Supplementary Material 1 – Influence of host genetics on tuberculosis
susceptibility in mice and humans.
Mouse Studies
Studies in inbred mice over the last 30 years led to the identification and
positional cloning of the first identified putative gene associated with
susceptibility to M. bovis BCG, the natural resistance associated macrophage
protein 1 (Nramp1) previously known as Lsh/Ity/Bcg and now renamed
Scl11A1 (solute carrier family 11 member 1) (Vidal et al. 1993; Malo et al.
1993; Jabado et al. 2000). The Nramp1 gene encodes a 90-100 kDa protein
composed of 12 hydrophobic transmembrane domains. Nramp1 is primarily
expressed on the surface of macrophage phagosomes and is a divalent ion
transporter (Govoni et al. 1995). The actual mutation conferring differences in
susceptibility to M. bovis BCG between mouse strains converts a glycine
residue 169 in transmembrane domain 4 to an aspartate residue, causing
decreased protein stability and absence of the mature form in the
macrophage phagosomes (Vidal et al. 1996). However, further studies
involving deletion of Nramp1 suggested that this gene may not, after all, be
associated with susceptibility to M. tuberculosis infections in mice and
consequently may be of limited importance in resistance to TB (North et al.
1999). It has been argued that the reason that Nramp1 may not play a role in
phagosomes infected with M. tuberculosis is that its role as cation transporter
appears late in the endosomal maturation process. Since M. tuberculosis
arrests this maturation, any benefit derived from Nramp1 activity is
circumvented (Kramnik et al. 2000).
A second candidate gene has been recently mapped to chromosome 1,
10 cM upstream from the Nramp1 gene. The locus, identified by linkage
analysis, was initially named sst1 (susceptibility to TB) (Kramnik et al. 2000).
Further work determined that the Ipr1 gene (Intracellular pathogen resistance
1) resided at the sst1 locus and that the gene encoded a transcription factor
that showed high homology with the human transcription factor SP110 (Pan et
al. 2005). Variants in the human homologue, SP110, have been investigated
in various populations with only one study finding an association with TB
susceptibility ( Thye et al. 2006; Tosh et al. 2006; Babb et al. 2007; Szeszko
et al. 2007). SP110 is activated by interferon and is known to interact with
pathogen protein motifs.
It is evident from both mouse and human studies
that variability in
response to TB is multigenic and should be studied as a quantitative trait
under the control of multiple genes, using a genome scan approach. In mice
several quantitative trait loci (QTL) associated with resistance (named Trl-1,
Trl-2, Trl-3 and Trl-4) and associated with susceptibility (named sst1) have
been identified (Bellamy 2006; Yan et al. 2006). The murine MHC (H-2) region
has also been associated with susceptibility/resistance to TB (Yan et al.
2006).
Human studies
Compelling evidence of a genetic component to TB susceptibility was
originally provided by studies on twins, where the concordance rate for TB is
2.5 times higher for monozygotic twins than for dizygotic twin pairs (Comstock
1978).
A recent re-appraisal of this study has however indicated that an
environmental component (intensity of exposure) was underestimated in this
study and that consequently the genetic component of susceptibility may have
been overestimated (van der Eijk et al. 2007). The latter does not disprove
however that a substantial amount of the variation in the human response to
TB is under genetic control. Indeed, the genetic component of resistance or
susceptibility to TB has been extensively elucidated in humans (Hill 2006;
Fortin et al. 2007), and a brief summary of the evidence will follow based on
these reviews.
Mendelian predisposition and Candidate genes
Mendelian predisposition to mycobacterial infections, caused by the
inheritance in family pedigrees of rare, deleterious mutations affecting crucial
biochemical pathways, have been found in children with IL-12/23-INF
pathway defects. This is perhaps unsurprising in light of the central role this
cytokine network plays in the immune response to the pathogen. Results have
shown an association with the IL-12R1 deficiency and with the gene
encoding the IFN- receptor ligand binding chain IFNR1 (Jouanguy et al.
1997). However, these rare mutations do not explain the population-wide
general variability in susceptibility to TB that is most probably under polygenic
control.
The candidate gene approach has been widely used in an attempt to
dissect this polygenic variation and several associations have been found with
susceptibility/resistance to TB. The main genes studied have been chosen
based on their known function and role in immune response against
mycobacterial infections or because they are homologues of the murine
genes involved in resistance to TB. The most studied genes have been
NRAMP1 (Greenwood et al. 2000), SP110 (Tosh et al. 2006; Thye et al.
2006), class II HLA DR2 serotype and HLA-DQB variants (Goldfeld et al.
1998; Delgado et al. 2006), the DC-SIGN encoding CD209 gene (Barreiro et
al. 2006), the MCP-1 gene encoding for the monocyte chemoattractant
protein-1 (Flores-Villanueva et al. 2005), NOD2 (Austin et al. 2008), the
vitamin D receptor (Bellamy et al. 1999) and the Toll Like Receptors (TLRs),
which have recently been revealed to contribute to human susceptibility to TB
(Smith et al. 2006b; Ma et al. 2007; Davila et al. 2008).
In general, these genes explain only a relatively small proportion of the
genetic variability to TB, indicating not only that the pathology is under
polygenic control but also that further research is needed to identify variation
in other genes. The human NRAMP gene will be discussed in detail in order
to illustrate the difficulties in repeating and validating results across
populations.
The human NRAMP gene
The human homologue of the murine Nramp1 gene (referred to as
NRAMP1 in humans) was nominated as the major candidate gene following
the characterisation of the murine Nramp1 gene as a possible contributor to
mycobacterial infection susceptibility. Interestingly, a large family-based
linkage study of aboriginal Canadians determined that the chromosomal
region harbouring the human NRAMP1 gene is linked to TB incidence
(Greenwood et al. 2000).
Several NRAMP1 polymorphisms have been found and investigated in
association studies across populations. Most commonly utilised has been a
microsatellite in the 5’UTR, identifed by Liu et al. (1995). Interestingly, in vitro
studies have revealed that promoter polymorphism variability is associated
with NRAMP1 expression (Searle & Blackwell 1999).
The first study of human NRAMP1 gene variants and their effect on TB
susceptibility was carried out in a Gambian population by Bellamy et al.
(1998). In a case-control study of approximately 400 TB-affected and 400
unaffected individuals four polymorphisms, 5’(GT)n, INT4, D543N and 3’UTR
(TGTG), were significantly associated with pulmonary TB and appeared to
confer a greater risk of contracting the disease (Bellamy et al., 1998). Another
independent case-control study in Gambia confirmed these results showing
that the same polymorphism in the promoter region of the gene was
associated with pulmonary TB (Awomoyi et al. 2002). Similarly, a South
African case-control study demonstrated that a microsatellite in the promoter
region of NRAMP1 was associated with enhanced susceptibility and
protection against pulmonary TB infection (Hoal et al. 2004). In addition,
homozygous individuals for the (1729+55del4) deletion in the 3’UTR (TGTG)
were over represented in the case population, but the effects were considered
to be weaker than those for the promoter microsatellite. Similar results have
been found in Korean (Ryu et al. 2000) and Japanese populations (Gao et al.
2000).
However, contrary to these findings, several studies revealed no
associations of the NRAMP1 polymorphisms with TB. A Moroccan casecontrol study observed no linkage or associations for the variants described.
The study used a family-based method, where the criteria were based on
chest X-ray, smear microscopy and culture examination, and the cohort
comprised 211 cases (Baghdadi et al. 2003). Similar findings have been
observed in separate Brazilian, South African and West African populations
(Selvaraj 2004), indicating a genetic heterogeneity in TB susceptibility and
possible confounding environmental effects. As an example, the Moroccan
study was performed in an area where TB is endemic and all families enrolled
had been vaccinated with BCG, whereas the study on the large aboriginal
Canadian family comprised a naïve cohort.
Genome-wide linkage studies
Four genome-wide linkage studies have identified possible chromosomal
regions associated with TB. A two stage genome-wide linkage study was
conducted on sib pair families from Gambia and from KwaZulu-Natal in South
Africa. The study identified putative regions on chromosomes 15 and X (LOD
= 2.00 and 1.77, respectively) (Bellamy et al. 2000). The region on
chromosome 15 was then followed up by fine mapping and a marginally
significant association with a 7 bp deletion in UBE3A (P = 0.01) was found.
A second genome-wide study conducted in a Brazilian population
identified putative regions on chromosome 20 and 11 (Miller et al. 2004). The
third genome-wide scan was conducted in a Moroccan population. This study
indicated the presence of a major TB susceptibility locus on chromosome 8,
which appeared to have a dominant mode of inheritance (Baghdadi et al.
2006).
Recently, two new putative loci for TB have been identified on
chromosome 6 and 20 in populations from South Africa and Malawi. These
findings have been followed by a large independent case-control study in a
West African population. The region on chromosome 20q13.31-33 was
mapped in detail, suggesting the possible involvement of two new genes in
TB susceptibility; the melanocorticoid 3 receptor (MC3R) and cathepsin Z
gene (CTSZ) (Cooke et al. 2008).
Unlike candidate gene approaches, genome-wide scans can reveal, as
in the latter case, genes that might not have been considered a priori to be
involved in susceptibility to the disease. Despite some differences, the studies
performed in humans clearly indicate that there is a genetic component to
resistance / susceptibility, and have highlighted many of the genes found to
be candidates in mice. This augurs well for future research in other species,
suggesting that common mechanisms may exist.
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