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

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DOI: 10.1155/2013/901740 · Source: PubMed
Abstract
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- γ ) and nitric oxide synthase 2 (NOS2) are expressed on alveolar 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 these polymorphisms influence serum nitrite and NO x (-) 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 NO x (-). These results indicate that variants of IFNG+874T/A SNP and NOS2A-954G/C SNP do not influence TB susceptibility or the secretion of nitric oxide radicals in the study population.

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BioMed Research International
Volume , Article ID , pages
http://dx.doi.org/.//
Clinical Study
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,
1,2
Márcia Andrade Rocha,
1
Andreia Lamoglia-Souza,
1
John L. VandeBerg,
2
Valeria Cavalcanti Rolla,
3
and Maria da Gloria Bonecini-Almeida
1
1
Immunology and Immunogenetics Laboratory, Evandro Chagas Clinical Research Institute, Oswaldo Cruz Foundation,
Avenida Brasil 4365, Manguinhos, 21045-900 Rio de Janeiro, RJ, Brazil
2
Department of Genetics and Southwest National Primate Research Center, Texas Biomedical Research Institute, 7620 NW Loop 410,
78227-5301 San Antonio, TX, USA
3
Tuberculosis 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; gloria.bonecini@ipec.ocruz.br
Received April ; Revised June ; Accepted July 
Academic Editor: Helder I. Nakaya
Copyright ©  Ana Cristina C. S. Leandro et al. is 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
properly cited.
Tuberculosis (TB) is one of the most common infectious diseases in the world. Mycobacterium tuberculosis infection leads to
pulmonary active disease in approximately –% of exposed individuals. Both bacteria- and host-related characteristics inuence
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-𝛾) and nitric oxide synthase  (NOS) are expressed on alveolar
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
these polymorphisms inuence serum nitrite and NO
𝑥
production. is case-control study enrolled  TB patients and 
healthy controls. Neither polymorphism was associated with susceptibility to TB. NOS2A-954G/C SNP was not associated with
serum levels of nitrite and NO
𝑥
. ese results indicate that variants of IFNG+874T/A SNP and NOS2A-954G/C SNP do not
inuence TB susceptibility or the secretion of nitric oxide radicals in the study population.
1. Introduction
Tuberculosis (TB) has been declared as a major global
healthy threat by the World Health Organization since .
Pulmonary TB is highly prevalent in Brazil [], mainly, in
Rio de Janeiro, where , new cases were reported in 
[]. Host factors play a major role in determining risk for
active TB. Among them, IFN-𝛾 production is critical in the
intracellular control of M. tuberculosis infection, as previously
demonstrated in vitro [, ] in experimental infection [, ].
In addition, interferon gamma (IFN-𝛾)inducesapoptosisin
mycobacteria-infected macrophages in a nitric oxide (NO)
dependent environment [, ]. Polymorphism in the rst
intron of human IFNG gene is associated with higher in vitro
production of this cytokine and is correlated with a gene
dosage eect in the presence of the IFNG+874T allele []. is
common polymorphism is associated with TB susceptibility
in African, Turkish, Tunisian, and Central West Brazilian
populations [], but not in African-Americans, or people
of Iranian, Hispanic, or Chinese origin [].
NOisafreeradicalandsecondmessengerthathasbeen
showntobeimportantinthedevelopmentofseveraldiseases,
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
BioMed Research International
intheimmuneresponseandkillsM. tuberculosis in vitro
[]. NO production and NOS expression in rat alveolar
macrophages are upregulated in response to heat-killed M.
tuberculosis [].However,theroleofNOinkillingor
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/NOS) and may control mycobacteria growth in vivo
[]. us, the NOS2A-954G/C SNP may represent a pivotal
protective locus against TB. Investigation of this possibility is
hampered by diculty 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 NOS in the immune system,
SNPs have been reported in many populations worldwide
[]. e 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
inuence of the IFNG+874T/A (rs) and NOS2A-
954G/C (rs) SNPs on TB susceptibility in a highly
exposed and admixed population of TB patients. We also
conducted functional studies to determine whether the mod-
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  years, and provided written informed consent.
Cases and control groups were matched by age, socioeco-
nomic class and area of residence. Control groups individuals
were excluded if they had a history of prior antituberculosis
therapy, signs and symptoms of suggestive active TB. e
diagnostic criteria for TB were dened 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
inthestudy.eprotocolwasapprovedbytheResearch
Ethics Committees in Brazil (IPEC REC ref. ...-
) and Rio de Janeiro Municipal Health Centre (REC
ref. S/CRH/DRH/DIC). Ethnic background was determined
for each case and control volunteers by self-identication.
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 signicant dierences between the two groups.
All TB patients and control groups were negative for HIV /
infection (following standard diagnosis from e Brazilian
Ministry of Health). Tuberculin skin test (TST) response
to UT RT- (Statens Serum Institute, Denmark) was
performed, and the skin test response was measured at the
diameter of induration  h aer the injection. Positive results
were obtained when induration was  mm. Control group
was classied into those who were naturally infected with M.
tuberculosis (latency) and those who were uninfected (TST <
 mm). BCG vaccination status was determined by the pres-
ence of the scar tissue. Blood samples were taken aer in-
formed consent was obtained from each subject. Patients and
controls from the same family were not enrolled in the study.
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 purication kit (QIamp
DNA mini Kit, Qiagen, USA) according to the manufacturer’s
instructions. e IFNG+874T/A SNP was detected by ampli-
cation refractory mutational system (ARMS-PCR) []. e
NOS2A-954G/C SNP was detected by restriction fragment
length polymorphism (RFLP) []. Amplications were per-
formed in a  ermocycler (-Well GeneAmp PCR Sys-
tem , Applied Biosystems, USA) using . UI and . UI
for IFNG and NOS2A of Taq DNA polymerase, respectively
(GoTaq exi DNA polymerase, Promega, USA). Cycling PCR
conditions for IFNG+874T/A were minutes at 
Cfollowed
by  cycles at 
Cfors,
Cfors,and
Cfors;
cycles at
Cfors,
Cfors,and
Cfors;
C
for minutes and
C until use. Cycling PCR conditions for
NOS2A-954G/C were minutes at 
C followed by cycles
at 
Cfors,
Cfors,and
Cfors;
Cfor
minutes and
C until use. e amplied products were eval-
uated by electrophoresis on a .% (IFNG)and.%(NOS2A)
agarose gel containing ethidium bromide (. 𝜇g/mL).
2.3. Detection of Serum Nitric Oxide Radicals Concentration in
TB Patients. e two primary stable nonvolatile breakdown
products of NO are nitrite (NO
2
)andnitrate(NO
3
). First,
the serum NO
2
levels were determined using a commer-
cial ready to use Griess reaction kit (Promega) according
to the manufacturers instruction. Briey, serum samples
were mixed with sulfanilamide solution following incubation
at room temperature. N--Naphthylethylenediamine dihy-
drochloridewasadded,andabsorbancewasmeasuredin
a plate reader with a lter of  nm. Values were plotted
in accordance with a NaNO
2
standard curve (.– mM).
Further analysis was performed to determine the total levels
of NO
2
by reduced NO
3
, in a Vanadium III reduction assay,
following a protocol previously described by Miranda et al.
[]. Vanadium III (Sigma,  mg in  mL of . N HCl)
was added to each well, incubated for  minutes at 
C,
and read at  nm to determine the total amount of NO
2
+
NO
3
(aer NO
3
reduction to NO
2
), which was named
NO
𝑥
.
BioMed Research International
2.4. Statistical Analysis. Deviation from Hardy-Weinberg
equilibrium for the genetic variants was assessed by the chi-
square test (𝜒
2
)inbothcaseandcontrolgroups.Weused
the 𝜒
2
test to compare the dierences in each genotype,
allele, and combined genotype of IFNG+874T/A SNP and
NOS2A-954G/C SNP frequency. Additionally, unconditional
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 % condence intervals (CIs) with and without
adjustment for gender, ethnicity, TST status, and previous
BCG vaccination between TB and control groups. All
statistical tests were two-sided, a 𝑃 value of . was
considered signicant, and analyses were performed using
Epi Info  (Version ., July , CDC, Atlanta, GA,
USA), SNPStats (http://bioinfo.iconcologia.net/SNPstats),
and SPSS (Version , September ). Additionally, the
distributions of IFNG+874T/A and NOS2A-954G/C SNPs
were compared among patients and in whom TST was
positive in the control group by the 𝜒
2
or Fisher’s exact
test. Subgroup analyses for genotype, allele, and combined
genotype associations to tuberculosis were also conducted
among TST-positive individuals. e 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
NO
𝑥
levels in association with NOS2A-954G/C SNP and
combined IFNG+874T/A/NOS2A-954G/C SNPs genotypes
with the level of signicance set at 𝑃 < 0.05.
3. Results
3.1. Study Population. TB patients and control group were
enrolled consecutively and included  males (.%) and
 females (.%) with a mean age of . ± . years
(mean ± standard deviation) in the TB group, and  males
(.%) and  females (.%) with a mean age of . ± .
years in the control group. Age was not signicantly dierent
between the groups. Each volunteer dened his or her own
ethnic group as White (Caucasian) or non-White (Afro-
descendants). No Indians or people with Asian background
were identied in the included subjects. In the TB group, 
of  (.%) individuals dened their ethnic group as White
(, .%) or non-White (, .%), and among control
group, (.%) and  (.%) of  were White or non-
White, respectively. TST ranged from to  (. ±.) mm
and from to  (. ± .) mm in TB and control groups,
respectively. TST-positive ( mm) reaction was identied
in  of  (.%) tested subjects from control group,
conrming the highest M. tuberculosis exposure in Rio de
Janeiro. No statistical signicance in relation to TST positivity
and ethnicity or gender was observed in either TB patients or
control groups (Table ).
3.2. IFNG+874T/A SNP Distribution Is Not Associated with
Tuberculosis. e genotype distribution of IFNG+874T/A
SNP was in Hardy-Weinberg equilibrium (𝑃 > 0.05)inboth
TB and control groups. TB patients and control group had
T : Clinical data from Brazilian tuberculosis patients and
control group.
Tuberculosis patients
𝑛 = 172 (%)
Control group
𝑛 = 179 (%)
Sex
M
 (.)  (.)
F
 (.)  (.)
Age (years)
36.9 ± 12.7 35.1 ± 11.5
Skin color
White
 (.)  (.)
Non-White
 (.)  (.)
Missing
 (.)  (.)
BCG vaccine
Ye s
 (.)  (.)
No
 (.) (.)
Missing
 (.)  (.)
TST
Positive
 (.)  (.)
Negative
 (.)  (.)
Missing
 (.) (.)
Diameter (mm)
(14.9 ± 10.3)(12.1 ± 12.2)
TST: tuberculin skin test.
very similar genotype and allele distributions (𝑃 > 0.05)
(Table ). No statistical dierence in genotypes was observed
between TB patients and control subgroup who were TST
positive (Table ). e ability to respond to TST did not
correlate with IFNG+874T/A SNP genotypes in either TB
patients or control group. ere is no statistical dierence in
IFNG+874T/A SNP genotypes between TB patients and con-
trol groups from univariate or multivariate analysis regarding
ethnicity, age, and BCG vaccination status.
3.3. NOS2-954G/C SNP Distribution Is Not Associated with
Tuberculosis. e genotype distributions of NOS2A-954G/C
SNP were in Hardy-Weinberg equilibrium (𝑃 > 0.05)in
both TB and control groups. No association was seen in the
genotypes and alleles frequencies between TB patients and
control groups and from those positives TST (control sub-
group) (Table ). When univariate analysis was performed
by age, gender, ethnicity, and BCG vaccination, no statistical
dierence was observed (𝑃 > 0.05)(Table ).
3.4. Serum Nitrite and 𝑁𝑂
𝑥
Levels Are Not Associated with
NOS2-954G/C Polymorphism in Tuberculosis Patients. e
Griess reaction was performed in  TB patients and 
control group, in proportion to sample sizes of the two
groups.emeanserumconcentrationofnitriteandNO
𝑥
exhibited no statistical dierence between TB patients (.
± . 𝜇Mand.± . 𝜇M) and control group (.
± . 𝜇Mand.± . 𝜇M), respectively. It was inves-
tigated whether serum levels of nitrite and NO
𝑥
could vary
according to NOS2A-954G/C SNP genotypes in TB patients
BioMed Research International
T : Distribution of genotypes and alleles frequencies of the IFNG+874T/A and NOS2A-954G/C SNPs in tuberculosis patients and control
groups.
Genotype/allele
Tuberculosis patients
𝑛 = 172 (%)
Control group
𝑛 = 179 (%)
𝑃
1
OR
TB latent
infection (TST+)
𝑛=98(%)
𝑃
2
OR
A/A
 (.)  (.)
.
.
 (.)
.
.
A/T
 (.)  (.)
.
 (.)
.
T/T
 (.)  (.)
.
 (.)
.
T/T+A/T versus A/A
 (.)  (.)
. . (.–.)
 (.)
. . (.–.)
T/T versus A/A+A/T
 (.)  (.)
. . (.–.)
 (.)
. . (..)
Alleles
A
 (.)  (.)
.
. (.–.)
 (.)
.
. (.–.)
T
 (.)  (.)  (.)
G/G
 (.)  (.)
.
.
 (.)
.
.
G/C
 (.)  (.)
.
 (.)
.
C/C
(.) (.)
.
NA
C/C versus G/C+G/G
 (.)  (.)
. . (.–.)
 ()
.
G/G versus C/C+G/C
 (.)  (.)
. . (.–.)
 (.)
. . (.–.)
Alleles
G
 (.)  (.)
.
.
 (.)
.
.
C
 (.)  (.)
(.–.)
 (.)
(.–.)
𝑃 value considered 𝑃 0.05;OR,oddsratio
𝑃
1
value from TB patients and control group. 𝑃
2
value from TB patients and control TST-positive subgroup. TST+: tuberculin skin test positive.
and control group. No statistical association of serum nitrite
(. ± . versus . ± . and . ± . versus
. ±., in TB patients and control group, resp.) or NO
𝑥
levels (. ± . versus . ± . and . ± .
versus . ± ., in TB patients and control group, resp.)
was observed with GG and GC genotypes of NOS2A-954G/C
SNP (Figure ) even in the TST-positive control subgroup
(data not shown) was found. ese 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 NO
𝑥
radical production in either
TB patients or control group. e frequency of higher nitric
oxide radical producers NOS2A-954C/C SNP was rare in our
population.
3.5. Combined Genotype of IFNG+874T/A and NOS2A-
954C/G SNPs Is Not Associated with Either Tuberculosis or
Nitrogen Radicals. To determine whether the combination of
SNPs in these two genes was associated with susceptibility
to TB, polymorphisms association was evaluated between
pulmonary TB and control groups. e rare NOS2A-954C/C
SNP was not taken into account in assessing the com-
bined genotypes with the IFNG+874T/A SNP. No statistical
signicance was seen between TB and control groups in
IFNG+874T/A and NOS2A-954G/C SNPs genotypic distribu-
tions (Table ) when they were compared. In both groups,
the most prevalent combined genotypes were AT/GG (.%
and .%) and AA/GG (.% and .%) in TB and
control groups, respectively, with no statistical dierence. e
secretion of reactive nitrogen radicals was not related with
(𝜇M)
120
90
60
30
0
GG NO
2
GC NO
2
GC NO
x
GG NO
x
F : Comparative analysis of serum nitrite (NO
2
)andNO
𝑥
(NO
2
+NO
3
) concentrations and NOS2A-954G/C genotype
association in tuberculosis patients (black symbols) and control
group (open symbols) (𝑃 > 0.05).
the combined genotypes when TB and control groups were
compared.emostfrequentcombinedgenotypeAT/GG in
TB (. ±. and . ± .) and control groups (. ±
. and . ± .) did not induce dierent serum levels of
nitrite and NO
𝑥
. ese results demonstrated in our popula-
tion that the combined genotypes proles 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
BioMed Research International
T : C ombined genotype analysis of IFNG+874T/A and NOS2A-954G/C SNPs in tuberculosis patients and control groups.
Combined
genotypes
Tuberculosis patients
𝑛 = 172 (%)
Control groups
𝑛 = 179 (%) 𝑃
1
OR TST+ 𝑛=98(%) 𝑃
2
OR
AA/GG  (.)
 (.)
.
.  (.)
.
.
AA/GC (.)
(.) . (.) .
AA/CC (.)
(.) . (.) .
AT/GG  (.)
 (.) . (.) .
AT/GC (.)
 (.) . (.) .
TT/GG  (.)
 (.) .  (.) .
TT/GC (.)
(.) . () ND
e AA/GG combined genotype was used as reference in a × 𝜒
2
for trend table. e combined genotypes TT/GG and AT/GG were not present.
𝑃-value considered 𝑃 0.05; 𝑃
1
-value comparing TB patients and control group.
𝑃
2
-value comparing TB patients and TST+ control subgroup.
TST+: tuberculin skin test positive; OR: odds ratio; ND: not determined .
nitrite and NO
𝑥
radicals in both TB patients and control
group or in TST-positive control subgroup.
4. Discussion
IFN-𝛾 mediated immune activation has an important role
in immunity to intracellular pathogens. IFN-𝛾 is critical to
macrophage activation, and measurable levels are lower in
patients with active TB than in control group [, ], but
they are not correlated with protection []. It has been
more formally suggested that IFN-𝛾 activity is a continuous,
genetically controlled trait with genetic variability in both
production and responsiveness to IFN-𝛾 [], although there
is little evidence to support a role for variability in IFN-𝛾
production. For the IFNG gene,therearetwointronicSNPs
that contribute to its expression phenotypes: +874T/A SNP
and +2109G/A SNP []. e AA genotype of IFNG+874T/A
SNPisthoughttoconferalow-secretorphenotypeof
IFN-𝛾. Conversely, the TT genotype of IFNG+874T/A SNP
is thought to confer a high-secretor phenotype [, ].
Some controversy exists concerning association of IFNG gene
polymorphisms and susceptibility to pulmonary TB [, ,
]. Our results showed no evidence of an association
between IFNG gene polymorphism and susceptibility to TB.
e AA genotype frequency of IFNG+874 in the control
group (.%) was a little higher than that in the Sicilian
(%) [], Spanish (%) [], and Indian populations
(%) [] but was lower than that in South African (%)
[], Hong Kong Chinese (%) [], and South Korean
populations (%) []. Reports describing the T allele
frequency (IFNG+874T/A SNP) revealed that there are ethnic
dierences at this allele distribution. It has been reported
that the T allele frequency is signicantly lower in a Japanese
population (%) []thaninaSouthAfricanpopulation
(%) [] and in an Italian Caucasian population (%) [
].
Rossouw et al. []reportedasignicantlylowerT 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. []andVallinotoetal.[] found that the
TT genotype was relatively rare in TB patients from Sicilian
and Brazilian populations. However, a study conducted in
a Central West Brazilian population by Amim et al. []
showed dierent 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 dierent
populations in their study: one from Rio de Janeiro (patients)
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
dierent countries into dierent regions of Brazil. During
the last  years these populations became mixed with
native Indians, and each Brazilian region has its own admixes
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. us,
thepopulationinthiscityhasageneticbackgroundthat
represents a mix of all regions of the country.
ere is convincing evidence that NO and related reactive
nitrogen intermediate (RNI) can kill and/or inhibit intracel-
lular pathogens such as mycobacteria. IFN-𝛾 knockout mice
which are not capable of producing NO and RNI in response
to the bacilli develop tuberculosis quickly, suggesting a role
for NO and RNI in the defense mechanism against M.
tuberculosis []. In contrast to murine models of TB, there is
greater controversy about the role of NO in killing or limiting
the growth of M. tuberculosis in humans. Nevertheless, alve-
olar macrophages from TB patients express higher amounts
of NOS compared to control group [], demonstrating a
possible role in aecting bacilli growth. Several SNPs have
been described in this gene [] given the importance of this
gene in the immune response to TB. Our study veried the
inuence 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 dierence between the
NOS2A-954G/C SNP genotype in our TB patients and control
group. e frequency of NOS2A-954G/C SNP may dier
among populations. e C allele of NOS2A-954G/C SNP has
beenshowntobeabsentfromCaucasianpopulations[]
andinPeruvianpopulation[] and in low frequency in Asia
[]. However, the C allele has high frequency in African
populations []. A single description of NOS2A-954G/C
BioMed Research International
SNP genotype study in Brazilian population, studying gastric
cancer, showed dierent frequencies for GG, GC, and CC
genotypes in NOS2A-954G/C SNP (.%, .%, and
.%, resp.) [] compared with our study. In Mexicans, this
SNP was not associated with TB and C allele frequency was
% [].
e involvement of NOS2A SNPs has been studied in
dierent 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 NO
𝑥
radicals
were analyzed. No association was identied between TB
and control groups or between dierent genotype proles or
combined genotypes regarding nitrite and NO
𝑥
production.
Our results indicate that in our admixed population the asso-
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
prolesbutcanleadtodierentapproachestoevaluatethe
tuberculosis immunopathology genetics background in the
near future. Tuberculosis is a multifactorial disease and the
pulmonary milieu involving the immune response and bacilli
interaction in dierent genetic background and environment
factors should be addressed to answer the question whether
M. tuberculosis growth is controlled by other interaction
candidate genes and/or several risk factors.
Acknowledgments
is work was supported by funds provided by the PAPES V-
Fiocruz/CNPq and IPEC/Fiocruz. A. C. C. S. Leandro and M.
A. Rocha received fellowships from CAPES/Brazil.
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    • "The allelic and genotypic frequencies for the IFNG+874 polymorphism agreed with those found for the Brazilian population [18,24,25] and elsewhere [26,27]. The A allele was the most frequent and there were no differences in its frequency between the groups evaluated (Table 1). "
    [Show abstract] [Hide abstract] ABSTRACT: In trials evaluating the immune responses to Bacille of Calmette-Guérin (BCG), the genetic background and the nutritional status are host-related factors that could affect the heterogeneity in these parameters. The IFNG+874 A/T (rs 62559044) polymorphism has been reported to influence the IFN-γ production by BCG-vaccinated individuals challenged in vitro with mycobacterial antigens. The body mass index (BMI) is a proxy for the nutritional status and has been associated both with the susceptibility to tuberculosis and with the IFN-γ response. We show that although the IFNG+874 A/T polymorphism was not associated with the heterogeneity of IFN-γ production in a randomized controlled trial that evaluated long-term immune responses to BCG revaccination previously conducted in Salvador, Bahia, Brazil, the effect of this polymorphism on the observed increase in IFN-γ production among revaccinated subjects was adjusted in individuals with a low BMI.
    Full-text · Article · Jul 2016
    • "In Brazil, the NOS2A-954G/C polymorphism have been reported in studies with tuberculosis and leprosy. In both studies, the allelic and genotypic frequencies were similar to the one found in this study, even though their population were from South and Southeast re- gion [50,51]. Although no association has been found, this study is the first to report NOS2A-954G/C polymorphism and NO levels in malaria exposed individuals in endemic region of Brazil. "
    Dataset · Feb 2015 · Malaria Journal
    • "In Brazil, the NOS2A-954G/C polymorphism have been reported in studies with tuberculosis and leprosy. In both studies, the allelic and genotypic frequencies were similar to the one found in this study, even though their population were from South and Southeast re- gion [50,51]. Although no association has been found, this study is the first to report NOS2A-954G/C polymorphism and NO levels in malaria exposed individuals in endemic region of Brazil. "
    [Show abstract] [Hide abstract] ABSTRACT: Cytokines play an important role in human immune responses to malaria and variation in their production may influence the course of infection and determine the outcome of the disease. The differential production of cytokines has been linked to single nucleotide polymorphisms in gene promoter regions, signal sequences, and gene introns. Although some polymorphisms play significant roles in susceptibility to malaria, gene polymorphism studies in Brazil are scarce. A population of 267 individuals from Brazilian Amazon exposed to malaria was genotyped for five single nucleotide polymorphisms (SNPs), IFNG + 874 T/A, IL10A-1082G/A, IL10A-592A/C, IL10A-819 T/C and NOS2A-954G/C. Specific DNA fragments were amplified by polymerase chain reaction, allowing the detection of the polymorphism genotypes. The polymorphisms IL10A-592A/C and IL10A-819 T/C were estimated by a single analysis due to the complete linkage disequilibrium between the two SNPs with D’ = 0.99. Plasma was used to measure the levels of IFN-γ and IL-10 cytokines by Luminex and nitrogen radicals by Griess reaction. No differences were observed in genotype and allelic frequency of IFNG + 874 T/A and NOS2A-954G/C between positive and negative subjects for malaria infection. Interesting, the genotype NOS2A-954C/C was not identified in the study population. Significant differences were found in IL10A-592A/C and IL10A-819 T/C genotypes distribution, carriers of IL10A -592A/-819 T alleles (genotypes AA/TT + AC/TC) were more frequent among subjects with malaria than in negative subjects that presented a higher frequency of the variant C allele (p < 0.0001). The presence of the allele C was associated with low producer of IL-10 and low parasitaemia. In addition, the GTA haplotypes formed from combinations of investigated polymorphisms in IL10A were significantly associated with malaria (+) and the CCA haplotype with malaria (−) groups. The IL10A-1082G/A polymorphism showed high frequency of heterozygous AG genotype in the population, but it was not possible to infer any association of the polymorphism because their distribution was not in Hardy Weinberg equilibrium. This study shows that the IL10A-592A/C and IL10A-819 T/C polymorphisms were associated with malaria and decreased IL-10 levels and low parasite density suggesting that this polymorphism influence IL-10 levels and may influence in the susceptibility to clinical malaria.
    Full-text · Article · Jan 2015
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