[Show abstract][Hide abstract] ABSTRACT: Development of tuberculosis (TB) disease is an outcome of complex host-pathogen interactions. The purinergic P2X(7) receptors are adenosine triphosphate gated molecules shown to induce killing of intracellular Mycobacterium tuberculosis, followed by apoptosis of the infected macrophage. A single nucleotide polymorphism in exon 13 of the P2X(7) receptors gene at +1513 position has been shown to abolish the function of this receptor and to be associated with increased susceptibility to TB in some ethnic groups.
To explore the association of +1513 (A-->C) polymorphism in TB patients in Punjab, North India.
A case-control study was conducted by studying peripheral blood samples from 204 TB patients (181 pulmonary, 23 extra-pulmonary) and 177 controls with no history of TB. P2X(7) +1513 (A-->C) polymorphism was studied using amplification refractory mutation system analysis.
The distribution of +1513 A/C genotypes in the TB patient and the control groups revealed a statistically significant association with TB (P = 0.002).
The +1513C allele is a risk factor for the development of TB in the North Indian Punjabi population.
Full-text · Article · Sep 2010 · The International Journal of Tuberculosis and Lung Disease
[Show abstract][Hide abstract] ABSTRACT: Extra pulmonary tuberculosis (EPTB) constitutes around 20% of all tuberculosis cases in India. Conventional methods are of limited use in diagnosing this form of the disease. Polymerase chain reaction (PCR) has emerged as a sensitive and specific tool for documenting the presence of Mycobacterium tuberculosis in clinical samples but lacks quantitative ability. The present study evaluates peripheral blood as an alternative clinical specimen for diagnosing EPTB. Peripheral blood samples from 38 EPTB and 89 non tuberculous subjects were analyzed for the presence of tubercle bacilli by MPB 64 gene based PCR method. The assay gave an overall sensitivity of 60.53% with negative predictive value of 76.92% which is superior to present gold standard of mycobacterial culture (10.53 and 72.36%). Additionally, 43.82% of non tuberculous subjects gave positive results with the PCR, thus mitigating the clinical utility of this test. An in-house Competitive PCR (C-PCR) assay was used to determine the mycobacterial load in peripheral blood from culture positive, culture negative EPTB patients and non tuberculous controls which ranged from 7498-12498, 602-4797 and 101-800 genome equivalent (ge)/mL, respectively. The data clearly demonstrated that C-PCR assay can furnish insightful information in diagnosing extra pulmonary disease.
Preview · Article · Jul 2009 · Indian journal of experimental biology
[Show abstract][Hide abstract] ABSTRACT: Two hospitals, Sri Guru Ram Das Institute of Medical Sciences and Research and the TB and Chest Hospital, Amritsar, Punjab.
To explore genetic diversity among the clinical Mycobacterium tuberculosis isolates prevalent in Punjab.
Fifty-six random clinical isolates of M. tuberculosis were cultured from the sputum specimens of pulmonary tuberculosis patients. DNA was extracted from cultured biomass and analysed using the mycobacterial interspersed repetitive units (MIRU) typing method.
MIRU typing of 51 isolates revealed 45 different patterns, with a combined Hunter-Gaston discriminatory index (HGDI) of 0.990. Five clinical isolates failed to amplify for one or more MIRUs and were excluded from the analysis. The remaining isolates were categorised in three groups based on the allelic heterogeneity of individual MIRUs. MIRU 10, 16, 26 and 31 were highly discriminant, with an HGDI value >0.6; MIRU 4, 23, 24, 39 and 40 were designated as moderately discriminant (HGDI value 0.6-0.3) and MIRU 2, 20 and 27 were poorly discriminant (HGDI value <0.3).
MIRU typing and the HGDI values revealed that M. tuberculosis strains from Punjab are genetically quite heterogeneous.
No preview · Article · Oct 2008 · The International Journal of Tuberculosis and Lung Disease
[Show abstract][Hide abstract] ABSTRACT: Bronchial asthma is a complex genetic disorder regulated by the release of cytokines and inflammatory mediators. Tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta1) cytokines play pivotal roles in the inflammatory response of the airways. Differential production of these two cytokines is associated with allelic variations in the transcriptional regulatory region of these genes.
The objective of the present study was to investigate G-308A TNF-alpha and C-509T TGF- beta1 polymorphisms for their association with Bronchial Asthma.
DNA isolated from 123 asthmatics and 100 normal healthy controls were screened for these polymorphisms using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) methods, developed in our laboratory.
Significant allelic association was observed between G-308A TNF-alpha polymorphism and asthma (P = 0.031) while no association was observed with C-509T TGF- beta1 polymorphism (P = 0.207). Further sub-grouping based on either allergic response or family history failed to reveal any statistical significance among the groups or with controls. The interaction between these polymorphisms revealed statistically significant association between the high producer genotype alleles of TNF-alpha and TGF-beta (A/T) and asthma (P = 0.016).
The present study reports, for the first time, the role of two polymorphisms, in concert, for their association with asthma in an Indian population. Our study supports the findings that the G-308A TNF-alpha promoter polymorphism is a risk factor for asthma and furthermore suggests that the patients with high producer alleles for TNF-alpha (-308) and TGF-beta (-509) have the highest risk of getting this disease in the Punjabi population.
Full-text · Article · Sep 2008 · Indian Journal of Medical Sciences
[Show abstract][Hide abstract] ABSTRACT: Tumour necrosis factor (TNF)-alpha is a pleiotropic, pro-inflammatory cytokine of 17 kDa, whose gene is localized on the short arm of chromosome 6. It has a G-308A polymorphism in the promoter region, which is known to be associated with its differential production; the A allele being the high producer. The circulating level of TNF-alpha is under genetic control and implicated in the pathophysiology of asthma and tuberculosis. Since raised levels of TNF-alpha have been found in asthma and tuberculosis, we looked for the association of TNF-alpha G-308A polymorphism in patients with these diseases.
A total of 300 blood samples from patients (155 with asthma, 145 tuberculosis) and 211 normal healthy controls were collected. The G-308A polymorphism was studied using amplification refractory mutation system analysis.
The distribution of G/A alleles in the two patient groups when compared with normal controls revealed a statistically significant association with asthma (p = 0.016) but not with tuberculosis (p = 0.178).
The data support the common variant common disease hypothesis, which emphasizes that common genetic variations may participate as critical players in inciting common diseases.
Full-text · Article · May 2008 · The National medical journal of India
[Show abstract][Hide abstract] ABSTRACT: Tuberculosis (TB) is a curable infectious disease that kills around half million individuals every year in India. Cytokines play a vital role in the pathogenesis of this deadly disease. Transforming growth factor-beta 1 (TGF-β1) is one of the important anti-inflammatory cytokine found to be elevated in TB patients. Single nucleotide polymorphisms (SNP) in the promoter of TGF-β1 cytokine gene are known to alter the production of this important cytokine. Given its immunosuppressive nature increased production of this cytokine is implicated towards susceptibility to TB. The C/T polymorphism in TGF-β1 promoter at -509 site is strongly associated with circulating levels of this cytokine and T allele is shown to be associated with its high production. Thus, in the present study we looked for association of TGF-β1 (C-509T) SNP with TB. A total of 245 subjects (145 TB patients and 100 normal healthy controls) were recruited for study. The -509 polymorphism was studied using Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR). The distribution of C/T alleles in TB patients and normal healthy controls did not reveal any statistically significant association of this polymorphism with TB (χ 2 = 0.04; p = 0.85).
Full-text · Article · Jan 2007 · International Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: Bronchial asthma is an inflammatory disorder in which genetic and environmental factors play an important role. Several susceptible genes have been identified using whole genome scan and candidate gene approaches. Tumor necrosis factor alpha, a pro-inflammatory cytokine, is one such gene that figures prominently in such investigations. The secreted levels of this cytokine are under genetic control and attributed to the presence of single nucleotide polymorphism G-308 A in the promoter region of its gene. However, the association of this polymorphism varies from population to population. As there are no data available on North Indians, the present study aims to fill this void. For this, 366 subjects (155 asthmatic and 211 normal control subject) were genotyped using Amplification Refractory Mutation System Analysis (ARMS-PCR) and agarose gel electrophoresis. The distribution of G/A alleles between the two groups revealed statistically significant differences (p = 0.016). Furthermore, the asthma patients categorized on the basis of pattern (Seasonal versus Perennial) and age of onset of disease (Childhood versus Late Onset) revealed significant association with only seasonal (p = 0.021) and late onset asthmatic groups (p = 0.039). The data from the present study strongly suggest an association between TNF-alpha and asthma in the North Indian population.
No preview · Article · Jan 2006 · Journal of Asthma
[Show abstract][Hide abstract] ABSTRACT: Background: The wide variation in sensitivity and specificity of PCR test for the detection of Mycobacterium tuberculosis is a major concern for clinicians and is often ascribed to variations in the components of PCR reaction mixture, besides other steps involved in its amplification. Aim: The aim of this study was to demonstrate the need to optimize a PCR based test using the 38 kDa segment of Mycobacterium tuberculosis complex for its detection. Material and Methods: Optimization of thermal cycling parameters and reaction conditions of 38 kDa based PCR was performed after DNA isolation from Mycobacterial cultures and sputum samples isolated from suspected tuberculosis patients. Results: Effect of different thermal cycling parameters revealed that annealing temperature of 57 o C and annealing time of 30 seconds was ideal for amplification of 239 bp product of 38 kDa gene. Further, the effects of different concentrations of reaction components were seen on the amplification. It was found that 2.0 mM MgCl 2 , 200 ìM dNTPs, 200 nM primer concentrations and 0.5 U of Taq polymerase per 25 ìl of reaction mixture yielded the 239 bp product with no artifacts. The optimized PCR protocol was then used to test 90 clinical samples of sputum and it was found that of the tested samples, 77 were positive with the PCR based assay. Before applying any PCR based test to clinical settings, it is necessary to optimize the thermal cycling parameters and reaction conditions in individual laboratories.
Preview · Article · Jan 2003 · The Indian journal of tuberculosis