**3. Results**

In this work, a partial mapping of the promoter regions of *IFNG* and *TNF-α* genes was performed by direct PCR sequencing approach in 265 TB patients and 235 healthy controls residents in Rio de Janeiro, Brazil. Sequencing approach allowed the identification of new SNPs and consequently new haplotypes for both genes. Expected genotype frequencies were calculated from respective single allele frequencies and were consistent with Hard Weinberg Equilibrium using χ<sup>2</sup> test.

#### **3.1. Partial mapping of the** *IFNG* **promoter region in samples from Brazilians residents in Rio de Janeiro**

Sequence analysis of the proximal portion of *IFNG* promoter region (863 bp upstream of the transcription starting site) revealed the presence of seven SNPs, of which, four were new, and located at positions (-787C>T, -599C>G, -517C>T, and -255A>G). The three remaining SNPs, already deposited in GenBank-Entrez SNP database, were located at positions (-785C>T, -200G>T, reported as (-183 and -179) and -172A>G (reported as -155). Table 1 show the allele and genotype frequencies of the identified SNPs in the whole studied population (500 samples). All SNPs were found in a very low frequency, sometimes as a singleton (-255A>G). In this case, the SNP was confirmed by new PCR amplification and re-sequencing. The two more frequent SNPs were the ones located at positions -599C>G and -200G>T, both with 1.4%. No homozygosity was identified in these positions.

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 83

Applied BioSystems). All singletons and even new/rare mutation identified were confirmed

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

Pair-wise linkage disequilibrium was tested for the loci studies. The Hardy-Weinberg equili‐

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

In this work, a partial mapping of the promoter regions of *IFNG* and *TNF-α* genes was performed by direct PCR sequencing approach in 265 TB patients and 235 healthy controls residents in Rio de Janeiro, Brazil. Sequencing approach allowed the identification of new SNPs and consequently new haplotypes for both genes. Expected genotype frequencies were calculated from respective single allele frequencies and were consistent with Hard Weinberg

**3.1. Partial mapping of the** *IFNG* **promoter region in samples from Brazilians residents in**

Sequence analysis of the proximal portion of *IFNG* promoter region (863 bp upstream of the transcription starting site) revealed the presence of seven SNPs, of which, four were new, and located at positions (-787C>T, -599C>G, -517C>T, and -255A>G). The three remaining SNPs, already deposited in GenBank-Entrez SNP database, were located at positions (-785C>T, -200G>T, reported as (-183 and -179) and -172A>G (reported as -155). Table 1 show the allele and genotype frequencies of the identified SNPs in the whole studied population (500 samples). All SNPs were found in a very low frequency, sometimes as a singleton (-255A>G). In this case, the SNP was confirmed by new PCR amplification and re-sequencing. The two more frequent SNPs were the ones located at positions -599C>G and -200G>T, both with 1.4%.

Info version 3.5.1 2008 (Centers for Disease Control and Prevention, USA).

test. Statistics were performed in XLSTAT 2008.7 (Addinsoft Software Inc -

by re-amplification and re-sequencing.

82 Tuberculosis - Current Issues in Diagnosis and Management

**2.3. Computational analysis**

software [21, 22].

brium using χ<sup>2</sup>

**3. Results**

Equilibrium using χ<sup>2</sup>

**Rio de Janeiro**

test.

No homozygosity was identified in these positions.

**2.4. Statistical analysis**


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

### **3.2.** *IFNG* **haplotypes characterization**

Haplotype reconstruction was achieved from genotyping data by using Phase Vs. 2.1.1 software. A total of eight different haplotypes were characterized with basis on the combina‐ tion of the seven SNPs identified within the *IFNG* promoter. Table 2 shows the frequencies of the identified haplotypes in the total population. The haplotype 4 was the, more frequent among the whole samples analyzed.

**Locus Genotype Subjects**

**(n=500)**

**Absolute Frequency**

GG 495 0.990 -

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

A 5 \_ 0.005

C 5 \_ 0.005

T 1 \_ 0.001

GG 471 0.942 \_

AA 1 0.002 \_

GG 418 0.836 \_

AA 5 0.010 \_

GG 489 0.978 \_

A 87 \_ 0.087

A 11 \_ 0.011

GG 453 0.906 \_

AA 3 0.006 \_

A 50 \_ 0.050

A 30 \_ 0.030

CC 499 0.998 \_

AA 495 0.990 -








**Table 3.** Genotype and allele frequencies of SNPs within TNF-α promoter in Brazilians from Rio de Janeiro.

**Allele Frequency** 85

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**Table 2.** Characterization of the identified haplotypes within *IFNG* proximal promoter region in Brazilians from Rio de Janeiro (n=500).

#### **3.3. Partial mapping of the** *TNF***–***α* **promoter region in samples from Brazilians residents in Rio de Janeiro**

The partial mapping of the proximal portion (855 bp upstream of the transcription starting site) of *TNF*-α promoter was also performed by direct sequencing of PCR products. Upon analysis of the generated sequences seven SNPs, all described in the literature, and were identified in a total of 500 samples. Table 3 shows the allele and genotype frequencies. With the exception of the most studied SNPs (-238 -308, and -376) presenting frequencies higher than 3%, all others were present in less than one percent.

#### **3.4.** *TNF***–***α***haplotypes characterization**

A total of fourteen different haplotypes were characterized. Except for the wild-type, haplo‐ type 1, the higher frequent was the haplotype 3, presenting a mutant variation only at -308 position. As expected, the rare combination presenting polymorphisms only at positions -238 and -308 was present in the sample studied although in a low frequency (Table 4).

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 85

**3.2.** *IFNG* **haplotypes characterization**

84 Tuberculosis - Current Issues in Diagnosis and Management

among the whole samples analyzed.

Janeiro (n=500).

**Rio de Janeiro**

Haplotype reconstruction was achieved from genotyping data by using Phase Vs. 2.1.1 software. A total of eight different haplotypes were characterized with basis on the combina‐ tion of the seven SNPs identified within the *IFNG* promoter. Table 2 shows the frequencies of the identified haplotypes in the total population. The haplotype 4 was the, more frequent

**Haplotypes -787 -785 -599 -517 -255 -200 -172 Frequency**

1 C C C C A G A 0.916

2 C **T** C C A G A 0.010

3 C C G C A G A 0.024

4 C C C C A **T** A 0.028

5 **T** C C C A G A 0.010

6 C C C C **G** G A 0.002

7 C C C C A G **G** 0.004

8 C C C **T** A G A 0.006

**Table 2.** Characterization of the identified haplotypes within *IFNG* proximal promoter region in Brazilians from Rio de

**3.3. Partial mapping of the** *TNF***–***α* **promoter region in samples from Brazilians residents in**

The partial mapping of the proximal portion (855 bp upstream of the transcription starting site) of *TNF*-α promoter was also performed by direct sequencing of PCR products. Upon analysis of the generated sequences seven SNPs, all described in the literature, and were identified in a total of 500 samples. Table 3 shows the allele and genotype frequencies. With the exception of the most studied SNPs (-238 -308, and -376) presenting frequencies higher

A total of fourteen different haplotypes were characterized. Except for the wild-type, haplo‐ type 1, the higher frequent was the haplotype 3, presenting a mutant variation only at -308 position. As expected, the rare combination presenting polymorphisms only at positions -238

and -308 was present in the sample studied although in a low frequency (Table 4).

than 3%, all others were present in less than one percent.

**3.4.** *TNF***–***α***haplotypes characterization**


**Table 3.** Genotype and allele frequencies of SNPs within TNF-α promoter in Brazilians from Rio de Janeiro.


**Loci Genotype**






Occurrence of TBactive

Occurrence pulmonary TB

disease severity

latent infection

**Patientes (N=140)**

CC 138 96

CT 2 0

CC 140 94

CT 0 2

CC 135 93

CG 5 3

CC 140 93

CG 0 3

GG 138 89

GT 2 7

**Table 5.** Genotype distribution of the *IFNG* SNPs among TB patients and healthy controls (TST+).

Given that the SNP -200 *IFNG* was the only one that was associated with any of the studied outcomes at genotype level, allele frequency was also tested for the same outcomes. Table 6 shows the comparison of the -200T variant between the stratified groups. The results confirm the association with protection to the occurrence of active TB and, additionally to TBP. Association of the -200T variant was also seen to occurrence of latent infection (*p*=0.035).

**Different outcomes Groups SNP IFNG -200**

TST+ 0.036

TST+ 0.036

> TBE 0.00

TST-0.000

Finally, the more prevalent *IFNG* polymorphisms (-599C>G and-200G>T) were tested against demographic variables, such as, gender and age. No significant association was found after

**Table 6.** Distribution of allele frequencies of-200T variant mutant groups according to the different outcomes.

Pacientes 0.0071

> TBP 0.0082

> TBP 0.0082

TST+ 0.036

stratified analysis at allele, genotype or haplotype levels (data not shown).

**Controls TST+\* (N=**

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

**96) <sup>χ</sup><sup>2</sup>** *p-value* **OR IC**

1.383 0.515 # #

2.942 0.16 # 0.00<2.79

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87

0.035 NS 1.15 0.23<6.23

2.29 0.066 # 0.00<1.52

3.86 0.033 0.18 0.03<1.00

*P-valor\** **OR IC**

0033 0.19 0.03<1.01

0.043 0.22 0.033<1.17

1.00 # #

0.035 # #

**Table 4.** Haplotypes description and frequencies within TNF-α promoter in Brazilians from Rio de Janeiro.

#### **3.5. Association of the** *IFNG* **SNPs and TB outcomes**

Association of the identified SNPs variations within the analyzed region of*IFNG* with different TB outcomes (susceptibility *per se*, protection, severity and susceptibility to latent *M. tubercu‐ losis* infection) was assessed based in the comparison of allele, genotype and haplotype frequencies between the stratified groups. The groups used for each evaluation were as follow: a) susceptibility *per se* to TB (TB patients versus TST+ controls), b) disease severity (PTB versus TBE) and c) susceptibility to the latent infection (healthy controls TST+ versus TST-).

As previously stated, for this analysis, the sample size was reduced in groups, (patients and controls) because of the exclusion criteria of TB-HIV co-infection and consanguinity. After exclusions, because of the very low frequency of the -255 A>G and -172 A>G these SNPs were also excluded.

Results of the association study upon comparison of genotype frequencies of the five remaining SNPs between TB patients (TBP/EPTB) versus TST+ controls are shown in Table 5. Only the SNP -200G>T presented a significantly higher frequency of the GT genotype in the control group indicating an association of this genotype with protection to the occurrence of active TB (χ<sup>2</sup> = 3.86, *p* = 0.033, OR = 0.18 CI = 0.03 -1.00). Evaluation of the identified SNPs with the other outcomes did not show any association (data not shown).

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 87


**Table 5.** Genotype distribution of the *IFNG* SNPs among TB patients and healthy controls (TST+).

**Haplotype -646 -572 -422 -376 -308 -238 -244 Frequency**

 G A C G G G G 0.710 G A C **A** G G G 0.006 G A C G **A** G G 0.146 G **C** C G G G G 0.006 G A C G G G **A** 0.020 G A C G G **A** G 0.040 **A** A C G G G G 0.008 G A **T** G G G G 0.002 G A C **A** G **A** G 0.044 G A C **A A A** G 0.060 G A C **A A** G G 0.002 G A C G **A** G **A** 0.002 G A C G **A A** G 0.004 G **C** C G **A** G G 0.004

**Table 4.** Haplotypes description and frequencies within TNF-α promoter in Brazilians from Rio de Janeiro.

TBE) and c) susceptibility to the latent infection (healthy controls TST+ versus TST-).

Association of the identified SNPs variations within the analyzed region of*IFNG* with different TB outcomes (susceptibility *per se*, protection, severity and susceptibility to latent *M. tubercu‐ losis* infection) was assessed based in the comparison of allele, genotype and haplotype frequencies between the stratified groups. The groups used for each evaluation were as follow: a) susceptibility *per se* to TB (TB patients versus TST+ controls), b) disease severity (PTB versus

As previously stated, for this analysis, the sample size was reduced in groups, (patients and controls) because of the exclusion criteria of TB-HIV co-infection and consanguinity. After exclusions, because of the very low frequency of the -255 A>G and -172 A>G these SNPs were

Results of the association study upon comparison of genotype frequencies of the five remaining SNPs between TB patients (TBP/EPTB) versus TST+ controls are shown in Table 5. Only the SNP -200G>T presented a significantly higher frequency of the GT genotype in the control group indicating an association of this genotype with protection to the occurrence of active TB (χ<sup>2</sup> = 3.86, *p* = 0.033, OR = 0.18 CI = 0.03 -1.00). Evaluation of the identified SNPs with the other

**3.5. Association of the** *IFNG* **SNPs and TB outcomes**

86 Tuberculosis - Current Issues in Diagnosis and Management

outcomes did not show any association (data not shown).

also excluded.

Given that the SNP -200 *IFNG* was the only one that was associated with any of the studied outcomes at genotype level, allele frequency was also tested for the same outcomes. Table 6 shows the comparison of the -200T variant between the stratified groups. The results confirm the association with protection to the occurrence of active TB and, additionally to TBP. Association of the -200T variant was also seen to occurrence of latent infection (*p*=0.035).


**Table 6.** Distribution of allele frequencies of-200T variant mutant groups according to the different outcomes.

Finally, the more prevalent *IFNG* polymorphisms (-599C>G and-200G>T) were tested against demographic variables, such as, gender and age. No significant association was found after stratified analysis at allele, genotype or haplotype levels (data not shown).

#### **3.6. Association of the** *TNF***–α SNPs and TB outcomes**

Table 7 summarizes the distribution and comparison of genotype frequencies of each indi‐ vidual SNP among TB patients and TST+ controls. No significant difference was observed. The evaluation of the possible association of different genotypes of *TNF*-α gene with susceptibility to the occurrence of TBP or TBE was also carried out separately, however, no association was found (data not shown).

**Locus Genotype**








Occurrence of activeTB

Occurrence of PTB

Severity of disease

Latent infection

studied.

**PTB N=121**

GG 120 19

AA 121 17

AC 0 2

GG 111 17

AA 0 1

GG 101 17

AA 2 1

GG 117 19

GA 4 0

GG 106 17

AA 1 1

**Table 8.** Genotypes distribution of the *TNF*-α SNPs among TBP and TBE.

association with any of the variants tested, (data not shown).

Patients (*fa*) 0.054

PTB (*fa*) 0.041

PTB (*fa*) 0.041

TST+ (*fa*) 0.016

significant difference was found (data not shown).

**TBE**

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

1.00 0.00 0,00<115.2 GA <sup>1</sup> <sup>0</sup>

CC <sup>121</sup> <sup>19</sup> - - - CT <sup>0</sup> <sup>0</sup>

GA 10 1 0.506 1.25 0.33<4.79

GA 18 1 0.392 0.63 0.16<2.54

GA 14 1 0.585 0.85 0.22<3.37

The association between the *TNF*-α genotypes with latent infection was also evaluated. No

The final evaluation of independent SNPs with the different TB outcomes was performed based in the allele frequencies comparison for the most common *TNF*-α SNPs (-376G>A, -308G>A, -244G>A and -238G>A). The SNP -376G>A, allele variant -376A, showed a significant association with susceptibility to the occurrence of active TB (*p* = 0.035, OR = 3.57, CI = 0.95 < 15.72) and severity (*p* = 0.038 and RR = 2.68) (Table 9). All other outcomes showed no significant

**Different outcomes Study Group SNP TNF-α -376A**

TST+ 0.016

TST+ 0.016

TBE 0.052

TST-0.017

**Table 9.** Distribution of allele frequency of the *TNF*-α -376A variant and association analysis with different outcomes

**N=19** *p-value* **RR IC**

0.0175 8.12 5.20<12.67

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89

0.554 # #

*p-valor* **OR IC**

0.035 3.57 0.95<15.72

0.201 2.72 0,68<12.62

0.038 2.68\* 1.22<5.86

0.90 0.623 0.12<7.86


**Table 7.** Genotype distribution of the *TNF*-α SNPs among PTB patients and healthy controls (TST+).

Comparison of the *TNF*-α SNPs frequencies between TBP and TBEis shown in (Table 8). Only the -572A>C (CA genotype) presented a significant difference between these groups, being absent among the 121 TBP subjects, (RR = 8.12, CI = 5.20 <12.67 and *p*-value = 0.0175). The results indicate a risk for disease severity. This association was confirmed upon allele frequency evaluation (RR = 7.72, CI = 5.69 <10.47 and *p* = 0.0179) data not shown.

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 89


**Table 8.** Genotypes distribution of the *TNF*-α SNPs among TBP and TBE.

**3.6. Association of the** *TNF***–α SNPs and TB outcomes**

88 Tuberculosis - Current Issues in Diagnosis and Management

**Patients (N=140)**

GG 139 95

AA 138 95

CC 140 96

GG 127 93

AA 2 0

GG 118 83

AA 3 2

GG 136 92

GG 123 88

AA 2 0

**Table 7.** Genotype distribution of the *TNF*-α SNPs among PTB patients and healthy controls (TST+).

evaluation (RR = 7.72, CI = 5.69 <10.47 and *p* = 0.0179) data not shown.

found (data not shown).

**Locus Genotype**








Table 7 summarizes the distribution and comparison of genotype frequencies of each indi‐ vidual SNP among TB patients and TST+ controls. No significant difference was observed. The evaluation of the possible association of different genotypes of *TNF*-α gene with susceptibility to the occurrence of TBP or TBE was also carried out separately, however, no association was

**Controls**

GA 1 1 1.00 0.68 0.02<25.33

CA 2 1 1.00 1.38 0.10<38.92

GA 11 3 0.11 3.17 0.81<14.46

GA 19 11 0.78 1.19 0.54<2.67

GA 4 4 0.71 0.68 0.14<3.31

GA 15 8 0.47 1.50 0.58<3.97

Comparison of the *TNF*-α SNPs frequencies between TBP and TBEis shown in (Table 8). Only the -572A>C (CA genotype) presented a significant difference between these groups, being absent among the 121 TBP subjects, (RR = 8.12, CI = 5.20 <12.67 and *p*-value = 0.0175). The results indicate a risk for disease severity. This association was confirmed upon allele frequency

CT 0 0 - - -

**TST+ (N= 96)** *p-valor* **OR IC**

The association between the *TNF*-α genotypes with latent infection was also evaluated. No significant difference was found (data not shown).

The final evaluation of independent SNPs with the different TB outcomes was performed based in the allele frequencies comparison for the most common *TNF*-α SNPs (-376G>A, -308G>A, -244G>A and -238G>A). The SNP -376G>A, allele variant -376A, showed a significant association with susceptibility to the occurrence of active TB (*p* = 0.035, OR = 3.57, CI = 0.95 < 15.72) and severity (*p* = 0.038 and RR = 2.68) (Table 9). All other outcomes showed no significant association with any of the variants tested, (data not shown).


**Table 9.** Distribution of allele frequency of the *TNF*-α -376A variant and association analysis with different outcomes studied.


Table 10 shows the distribution of the 14 identified haplotypes in the different groups used for the association study. No significant difference was observed in the haplotypes frequencies between groups (data not shown) and their distribution was quite homogeneous.

The establishment of an efficient immune response involves many different molecules, among which, cytokines and their receptors play an extremely important role. Thus, any genetic alteration leading to changes in the regulation of gene expression may reflect this response. It is known that the interindividual variation in the production of these molecules is directly related to the genetic "background". Literature data have clearly demonstrated that genetic variability of the genes encoding these molecules can affect the regulation of gene expression positively or negatively influencing the final yield of the molecule in question. In the last decade, several single nucleotide polymorphisms (SNPs) in the regulatory region of different cytokine genes have been described and associated with susceptibility, severity or protection for a growing number of diseases of different etiologies including tuberculosis [7, 34-35].

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

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91

Among the possible genetic variations associated with an increased risk of developing TB, there are several polymorphisms, mainly SNPs, in genes coding for cytokines, cytokine receptors and several other molecules such as vitamin D receptor, NRAMP1 (SLC11A1), HLA

The immune defense against *M*. *tuberculosis* is complex and involves the interaction between

Convincing evidence indicating the importance of IFN-γ in particular, in the control of mycobacterial infections has been found in both experimental and clinical studies [37-38]. Among the mainly important cytokines involved in TB progress after infection with *M. tuberculosis*, TNF-α plays a key role. It is also a potent proinflammatory cytokine acting in

The genetic variability of *TNF*-*α* and *IFNG* has been described in the last decade [41-46] including association studies with tuberculosis. However, the frequencies of the polymor‐ phisms already described varies according to the ethnicity of the population studied, ham‐ pering the better interpretation of the value of association studies. Unfortunately, most of these are performed in ethnically homogeneous populations, and therefore, many of the associations described for a particular allelic variant in a certain gene may not represent genetic risk factor in other populations. In Brazil, a country characterized by ethnically mixed population, there are few data regarding the frequency of single nucleotide polymorphisms in these genes (*IFNG*,*TNF*-*α*) and the few existing studies refers to one or two SNPs only. In view of the importance of the promoter region with respect to regulation of gene expression, the major goal of this work was to proceed a partial mapping of the promoter region of IFN*G* and *TNFα* genes (approximately, 800bp upstream of the transcription starting site) through PCRsequencing approach in samples from TB patients and healthy controls from Rio de Janeiro, Brazil. Subsequently, based on frequencies of the different SNPs found individually for each

cytokines, such as interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) [36].

gene, we performed an association study with different TB outcomes.

**4.1. Polymorphisms in the promoter region of IF***NG* **and its association with TB**

Characterization of the important portion within the *IFNG*was firstly identified two decades ago bydeletion analysisstudies [47-48]. According to authors, it comprises a highly conserved

lymphocytes, macrophages, and monocytes along with the production of

genes, etc.

T CD4+

, T CD8+

protection against intracellular pathogens [39-40].

**Table 10.** Frequency of *TNF*-α haplotypes in the different groups studied

After the genotyping of all samples and evaluation of the possible association with the different TB outcomes, the most frequent polymorphisms (-376G>A; -308G>A; -244G>A and -238G>A) were tested in a stratified analysis against the demographic variables gender and age. No significant differences were found for gender or age (data not shown).
