Genome-Wide Expression Analysis Suggests Hypoxia-Triggered Hyper-Coagulation Leading to Venous Thrombosis at High Altitude.

Venous thromboembolism (VTE), a multi-factorial disease, is the third most common cardiovascular disease. Established genetic and acquired risk factors are responsible for the onset of VTE. High altitude (HA) also poses as an additional risk factor, predisposing individuals to VTE; however, its molecular mechanism remains elusive. This study aimed to identify genes/pathways associated with the pathophysiology of deep vein thrombosis (DVT) at HA. Gene expression profiling of DVT patients, who developed the disease, either at sea level or at HA-DVT locations, resulted in differential expression of 378 and 875 genes, respectively. Gene expression profiles were subjected to bioinformatic analysis, followed by technical and biological validation of selected genes using quantitative reverse transcription-polymerase chain reaction. Both gene ontology and pathway analysis showed enrichment of genes involved in haemostasis and platelet activation in HA-DVT patients with the most relevant pathway being 'response to hypoxia'. Thus, given the environmental condition the differential expression of hypoxia-responsive genes (angiogenin, ribonuclease, RNase A family, 5; early growth response 1; lamin A; matrix metallopeptidase 14 [membrane-inserted]; neurofibromin 1; PDZ and LIM domain 1; procollagen-lysine 1, 2-oxoglutarate 5-dioxygenase 1; solute carrier family 6 [neurotransmitter transporter, serotonin], member 4; solute carrier family 9 [sodium/hydrogen exchanger], member 1; and TEK tyrosine kinase, endothelial) in HA-DVT could be a determining factor to understand the pathophysiology of DVT at HA.

Understanding gene expression in coronary artery disease through global profiling, network analysis and independent validation of key candidate genes.

Molecular mechanism underlying the patho-physiology of coronary artery disease (CAD) is complex. We used global expression profiling combined with analysis of biological network to dissect out potential genes and pathways associated with CAD in a representative case-control Asian Indian cohort. We initially performed blood transcriptomics profiling in 20 subjects, including 10 CAD patients and 10 healthy controls on the Agilent microarray platform. Data was analysed with Gene Spring Gx12.5, followed by network analysis using David v 6.7 and Reactome databases. The most significant differentially expressed genes from microarray were independently validated by real time PCR in 97 cases and 97 controls. A total of 190 gene transcripts showed significant differential expression (fold change>2,P<0.05) between the cases and the controls of which 142 genes were upregulated and 48 genes were downregulated. Genes associated with inflammation, immune response, cell regulation, proliferation and apoptotic pathways were enriched, while inflammatory and immune response genes were displayed as hubs in the network, having greater number of interactions with the neighbouring genes. Expression of EGR1/2/3, IL8, CXCL1, PTGS2, CD69, IFNG, FASLG, CCL4, CDC42, DDX58, NFKBID and NR4A2 genes were independently validated; EGR1/2/3 and IL8 showed >8-fold higher expression in cases relative to the controls implying their important role in CAD. In conclusion, global gene expression profiling combined with network analysis can help in identifying key genes and pathways for CAD.

CELSR2-PSRC1-SORT1 gene expression and association with coronary artery disease and plasma lipid levels in an Asian Indian cohort.

BACKGROUND: Genetic regulation of plasma lipids has been shown to influence the risk of coronary artery disease (CAD). We analyzed the relationship between rs599839 and rs646776 single nucleotide polymorphisms (SNPs) present in the CELSR2-PSRC1-SORT1 gene cluster, candidate gene expression, and their association with CAD and circulating lipid levels in a representative cohort of Asian Indians selected from the Indian Atherosclerosis Research Study. METHODS: SNPs rs599839 and rs646776 were genotyped by Taqman assay in 1034 CAD patients (cases) and 1034 age- and gender-matched controls. Expression of CELSR2, PSRC1, and SORT1 genes was measured in 100 cases and 100 controls. Plasma levels of total cholesterol (TC), triglycerides, high-density lipoprotein-cholesterol, and low-density lipoprotein-cholesterol (LDL-c) were measured by enzymatic assay. RESULTS: Both rs646776 and rs599839 were in strong linkage disequilibrium (r = 0.98) and showed significant protective association with CAD (OR = 0.315, 95% CI 0.136-0.728, p<0.007 and OR = 0.422, 95% CI 0.181-0.981, p = 0.045, respectively). Haplotype TA showed 72% frequency and was associated with CAD (OR 0.77, 95% CI 0.67-0.88, p = 0.0002). PSRC1 gene expression was lower in the cases than in the controls (0.75 ± 0.405 versus 1.04 ± 0.622, p = 2.26 × 10(-4)). The homozygous variant and heterozygous genotypes showed 30% and 15% higher PSRC1 expression, respectively. Correspondingly, the minor alleles were associated with lower plasma TC and LDL-c levels. CONCLUSION: PSRC1 in the cholesterol gene cluster shows a significant association with CAD by virtue of the two SNPs, rs646776 and rs599839 that also regulate plasma cholesterol levels.

Genetic analysis of the 9p21.3 CAD risk locus in Asian Indians.

The 9p21.3 locus is the best replicated region to date for coronary artery disease (CAD). We investigated the association of 9p21.3 common variants with CAD, candidate gene expression including ANRIL, a non-coding RNA, followed by in vitro validation. Five variants, rs10757278, rs10757274, rs2383206, rs1333049 and rs4977574 were genotyped in 1,034 cases and 1,034 controls. Gene expression of C9orf5, MTAP1, MTAP 2, p16INK4a, p14ARF, p15INK4b and two ANRIL splice variants, NR_003529 and EU741058, were measured in 100 cases and 100 controls. Human aortic smooth muscle cells (HuAoSMCs) were transfected with siRNA targeting ANRIL exon 19 (siRNA-1) or exon 2 (siRNA-2) and consequent effect determined. rs2383206 showed the highest association with CAD (odds ratio [OR] 2.02, 95% confidence interval [CI] 1.56 -2.62) and an adjusted OR of 2.55, 1.33-2.88 along with rs10757278. Conventional risk factors (conventional RFs), rs2383206 and rs10757278 variants together yielded a higher c index (OR 0.790, 95% CI 0.770 -0.810) as compared to conventional RFs (OR 0.783, 95% CI 0.763-0.803) or genetic variants (OR 0.561, 95% CI 0.536-0.586) alone. GAAAA haplotype showed significant protective association with CAD compared to CGGGG risk haplotype (OR 0.45, 95% CI 0.27-0.77). Expression of p16INK4a, p14ARF and p15INK4b as well as plasma CDKN2A levels were lower in cases than controls. GG genotype was associated with higher EU741058 expression and lower p16INK4a expression. HuAoSMCs transfected with siRNA-1 showed lower NR_003529, p16INK4aand p14ARFexpression. Our study provides further evidence on the significance of 9p21.3 locus for CAD wherein the risk allele regulate the expression of ANRIL and adjacent tumour suppressor genes which in turn alter smooth muscle proliferation, a fundamental process in atherosclerosis.

Expression analysis of leukotriene-inflammatory gene interaction network in patients with coronary artery disease.

AIM: Leukotrienes are important lipid inflammatory mediators that play a pivotal role in the pathogenesis of atherosclerosis. We aimed to construct a network of interactions between leukotrienes and inflammatory biomarkers and evaluate the expression of key members of the leukotriene pathway and leukotriene-induced inflammatory molecules in patients with coronary artery disease (CAD) and healthy controls. METHODS: Leukotrienes and their regulatory inflammatory molecules reported in the literature were used to construct a biological network employing Gene spring GX v12.5. Key leukotriene genes and their closely interacting members were selected for expression study in 64 patients and 64 matched controls. Four single nucleotide polymorphisms(SNPs) (rs6538697, rs2660898, rs17525495 and rs1978331) in the leukotriene A4 hydrolase(LTA4H) gene were genotyped using SYBR green method, and plasma leukotriene B4 (LTB4) levels were measured using ELISA. RESULTS: The expression levels of arachidonate 5-lipoxygenase(ALOX5), LTA4H, tumor necrosis factor (TNF) and interleukin-8 (IL-8) genes were significantly higher in patients than in the controls(p＜0.05). IL-8(r=0.35-0.47) and TNF (r=0.42-0.53) expression levels exhibited strong correlations with the leukotriene genes. The SNPs rs17525495 and rs1978331 were associated with LTA4H mRNA expression, while LTA4H and IL-8 levels were associated with CAD. The addition of these two markers to the conventional risk factors improved the c-statistics(area under the receiver operating characteristic(ROC) curve) from 0.75 to 0.93(p＜0.01), with a Net Reclassification Index of 0.45(p＜0.01) and Integrated Discrimination Improvement of 0.26(p＜0.01). CONCLUSIONS: Leukotrienes and inflammatory genes, in particular, LTA4H and IL-8, exhibit close association in subjects with cardiovascular disease. Assessing these markers may provide incremental value for predicting cardiovascular risk beyond that obtained with classical risk factors.

Relationship between periodontal disease, Porphyromonas gingivalis, peripheral vascular resistance markers and coronary artery disease in Asian Indians.

INTRODUCTION: A close association exists between oral health and cardiovascular disease. Periodontal disease induces early vascular changes while oral pathogens have been detected in sub gingival and atheromatous plaques. We examined the interrelationship between Periodontal disease, oral bacteria, surrogate sub-clinical markers and coronary artery disease (CAD) in a representative Asian Indian cohort. MATERIALS AND METHODS: 532 Gingivitis cases and 282 Periodontitis cases were assessed for early peripheral vascular changes, namely pulse wave velocity (PWV), arterial stiffness index (ASI) and ankle brachial index (ABI) using computerized oscillometry method. Relative quantitation (RQ) of Porphyromonas gingivalis (Pg) was estimated in saliva samples of 54 Periodontitis, 25 Gingivitis and 51 CAD cases (38 also had oral disease) by Taqman assay by amplifying pathogen-specific gene targets, 16srRNA and IktA, respectively, and 16s universal bacterial rRNA as endogenous control. RESULTS: PWV and ASI were elevated in Periodontitis compared to Gingivitis cases (p<0.0001) and in those with diabetes and hypertension. Cases with Periodontitis showed higher mean expression of Pg than Gingivitis (0.37±0.05 versus 0.15±0.04, p<0.0001), while CAD patients with oral disease (N=38) showed lower mean Pg expression than those without oral disease (N=13) (0.712±0.119 versus 1.526±0.257, p=0.008). Higher Pg expression was recorded in subjects with diabetes and hypertension. CONCLUSION: Oral disease induces early changes in the peripheral blood vessels. Further, common presence of Pg in subjects with oral disease, in those with established cardiovascular risk factors and in patients with symptomatic CAD reflects the importance of oral hygiene in the development of Coronary Artery Disease in Asian Indians.

Integrative bioinformatics analysis of genomic and proteomic approaches to understand the transcriptional regulatory program in coronary artery disease pathways.

Patients with cardiovascular disease show a panel of differentially regulated serum biomarkers indicative of modulation of several pathways from disease onset to progression. Few of these biomarkers have been proposed for multimarker risk prediction methods. However, the underlying mechanism of the expression changes and modulation of the pathways is not yet addressed in entirety. Our present work focuses on understanding the regulatory mechanisms at transcriptional level by identifying the core and specific transcription factors that regulate the coronary artery disease associated pathways. Using the principles of systems biology we integrated the genomics and proteomics data with computational tools. We selected biomarkers from 7 different pathways based on their association with the disease and assayed 24 biomarkers along with gene expression studies and built network modules which are highly regulated by 5 core regulators PPARG, EGR1, ETV1, KLF7 and ESRRA. These network modules in turn comprise of biomarkers from different pathways showing that the core regulatory transcription factors may work together in differential regulation of several pathways potentially leading to the disease. This kind of analysis can enhance the elucidation of mechanisms in the disease and give better strategies of developing multimarker module based risk predictions.

Role of vitamin D levels and vitamin D receptor polymorphisms in relation to coronary artery disease: the Indian atherosclerosis research study.

OBJECTIVE: Assessment of association between plasma vitamin D levels, vitamin D receptor (VDR) gene polymorphisms, and coronary artery disease (CAD) in a predisposed Asian Indian cohort. MATERIALS AND METHODS: Patients with angiographically proven CAD having age at onset less than 60 years for men and less than 65 years for women were recruited in the Indian Atherosclerosis Research Study and treated as cases (N=287), whereas asymptomatic healthy matched individuals were enrolled from the population, who showed normal electrocardiogram and acted as controls (N=241). Plasma [vitamin D (25-hydroxy vitamin D)] levels were measured by enzyme-linked immunosorbent assay, and five haplotype-tagging single nucleotide polymorphisms were genotyped by ABI Taqman assays. RESULTS: Mean vitamin D levels were significantly lower in patients with CAD (10.59 ng/ml) than in controls (11.82 ng/ml) (P=0.036). Vitamin D showed protective association against CAD (odds ratio: 0.54, 95% confidence interval: 0.34-0.84, P=0.007) after adjusting for conventional risk factors. Patients in the first vitamin D quartile showed 2.54 times greater risk for CAD than those in the fourth quartile. There was no significant association of VDR single nucleotide polymorphisms/haplotypes with either vitamin D or CAD. Vitamin D levels were significantly lower in vegetarians than in nonvegetarians (P=0.048) and showed inverse association with body weight (P=0.054), triglyceride (P=0.031), and body mass index (P=0.020). CONCLUSION: Low vitamin D level was associated with an enhanced risk for incident CAD. VDR genotypes did not show any association with either vitamin D levels or CAD.

The Soliga, an isolated tribe from Southern India: genetic diversity and phylogenetic affinities.

India's role in the dispersal of modern humans can be explored by investigating its oldest inhabitants: the tribal people. The Soliga people of the Biligiri Rangana Hills, a tribal community in Southern India, could be among the country's first settlers. This forest-bound, Dravidian speaking group, lives isolated, practicing subsistence-level agriculture under primitive conditions. The aim of this study is to examine the phylogenetic relationships of the Soligas in relation to 29 worldwide, geographically targeted, reference populations. For this purpose, we employed a battery of 15 hypervariable autosomal short tandem repeat loci as markers. The Soliga tribe was found to be remarkably different from other Indian populations including other southern Dravidian-speaking tribes. In contrast, the Soliga people exhibited genetic affinity to two Australian aboriginal populations. This genetic similarity could be attributed to the 'Out of Africa' migratory wave(s) along the southern coast of India that eventually reached Australia. Alternatively, the observed genetic affinity may be explained by more recent migrations from the Indian subcontinent into Australia.

Understanding the expression of Toll-like receptors in Asian Indians predisposed to coronary artery disease.

INTRODUCTION: Toll-like receptors (TLRs) are an important link between innate and adaptive immunity. MATERIAL AND METHODS: Expression of TLR-2, TLR-4, and TLR-9 genes was assessed in 60 coronary artery disease (CAD) patients and 79 controls by SYBR Green 1 based real time PCR assay. RESULTS: Expression of the TLR-2 gene was found to be significantly elevated in cases (1.295 ±0.09) compared to the controls (1.033 ±0.08) (p = 0.015) whereas expression of the TLR-9 gene was significantly lower in cases (1.522 ±0.18) than in the controls (2.165 ±0.16) (p = 0.032). There was no difference in TLR-4 expression levels (p = 0.174). A significant correlation of TLR-2 was observed with TLR-4 (r = 0.803, p<0.0001) and TLR-9 (r = 0.264, p = 0.003) as well as between TLR-4 and TLR-9 (r = 0.303, p = 0.001). A significant association was seen between TLR 2 (OR 3.94, 95% CI 1.73-8.99, p = 0.001) and TLR-9 (OR 0.297, 95% CI 0.131-0.672, p = 0.004) with CAD after adjustment for age and gender. Statins did not affect TLR gene expression. CONCLUSIONS: The TLR-2, TLR-4 and TLR-9 genes exhibit a differential pattern of expression between CAD patients and controls in this Asian Indian cohort. This observation warrants further investigation, keeping in mind the infectious and inflammatory elements in perspective, in order to understand the true implications of TLR in the aetiopathology of CAD and consequent therapeutic implications.