A genome wide association study of pulmonary tuberculosis susceptibility in Indonesians.

BACKGROUND: There is reason to expect strong genetic influences on the risk of developing active pulmonary tuberculosis (TB) among latently infected individuals. Many of the genome wide linkage and association studies (GWAS) to date have been conducted on African populations. In order to identify additional targets in genetically dissimilar populations, and to enhance our understanding of this disease, we performed a multi-stage GWAS in a Southeast Asian cohort from Indonesia. METHODS: In stage 1, we used the Affymetrix 100 K SNP GeneChip marker set to genotype 259 Indonesian samples. After quality control filtering, 108 cases and 115 controls were analyzed for association of 95,207 SNPs. In stage 2, we attempted validation of 2,453 SNPs with promising associations from the first stage, in 1,189 individuals from the same Indonesian cohort, and finally in stage 3 we selected 251 SNPs from this stage to test TB association in an independent Caucasian cohort (n = 3,760) from Russia. RESULTS: Our study suggests evidence of association (P = 0.0004-0.0067) for 8 independent loci (nominal significance P < 0.05), which are located within or near the following genes involved in immune signaling: JAG1, DYNLRB2, EBF1, TMEFF2, CCL17, HAUS6, PENK and TXNDC4. CONCLUSIONS: Mechanisms of immune defense suggested by some of the identified genes exhibit biological plausibility and may suggest novel pathways involved in the host containment of infection with TB.

Association of polymorphisms in IL-12/IFN-gamma pathway genes with susceptibility to pulmonary tuberculosis in Indonesia.

Upon infection with mycobacteria the IL-12/IFN-gamma axis plays an essential role in the activation of cell-mediated immunity required for the elimination of pathogens. Mutations in genes of the IL-12/IFN-gamma axis are known to cause extreme susceptibility to infection with environmental mycobacteria, and subtle variations in these genes may influence susceptibility to more virulent mycobacteria. We analyzed the distribution of polymorphisms in four essential genes from the IL-12/IFN-gamma axis, IL12B, IL12RB1, IFNG and IFNGR1, in 382 pulmonary tuberculosis patients and 437 healthy controls from an endemic region in Jakarta, Indonesia. The IL12RB1 gene was sequenced in a subset of individuals. Nine known single nucleotide polymorphisms (SNPs) and two new silent variations, 135G>A and 1056C>T, were detected in IL12RB1. Six functional SNPs (-2C>T, 467G>A, 641A>G, 1312C>T, 1573G>A, 1781G>A) in IL12RB1, an IL12B promoter insertion/deletion polymorphism and CA repeats in IFNG and IFNGR1 were analyzed in the cohort. The IFNGR1 allele CA(12) (p=0.004) and genotype CA(12)/CA(12) (p=0.01; OR 0.5) were associated with protection from pulmonary tuberculosis. Interestingly, IL12B promoter heterozygosity was associated with protection from tuberculosis in BCG-vaccinated individuals (p=0.03; OR=0.6). This new finding supports the role that IL-23-of which IL12B encodes a subunit--plays in generation of memory T cells.

Polymorphisms in SP110 are not associated with pulmonary tuberculosis in Indonesians.

Despite being high transmissible, Mycobacterium tuberculosis (M. tuberculosis) infection causes active disease in only 5-10% of disease-susceptible individuals. This has instigated interest in studying potentially underlying genetic host factors and mechanisms in tuberculosis (TB). The recent identification of the Intracellular pathogen resistance 1 (Ipr1) gene, which plays a major role in controlling M. tuberculosis susceptibility and infection severity in mice (Pan et al., 2005), has prompted studies on its human homolog; SP110 in humans. Association of SP110 SNPs with pulmonary TB were first reported in a study on West African families (Tosh et al., 2006). Subsequent attempts to replicate these findings in other populations, including another West African (Ghanaian) cohort (Thye et al., 2006), however, were unsuccessful. Here we have genotyped 20 SNPs located in the SP110 gene, including the previously TB associated variants; rs2114592 and rs3948464, for the first time in a South East Asian cohort from Indonesia. Our study did not reveal any statistically significant associations between SP110 SNPs and pulmonary TB. In addition, a meta-analysis of the two previously TB associated SNPs revealed that these are not associated with TB, further confirming the lack of convincing evidence for SP110 to be implicated in TB susceptibility, as yet in humans.

Iron deficiency and NRAMP1 polymorphisms (INT4, D543N and 3'UTR) do not contribute to severity of anaemia in tuberculosis in the Indonesian population.

Fe-deficiency anaemia is the most common cause of anaemia in developing countries. In these settings, many chronic infections, including tuberculosis (TB), are highly prevalent. Fe is an essential nutrient for both host and mycobacteria that play a pivotal role in host immunity and mycobacterial growth. A case-control study was performed in a TB-endemic region in Jakarta, Indonesia, among 378 pulmonary TB patients and 436 healthy controls from the same neighbourhood with the same socio-economic status. In a number of these subjects the Fe status could be explored. The distribution of three polymorphisms in the natural resistance-associated macrophage protein gene (NRAMP1) including INT4, D543N and 3'UTR was examined for a possible association with susceptibility to TB. Anaemia (corrected for sex) was present in 63.2 % of active TB compared with 6.8 % of controls, with female patients more often affected. Anaemia was more pronounced in advanced TB as diagnosed by chest radiography. Lower Hb concentrations in TB patients were accompanied by lower plasma Fe concentrations, lower Fe-binding capacity and higher plasma ferritin. After successful TB therapy, Fe parameters improved towards control values and Hb levels normalised, even without Fe supplementation. NRAMP1 gene polymorphisms were not associated with TB susceptibility, TB severity or anaemia. In conclusion, most active TB patients had anaemia, which was probably due to inflammation and not to Fe deficiency since TB treatment without Fe supplementation was sufficient to restore Hb concentration.

Dynamic changes in pro- and anti-inflammatory cytokine profiles and gamma interferon receptor signaling integrity correlate with tuberculosis disease activity and response to curative treatment.

Pro- and anti-inflammatory cytokines and their signaling pathways play key roles in protection from and pathogenesis of mycobacterial infection, and their balance and dynamic changes may control or predict clinical outcome. Peripheral blood cells' capacity to produce proinflammatory (tumor necrosis factor alpha [TNF-alpha], interleukin-12/23p40 [IL-12/23p40], and gamma interferon [IFN-gamma]) and anti-inflammatory (IL-10) cytokines in response to Mycobacterium tuberculosis or unrelated stimuli (lipopolysaccharide, phytohemagglutinin) was studied in 93 pulmonary tuberculosis (TB) patients and 127 healthy controls from Indonesia. Their cells' ability to respond to IFN-gamma was examined to investigate whether M. tuberculosis infection can also inhibit IFN-gamma receptor (IFN-gammaR) signaling. Although there was interindividual variability in the observed responses, the overall results revealed that M. tuberculosis-induced TNF-alpha and IFN-gamma levels showed opposite trends. Whereas TNF-alpha production was higher in active-TB patients than in controls, IFN-gamma production was strongly depressed during active TB, correlated inversely with TB disease severity, and increased during therapy. By contrast, mitogen-induced IFN-gamma production, although lower in patients than in controls, did not change during treatment, suggesting an M. tuberculosis-specific and reversible component in the depression of IFN-gamma. Depressed IFN-gamma production was not due to decreased IL-12/IL-23 production. Importantly, IFN-gamma-inducible responses were also significantly depressed during active TB and normalized during treatment, revealing disease activity-related and reversible impairment in IFN-gammaR signaling in TB. Finally, IFN-gamma/IL-10 ratios significantly correlated with TB cure. Taken together, these results show that M. tuberculosis-specific stimulation of IFN-gamma (but not TNF-alpha) production and IFN-gammaR signaling are significantly depressed in active TB, correlate with TB disease severity and activity, and normalize during microbiological TB cure. The depression of both IFN-gamma production and IFN-gammaR signaling may synergize in contributing to defective host control in active TB.