Hindawi Publishing Corporation
BioMed Research International
Volume 2013, Article ID 901740, 7 pages
No Association of IFNG+874T/A SNP and NOS2A-954G/C SNP
Variants with Nitric Oxide Radical Serum Levels or
Susceptibility to Tuberculosis in a Brazilian Population Subset
Ana Cristina C. S. Leandro,1,2Márcia Andrade Rocha,1Andreia Lamoglia-Souza,1
John L. VandeBerg,2Valeria Cavalcanti Rolla,3and Maria da Gloria Bonecini-Almeida1
1Immunology and Immunogenetics Laboratory, Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation,
Avenida Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil
2Department of Genetics and Southwest National Primate Research Center, Texas Biomedical Research Institute, 7620 NW Loop 410,
78227-5301 San Antonio, TX, USA
3Tuberculosis Clinical Laboratory, Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation,
Avenida Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil
Correspondence should be addressed to Maria da Gloria Bonecini-Almeida; firstname.lastname@example.org
Received 1 April 2013; Revised 5 June 2013; Accepted 5 July 2013
Academic Editor: Helder I. Nakaya
Copyright © 2013 Ana Cristina C. S. Leandro et al. This is an open access article distributed under the Creative Commons
Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
Tuberculosis (TB) is one of the most common infectious diseases in the world. Mycobacterium tuberculosis infection leads to
pulmonary active disease in approximately 5–10% of exposed individuals. Both bacteria- and host-related characteristics influence
latent infection and disease. Host genetic predisposition to develop TB may involve multiple genes and their polymorphisms.
It was reported previously that interferon gamma (IFN-훾) and nitric oxide synthase 2 (NOS2) are expressed on alveolar
these polymorphisms influence serum nitrite and NO푥
influence TB susceptibility or the secretion of nitric oxide radicals in the study population.
macrophages from TB patients and are responsible for bacilli control; thus, we aimed this study at genotyping single nucleotide
polymorphisms IFNG+874T/A SNP and NOS2A-954G/C SNP to estimate their role on TB susceptibility and determine whether
−production. This case-control study enrolled 172 TB patients and 179
healthy controls. Neither polymorphism was associated with susceptibility to TB. NOS2A-954G/C SNP was not associated with
serum levels of nitrite and NO푥
−. These results indicate that variants of IFNG+874T/A SNP and NOS2A-954G/C SNP do not
Tuberculosis (TB) has been declared as a major global
healthy threat by the World Health Organization since 1993.
Pulmonary TB is highly prevalent in Brazil , mainly, in
Rio de Janeiro, where 11,155 new cases were reported in 2011
. Host factors play a major role in determining risk for
active TB. Among them, IFN-훾 production is critical in the
demonstrated in vitro [3, 4] in experimental infection [5, 6].
In addition, interferon gamma (IFN-훾) induces apoptosis in
mycobacteria-infected macrophages in a nitric oxide (NO)
dependent environment [7, 8]. Polymorphism in the first
intron of human IFNG gene is associated with higher in vitro
production of this cytokine and is correlated with a gene
common polymorphism is associated with TB susceptibility
in African, Turkish, Tunisian, and Central West Brazilian
of Iranian, Hispanic, or Chinese origin [14–17].
NO is a free radical and second messenger that has been
including TB. NO plays a major role in the pulmonary
host-defense mechanism in response to infections and is
implicated in bacteriostatic and bactericidal processes. NO
is vital for macrophage function and granuloma formation
2BioMed Research International
in the immune response and kills M. tuberculosis in vitro
. NO production and NOS2 expression in rat alveolar
macrophages are upregulated in response to heat-killed M.
tuberculosis . However, the role of NO in killing or
limiting the growth of M. tuberculosis in humans is still
unclear. It has been proposed that NO produced by TB-
infected human macrophages and by epithelial cells exhibits
antimycobacterial behavior against M. tuberculosis . It
was previously reported that the alveolar macrophages from
active TB patients express inducible nitric oxide synthase
(iNOS/NOS2) and may control mycobacteria growth in vivo
. Thus, the NOS2A-954G/C SNP may represent a pivotal
protective locus against TB. Investigation of this possibility is
hampered by difficulty in estimating the production of NO
in vivo mainly in lung tissues, but genetic analysis provides a
potential means of examining the relation between NOS2A
expression and disease outcome. Given the biological and
genetic validity of the role of NOS2 in the immune system,
SNPs have been reported in many populations worldwide
[22–24]. The NOS2A-954G/C SNP variant was originally
reported in a malaria endemic area in Africa , suggesting
that this mutation might have originated as a consequence
of selective pressure of Plasmodium infection. In a Mexican
admixed population, this functional SNP was not associated
with TB  and no further reports have associated it with
NO radical levels.
In this present case-control study, we investigated the
influence of the IFNG+874T/A (rs2430561) and NOS2A-
954G/C (rs1800482) SNPs on TB susceptibility in a highly
exposed and admixed population of TB patients. We also
ulation of nitric oxide radical secretion varies according to
NOS2A-954G/C or IFNG/NOS2A combined genotypes.
2. Materials and Methods
2.1. Study Population. Patients and control groups were re-
cruited from Evandro Chagas Clinical Research Institute at
Fiocruz and from Municipal Health Centers, Rio de Janei-
ro, Brazil. All volunteers included in this study lived in the
metropolitan area of Rio de Janeiro City (RJ, Brazil), were
older than 18 years, and provided written informed consent.
Cases and control groups were matched by age, socioeco-
were excluded if they had a history of prior antituberculosis
therapy, signs and symptoms of suggestive active TB. The
diagnostic criteria for TB were defined as the presence
of a positive smear for acid fast bacilli  and/or culture
positivity for M. tuberculosis in a sample from sputum
and/or bronchial lavage and/or other clinical specimens
according to . HIV-infected people and those taking
immunosuppressant drugs were excluded from participation
in the study. The protocol was approved by the Research
Ethics Committees in Brazil (IPEC REC ref. 0008.0.009.000-
04) and Rio de Janeiro Municipal Health Centre (REC
for each case and control volunteers by self-identification.
We recognize the inherent inaccuracy and potential bias in
dichotomous self-assessment of ethnic origin, in an admixed
population, but self-assessment might nevertheless lead to
statistically significant differences between the two groups.
All TB patients and control groups were negative for HIV 1/2
infection (following standard diagnosis from The Brazilian
Ministry of Health). Tuberculin skin test (TST) response
to 5UT RT-23 (Statens Serum Institute, Denmark) was
performed, and the skin test response was measured at the
tuberculosis (latency) and those who were uninfected (TST <
controls from the same family were not enrolled in the study.
were obtained when induration was ≥10mm. Control group
10mm). BCG vaccination status was determined by the pres-
ence of the scar tissue. Blood samples were taken after in-
was classified into those who were naturally infected with M.
2.2. Genotyping of IFNG+874T/A and NOS2A-954G/C Gene
Polymorphisms. Genomic DNA was extracted from fresh or
frozen EDTA blood using a DNA purification kit (QIamp
instructions. The IFNG+874T/A SNP was detected by ampli-
fication refractory mutational system (ARMS-PCR) . The
NOS2A-954G/C SNP was detected by restriction fragment
length polymorphism (RFLP) . Amplifications were per-
tem 9700, Applied Biosystems, USA) using 2.5UI and 1.5UI
for IFNG and NOS2A of Taq DNA polymerase, respectively
agarose gel containing ethidium bromide (0.5휇g/mL).
TB Patients. The two primary stable nonvolatile breakdown
products of NO are nitrite (NO2
cial ready to use Griess reaction kit (Promega) according
to the manufacturer’s instruction. Briefly, serum samples
at room temperature. N-1-Naphthylethylenediamine dihy-
drochloride was added, and absorbance was measured in
a plate reader with a filter of 420nm. Values were plotted
in accordance with a NaNO2standard curve (0.8–100mM).
following a protocol previously described by Miranda et al.
. Vanadium III (Sigma, 400mg in 50mL of 0.1NHCl)
and read at 540nm to determine the total amount of NO2
for 7 minutes and 4∘C until use. Cycling PCR conditions for
minutes and 4∘C until use. The amplified products were eval-
cycles at 95∘C for 20s, 55∘C for 50s, and 72∘C for 50s; 72∘C
at 94∘C for 10s, 60∘C for 30s, and 72∘C for 30s; 72∘C for 7
−) and nitrate (NO3
the serum NO2
−levels were determined using a commer-
Further analysis was performed to determine the total levels
was added to each well, incubated for 90 minutes at 37∘C,
−reduction to NO2
−), which was named
BioMed Research International3
2.4. Statistical Analysis. Deviation from Hardy-Weinberg
equilibrium for the genetic variants was assessed by the chi-
the 휒2test to compare the differences in each genotype,
univariate and multivariate logistic regression analyses
were used to examine the associations between the selected
SNPs and tuberculosis risk by estimating odds ratios (ORs)
and 95% confidence intervals (CIs) with and without
adjustment for gender, ethnicity, TST status, and previous
BCG vaccination between TB and control groups. All
square test (휒2) in both case and control groups. We used
allele, and combined genotype of IFNG+874T/A SNP and
NOS2A-954G/C SNP frequency. Additionally, unconditional
statistical tests were two-sided, a 푃 value of ≤0.05 was
USA), SNPStats (http://bioinfo.iconcologia.net/SNPstats),
and SPSS (Version 16, September 2007). Additionally, the
distributions of IFNG+874T/A and NOS2A-954G/C SNPs
were compared among patients and in whom TST was
considered significant, and analyses were performed using
Epi Info 6 (Version 6.04, July 1996, CDC, Atlanta, GA,
positive in the control group by the 휒2or Fisher’s exact
among TST-positive individuals. The analysis of the skin
test positive group was planned because it was thought
that this would represent people with probable latent TB
infection. ANOVA test was used to compare the nitrite and
test. Subgroup analyses for genotype, allele, and combined
genotype associations to tuberculosis were also conducted
−levels in association with NOS2A-954G/C SNP and
combined IFNG+874T/A/NOS2A-954G/C SNPs genotypes
with the level of significance set at 푃 < 0.05.
3.1. Study Population. TB patients and control group were
enrolled consecutively and included 105 males (61.0%) and
ethnic group as White (Caucasian) or non-White (Afro-
descendants). No Indians or people with Asian background
were identified in the included subjects. In the TB group, 136
(52, 30.3%) or non-White (84, 48.8%), and among control
group, 78 (43.6%) and 82 (45.8%) of 160 were White or non-
respectively. TST-positive (≥10mm) reaction was identified
control groups (Table 1).
67 females (39.0%) with a mean age of 36.9 ± 12.7 years
years in the control group. Age was not significantly different
between the groups. Each volunteer defined his or her own
(mean ± standard deviation) in the TB group, and 63 males
in 98 of 155 (54.7%) tested subjects from control group,
confirming the highest M. tuberculosis exposure in Rio de
and from 0 to 57 (12.1 ± 12.2) mm in TB and control groups,
3.2. IFNG+874T/A SNP Distribution Is Not Associated with
Tuberculosis. The genotype distribution of IFNG+874T/A
SNP was in Hardy-Weinberg equilibrium (푃 > 0.05) in both
TB and control groups. TB patients and control group had
Table 1: Clinical data from Brazilian tuberculosis patients and
푛 = 172 (%)
TST: tuberculin skin test.
very similar genotype and allele distributions (푃 > 0.05)
positive (Table 2). The ability to respond to TST did not
correlate with IFNG+874T/A SNP genotypes in either TB
patients or control group. There is no statistical difference in
IFNG+874T/A SNP genotypes between TB patients and con-
ethnicity, age, and BCG vaccination status.
Tuberculosis patientsControl group
푛 = 179 (%)
36.9 ± 12.735.1 ± 11.5
(14.9 ± 10.3)(12.1 ± 12.2)
(Table 2). No statistical difference in genotypes was observed
between TB patients and control subgroup who were TST
3.3. NOS2-954G/C SNP Distribution Is Not Associated with
Tuberculosis. The genotype distributions of NOS2A-954G/C
SNP were in Hardy-Weinberg equilibrium (푃 > 0.05) in
control groups and from those positives TST (control sub-
group) (Table 2). When univariate analysis was performed
by age, gender, ethnicity, and BCG vaccination, no statistical
3.4. Serum Nitrite and 푁푂푥
control group, in proportion to sample sizes of the two
groups. The mean serum concentration of nitrite and NO푥
tigated whether serum levels of nitrite and NO푥
both TB and control groups. No association was seen in the
genotypes and alleles frequencies between TB patients and
difference was observed (푃 > 0.05) (Table 2).
NOS2-954G/C Polymorphism in Tuberculosis Patients. The
Griess reaction was performed in 75 TB patients and 78
−Levels Are Not Associated with
exhibited no statistical difference between TB patients (23.68
± 15.74휇M and 32.61 ± 16.97휇M) and control group (23.99
according to NOS2A-954G/C SNP genotypes in TB patients
± 17.29휇M and 34.71 ± 19.39휇M), respectively. It was inves-
4 BioMed Research International
푛 = 172 (%)
78 (45.3)91 (50.8)
22 (12.8)26 (14.6)
T/T+A/T versus A/A
100 (58.1) 117 (65.3)
T/T versus A/A+A/T
150 (87.2)153 (85.4)
222 (64.5)215 (60.1)
122 (35.5) 143 (39.9)
152 (88.4) 160 (89.4)
19 (11.0)18 (10.0)
1 (0.6)1 (0.6)
C/C versus G/C+G/G
171 (99.4)178 (99.4)
G/G versus C/C+G/C
20 (11.6) 19 (10.6)
323 (93.9)338 (94.4)
21 (6.1)20 (5.6)
푃1value from TB patients and control group. 푃2value from TB patients and control TST-positive subgroup. TST+: tuberculin skin test positive.
and control group. No statistical association of serum nitrite
Tuberculosis patients Control group
푛 = 179 (%)
푛 = 98 (%)
72 (41.8)62 (34.6)
푃 value considered 푃 ≤ 0.05; OR, odds ratio
(23.77 ± 15.59 versus 23.49 ± 17.39 and 23.13 ± 17.78 versus
was observed with GG and GC genotypes of NOS2A-954G/C
SNP (Figure 1) even in the TST-positive control subgroup
(data not shown) was found. These results suggested that
the low NOS2A-954G/G SNP or moderate NOS2A-954G/C
SNP nitric oxide producers were not associated with the
modulation of nitrite and NO푥
oxide radical producers NOS2A-954C/C SNP was rare in our
levels (32.40 ± 16.6 versus 34.22 ± 19.59 and 34.34 ± 20.99
versus 38.12 ± 18.67, in TB patients and control group, resp.)
−radical production in either
TB patients or control group. The frequency of higher nitric
3.5. Combined Genotype of IFNG+874T/A and NOS2A-
954C/G SNPs Is Not Associated with Either Tuberculosis or
SNPs in these two genes was associated with susceptibility
to TB, polymorphisms association was evaluated between
pulmonary TB and control groups. The rare NOS2A-954C/C
SNP was not taken into account in assessing the com-
bined genotypes with the IFNG+874T/A SNP. No statistical
significance was seen between TB and control groups in
tions (Table 3) when they were compared. In both groups,
the most prevalent combined genotypes were AT/GG (40.7%
and 44.7%) and AA/GG (36.0% and 31.3%) in TB and
secretion of reactive nitrogen radicals was not related with
−) concentrations and NOS2A-954G/C genotype
association in tuberculosis patients (black symbols) and control
−) and NO푥
Figure 1: Comparative analysis of serum nitrite (NO2
group (open symbols) (푃 > 0.05).
the combined genotypes when TB and control groups were
compared. The most frequent combined genotype AT/GG in
−. These results demonstrated in our popula-
tion that the combined genotypes profiles of IFNG+874T/A
SNP (low IFNG+874AA, moderate IFNG+874AT, or high
IFNG+874TT producers) and NOS2A-954G/C SNP (low
NOS2A-954GG or moderate NOS2A-954GC producers) are
not associated with the modulation of the production of
1.90 and 34.83 ± 2.21) did not induce different serum levels of
nitrite and NO푥
BioMed Research International5
Table 3: Combined genotype analysis of IFNG+874T/A and NOS2A-954G/C SNPs in tuberculosis patients and control groups.
푛 = 172 (%)
푃-value considered 푃 ≤ 0.05; 푃1-value comparing TB patients and control group.
TST+: tuberculin skin test positive; OR: odds ratio; ND: not determined .
−radicals in both TB patients and control
group or in TST-positive control subgroup.
푛 = 179 (%)
TST+ 푛 = 98 (%)
The AA/GG combined genotype was used as reference in a 7 × 2 휒2for trend table. The combined genotypes TT/GG and AT/GG were not present.
푃2-value comparing TB patients and TST+ control subgroup.
nitrite and NO푥
IFN-훾 mediated immune activation has an important role
patients with active TB than in control group [28, 29], but
they are not correlated with protection . It has been
production and responsiveness to IFN-훾 , although there
that contribute to its expression phenotypes: +874T/A SNP
and +2109G/A SNP . The AA genotype of IFNG+874T/A
SNP is thought to confer a low-secretor phenotype of
in immunity to intracellular pathogens. IFN-훾 is critical to
macrophage activation, and measurable levels are lower in
more formally suggested that IFN-훾 activity is a continuous,
genetically controlled trait with genetic variability in both
is little evidence to support a role for variability in IFN-훾
IFN-훾. Conversely, the TT genotype of IFNG+874T/A SNP
polymorphisms and susceptibility to pulmonary TB [14, 16,
35–37]. Our results showed no evidence of an association
between IFNG gene polymorphism and susceptibility to TB.
The AA genotype frequency of IFNG+874 in the control
group (35.2%) was a little higher than that in the Sicilian
(26%) , Spanish (28%) , and Indian populations
(11%)  but was lower than that in South African (47%)
, Hong Kong Chinese (46%) , and South Korean
populations (74%) . Reports describing the T allele
differences at this allele distribution. It has been reported
that the T allele frequency is significantly lower in a Japanese
population (9%)  than in a South African population
Rossouw et al.  reported a significantly lower T allele
frequency of INFG+874T/A in patients with M. tuberculosis
infection than that in control groups in a South African
population with a high annual incidence of TB.
Lio et al.  and Vallinoto et al.  found that the
TT genotype was relatively rare in TB patients from Sicilian
production. For the IFNG gene, there are two intronic SNPs
is thought to confer a high-secretor phenotype [33, 34].
and Brazilian populations. However, a study conducted in
a Central West Brazilian population by Amim et al. 
showed different results from our study; in that population,
the AA genotype of IFNG+874T/A SNP was associated with
TB susceptibility. Amim et al.  compared two different
and the other from Central West of Region Brazil (control
group), which can be characterized as bias because Brazil
was colonized by several ethnic groups that migrated from
different countries into different regions of Brazil. During
the last 500 years these populations became mixed with
ethnic group descendants. Our patients and control group
were enrolled from Rio de Janeiro City, which is composed
of migratory populations from all over the country. Thus,
the population in this city has a genetic background that
represents a mix of all regions of the country.
nitrogen intermediate (RNI) can kill and/or inhibit intracel-
lular pathogens such as mycobacteria. IFN-훾 knockout mice
for NO and RNI in the defense mechanism against M.
the growth of M. tuberculosis in humans. Nevertheless, alve-
olar macrophages from TB patients express higher amounts
of NOS2 compared to control group , demonstrating a
possible role in affecting bacilli growth. Several SNPs have
been described in this gene  given the importance of this
gene in the immune response to TB. Our study verified the
influence of NOS2A-954G/C SNP on the risk of developing
TB in a Brazilian population from a highly endemic area.
We did not observe a statistical difference between the
group. The frequency of NOS2A-954G/C SNP may differ
among populations. The C allele of NOS2A-954G/C SNP has
been shown to be absent from Caucasian populations 
. However, the C allele has high frequency in African
populations . A single description of NOS2A-954G/C
which are not capable of producing NO and RNI in response
to the bacilli develop tuberculosis quickly, suggesting a role
6BioMed Research International
SNP genotype study in Brazilian population,studying gastric
cancer, showed different frequencies for GG, GC, and CC
genotypes in NOS2A-954G/C SNP (64.77%, 28.69%, and
SNP was not associated with TB and C allele frequency was
The involvement of NOS2A SNPs has been studied in
different pathologies and still has controversy results. In
order to assess the nitric oxide radical levels and to compare
them with NOS2A-954G/C SNP and combined genotypes of
IFNG+874T/A SNP/NOS2A-954G/C SNP in TB patients and
control groups, the secretions of nitrite and NO푥
and control groups or between different genotype profiles or
ciation between disease development and IFNG+874T/A and
NOS2A-954G/C SNPs does not exert selective pressure on
M. tuberculosis via immune response surveillance, as shown
by the absence of correlation of active TB with these SNPs
profiles but can lead to different approaches to evaluate the
tuberculosis immunopathology genetics background in the
near future. Tuberculosis is a multifactorial disease and the
interactionin different geneticbackgroundandenvironment
factors should be addressed to answer the question whether
M. tuberculosis growth is controlled by other interaction
candidate genes and/or several risk factors.
were analyzed. No association was identified between TB
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