Identification of genetic markers of susceptibility to Tuberculosis in signalling pathways that regulates IFNg; production

GUADALUPE INÉS ALVAREZ; RODRIGO HERNÁNDEZ DEL PINO; ANGELA BARBERO; VERÓNICA GARCÍA; VIRGINIA PASQUINELLI

Medellín

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

IMMUNOCOLOMBIA 2015

Tuberculosis(TB) a disease caused by Mycobacterium tuberculosis has a large impact onpublic health causing 9 million of new cases per year and almost 1.5 milliondeaths. Although more than one­third of the world  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 indetermining susceptibility and progression to TB disease. Moreover, the geneticbasis for pulmonary tuberculosis susceptibility has been demonstrated by thegreater concordance of pulmonary tuberculosis among monozygotic than dizygotictwins. The response triggered by IFN­g is a key component for the generation ofa protective immune response against M. tuberculosis. A significant associationbetween +874A/T (rs2430561) single nucleotide polymorphism (SNP) in IFNG andprotection against TB has been demonstrated in several populations (Lopez­Maderuelo and col., 2003,Cooke and col., 2006, Amim and col.,2008, Hashemi et al.,2011).We have previously shown that signaling lymphocytic activation molecule(SLAM) engagement up­regulate IFN­g production in patients with TB. However,the expression of the SLAM associated protein (SAP) on cells from TB patientsis inversely correlated with IFNg production (Pasquinelli and col., 2004). Ourresults demonstrate that expression of SAP interferes with Th1 responseswhereas SLAM expression contributes to Th1 cytokine responses in TB. Several SNPs of SLAM has been studied during the humoral response against measles virusvaccination (Dhiman , 2007 and col.,Ovsyannikova and col., 2011). Moreover SNPs of SLAM has been studied in rheumatoid arthritis patients and in a murine model oflupus (Wandstrat and col., 2004,Chabchoub and col., 2006). However, there are nostudies 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 ofautoimmunity/lymphoproliferation in males with defective Fas function. Furtheranalyses showed that ­346C was a methylation site and SAP expression was higherin ­346T than in ­346C males (Boggio and col., 2012). In this work we studied several SNPs in the candidate genes mentioned above with the goal ofcharacterize new genetic markers of TB susceptibility. DNA was extracted fromwhole blood of TB patients (PTB) and Healthy Donors (HD). +874 A/T SNP at the IFN­g gene was studied by Amplification­refractory mutation system­PCR. We also studied the ­262 A/T, ­188 A/G (by PCR­ Restriction fragment lengthpolymorphism (PCRRFLP)) 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 IFN­g, we found anelevated frequency of the AA genotype in PTB. Our results showed significantdifferences in the frequency distributions among the groups under studied (PTBand HD, X2= 20.88, p< 0.0001). The AA genotype was the most frequent inpatients 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 associatedwith lower IFN­γ production, we perform functional studies. To this end, PBMCs were stimulated for 48h with a cell lysate from M. tuberculosis (Mtb­Ag). IFNg production was determined in the culture supernatant by ELISA. However, theresults obtained so far did not show significant differences on IFN­g production among the different genotypes analyzed. As described above we alsostudied SNPs at the SLAM gene, a molecule known to positive regulate IFN­g production. Our results shown that the AA genotype of the ­262 A/T SNP of SLAMis 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 significantdifferences in the frequency distribution (X2= 11.68, p= 0.0029). To evaluatethe impact of this SNP on SLAM expression, PBMCs were stimulated with Mtb­Agfor 5 days and SLAM expression was determined by flow cytometry on CD3 positiveT cells. No significant differences were found on SLAM expression among thedifferent genotypes for this SNP. Regarding the ­188 A/G SNP, the GG genotypewas 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 ishighly frequent in TB patients and HD. Then, we decided to study the +1343 G/TSNP at the SLAM gene, since this SNP has been associated with significantlylower humoral immunity after measles vaccine (Dhiman and col., 2007). Moreover,this SNP leads to a non­synonymous mutation, with a change of a threonine for aproline in the 333 position of the protein. We speculate that this change could affect SLAM function and therefore IFN­g production. By analyzing 112 samplesfrom PTB and 146 samples from HD we found that the GG genotype is the mostfrequent in both groups (PTB= 74% vs HD= 84%) with no significantdifferences.Since, SLAM induce IFN­g production in M. tuberculosis stimulatedcells, but this signaling pathway is strongly influenced by the presence orabsence 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 frequencydistribution 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% (X2=31,32, p< 0,0001), ­494 A/G SNP, GG frequencies PTB= 87% vs HD=56% (X2= 29,72, p< 0,0001) and ­346 C/T SNP, TT frequencies PTB= 79% vs HD=44% (X2= 31,05, p< 0,0001)). Interestingly, all three genotypes of SAP thatwere more frequently found in PTB were associated with significantly lowerlevels of IFN­g production in vitro. Moreover, the AA susceptible genotype of IFNG is the most frequent on TB patients and the majority of them have theTT/CT susceptible genotype of SAP.The results shown that the AA/TT haplotype (IFNG +874 A/T and SAP ­346 C/T SNP) could be a genetic marker of TB susceptibility. Moreover, the TT genotype of ­346 SNP of SAP could be associated with thehypomethylation of the SAP promoter leading to reduced IFN­g production ontuberculosis patients. Further studies are needed to determine the role ofthese SNP on the generation of the immune response against M. tuberculosis, and the impact of the epigenetics modifications on IFN­g responses during active disease.