Tulika Mittal

Investigative Role of Pre-MicroRNAs in Bladder Cancer Patients: A Case–Control Study in North India

MicroRNAs (miRNA) are a class of small noncoding RNA molecules that have been implicated in a wide variety of cellular functions through post-transcriptional regulations on target genes. Common genetic variants (single-nucleotide polymorphisms, SNPs) in pre-miRNA genes may alter their expression and/or maturation effecting thousands of target mRNAs, resulting in varied functional consequences. Three common SNPs (hsa-mir-146a G>C rs2910164, hsa-mir-196a2 C>T rs11614913, and hsa-mir-499 T>C rs3746444) in pre-miRNAs were investigated to evaluate their association with urinary bladder cancer risk. The hospital-based case-control study comprised of 212 histologically confirmed patients with urinary bladder cancer and 250 healthy controls who were unrelated, of similar ethnicity, and age and gender matched. Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism methodology. Our results showed that the heterozygous genotype of rs11614913 was higher in cases than controls but the results were marginally significant (p = 0.055; odds ratio, 1.44). Smoking had no impact in modulating the effect of any of the three miRNA SNPs studied. No association was observed with either the tumor stage or grade in patients with bladder cancer. Even though there was no association between the individuals carrying the variant genotypes of the three miRNA studied and bladder cancer risk, marginal significance of heterozygousity in rs11614913 suggested further characterization of miRNA SNPs in a large cohort of varied ethnicity. This could further provide new prospects for understanding the underlying mechanisms between miRNAs and disease etiology.

Therapeutic Dosing of Acenocoumarol: Proposal of a Population Specific Pharmacogenetic Dosing Algorithm and Its Validation in North Indians

Objectives To develop a population specific pharmacogenetic acenocoumarol dosing algorithm for north Indian patients and show its efficiency in dosage prediction. Methods Multiple and linear stepwise regression analyses were used to include age, sex, height, weight, body surface area, smoking status, VKORC1 -1639 G>A, CYP4F2 1347 G>A, CYP2C9*2,*3 and GGCX 12970 C>G polymorphisms as variables to generate dosing algorithms. The new dosing models were compared with already reported algorithms and also with the clinical data for various performance measures. Odds ratios for association of genotypes with drug sensitive and resistant groups were calculated. Results The pharmacogenetic dosing algorithm generated by multiple regression analysis explains 41.4% (p-value <0.001) of dosage variation. Validation of the new algorithm showed its predictive ability to be better than the already established algorithms based on similar variables. Its validity in our population is reflected by increased sensitivity, specificity, accuracy and decreased rates of over- and under- estimation in comparison to clinical data. The VKORC1-1639 G>A polymorphism was found to be strongly associated with acenocoumarol sensitivity according to recessive model. Conclusions We have proposed an efficient north India specific pharmacogenetic acenocoumarol dosing algorithm which might become a baseline for personalised medicine approach for treatment of patients in future.

Role of functional polymorphisms of P53 and P73 genes with the risk of prostate cancer in a case-control study from Northern India.

BACKGROUND AND AIMS P53 gene variants BstUI RFLP at codon 72 in exon 4, 16-bp tandem repeat in intron 3 and Msp I RFLP in intron 6 and P73 gene variants of G4C14-to-A4T14 (GC/AT), exon 2 polymorphism, which respectively codes for four functionally different protein isoforms, have been shown to modulate susceptibility to different types of human neoplasms. We undertook this study to evaluate the role of P53 and P73 SNPs in prostate cancer in a Northern Indian population. METHODS P53 and P73 genotypes were assessed in a hospital-based case-control study comprised of 177 prostate cancer cases and 265 healthy controls. After the extraction of genomic DNA from blood, genotyping was done using PCR-RFLP and PCR-CTPP methods, respectively. RESULTS A significant association was found in P53 intron 6 G>A and P53 R72P G>C polymorphism with PCa risk. In P53 intron 6 G>A polymorphism the heterozygous genotype (GA) showed marginal risk with the disease (OR = 1.48, 95% CI = 0.999-2.220). Individuals with heterozygous genotype only (GC) of P53 R72P G>C polymorphism demonstrated PCa risk (OR = 1.5, 95% CI = 1-2.199). Haplotypes G-C-D and A-G-D (OR = 1.58, 95% CI = 1.125-2.241 and OR = 2.70, 95% CI = 1.767-4.143, respectively) were also found to be associated with an increased risk of PCa. CONCLUSIONS Our study provided evidence that the P53 intron 6 G>A and R72P G>C polymorphisms were associated with a higher risk of prostate cancer in a Northern Indian population.

Association of matrix metalloproteinases (MMP2, MMP7 and MMP9) genetic variants with left ventricular dysfunction in coronary artery disease patients.

BACKGROUND Left ventricular dysfunction (LVD) is a condition resulting from clustered structural or functional cardiac disorder that reduces the ability of the ventricle to fill with or eject blood. The impaired ventricular function can be attributed to unfavorable ventricular remodeling. Among the pathways that contribute to remodeling process, matrix metalloproteinases (MMPs) appear to be of particular interest. We explored the association of MMP2 (C-735T, rs2285053), MMP7 (A-181G, rs11568818) and MMP9 (R279Q, rs17576), (P574R, rs2250889), (R668Q, rs17577) genetic variants with LVD in coronary artery disease (CAD) patients. METHODS The study included 310 consecutive patients with angiographically confirmed CAD and 230 healthy controls. Among patients with CAD, 95 with reduced left ventricle ejection fraction (LVEF ≤ 45) were categorized as LVD. Polymorphisms were determined by PCR-RFLP. RESULTS The MMP9 R668Q genetic variant was significantly associated with LVD (LVEF ≤ 45) (p value=0.009; OR=3.82). To validate our results, we performed a replication study in additional 200 cases with similar clinical characteristics and results again confirmed consistent findings (p value=0.033; OR=3.59). Also the frequency of haplotype R,P,Q comprising R668Q variation in MMP 9 was significantly higher in reduced LVEF subjects (p value=0.008; OR=1.83). CONCLUSION MMP9 R668Q plays important role in conferring susceptibility of LVD.

Role of inflammatory gene polymorphisms in left ventricular dysfunction (LVD) susceptibility in coronary artery disease (CAD) patients.

RATIONALE Inflammation exacerbates a number of deleterious effects on the heart, most notable being left ventricular dysfunction (LVD). A promoter polymorphism of the NFKB1 gene (encodes p50 subunit) results in lower protein levels of NFkB p50 subunits, which in its dimmer (p50) form has anti-inflammatory effects. The active NFkB transcription factor promotes the expression of over 150 target genes including IL6 and TNF-α. Therefore, the aim of the present study was to assess the association of NFKB1, IL6 and TNF-α gene polymorphisms with LVD in coronary artery disease (CAD) patients. METHODS AND RESULTS The present study included a total of 830 subjects (600 CAD patients and 230 controls) and was carried out in two (primary and replication) cohorts. CAD patients with reduced left ventricle ejection fraction (LVEF ≤45%) were categorized having LVD. The NFKB1 -94 ATTG ins/del (rs28362491), IL6 -174 G/C (rs1800795) and TNF-α -308 G/A (rs1800629) polymorphisms were genotyped by PCR/ARMS-PCR methods. The results of the primary cohort were validated in a replicative cohort and pooled by meta-analysis using Fishers and Mantel-Haenszel test. The analysis showed that NFKB1 ATTG/ATTG genotype was significantly associated with LVD (Fishers method p-value=0.007, Mantel-Haenszel OR=2.34), LV end diastole (p-value=0.013), end systole (p-value=0.011) dimensions, LV mass (p-value=0.024), mean LVEF (p-value=0.001) and myocardial infarction (p-value=0.043). CONCLUSION Our data suggests that NFKB1 -94 ATTG ins/del polymorphism plays significant role in conferring susceptibility of LVD and ATTG/ATTG genotype may modulate risk of heart failure by increasing ventricular remodeling and worsening LV function.

Impact of Renin-Angiotensin-Aldosterone System Gene Polymorphisms on Left Ventricular Dysfunction in Coronary Artery Disease Patients

Background: Left ventricular dysfunction (LVD), followed by fall in cardiac output is one of the major complications in some coronary artery disease (CAD) patients. The decreased cardiac output over time leads to activation of the renin-angiotensin-aldosterone system which results in vasoconstriction by influencing salt-water homeostasis. Therefore, the purpose of the present study was to explore the association of single nucleotide polymorphisms (SNPs) in angiotensin I converting enzyme; ACE (rs4340), angiotensin II type1 receptor; AT1 (rs5186) and aldosterone synthase; CYP11B2 (rs1799998) with LVD. Methods and results: The present study was carried out in two cohorts. The primary cohort included 308 consecutive patients with angiographically confirmed CAD and 234 healthy controls. Among CAD, 94 with compromised left ventricle ejection fraction (LVEF ≤ 45) were categorized as LVD. The ACE I/D, AT1 A1166C and CYP11B2 T-344C polymorphisms were determined by PCR. Our results showed that ACE I/D was significantly associated with CAD but not with LVD. However, AT1 1166C variant was significantly associated with LVD (LVEF ≤ 45) (p value=0.013; OR=3.69), but CYP11B2 (rs1799998) was not associated with either CAD or LVD. To validate our results, we performed a replication study in additional 200 cases with similar clinical characteristics and results again confirmed consistent findings (p value=0.020; OR=5.20). Conclusion: AT1 A1166C plays important role in conferring susceptibility of LVD.

Association of 25 bp Deletion in MYBPC3 Gene with Left Ventricle Dysfunction in Coronary Artery Disease Patients

Rationale Mutations in MYBPC3 encoding cardiac myosin binding protein C are common genetic cause of hereditary cardiac myopathies. An intronic 25-bp deletion in MYBPC3 at 3′ region is associated with dilated (DCM) and hypertrophic (HCM) cardiomyopathies in Southeast Asia. However, the frequency of MYBPC3 25 bp deletion and associated clinical presentation has not been established in an unrelated cohort of left ventricular dysfunction (LVD) secondary to coronary artery disease (CAD) patients. Objective We sought to determine the role of MYBPC3 25 bp polymorphism on LVD in two cohorts of CAD patients. Methods and Results The study included 265 consecutive patients with angiographically confirmed CAD and 220 controls. MYBPC3 25 bp polymorphism was determined by polymerase chain reaction. Our results showed that carrier status of MYBPC3 25 bp deletion was associated with significant compromised left ventricle ejection fraction (LVEF ≤45) in CAD patients (p value  =  <0.001; OR = 4.49). To validate our results, we performed a replication study in additional 140 cases with similar clinical characteristics and results again confirmed consistent findings (p = 0.029; OR = 3.3). Also, presence of the gene deletion did not have significant association in CAD patients with preserved ejection fraction (LVEF>45) (p value  = 0.1; OR  = 2.3). Conclusion The frequency of MYBPC3 DW genotype and D allele was associated with compromised LVEF implying that genetic variants of MYBPC3 encoding mutant structural sarcomere protein could increase susceptibility to left ventricular dysfunction. Therefore, 25 bp deletion in MYBPC3 may represent a genetic marker for cardiac failure in CAD patients from Southeast Asia.

Association of Caspases with an Increased Prostate Cancer Risk in North Indian Population

Dysregulation of apoptosis plays a crucial role in carcinogenesis. Thus, genetic alterations within caspase genes would be expected to provoke a deficient apoptotic signaling thereby facilitating the development of prostate cancer (PCa). In the present study we investigated whether three different polymorphisms in the caspase-5 and -3 genes are differentially expressed in PCa. In a hospital-based case control study in northern India, we genotyped 192 PCa patients and 225 unrelated healthy controls for caspase-5 (G>C) (T>C) and caspase-3 (G>A) polymorphisms using amplification refractory mutation system and polymerase chain restriction fragment length polymorphism methods. Data were statistically analyzed and variant genotype GG of caspase-3 demonstrated increased risk for PCa (odds ratio [OR]=2.72, p=0.005). Similarly variant allele carrier (AG+GG) (OR=1.53, p=0.034) and G allele (OR=1.54, p=0.005) were also statistically associated with PCa risk. High risk for PCa was also observed with respect to caspase-5 (CC) diplotypes (OR=21.67, p=0.012, Pc=0.048). We observed significantly enhanced risk for PCa due to interaction between caspase-3 and -5 gene polymorphisms. In association of genotypes with clinical characteristics, heterozygous TC genotype of caspase-5 (T>C) conferred risk with high Gleason grade tumor (OR=2.35, p=0.042). In case-only analysis, interaction of environmental risk factors and genotypes did not further modulate the risk for PCa. Our observations suggested positive association of caspase-3 and diplotype analysis of caspase-5 to be associated with PCa risk. Interaction of caspase-3 and -5 genotypes also modulated the PCa risk.

Significant role of ADRB3 rs4994 towards the development of coronary artery disease.

BackgroundCoronary artery disease (CAD) is the most common type of heart disease and cause of heart attacks. It has been proposed that both the susceptibility to disease and the interindividual variability in response to treatment relates in part to genetic polymorphisms, particularly those polymorphisms for neurotransmitter and drug receptors. Common functional polymorphisms in &bgr;-adrenergic receptor genes (ADRB) have been associated with heart failure phenotypes. Therefore, the purpose of the present study was to explore the association of genetic variants in ADRB3 (C190T or Trp64Arg) ADRB1 (C1165G or Arg389Gly), and ADRA2A (C-1291G) with CAD. Materials and methodsThe present study recruited a total of 600 consecutive patients with angiographically confirmed CAD and 200 population-matched controls (173 men and 27 women) (mean age 54.10±8.30 years). The ADRB3 T190C, ADRA2A C-1291G, and ADRB1 C1165G polymorphisms were determined by PCR-restriction fragment length polymorphism. The putative functional effects were determined in the coding region of the ADRD3 gene by online web servers FASTSNP and F-SNP. ResultsOn comparing the genotype frequency distribution in CAD patients with that of healthy individuals, significant association was observed with the CC genotype of the ADRB3 T190C polymorphism (P=0.040, odds ratio=1.5). Also, at the allelic level the C allele of ADRB3 T190C conferred risk for CAD (P=0.005, odds ratio=1.7). The ADRA2A C-1291G and ADRB1 C1165G polymorphisms were not found to be a risk for CAD when compared with controls. ConclusionThe present study finding suggests that ADRB3 C190T may also be involved in the complex pathophysiology of CAD.

Genetic predisposition to left ventricular dysfunction: a multigenic and multi-analytical approach.

BACKGROUND Left ventricular dysfunction (LVD) is a complex, multifactorial condition, caused by mechanical, neurohormonal, and genetic factors. We have previously observed association of renin-angiotensin-aldosterone system (RAAS), matrix metalloproteinases (MMPs) and inflammatory pathway genes with LVD. Therefore the present study was undertaken to identify the combination of genetic variants and their possible interactions contributing towards genetic susceptibility to LVD in the background of coronary artery disease (CAD). METHODS AND RESULTS The study included 230 healthy controls and 510 consecutive patients with angiographically confirmed CAD. Among them, 162 with reduced left ventricle ejection fraction (LVEF≤45%) were categorized as having LVD. We analyzed 11 polymorphisms of RAAS, MMPs and inflammatory pathways. Single locus analysis showed that AT1 A1166C (p value<0.001; OR=3.67), MMP9 R668Q (p value=0.007; OR=3.48) and NFKB1-94 ATTG ins/del (p value=0.013; OR=2.01) polymorphisms were independently associated with LVD when compared with both non-LVD patients and healthy controls. High-order gene-gene interaction analysis, using classification and regression tree (CART) and multifactor dimensionality reduction (MDR) revealed that AT1 A1166C and NFKB1-94 ATTG ins/del polymorphisms jointly increased the risk of LVD to great extent (p-value=0.001; OR=8.55) and best four-factor interaction model consisted of AT1 A1166C, MMP7 A-181G, MMP9 R668Q and NFKB1-94 ATTG ins/del polymorphisms with testing accuracy of 0.566 and cross validation consistency (CVC)=9/10 (permutation p<0.001) showed increased risk for LVD respectively. CONCLUSION AT1 A1166C independently and in combination with MMP9 R668Q and NFKB1-94 ATTG ins/del polymorphisms plays important role in conferring genetic susceptibility to LVD in CAD patients.