**1. Introduction**

Tuberculosis (TB) is a major public concern and is the most important single infectious cause of mortality and morbidity worldwide. According the World Health Organization (WHO) records, in 2009, there were an estimated 9.4 million new cases, 14 million prevalent cases, and approximately 1.7 million deaths by TB [1]. Additionally, approximately one third of the world's population is infected with the causative bacterium, *Mycobacterium tuberculosis* (*Mtb*), and is at risk for developing active tuberculosis. Interestingly, while approximately 9 million people develop active TB each year, the majority remain asymptomatically (latently) infected with the pathogen presumably due to a protective immune response. Without intervention, approximately five to ten percent of those latently infected will develop overt disease and the potential to transmit *Mtb* to others [2].

Familial clustering data, twin studies and complex segregation analysis have all suggested a strong genetic component in the human susceptibility to the chronic mycobacterial diseases [3-7] but also a complex picture of geographic heterogeneity in genetic effects on the different

© 2013 Lopes et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

mycobacterial infections is involved [8, 9]. Several non-HLA genes have been implicated in TB susceptibility. However, the discrepant data reported may be attributed to a number of different factors, such as the types of studies, ethnicity, genetic background, and clinical status of patients with tuberculosis that may be associated with a particular genetic profile. The interaction among lung cells with pro and anti-inflammatory mediators during the infection with *Mtb* have been deeply investigated [10]. Among involved cytokines, the key role of interferon-gamma (IFN-γ) and tumor necrosis factor (TNF-α) in eliciting an inflammatory response against *Mtb* have been emphasized [11-13].

For the control group, a complete questionnaire to document TB risk factors since baseline testing was used. Individuals were eligible as controls if they had no previous TB history, consanguinity and negative HIV status. In formations concerning Tuberculin Skin Test (TST) response were available for all controls. They comprised 235 individuals, to be used for descriptive genetic analysis. For the association study, after application of the exclusion criteria, 154 individuals were included in this group, of which, 96 were TST positive (TST+) and 58 TST negative (TST-). The mean age in this group was ± 50 (range 18 - 82 years) and

Influence of the Interferon–Gamma (IFN–γ) and Tumor Necrosis Factor Alpha (TNF–α) Gene Polymorphisms in TB...

http://dx.doi.org/10.5772/55099

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A volume of 3 mL of venous blood was collected from each volunteer and stored at -20°C. Genomic DNA was isolated from 100 μL of frozen whole blood using the FlexiGene DNA Kit (Qiagen Inc., USA), according to the manufacturer's specifications. After extraction, DNA

Genotyping of the proximal portion of the promoter region in *TNF*-α and *IFNG* genes was achieved by direct sequencing of PCR products. Two sets of primers for PCR amplifica‐ tion and sequencing of *IFNG*, DNA fragment of 863bp, (IFN-EF: 5' GGAACTCCCCCTGG‐ GAATATTCT 3`, IFNER: 5´AGCTGATCAGGTCCAAAGGA3´, IFNIF: 5 ´CGAAGTGGGGAGGT ACAAAA 3´ and IFNIR: 5´ CCCAGGAAACTGCTACTCTG 3´), and *TNF*-α, DNA fragment of 855bp (TNFEF: 5´CAGGACCTCCAGGTATGGAA3´, TNFER: 5' TAGCTGGTCCTCTGCTGTCC3', TNFIF: 5´CCTGCATCCTGTCTGGAAGT 3´ and TNF-IR: 5'TTTCAACCCCTGTGTGTTCG 3') were designed by using the Primer3 software [20].

For PCR-mediated DNA amplification of *IFNG*, 100 ng of genomic DNA were added to a 50μL reaction mixture containing 200ng of each primer (IFN-EF and IFN-ER), 0.2mM of each dNTPs, 2.0mM MgCl2 and 1U *Taq* DNA polymerase (Invitrogen by Life Technolo‐ gies, USA) and submitted to an initial denaturation at 94°C for 5 min., followed by 35 cycles of 1 min. at 94°C, 1 min. at 65.3°C and 1 min at 72°C. Final extension was per‐ formed for 5 min. at 72°C. Likewise, for amplification of the 855pb *TNF*-α fragment, 100ng of genomic DNA were added to a 25μL reaction mixture containing 200ng of each primer (TNF-EF and TNF-ER), 2mM MgCl2, 0.2mM of each dNTPs and 0.5U of *Taq* gold DNA polymerase (PE Applied BioSystems) and submitted to initial denaturation at 94°C for 15 min, followed by 35 cycles of 1 min. at 94°C, 1 min. at 65.9°C and 1 min at 72°C with a final extension at 72°C for 5 min. Evaluation of PCR products was done by electrophore‐

For sequencing, PCR products were purified with ChargeSwitch Kit (Invitrogen Life Tech‐ nologies), according to the manufacturer's recommendations. Sequencing of the amplified fragments was performed in both DNA strands using a combination of the internal and external primers using ABI PRISM Big Dye Terminator v. 3.1 Kit (PE Applied BioSystems), according to the manufacturer's recommendations, on an ABI PRISM 3730 DNA Analyser (PE

sis on 1.2% agarose gel followed by ethidium bromide staining.

included 55 males and 99 females.

Sample Collection and handling

samples were stored at -20°C.

**2.2.** *IFNG* **and** *TNF***–α genotyping**

In human studies, the crucial role of TNF-α in protective host immunity against reactiva‐ tion of latent TB was highlighted by the observation that the relapse and severe course of TB is over-represented in rheumatoid arthritis patients following the use of anti-TNF-α antibodies [14]. Concerning the IFN-γ, it is well established that deficiency in IFN-γ gene expression is associated with severe impairment of resistance to infections, in particular those that are normally killed by activated macrophages [15, 16]. Low synthesis of this cytokine has been associated with active tuberculosis [17]. However, on the contrary of TNF-α, the Interferon gamma conding gene (*IFNG*) is highly conserved and few single nucleotide polymorphisms (SNPs) are found in the intragenic region. Several case-control studies to evaluate association of SNPs in these genes with TB have produced mixed results, with little consensus in most cases on whether any TNF polymorphisms are actually associated with active TB disease [18, 19].

In the present study we aimed to analyse the existing promoter variability of the *IFNG* and *TNF*-α genes by partial mapping of this region in samples from Brazilians, followed by an association study of the identified SNPs and TB outcome after infection with *Mtb*.
