**2. Method used**

#### **2.1. Study population**

In a case-control design, five hundred consanguineously unrelated individuals admitted at the University Hospital Complex: Thoracic Institute/ClementinoFraga University Hospital from Federal University of Rio de Janeiro-UFRJ were enrolled in this study after signing informed consent approved by the local Ethics Committee of HUCFF-UFRJ.

Demographic, clinical, and microbiological data as well as the HIV status of the subjects (age > 18 years old) were collected. Active TB cases (n=265) were defined as those after a positive culture confirmation in clinical specimen or with clinical, radiographic and laboratory improvement according to the American Thoracic Statements.They comprised 265 TB patients to be used for the descriptive genetic analysis. For the association study, TB-HIV comorbity was considered as an exclusion criteria and sample size was reduced as follow: 140 TB patients, being 121 with pulmonary TB (PTB) and 19 extrapulmonary forms of TB (TBE). The mean age of TB patients was ± 51 years (range 18-84 years) including 73 males and 67 females.

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 included 55 males and 99 females.

#### Sample Collection and handling

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

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

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

In a case-control design, five hundred consanguineously unrelated individuals admitted at the University Hospital Complex: Thoracic Institute/ClementinoFraga University Hospital from Federal University of Rio de Janeiro-UFRJ were enrolled in this study after signing

Demographic, clinical, and microbiological data as well as the HIV status of the subjects (age > 18 years old) were collected. Active TB cases (n=265) were defined as those after a positive culture confirmation in clinical specimen or with clinical, radiographic and laboratory improvement according to the American Thoracic Statements.They comprised 265 TB patients to be used for the descriptive genetic analysis. For the association study, TB-HIV comorbity was considered as an exclusion criteria and sample size was reduced as follow: 140 TB patients, being 121 with pulmonary TB (PTB) and 19 extrapulmonary forms of TB (TBE). The mean age

of TB patients was ± 51 years (range 18-84 years) including 73 males and 67 females.

association study of the identified SNPs and TB outcome after infection with *Mtb*.

informed consent approved by the local Ethics Committee of HUCFF-UFRJ.

response against *Mtb* have been emphasized [11-13].

80 Tuberculosis - Current Issues in Diagnosis and Management

associated with active TB disease [18, 19].

**2. Method used**

**2.1. Study population**

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 samples were stored at -20°C.

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

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

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 Applied BioSystems). All singletons and even new/rare mutation identified were confirmed by re-amplification and re-sequencing.

**Locus**

*IFNG* **Genotype Subjects**

(f) G

**(n = 500) Absolute Frequency**

CC 495 0.99

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


(f) T 5 \_

CC 495 0.99


(f) T 5 \_

(f) G 2 -


(f)T 3 -

AA 499 0.998

GG 486 0.972


(f) T 14 -

AA 498 0.996


(f) G 2

**Table 1.** Genotype and allele frequencies of SNPs within *IFNG* promoter in Brazilians from Rio de Janeiro.


CC 497 0.994


CC 487 0.974

GG 1 0.002 0.014

1 -

**Allele frequency** 83

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

#### **2.3. Computational analysis**

The SNPs identification in each individual sample was achieved after alignment of the generated sequences with the GenBank reference sequences AF3757790 and AB088112 for *IFNG* and *TNF-α* respectively. Transcription starting site sequence definition adopted for both genes considered as starting point, the first nucleotide immediately preceding position (-1) out of mRNA. Sequence analysis was carried out through SeqScapev. 2.6 software (Applied Biosystem). Haplotype reconstruction was achieved through the use of PHASE Vs. 2.1.1 software [21, 22].

#### **2.4. Statistical analysis**

Pair-wise linkage disequilibrium was tested for the loci studies. The Hardy-Weinberg equili‐ brium using χ<sup>2</sup> test. Statistics were performed in XLSTAT 2008.7 (Addinsoft Software Inc - New York USA). The magnitude of the associations was estimated by odds ratio values and the coefficient of associations. All tests were performed at the 0.05 level of significance by Epi Info version 3.5.1 2008 (Centers for Disease Control and Prevention, USA).
