Genotypic and haplotype analysis of Interleukin-6 and -18 gene polymorphisms in association with clinicopathological factors in breast cancer.

Cytokines are multifunctional glycoproteins that play a vital role in the tumor microenvironment and progression of breast cancer. Genetic polymorphisms may influence the immune responses restrained by pro- and anti-inflammatory cytokine expression in tumors. Hence, the present study evaluated the contribution of Interleukin (IL) 6 (rs1800797, rs1800796, and rs1800795) and IL18 (rs1946518, rs187238, and rs549908) genotypes and their haplotypes to the risk, progression of breast cancer in South Indian population. The polymorphisms of IL6 -597G > A, -572C > G & -174G > C, and IL18 -607C > A, -137G > C, 105A > T were genotyped through PCR-RFLP and As-PCR assays in the blood DNA of 600 subjects. We have performed haplotype, LD, univariate, multivariate logistic regression, and Kaplan-Meier analyses for the obtained data. The frequency of AA genotype & A-allele of IL6 -597G > A, and CC genotype & C-allele of IL6 -174G > C polymorphism was higher in breast cancer patients and was found to be significantly associated with late (advanced) stage, metastasis, etc. Further, IL18 -607C > A, -137G > C, and 105A > T polymorphisms were found to be associated with lobular carcinoma subtype, PgR -ve, and HER2 +ve breast cancer patients. In survival analysis, we have observed that the C-allele of IL6 -174G > C polymorphism to be significantly associated with 5 years of overall survival in breast cancer subjects. All SNPs of the IL6 and IL18 genes showed perfect LD; the G-C-C, A-G-G, and A-C-C haplotype combinations of IL6 gene conferred 2.09, 2.25, and 4.72 folds risk for breast cancer respectively. Hence, our results suggest the importance of genotypic and haplotype analysis of IL6 and IL18 gene variants in the progression and risk prediction of breast cancer.

PCSK9 genetic (rs11591147) and epigenetic (DNA methylation) modifications associated with PCSK9 expression and serum proteins in CAD patients.

INTRODUCTION: Proprotein convertase subtilisin/kexin type 9 (PCSK9) genetic polymorphisms play a significant role in cholesterol homeostasis. Therefore, we aimed to investigate the association of PCSK9 genetic variations NM_174936.3:c.137G>T (R46L, rs11591147) and NM_174936.3:c.1120G>T (D374Y, rs137852912), as well as promoter DNA methylation status, with mRNA expression and circulating serum protein levels in coronary artery disease (CAD) patients. METHODS: The present study includes 300 CAD cases and 300 controls from South India. Biochemical assays were performed using commercially available kits. PCSK9 rs11591147 and rs137852912 polymorphisms were analyzed by the polymerase chain reaction (PCR)-restriction fragment length polymorphism method, whereas promoter DNA methylation status and gene expression were determined using methylation specific PCR and quantitative PCR respectively. RESULTS: The genotypic distribution of PCSK9 rs11591147 revealed that individuals with the TT-genotype and T-allele have a reduced risk for CAD. Furthermore, patients with the PCSK9 rs11591147 TT genotype have a significantly lower total cholesterol and low-density lipoprotein-cholesterol levels and also higher high-density lipoprotein-cholesterol levels than individuals with the GG genotype. Logistic regression analysis has shown that the GG and GT (p = 1.51 × 10-8 , p = 1.47 × 10-9 ) genotypes predicted the risk for CAD with an odds ratio of 5.8 and 7.3 respectively. In addition, individuals with the TT genotype were hypermethylated at promoter DNA of PCSK9, resulting in lower mRNA expression and circulating serum proteins than in individuals with the GG genotype. In silico analyses revealed that rs11591147 T-allele has protein destabilizing capacity. CONCLUSIONS: In conclusion, the present study indicates that the PCSK9 gene expression and circulating serum protein levels are not only associated with rs11591147 genotype, but also with promoter DNA methylation. Furthermore, the findings with respect to both single nucleotide polymorphism and promoter DNA methylation may open avenues for novel treatment possibilities targeting PCSK9 for CAD management.

The rs1126616 Single Nucleotide Polymorphism of the Osteopontin Gene Is Independently Associated with Cardiovascular Events in a Chronic Kidney Disease Cohort.

Chronic kidney disease (CKD) is associated with a higher risk of cardiovascular events (CVE), partly due to the higher burden of atherosclerosis. Circulating Osteopontin (OPN) levels have been also shown to have a potential role in the development of atherosclerosis. Indeed, CKD patients show an increase in circulating OPN levels, but their effect of CKD-related atherosclerosis is not clear. Polymorphisms in the OPN gene (SPP1) have been studied in atheromatous disease, but reported results show conflictive findings. Thus, the main aim of the present study is to analyze the influence of SPP1 polymorphisms in CVE in CKD patients, taking into account circulating OPN levels. We followed 559 healthy controls and 2445 CKD patients without previous CVE from the National Observatory of Atherosclerosis in Nephrology study (NEFRONA study). After 48 months of follow-up 206 CVE were recorded. Genotyping for rs9138, rs1126616, rs1126772, rs11730582 and rs28357094 polymorphisms of the SPP1 gene was performed along with the measurements of plasma OPN levels. The group of patients with CVE showed higher incidence of atherosclerotic plaque (90.3% vs 64.5%; p < 0.001) and higher OPN levels (p < 0.001) at baseline. Patients with the heterozygous genotype of the rs1126616 polymorphism showed a higher hazard ratio of having a CVE, even after adjustment for multiple potential confounders. After adjustment, OPN levels were no longer associated with the incidence of CVE. We found that the rs1126616 single nucleotide polymorphism (SNP) of the SPP1 gene is independently associated with a higher incidence of CVE in a cohort of CKD patients and that it could be used to predict CVE risk.

Association of the rs495392 Klotho polymorphism with atheromatosis progression in patients with chronic kidney disease.

BACKGROUND: Prevalence of atherosclerotic cardiovascular disease and its rate of progression are higher in patients with chronic kidney disease (CKD) compared with the general population. Mineral metabolism parameters have been shown to be involved in the increased velocity of atheromatosis progression. The aim of this study is to determine the role of 11 single-nucleotide polymorphisms (SNPs) of the Klotho gene on the rate of atherosclerosis progression in CKD. METHODS: This was a multicentre, prospective, observational study of 1439 CKD patients from the NEFRONA cohort. Carotid and femoral ultrasounds were performed at baseline and after 24 months in 10 arterial territories. Progression of atheromatosis was defined as an increase in the number of territories with plaque. Genotyping of 11 SNPs of the Klotho gene was performed and its association with atheromatosis progression was determined by multivariate logistic regression. RESULTS: Bivariate analysis showed that none of the 11 SNPs was associated with atheroma plaque prevalence, but 3 of them (rs495392, rs562020 and rs567170) showed association with atheromatosis progression. The multivariate analysis revealed that only rs495392 showed a statistically significant association with atheromatosis progression, after adjustment for several parameters known to affect it in CKD patients. Thus, the presence of one allele T was associated with a reduction of 30% of the odds of progression, whereas the presence of the two T alleles was associated with a decrease close to 50%. CONCLUSIONS: The presence of the allele T of the SNP rs495392 of the Klotho gene is associated with a decrease in the odds of progression of atheromatosis in CKD patients.

Collagenase-1 (-1607 1G/2G), Gelatinase-A (-1306 C/T), Stromelysin-1 (-1171 5A/6A) functional promoter polymorphisms in risk prediction of type 2 diabetic nephropathy.

Type 2 Diabetic Nephropathy (DN) is a common multifactorial disorder. Degradation of glomerular basement membrane (GBM) by matrix metalloproteases (MMPs) is a key event in the progression of renal disease. A functional polymorphism at position -1607 1G/2G, -1306 C/T and -1171 5A/6A has been shown to alter the transcriptional activity of MMP-1, MMP-2, and MMP-3 respectively, and also associated with several diseases contributing to inter-individual differences in susceptibility to type 2 DN. The study population consisted of 310 type 2 DN patients and 310 healthy controls. Genotypes of MMP-1, 2 and 3 were determined by PCR-RFLP assay. Gene interactions, Linkage disequilibrium, and haplotype analysis were carried out by MDR analysis and Haploview software respectively. The promoter binding sites of MMP genes were determined by using Alibaba 2.1 and the gene-gene interactions of MMPs were analyzed by GeneMania. The individuals carrying 2G allele of -1607, C allele of -1306 and 5A/6A genotype of -1171 were associated with type 2 DN susceptibility and progression from stage 1 to stage 5. 2G-5A haplotypes of MMP-1 (-1607 1G/2G) and MMP-3 (-1171 5A/6A) gene polymorphisms were found to be significantly predominant in the disease group. MDR analysis revealed a strong interaction between the genes under study. 2G allele of MMP-1, C allele of MMP-2 and 5A/6A genotype of MMP-3 were associated with susceptibility and disease progression of type 2 DN and might be used as potential markers for risk prediction and prognosis of type 2 DN.

Synergistic effect of collagenase-1 (MMP1), stromelysin-1 (MMP3) and gelatinase-B (MMP9) gene polymorphisms in breast cancer.

BACKGROUND: Extracellular matrix degradation by matrix metalloproteinases (MMPs) is an important mechanism involved in tumor invasion and metastasis. Genetic variations of MMPs have shown association with multiple cancers. The present study is focused to elucidate the association of MMP-1, 3 and 9 genetic variants with respect to epidemiological and clinicopathological variables by haplotype, LD, MDR, survival in silico analyses among South Indian women. MATERIAL AND METHODS: MMP3-1171 5A/6A and MMP9-1562 C/T SNPs were genotyped by Allele specific polymerase chain reaction and MMP1-1607 1G/2G polymorphism by restriction fragment length polymorphism assays respectively, in 300 BC patients and age-matched 300 healthy controls. Statistical analysis was performed using the SNPStats and SPSS software. Linkage disequilibrium and gene-gene interactions were performed using Haploview and MDR software respectively. Further, transcription factor binding sites in the promoter regions of SNPs under study were carried out using AliBaba2.1 software. RESULTS: We have observed an increased frequency of 2G-allele of MMP1, 6A-allele of MMP3 and T-allele of MMP9 (p<0.05) respectively in BC subjects. The 2G-6A haplotype (minor alleles of MMP-1 and MMP-3 respectively) has shown an increased susceptibility to BC. Further, MMP polymorphisms were associated with the clinical characteristics of BC patients such as steroid hormone receptor status, lymph node involvement and metastasis. SNP combinations were in perfect LD in controls. MDR analysis revealed a positive interaction between the SNPs. 5-years survival rate and cox-regression analysis showed a significant association with clinicopathological variables. CONCLUSION: Our results suggest that MMP1-1607 1G/2G, MMP3-1171 5A/6A and MMP9-1562 C/T gene polymorphisms have synergistic effect on breast cancer. The interactions of MMPs clinical risk factors such as lymph node involvement has shown a strong correlation and might influence the 5-years survival rate, suggesting their potential role in the breast carcinogenesis.

Polymorphic variants of Caspase genes (8 & 3) in the risk prediction of Coronary Artery Disease.

Apoptosis has been involved in a number of pathological conditions including coronary artery disease (CAD). Caspases (CASP) are important regulators and executioners in both extrinsic and intrinsic apoptotic pathways. The aim of the present study is to examine the role of Caspase 8 and 3 polymorphisms in the pathogenesis of CAD. CAD patients (n=300) and healthy controls (n=300) were genotyped for polymorphisms in CASP8 (-652 6N del/ins, IVS12-19G>A), CASP3 (rs4647601;G>T) by PCR-RFLP. Splicing defects were determined by HSF. Gene interactions, Linkage disequilibrium and haplotype analysis were carried out by MDR analysis and Haploview software respectively. Molecular analysis revealed that insertion genotype (II) of CASP8 -652 6N del/ins and TT genotype of CASP3 rs4647601;G>T polymorphism conferred risk for the development of CAD. HSF analysis showed that intronic cryptic donor site for CASP8 -652 6N del/ins and a new ESE site for CASP3 rs4647601;G>T polymorphisms. SNP combinations of Caspase 8 and 3 were in perfect LD (D'=1) in controls. D-A, I-G haplotypes of Caspase 8 polymorphisms (-652 6N del/ins & IVS12-19G>A) were found to be significantly predominant in the disease group. The present study suggests that CASP8 & 3 polymorphic variants might be used as markers for susceptibility to CAD.

MMP 1 circulating levels and promoter polymorphism in risk prediction of coronary artery disease in asymptomatic first degree relatives.

Coronary artery disease (CAD) remains to be the prominent health problem in India, and its incidence is growing in developing countries as well. Matrix metalloproteinase 1 (MMP 1) is highly expressed in disruption-prone shoulder regions of the fibrous plaques. The present study aims to investigate association of MMP 1 gene polymorphisms (-1607 1G/2G) and serum circulating levels with CAD. The study includes 300 CAD patients, 100 FDRS, and 300 controls. ELISA and PCR-RFLP were performed to determine MMP 1 serum levels and genotypes respectively. MMP1 levels were high in CAD patients, followed by FDRS compared to controls (2.15±1.2ng/ml; 1.46±1.04ng/ml and 0.96±0.53ng/ml) respectively. ROC analysis showed the AUC at 95% CI of serum MMP-1 to be 0.83 and 0.73-0.94, respectively. The optimal cut-off point (sensitivity; specificity) of serum MMP 1 was >1.5ng/ml (0.74; 0.90). The 2G/2G genotype was associated with high MMP 1 circulating levels in CAD patients, and a similar trend was observed in FDRS and controls. The pre-mRNA secondary structure of the 2G allele is much more stable than 1G allele. Our results suggest MMP 1 serum levels and polymorphism as potential independent prognostic markers for future cardiovascular events. These may also help to stratify CAD patients and to identify susceptibility for CAD in asymptomatic healthy FDRS.

A Therapeutic Effects of Atorvastatin on Genetic Damage in Coronary Artery Disease.

INTRODUCTION: Coronary Artery Disease (CAD) a multifactorial chronic heart disease and the most frequent cause of death and disabling symptoms worldwide, occurs due to the formation of atheromatous lipid rich plaques in the arteries. Statins, which inhibit cholesterol biosynthesis, have both pleiotropic and Low-Density Lipoprotein (LDL)-lowering properties. Atorvastatin is one of the choices for the primary and secondary prevention of cardiovascular disease and management of hypercholesterolaemia. However, there is a paucity of data regarding the changes in the DNA damage in patients with coronary atherosclerosis after statin use. AIM: The aim of the present study was to evaluate atorvastatin treatment efficacy on lipid profiles and DNA damage in CAD patients. MATERIALS AND METHODS: The current observational study was conducted on 180 CAD patients between November 2011 to December 2013 at Durgabai Deshmukh Hospital and Research Centre, Vidya Nagar, Hyderabad, India. Atorvastatin administered and blood samples were collected at index hospitalization and after 6 months statin therapy and lipid profiles and DNA damage was compared with 200 healthy control. RESULTS: Lipid profiles and DNA damage were found to be significantly high (p < 0.01) in CAD patients before atorvastatin therapy compared to after 6 months statin therapy and healthy controls. CONCLUSION: The study suggests that atorvastatin might help in regression of lipid profile as well as DNA damage of CAD patients.

Interleukin 6(-174G/C) variant and its circulating levels in coronary artery disease patients and their first degree relatives.

Interleukin-6 (IL-6) a pleiotropic cytokine is a central mediator of inflammation in the pathogenesis of coronary artery disease (CAD). Our aim is to evaluate the serum levels of IL-6 and C-reactive protein (CRP) and to analyze the IL-6 polymorphism in CAD patients and to identify the first-degree relatives (FDRs) at risk of the disease in comparison with healthy controls. Estimation of IL-6 levels by enzyme-linked immunosorbent assay (ELISA) and CRP by latex reagent kit method, and genotyping of IL6 gene variants -174 (G>C) was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 600 subjects. IL-6 and CRP levels were significantly high in patients followed by FDRs compared to controls. The frequency of the IL-6 genotype was significantly different between cases, FDRs and controls and association of serum IL-6 levels with genotype found to be significant in CC genotype compared to GC and GG at p < 0.01 in CAD patients and FDRs, while there is no significant difference observed in controls. The study shows the importance of inflammation in the pathogenesis of CAD and predicts the risk of future coronary events in healthy asymptomatic FDRs.

Influence of gelatinase B polymorphic variants and its serum levels in atherosclerosis.

AIM: Atherosclerosis, the underlying pathology of cardiovascular disease, is a common, multifactorial disorder with both genetic and environmental components as risk factors. Gelatinase B, also known as MMP-9, is one of the matrix metalloproteinases that is highly expressed in the disruption-prone regions of atherosclerotic plaques. It has been hypothesized that a genetic variation affecting the expression or activity of MMP-9 influences the susceptibility and progression of atherosclerosis. The present study aims to ascertain the polymorphic variants of the MMP-9 gene promoter and its serum levels, which contribute to interindividual differences in susceptibility to atherosclerosis. The study population consisted of 200 individuals who include 100 cases with angiographically recorded coronary artery disease (CAD) and 100 age- and sex-matched healthy controls. Serum levels of MMP-9 were determined in these subjects using an enzyme-linked immunosorbent assay, and polymorphic genotypes of MMP-9 were determined by the polymerase chain reaction-restriction fragment length polymorphism assay. RESULTS: The MMP-9 levels among subjects with the TT genotype for controls (12.21±2.39) and CAD (22.86±2.45) were significantly higher than that of CC genotype (controls 10.37±1.42 and CAD 16.44±7.99), and the values were intermediate for the CT genotype (control 11.21±2.01 and CAD 18.80±3.17) and found to be significant at p<0.01. Genotypic analysis of -1562C/T polymorphism among patients and controls showed higher T allele frequencies in the patient group (0.36) than in the controls (0.29). CONCLUSIONS: It has been observed that increased MMP-9 expression in T allele carriers may contribute to the severity of coronary atherosclerosis. These findings not only are relevant to the understanding of the pathogenesis of atherosclerosis but also may provide a novel target for future development of predictive, preventive, and therapeutic measures.