Identification of genetic markers of susceptibility to Tuberculosis in signalling pathways that regulates IFN-g production

ALVAREZ GI; HERNÁNDEZ DEL PINO RE,; BARBERO AM; GARCÍA VE; PASQUINELLI V

Medellín

Congreso; IMMUNOCOLOMBIA2015; 11th Congress of the Latin American Association of Immunology; 10º Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología; 2015

Latin American Association of Immunology (ALAI) y Asociación Colombiana de Alergia, Asma e Inmunología

Tuberculosis (TB) a disease caused by Mycobacterium tuberculosis has a large impact on public health causing 9 million of new cases per year and almost 1.5 million deaths. Although more than one-third of the world?s population is potentially infected with M. tuberculosis, only 10 % of infected individuals eventually develop TB disease, indicating that host genetic factors may play essential but complex role in determining susceptibility and progression to TB disease. Moreover, the genetic basis for pulmonary tuberculosis susceptibility has been demonstrated by the greater concordance of pulmonary tuberculosis among monozygotic than dizygotic twins. The response triggered by IFN-γ is a key component for the generation of a protective immune response against M. tuberculosis. A significant association between +874A/T (rs2430561) single nucleotide polymorphism (SNP) in IFNG and protection against TB has been demonstrated in several populations (Lopez-Maderuelo et al., 2003;Cooke et al., 2006;Amim et al., 2008;Hashemi et al., 2011). We have previously shown that signaling lymphocytic activation molecule (SLAM) engagement up-regulate IFN-γ production in patients with TB. However, the expression of the SLAM-associated protein (SAP) on cells from TB patients is inversely correlated with IFN-γ production (Pasquinelli et al., 2004). Our results demonstrate that expression of SAP interferes with Th1 responses whereas SLAM expression contributes to Th1 cytokine responses in TB. Several SNPs of SLAM has been studied during the humoral response against measles virus vaccination (Dhiman et al., 2007;Ovsyannikova et al., 2011). Moreover SNPs of SLAM has been studied in rheumatoid arthritis patients and in a murine model of lupus (Wandstrat et al., 2004;Chabchoub et al., 2006). However, there are no studies of SLAM SNPs in TB. Recently it has been demonstrated that the -346T polymorphism of the SH2D1A gene (SAP) is a risk factor for development of autoimmunity/lymphoproliferation in males with defective Fas function. Further analyses showed that -346C was a methylation site and SAP expression was higher in -346T than in -346C males (Boggio et al., 2012). In this work we studied several SNPs in the candidate genes mentioned above with the goal of characterize new genetic markers of TB susceptibility. DNA was extracted from whole blood of TB patients (PTB) and Healthy Donors (HD). +874 A/T SNP at the IFN-γ gene was studied by Amplification-refractory mutation system-PCR. We also studied the -262 A/T; -188 A/G (by PCR-Restriction fragment length polymorphism (PCR-RFLP)) and +1343 G/T (by PCR and sequencing) SNPs at SLAM gene. The SNPs polymorphisms for SAP (-631 A/G, -494 A/G and -346 C/T) were also studied by PCR and sequencing. For the +874 A/T SNP of IFN-γ, we found an elevated frequency of the AA genotype in PTB. Our results showed significant differences in the frequency distributions among the groups under studied (PTB and HD, χ2= 20.88; p< 0.0001). The AA genotype was the most frequent in patients with active TB (PTB= 63.9% (N=161) vs HD= 31.9% (N=191)), demonstrating that the AA genotype could be a genetic marker of disease susceptibility to TB. Since, the presence of the A allele has been associated with lower IFN-γ production, we perform functional studies. To this end, PBMCs were stimulated for 48h with a cell lysate from M. tuberculosis (Mtb-Ag). IFN-γ production was determined in the culture supernatant by ELISA. However, the results obtained so far did not show significant differences on IFN-γ production among the different genotypes analyzed. As described above we also studied SNPs at the SLAM gene, a molecule known to positive regulate IFN-γ production. Our results shown that the AA genotype of the -262 A/T SNP of SLAM is overrepresented in both groups of individuals under study (PTB= 70.5% vs HD= 89.3%). However, was a 20% more frequent in HD, leading to significant differences in the frequency distribution (χ2= 11.68; p= 0.0029). To evaluate the impact of this SNP on SLAM expression, PBMCs were stimulated with Mtb-Ag for 5 days and SLAM expression was determined by flow cytometry on CD3 positive T cells. No significant differences were found on SLAM expression among the different genotypes for this SNP. Regarding the -188 A/G SNP, the GG genotype was highly represented in both groups of individuals (PTB= 84% vs HD= 93%) without differences in frequency distribution. Therefore, the two SLAM SNPs could not be used as markers of susceptibility, since the AG/AG haplotype is highly frequent in TB patients and HD. Then, we decided to study the +1343 G/T SNP at the SLAM gene, since this SNP has been associated with significantly lower humoral immunity after measles vaccine (Dhiman et al., 2007). Moreover, this SNP leads to a non-synonymous mutation, with a change of a threonine for a proline in the 333 position of the protein. We speculate that this change could affect SLAM function and therefore IFN-γ production. By analyzing 112 samples from PTB and 146 samples from HD we found that the GG genotype is the most frequent in both groups (PTB= 74% vs HD= 84%) with no significant differences. Since, SLAM induce IFN-γ production in M. tuberculosis stimulated cells, but this signaling pathway is strongly influenced by the presence or absence of SAP, we then evaluate three SNPs at the SAP gene (-631 A/G, -494 A/G and -346 C/T). Interestingly, we found significant differences in the frequency distribution between PTB and HD for the three SNPS studied. The most frequent genotypes found in PTB were AA, GG and TT for the -631 A/G, -494 A/G and -346 C/T, respectively. In all the cases we found significant differences when compared the frequency distributions against HD (-631 A/G SNP, AA frequencies PTB=79% vs HD=44% (χ2= 31,32; p< 0,0001); -494 A/G SNP, GG frequencies PTB= 87% vs HD= 56% (χ2= 29,72; p< 0,0001) and -346 C/T SNP, TT frequencies PTB= 79% vs HD= 44% (χ2= 31,05; p< 0,0001)). Interestingly, all three genotypes of SAP that were more frequently found in PTB were associated with significantly lower levels of IFN-γ production in vitro. Moreover, the AA susceptible genotype of IFNG is the most frequent on TB patients and the majority of them have the TT/CT susceptible genotype of SAP. 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