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 miceand 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-12R1 deficiency and with the gene encoding the IFN- receptor ligand binding chain IFNR1 (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, thesegenes 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 (Bellamyet 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 case-control 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 inSouth 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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