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The association between hla drb alleles with pulmonary tuberculosis in babil province, iraq

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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). References Amirzargar, AA. ; Yalda, A. ; Hajabolbaghi, M. ; Khosravi, F.; Jabbari, H.; Rezaei, N.; Niknam, MH.; Ansari, B.; Moradi, B. and Nikbin, B. (2004). The association of HLA-DRB; DQA1; DQB1 alleles and haplotype frequency in Iranian patients with pulmonary tuberculosis. Int J Tuberc Lung Dis 8(8):1017–1021. Dubaniewicz, A.; Lewko, B.; Moszkowska, G.; Zamorska, B. and Stepinski, J. (2000).Molecular subtypes of the HLA-DR antigens in pulmonary tuberculosis. Int J Infect Dis. ;4(3):129-33. Figueiredo, JF.; Rodrigues, Mde. L.; Deghaide, NH. and Donadi, EA. (2008). HLA profile in patients with AIDS and tuberculosis. Braz J Infect Dis. ;12(4):278-80. Goldfeld, AE. (2004). Genetic susceptibility to pulmonary tuberculosis in Cambodia. Tuberculosis (Edinb.);84:76-81. Kettaneh, A.; Seng, L.; Tiev, KP.; Tolédano, C.; Fabre, B. and Cabane, J. (2006). Human leukocyte antigens and susceptibility to tuberculosis: a meta-analysis of case-control studies. Int J Tuberc Lung Dis 10(7):717– 725. Lombard, Z.; Dalton, DL.; Venter, PA.; Williams, RC. and Bornman, L. (2006). Association of HLA-DR; -DQ; and vitamin D receptor alleles and haplotypes with tuberculosis in the Venda of South Africa. Hum Immunol.;67:643-54. Rajalingam, R.; Mehra, NK.; Jain, RC.; Myneedu, VP. and Pande, JN. (1996). Polymerase Chain Reaction Based Sequence-Specific Oligonucleotide Hybridization Analysis of HLA Class II Antigens in Pulmonary Tuberculosis: Relevance to Chemotherapy and Disease Severity. J Infect Dis. ;173(3):669-76. Ravikumar, M.; Dheenadhayalan, V.; Rajaram, K.; Lakshmi, SS.; Kumaran, PP.; Paramasivan, CN.; Balakrishnan, K. and Pitchappan, RM. (1999). Associations of HLA-DRB1; DQB1 and DPB1 alleles with pulmonary tuberculosis in south India. Tuber Lung Dis. ;79(5):309-17. Ruggiero, G.; Cosentini, E.; Zanzi, D.; Sanna, V.; Terrazzano, G.; Matarese, G.; Sanduzzi, A.; Perna, F. and Zappacosta, S. (2004). Allelic distribution of human leucocyte antigen in historical and recently diagnosed tuberculosis patients in Southern Italy. Immunology;111(3):318-22. Shen, J.; Ji, Y.; Guan, X.; Huang, R. and Sun, Y. (1999). The association of HLA-DRBI* 10 with chronic hepatitis B in Chinese patients. Chinese J Microb Immun. ;19(1):58–9. Shi, GL.; Hu, XL.; Yang, L.; Rong, CL.; Guo, YL. and Song, CX. (2011). Association of HLA-DRB alleles and pulmonary tuberculosis in North Chinese patients. Genet. Mol. Res. 10 (3): 1331-1336. Stern, LJ.; Brown, JH.; Jardetzky, TS.; Gorga, JC.; Urban, RG.; Strominger, JL. and Wiley, DC. (1994). Crystal structure of the human class II MHC protein HLA-DR1 complexed with an influenza virus peptide. Nature; 368: 215–21. Terán-Escandón, D.; Terán-Ortiz, L.; Camarena-Olvera, A.; González-Avila, G.; Vaca-Marín, MA.; Granados, J. and Selman, M. (1999). Human Leukocyte Antigen-Associated Susceptibility to Pulmonary Tuberculosis. Chest. ;115(2):428-33. Vanderborght, PR.; Pacheco, AG.; Moraes, ME.; Antoni, G.; Romero, M.; Verville, A.; Thai, VH.; Huong, NT.; Ba, NN.; Schurr, E.; Sarno, EN. and Moraes, MO. (2007). HLA-DRB1*04 and DRB1*10 are associated with resistance and susceptibility; respectively; in Brazilian and Vietnamese leprosy patients. Genes Immun. ;8(4):320-4. Wang, J.; Song, C. and Wang, S.( 2001). Association of HLA-DRB1 genes with pulmonary tuberculosis. Zhonghua Jie He He Hu Xi Za Zhi 24:302. World Health Organization. 2011. WHO report 2011. Global tuberculosis control. WHO/HTM/TB/2011.16. Geneva: WHO. 3
  • 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
  • 5. This academic article was published by The International Institute for Science, Technology and Education (IISTE). The IISTE is a pioneer in the Open Access Publishing service based in the U.S. and Europe. The aim of the institute is Accelerating Global Knowledge Sharing. More information about the publisher can be found in the IISTE’s homepage: http://www.iiste.org CALL FOR JOURNAL PAPERS The IISTE is currently hosting more than 30 peer-reviewed academic journals and collaborating with academic institutions around the world. There’s no deadline for submission. Prospective authors of IISTE journals can find the submission instruction on the following page: http://www.iiste.org/journals/ The IISTE editorial team promises to the review and publish all the qualified submissions in a fast manner. All the journals articles are available online to the readers all over the world without financial, legal, or technical barriers other than those inseparable from gaining access to the internet itself. Printed version of the journals is also available upon request of readers and authors. MORE RESOURCES Book publication information: http://www.iiste.org/book/ Recent conferences: http://www.iiste.org/conference/ IISTE Knowledge Sharing Partners EBSCO, Index Copernicus, Ulrich's Periodicals Directory, JournalTOCS, PKP Open Archives Harvester, Bielefeld Academic Search Engine, Elektronische Zeitschriftenbibliothek EZB, Open J-Gate, OCLC WorldCat, Universe Digtial Library , NewJour, Google Scholar