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1. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.3, No.14, 2013
www.iiste.org
The Association between HLA-DRB Alleles with Pulmonary
Tuberculosis in Babil Province, Iraq
Ammar Abbas Shalan (Corresponding author)
Biology department , Babylon University, Hilla, Babil province , Iraq
E-mail: ammar_shalan@yahoo.com.au
Habeeb S. Naher
Microbiology branch, Babylon University, Hilla, Babil province , Iraq
E-mail: habeebnaher@yahoo.com
Mohammed A.K. Al-Saadi
Microbiology branch, Babylon University, Hilla, Babil province , Iraq
E-mail: mbmc.kadhum70@gmail.com
Abstract
This study is a case-control association study to investigate the role of host genetic polymorphisms in Human
leucocytes antigen (HLA-DRB) genes in susceptibility/resistance of tuberculosis in Iraqi population. This is the
first attempt to reveal the role of these polymorphisms in Iraq. Whole blood and sputum samples were collected
from 124 tuberculosis patients in addition to 102 healthy contact control subjects at consultant clinic for
respiratory diseases in Hilla – Babil province during the period from May 2010 to June 2011. Real-time PCR,
acid fast smear and Lowenstein-Jensen culture were used to diagnose the tuberculosis cases. Sequence Specific
Primers (SSP) were used to genotype HLA-DRB polymorphisms, respectively.
The Allele frequencies of HLA-DRB1*04, DRB1*10 and DRB1*13 showed significant increase (p value=
0.018, 0.032 and 0.001, respectively) in PTB patients compared with control subjects. On the other hand, allele
frequencies of HLA-DRB1*07 and DRB1*15 significantly (p value= 0.022 and 0.001, respectively),
overrepresented in control subjects.
In conclusion, the HLA-DRB1*04, DRB1*10 and DRB1*13 alleles were found to be associated with increased
susceptibility to pulmonary tuberculosis, while the HLA-DRB1*07 and DRB1*15 were associated with
protection against pulmonary tuberculosis in this population.
Keywords: HLA-DRB, pulmonary TB, Association
1.
Introduction
Major histocompatibility complex Human leucocyte antigens class I and II are highly polymorphic and
present antigenic peptides to cytotoxic CD8 and helper CD4 T cells, respectively. The main chains of antigenic
peptides that form hydrogen bonds with residues are conserved in most HLA class I and II alleles, whereas the
side-chains are accommodated in the polymorphic-binding pockets that determine the peptide specificity for
different class I and II proteins (Stern et al., 1994). Hence, HLA genes have been studied extensively in
susceptibility genes for mycobacterial disease.
HLA class II molecules are crucial in modulating the adaptive immune response, and their association with
various diseases, including TB, has been described. However, results have been controversial concerning to TB
(Lombard et al., 2006), with ethnic and/or geographic variations (Goldfeld, 2004) apparently playing a major
role in such discrepancies.
Iraq is one of the countries with a relatively high TB incidence rate (64/100,000) and low case detection rate
(48%) (World Health Organization. 2011). The WHO assumed that the incidence rate of TB in Iraq is stable, but
there is no reliable data from which to assess incidence trends. Iraq has a higher incidence of TB than the
majority of neighboring countries (World Health Organization. 2011) , but knowledge about the influence of
host genetic polymorphism of genes that affect the immune response to M. tuberculosis in Iraqi population has
not yet been studied. Such information is essential planning the prevention, treatment and control strategies of
tuberculosis in Iraq . To our knowledge, the present study is the first study about the association of genetic
polymorphism in HLA-DRB genes with susceptibility/resistance to pulmonary tuberculosis in Iraqi population.
2. Subjects and methods
2.1 Patients and control subjects
Study population included patients with suspected pulmonary or extrapulmonary tuberculosis from Babil
province - Iraq. Whole blood and sputum samples were collected from 124 TB cases at consultant clinic for
respiratory diseases in Hilla – Babil province in the period between May 2010 to June 2011. The inclusion
criteria used in this study were that all the TB cases must meet the clinical diagnosis criteria and confirmed by at
least one of the laboratory diagnosis tests (Smear, PCR and culture). The clinical diagnosis were conducted by
1

2. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.3, No.14, 2013
www.iiste.org
the specialists in the consultant clinic for respiratory diseases. Acid fast smear and Lowenstein-Jensen culture
were used to laboratory diagnosis of the tuberculosis cases and confirmed by Real Time -PCR based M.
tuberculosis diagnosis using AccuPower® MTB&NTM Real-Time PCR Kit (Bioneer Co.,Korea).
Out of the 124 TB patients, there were 74 male and 50 female. The patients age range was from 12 to 80
years (the mean 38.7 years , median 34.5 years). There were 100 (80.6%) patients attended as a new TB patients.
The remaining 24 (19.4%) patients were attended to follow-up evaluation of the DOT (Directly observed
treatment) program.
The control specimens were collected from 102 healthy subjects. From those subjects there were 35 health
care workers who works at the subjects from consultant clinic for respiratory diseases in Hilla city – Babylon ,
Iraq. The rest 67 control samples were obtained from non-relative healthy contacts (patients non-relative
spouses). The control samples included 128 whole blood samples in addition to 102 sputum samples. 26 control
subjects didn't provide a sputum sample and so they excluded from the study.
2.2 Human genomic DNA purification
The human genomic DNA was extracted from whole blood samples of patients and control subjects using
Wizard® Genomic DNA Purification Kit (Promega, USA).
2.3 HLA-DR typing
The HLA-DRB typing was performed at molecular biology laboratory – Faculty of medicine and health
science – UPM university –Malaysia, by using AllSet+™ Gold SSP kits provided by Invitrogen, USA. This kit
resolved Alleles at the generic level, or allelic group level, which will in general agree with the serologically
defined specificities. The AllSet+™ Gold SSP kits utilize the PCR technique to amplify the HLA locus to be
investigated. The SSP method is a PCR based technique, which uses Sequence Specific Primers (SSP), for DNA
based tissue typing. The assignment of alleles consists of determining whether amplification has occurred or
not, i.e. visualization and detection of the amplification by agarose gel electrophoresis. The PCR-SSP technique;
each primer pair identifies two linked, cis-located polymorphic sites.
2.4 Statistical analysis
To determine whether any significant differences in polymorphisms frequencies occurred between the case
and the control populations the allele and genotype frequencies were compared, using the Chi square.
Associations between the disease and genotypes were assessed by calculating odds ratios and 95 confidence
intervals (CI). P value less than 0.05 was considered statistically significant.
3.
Results & discussion
Figure (1) shows a gel electrophoresis results of two samples. The HLA-DRB allele frequency in PTB
patients and controls are shown in table (1). Allele frequencies of DRB1*04, DRB1*10 and DRB1*13
exhibited significant increase in PTB patients compared to control subjects. On the other hand, allele
frequencies of DRB1*07 and DRB1*15 significantly overrepresented in control subjects. The other HLA-DRB
alleles show no significant difference between PTB patients and controls.
This study reported an association between HLA- DRB1*04, DRB1*10 and DRB1*13 with susceptibility to
PTB in Iraqi population. The association between HLA-DRB1*04 was reported In Italy, where HLA-DR4
antigen was associated with pulmonary tuberculosis (Ruggiero et al. 2004). By contrast, frequencies of antigens
DR4 and DR8 were significantly decreased in patients with PTB (Terán-Escandón et al. 1999). The HLADRB1*04 is associated with resistance to leprosy, both in the Brazilian and Vietnamese populations
(Vanderborght et al. 2007).
HLA-DRB1*10 allele was associated with susceptibility to tuberculosis in Brazilian patients with AIDS
(Figueiredo et al. 2008).Some reports pointed out that the association of HLA-DRB1*10 allele with other
infectious disease. A study in northern Chinese patients showed that susceptibility to chronic hepatitis B was
strongly associated with HLA-DRB1*10 allele (Shen et al. 1999). These studies reported that these alleles play
an important role in the activation of cellular immune responses against intracellular pathogens.
The HLA-DRB1*13 was found to be associated with PTB in this study. This association was reported in
other population. The HLA-DRB1*13 predispose to PTB Russia (Pospelova et al. 2005). In addition, in South
Africa, DRB1*1302 and DRB1*1101-1121 phenotype was significantly associated with TB occurring at a
significantly higher allele frequency in cases than controls (Lombard et al. 2006). By contrast, In Poland, a study
results suggest that the presence of HLA-DRB1*16 alleles may increase the risk of development of PTB,
whereas HLA-DRB1*13 alleles may be resistant to tuberculosis (Dubaniewicz et al. 2000).
The HLA-DRB1*15 was found to be associated with protection against PTB in Iraqi population in this study.
This result did not agree with those results obtained from other populations. In south India HLA DRB1*15
predisposed for pulmonary tuberculosis (odds ratio (OR) =2.68, 95% confidence interval (CI)=1.30–5.89, P
value (P)=0.013) (Ravikumar et al. 1999). In addition to that, In Mexico, the frequencies of alleles DRB1*15
(OR, 7.92; 95% CI, 2.71 to 23.14) were significantly increased in with PTB when compared with healthy
2

3. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.3, No.14, 2013
www.iiste.org
subjects (Terán-Escandón et al. 1999). Moreover, In North Chinese, HLA-DRB1*15 allele is associated with
pulmonary tuberculosis (Shi et al. 2011). In India, HLA-DR2 (encoded by DRB1*15 allele) was present more
frequently in PTB patients than in controls (51% vs. 36.3%; corrected P [Pc] = .029, relative risk [RR] = 1.8
(Rajalingam et al. 1995).
According to the results of this study, the DRB1*07 was also associated with protection against PTB in Iraqi
population. This is agreed with meta analysis study of 22 HLA studies that report lower risk of thoracic TB was
found in carriers of DR7 antigens (OR 0.65, 95%CI 0.53–0.80, P < 0.0001) (Kettaneh et al. 2006). In contrast, In
Iran, HLA-DRB1*07 appeared to be the predisposing alleles and in patients with TB (Amirzargar et al. 2004).
Other HLA allele were reported to be associated with susceptibility / protection to TB. HLA-DRB1*11
reported to be protective from PTB in China (Wang et al. 2001). A lower risk of thoracic TB was found in
carriers of DR3 (OR 0.72, 95% CI 0.59–0.89, P < 0.002),while carriers of DR8 were at higher risk for thoracic
TB (OR 1.72, 95%CI 1.21–2.46, P < 0.003) (Kettaneh et al. 2006).
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4. Journal of Biology, Agriculture and Healthcare
ISSN 2224-3208 (Paper) ISSN 2225-093X (Online)
Vol.3, No.14, 2013
www.iiste.org
Table 1. The frequency of HLA-DRB alleles in patients with pulmonary tuberculosis and controls.
HLA-DRB Allele
DRB1*01
DRB1*03
DRB1*04
DRB1*07
DRB1*10
DRB1*11
DRB1*13
DRB1*14
DRB1*15
DRB1*16
DRB3*01
DRB4*01
DRB5*01
PTB patients (%)
(n=63)
5 (8.1%)
19 (30.2%)
9 (14.3%)
2 (3.2%)
5 (8.1%)
16 (25.4%)
23 (36.5%)
36 (56.4%)
1 (1.6%)
5 (8.1%)
56 (88.6%)
20 (32.2%)
20 (32.2%)
Controls (%)
(n=60)
11 (18.3%)
13 (21.7%)
1 (1.7%)
10 (16.7)
0 (0.0%)
12 (20.3%)
4 (6.7%)
24 (40.7%)
14 (23.3%)
12 (20.3%)
49 (81.3%)
12 (20.3%)
24 (40.7%)
OR (Confidence intervals)
8.571 (1.054 - 69.720)
5.250 (1.105 - 24.955)
NA
5.476 (1.788 - 16.769)
14.700 (1.875 - 115.263)
P value
0.133
0.410
0.018
0.022
0.032
0.571
0.001
0.263
0.001
0.092
0.750
0.254
0.512
Figure 1. Agarose gel electrophoresis of HLA-DRB gene (2%, 0.5 X TBE). Lanes 1-23 represent different
alleles specificity of HLA-DRB gene. Lane 24 show the internal negative control. The bands sized
approximately 800 bp in lanes 1-23 represent the internal positive band. Lanes 3, 10, 11, 21,22,23 positive DRB
allele. Bands bellow 80bp show primer dimmers. The defused bands under 50 bp represent unused primers.
4

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