several genes in over 400 subjects with smearpositive pulmonary tuberculosis and 400 ethnically matched healthy controls. Polymorphisms in
genes encoding natural-resistance-associated macrophage protein, vitamin D receptor and
mannose-binding lectin were associated with tuberculosis. These results suggest that many genes
may be involved in determining host susceptibility to tuberculosis, and highlight the importance
of using several different study methods to locate them.
INTRODUCTION
It is estimated that one-third of the World’s population is
infected with Mycobacterium tuberculosis, but that only
one in ten of those infected ever develop clinical disease
[1,2]. There are many well known risk factors for
tuberculosis, including HIV infection, advanced age,
malnutrition, alcohol abuse, diabetes and corticosteroids.
However, many patients do not have any obvious
underlying risk factor, and there is now convincing
evidence that individual variability in tuberculosis susceptibility is partly determined by host genes [3]. Racial
differences in resistance to tuberculosis may be determined by history of exposure among previous
generations [4,5], and concordance for tuberculosis is
higher among identical than non-identical twins [6,7].
* This paper was presented at the Glaxo/MRS Young Investigator session at the MRS Meeting, Royal College of Physicians,
London, on 19 May 1999.
Key words : genetic susceptibility, genome screen, mannose-binding lectin, Nramp, tuberculosis, vitamin D receptor.
Abbreviations : MBL, mannose-binding lectin ; NRAMP\Nramp, natural-resistance-associated macrophage protein ; VDR, vitamin
D receptor.
Correspondence : e-mail bellamyrj!cardiff.ac.uk
# 2000 The Biochemical Society and the Medical Research Society
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R. Bellamy
M. tuberculosis is a facultative intracellular pathogen
which utilizes the macrophage as its host cell. Resistance
to M. tuberculosis involves a complex interaction between
the bacteria and the host immune system. Several
mechanisms have been proposed to explain how the
macrophage combats mycobacterial resistance. These
include the generation of reactive oxygen and reactive
nitrogen intermediates, acidification of phagosomes,
phagosome–lysosome fusion and the limitation of intraphagosomal iron [8]. It is uncertain how M. tuberculosis
evades these mechanisms. Hypotheses include invading
macrophages via C3b receptors to evade the production
of reactive oxygen intermediates [9], production of
ammonia to resist phagosome acidification [10], and
inhibition of phagosome–lysosome fusion [11]. Cytokines are believed to play a key role in mycobacterial
resistance. Macrophages are activated by interferon γ,
tumour necrosis factor α, vitamin D and interleukin 6
[11]. These cytokines may play an important role in
resistance to tuberculosis, although they may also be
responsible for much of the tissue destruction that occurs
in this disease [12].
Two approaches were used to investigate the role of
host genes in susceptibility to tuberculosis in Africans. A
linkage study was used to screen the whole human
genome for regions containing potential major
tuberculosis-susceptibility loci. This approach is systematic and comprehensive, but has a relatively low
power of detection and would fail to identify any genes
exerting only a moderate effect on disease susceptibility.
It has been calculated that if a susceptibility allele with a
population frequency of 0.5 exerts a 2-fold increased risk
of disease, 2498 sib-pairs would be required to provide
80 % power to detect the effect by linkage analysis [13].
An association (case–control) study would only require
340 cases to provide the same power [13], and can
therefore detect smaller population-wide genetic effects.
Association studies cannot currently be used to perform
genome-wide screens, as at least 3000 markers would
need to be typed. Our case–control study was therefore
restricted to candidate genes.
The study described in this review was approved by
the joint ethics committee of the Gambian Government
and the Medical Research Council. Verbal consent was
obtained from all study subjects.
RESULTS OF THE GENOME-WIDE SCREEN
The genome screen was carried out using sib-pair
families. These are families with two or more full siblings
affected by the same disease (tuberculosis in this case). A
family containing two affected siblings provides one sibpair, one containing three affected siblings provides two
fully independent sib-pairs, and if there are four affected
siblings this is equivalent to three independent sib-pairs,
# 2000 The Biochemical Society and the Medical Research Society
etc. If a genetic marker locus is linked to a major diseasesusceptibility gene, then the siblings will share the
parental alleles that they have inherited at that locus more
often than would be expected by chance. This nonparametric system of analysis is not dependent on the
mode of inheritance, and a major disease-susceptibility
locus should be identifiable regardless of whether it is Xlinked or autosomal, dominant or recessive.
The genome screen was carried out in two stages, with
approximately half of the sib-pairs in each stage. This is
more efficient than carrying out the whole genome-wide
screen on all of the families. In the first stage, 299
microsatellite markers covering all 23 chromosomes were
typed in 67 Gambian families including 73 fully independent sib-pairs, and in 16 KwaZulu–Natal families
including 19 independent sib-pairs. A total of seven
regions, on chromosomes 3, 5, 6, 8, 9, 15 and X, showed
nominal evidence for linkage to tuberculosis (R. Bellamy,
N. Beyers, C. Ruwende, H. C. Whittle, E. Hoal van
Helden, T. Corrah, R. Wilkinson, K. P. W. J. McAdam,
W. Amos, P. van Helden and A. V. S. Hill, unpublished
work). These results could have been due to genuine
linkage or simply to chance, because of the large number
of loci typed, and therefore needed replication in a
second series of families. No region showed evidence of
strong linkage, indicating that in these populations
genetic susceptibility to tuberculosis is not determined
by a single-gene effect.
In the second round of the screen, 22 markers from the
seven regions identified in the first stage of the screen
were typed in 12 Gambian sib-pair families and in 41
families including 69 independent sib-pairs from the
Western Cape, South Africa. Two regions, on chromosome Xq26 and the pericentromeric region of chromosome 15q, showed evidence of linkage to a putative
tuberculosis-susceptibility gene (R. Bellamy, N. Beyers,
C. Ruwende, H. C. Whittle, E. Hoal van Helden, T.
Corrah, R. Wilkinson, K. P. W. J. McAdam, W. Amos,
P. van Helden and A. V. S. Hill, unpublished work).
Several candidate genes lie in the region of interest on
Xq26, and the CD40 ligand in particular is worthy of
further investigation. It is of note that there are approximately twice as many male as female patients with
tuberculosis in The Gambia and South Africa (R.
Bellamy, unpublished work ; [14]). This significant male
excess has been observed in many different ethnic groups
[15–17], and our results suggest that it may be due to an
X-linked tuberculosis-susceptibility gene.
CANDIDATE GENE STUDY
Subjects for the case–control study were all recruited
from The Gambia. The relative ethnic homogeneity of
the population [18] make this an ideal population for
case–control studies. All patients were HIV-negative
Genetic susceptibility to tuberculosis
adults (over 16 years old) with smear-positive pulmonary
tuberculosis. The control group consisted of HIVnegative, healthy, unrelated blood donors retrospectively
matched to the patients by ethnic group as far as numbers
allowed.
To have 90 % power to detect a statistically significant
difference (P 0.05) between the number of tuberculosis
cases and controls with a genotype which has a population frequency of 10 % and which exerts an odds ratio
of 2.0 on a 2i2 contingency table would require 402
cases. Thus a greater number than this of cases and of
ethnically matched controls were recruited for
the candidate gene study to provide this power. Several
candidate gene polymorphisms have been typed in this
case–control study [19], and associations have been
detected for natural-resistance-associated macrophage
protein (NRAMP1), vitamin D receptor (VDR) and
mannose-binding lectin (MBL) gene variants [20–22].
NRAMP
Among inbred strains of mice, natural resistance to
infection with several antigenically unrelated intracellular
pathogens is controlled by a single dominant gene on
mouse chromosome 1, designated Bcg (also known as
Lsh\Ity) [23,24]. Two distinct non-overlapping phenotypes are recognized, Bcgs and Bcgr, which are respectively susceptible and resistant to the early stage of
infection with Mycobacterium bovis (BCG), M. intracellulare, M. lepraemurium, Leishmania donovani and
Salmonella typhimurium [23–28]. A candidate gene for
Bcg has been isolated by positional cloning and designated Nramp1 (murine natural-resistance-associated
macrophage protein) [29]. Nramp1 and Bcg\Lsh\Ity
were proven to be identical by the production of a genedisrupted Nramp1 knockout mouse [30], and an Nramp1
transgenic mouse which restored the wild-type resistant
phenotype [31]. It has been shown that Nramp1 is not the
sole major gene determining resistance to infection with
virulent M. tuberculosis [32] in mice, but it may have a
more complex role in murine M. tuberculosis immunity
in conjunction with other host genes.
The human homologue of the Nramp1 gene, designated NRAMP1, has been cloned and mapped to
chromosome 2q35 [33,34]. Several polymorphisms have
been described in NRAMP1, and it has been suggested
that they could influence NRAMP1 function [34,35].
Weak evidence of linkage has been found between
NRAMP1 markers and susceptibility to leprosy in 20
families from Vietnam (P 0.02), although this was not
confirmed in seven families from French Polynesia
[36,37]. Weak evidence of linkage to the NRAMP1 locus
was also found in our own study of tuberculosis in
families (P l 0.06) (R. Bellamy, unpublished work) and
in a study of 98 Brazilian families (P l 0.025) [38]. The
absence of strong evidence of linkage in these studies
Combined analysis of NRAMP1 genetic variants
The genetic variants are a single-base substitution (G C) in intron 4 (INT4) and
a deletion (del) in the 3h untranslated region (3h UTR). Odds ratios (with 95 %
confidence intervals in parentheses) and P values (Yates’ ; 1 degree of freedom)
shown are for Chi-square comparisons with the GG/jj genotype. An overall
4i2 Chi-square test on combined genotypes shows strong association with
tuberculosis (P l 0.000008).
Table 1
No. of subjects
INT4/3h UTR
genotype
Tuberculosis
cases
Controls
Odds ratio
P value
GG/jj
GC/jj
GG/jdel
GC/jdel
191
45
118
31
251
33
84
10
1.0
1.79 (1.07–3.00)
1.85 (1.30–2.62)
4.07 (1.86–9.12)
–
0.02
0.0005
0.0001
indicates that NRAMP1 is not the sole determinant of
host genetic susceptibility to mycobacterial infections in
humans.
Case–control studies have much greater power than
linkage studies for investigating the complex role of
candidate genes in multifactorial diseases. Six NRAMP1
polymorphisms were typed in our large Gambian case–
control study. Four polymorphisms were strongly
associated with tuberculosis in this population (overall
P l 0.000008 ; one gene variant was not associated and
the other was too rare to evaluate) [20]. Individuals who
possessed two variant alleles were 4-fold overrepresented among the tuberculosis cases compared with
the healthy controls (Table 1 ; [20]). This result demonstrates that, although NRAMP1 is not the sole determinant of host tuberculosis susceptibility, it is an
important mycobacteria-susceptibility gene in humans as
well as in mice. The functions of Nramp1\NRAMP1
remain unknown, although it is believed that they may
restrict the availability of iron to intraphagosomal mycobacteria [39].
VDR
Genetic variation in bone mineral density has been shown
to be associated with polymorphisms in the VDR gene in
many, although not all, populations studied [40].
Epidemiological evidence suggests a link between vitamin
D deficiency and susceptibility to tuberculosis [41,42],
and vitamin D has been shown to have beneficial effects
in cutaneous tuberculosis [43]. Vitamin D is an important
immunoregulatory hormone [44]. The active metabolite
of vitamin D, 1,25-dihydroxyvitamin D (1,25$
dihydroxycholecalciferol), activates monocytes, stimulates cell-mediated immunity and suppresses lymphocyte
proliferation, immunoglobulin production and cytokine
synthesis [44]. In vitro studies suggest that vitamin D
metabolites can enhance the ability of human monocytes
# 2000 The Biochemical Society and the Medical Research Society
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R. Bellamy
Tuberculosis and VDR genotype
The frequency of each genotype is given as an actual value and (in parentheses)
as a percentage. The overall distribution of VDR genotypes is significantly different
between cases and controls (3i2 Chi-square l 6.98 ; 2 degrees of freedom,
P l 0.03). The tt genotype was less common in tuberculosis cases compared with
controls (Yates’ Chi-square l 6.06 ; 1 degree of freedom, P l 0.01).
Table 2
Frequency (no.) (%)
Genotype
Tuberculosis cases
Controls
TT
Tt
tt
Total
204 (50)
177 (43)
27 (6.6)
408
188 (45)
177 (43)
49 (12)
414
to restrict the growth of intracellular M. tuberculosis [45].
Alveolar macrophages in the lungs of tuberculosis
patients have been shown to produce 1,25-dihydroxyvitamin D [46], which could therefore be involved in the
$
host immune response to M. tuberculosis in vivo.
A polymorphism in codon 352 of the VDR gene was
typed in our Gambian tuberculosis case–control study.
The genotype associated with low bone mineral density,
which has been designated tt, was significantly underrepresented in the tuberculosis cases compared with the
controls (P l 0.01) (Table 2 ; [21]). The odds ratio for
subjects of genotype tt having tuberculosis compared
with those of genotypes TT\Tt combined was 0.53 (95 %
confidence interval 0.31–0.88), suggesting that tt homozygotes may be resistant to clinical tuberculosis. Subjects
with this genotype have also been found to be less likely
to develop persistence of hepatitis B virus infection [21]
and the lepromatous form of leprosy [47]. Further studies
will be required to investigate how VDR polymorphism
may influence susceptibility to infectious diseases.
throughout Africa and we might therefore expect strong
selection against MBL variants.
It has been suggested that heterozygote advantage may
maintain MBL variant alleles at high frequency by
conferring resistance to mycobacterial diseases [50]. In
our case–control study in The Gambia it was found that
the frequency of the common African variant MBL allele
(codon 57) was lower among tuberculosis cases than
controls (P l 0.037) [22]. This result will require confirmation in other populations due to its borderline
statistical significance. However, it would be fascinating
if MBL deficiency, described as the world’s commonest
immune deficiency, has been selected for by conferring
resistance to tuberculosis.
CONCLUSIONS
Recent work on subjects with extreme susceptibility to
atypical mycobacteria and other intracellular pathogens
has identified rare immune deficiencies due to abnormalities of the genes encoding the interferon γ receptor,
interleukin 12 and the interleukin 12 receptor [51–54].
These studies provide important candidate genes for
investigation in large population-based studies of common, related infections. It is likely that a large number of
host genes are involved in susceptibility to mycobacteria
and other infectious diseases. A variety of study designs,
including family-based linkage studies, large-scale population case–control studies, investigation of unexplained
immune deficiencies and comparisons with animal
models of disease, will be required to identify further
host genes and investigate their interactions. Identifying
the critical determinants of host genetic susceptibility to
specific pathogens will further our understanding of the
pathogenesis of these diseases, and hopefully suggest new
treatment strategies.
MBL
MBL is a calcium-dependent serum lectin. It interacts
with the immune system by acting as an opsonin to
promote phagocytosis and by activating the complement
cascade. Three co-dominant single-base substitutions in
codons 52, 54 and 57 of the MBL gene result in reduced
serum MBL concentrations. Individuals with two variant
alleles have extremely low or undetectable levels of serum
MBL, and are predisposed to recurrent childhood
infections and possibly to infections in adult life [48].
However, MBL variant alleles are extremely common
and have a combined frequency of 22.5 % among a
healthy British population, with around 4.6 % of the
population possessing two mutant alleles [49]. Among
Gambians, MBL variant alleles have a combined frequency of 31 %, and around 10 % of the population carry
two mutant alleles [22]. This is surprisingly high, because
infections are a frequent cause of premature death
# 2000 The Biochemical Society and the Medical Research Society
ACKNOWLEDGMENTS
I am grateful to Professor A. V. S. Hill for helpful
discussions, and to the Wellcome Trust for funding.
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