The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

Preimplantation Genetic Testing for Couples with Balanced Chromosomal Rearrangements.

Background: Chromosomal rearrangements play an important role in infertility. Carriers of chromosomal rearrangements have a lower chance of producing normal or balanced gametes due to abnormal segregation of chromosomes at meiosis, which leads to recurrent spontaneous abortions and infertility. Preimplantation genetic testing for structural chromosome rearrangements (PGT-SR) is offered to couples who have balanced chromosomal rearrangements in order to select embryos with a balanced karyotype prior to implantation, thereby increasing the chances of pregnancy. The purpose of the current study was to assess the outcomes of PGT-SR in patients carrying various balanced chromosomal rearrangements and to assess their clinical pregnancy outcome after in vitro fertilization (IVF). Methods: In this study, infertile couples with balanced chromosomal abnormalities undergoing PGT-SR were retrospectively analyzed at a single fertility center from January 2016 to December 2019. Results: PGT-SR was performed on 87 embryos from 22 couples in whom one partner carried a balanced translocation or an inversion. Fifty-seven (65.5%) of these embryos had unbalanced or sporadic aneuploidies, 30 (34.5%) embryos were normal or chromosomally balanced, which were then transferred in 18 couples. A higher rate of unbalanced translocations in comparison to sporadic aneuploidies was observed in couples with reciprocal translocation. The live birth rate per embryo transfer was found to be 66.6% (12/18). Conclusion: PGT-SR is a useful tool in selecting normal or balanced embryos for transfer in IVF, which could lead to a pregnancy by reducing the chance of miscarriages due to chromosome aneuploidy in couples with balanced chromosomal rearrangements.

Preimplantation genetic testing for aneuploidy (PGT-A)-a single-center experience.

PURPOSE: The aim of this study was to determine the prevalence and nature of human embryonic aneuploidy based on the preimplantation genetic testing for aneuploidy (PGT-A), the distribution of aneuploidy across the individual chromosomes, and their relationship to maternal age. METHODS: This is a retrospective cohort study conducted at a single center. The study includes subjects who opted for PGT-A in their in vitro fertilization (IVF) cycle from 2016 to 2020. PGT-A was performed on 1501 embryos from 488 patients in 535 cycles. PGT-A was performed using NGS-based technique on Ion Torrent PGM (Life Technologies). Analysis was performed to determine the (i) frequency of the aneuploidy, (ii) the chromosome most commonly affected, (iii) relationship between maternal age and the rate of aneuploidy, and (iv) incidence of segmental aneuploidy. RESULTS: The overall frequency of aneuploidy was observed to be 46.8%. The incidence of aneuploidy rate was ~ 28% at maternal age < 30 years which steadily increased to ~ 67% in women above 40 years. High frequency of aneuploidy was observed in chromosomes 16, 22, 21, and 15. Segmental abnormalities, involving loss or gain of chromosomal fragments, were observed at a frequency of 5.3%, and highest incidence of segmental gain was observed on the q-arm of chromosome 9. CONCLUSION: The study provides important information regarding the frequency of the aneuploidy in IVF cohort and the most frequent chromosomal abnormality. The study further emphasizes the relationship between maternal age and aneuploidy. This study has important implications which help clinicians and genetic counselors in providing information in patient counseling.

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.

Integrative gene ontology and network analysis of coronary artery disease associated genes suggests potential role of ErbB pathway gene EGFR.

Coronary artery disease (CAD) is a major cause of mortality in India, more importantly the young Indians. Combinatorial and integrative approaches to evaluate pathways and genes to gain an improved understanding and potential biomarkers for risk assessment are required. Therefore, 608 genes from the CADgene database version 2.0, classified into 12 functional classes representing the atherosclerotic disease process, were analyzed. Homology analysis of the unique list of gene ontologies (GO) from each functional class gave 8 GO terms represented in 11 and 10 functional classes. Using disease ontology analysis 80 genes belonging to 8 GO terms, using FunDO suggested that 29 of them were identified to be associated with CAD. Extended network analysis of these genes using STRING version 9.1 gave 328 nodes and 4,525 interactions of which the top 5% had a node degree of ≥75 associated with pathways including the ErbB signaling pathway with epidermal growth factor receptor (EGFR) gene as the central hub. Evaluation of EFGR protein levels in age and gender­matched 342 CAD patients vs. 342 control subjects demonstrated significant differences [controls=149.76±2.47 pg/ml and CAD patients stratified into stable angina (SA)=161.65±3.40 pg/ml and myocardial infarction (MI)=171.51±4.26 pg/ml]. Logistic regression analysis suggested that increased EGFR levels exhibit 3­fold higher risk of CAD [odds ratio (OR) 3.51, 95% confidence interval [CI] 1.96­6.28, P≤0.001], upon adjustment for hypertension, diabetes and smoking. A unit increase in EGFR levels increased the risk by 2­fold for SA (OR 2.58, 95% CI 1.25­5.33, P=0.01) and 3.8­fold for MI (OR 3.82, 95% CI 1.94­7.52, P≤0.001) following adjustment. Thus, the use of ontology mapping and network analysis in an integrative manner aids in the prioritization of biomarkers of complex disease.

Comparative analysis of inflammatory gene expression levels in metabolic syndrome & coronary artery disease.

BACKGROUND & OBJECTIVES: Metabolic syndrome (MetS) increases the likelihood of developing coronary artery disease (CAD), and inflammation is involved in the pathogenesis of both these conditions. The present work was conducted to examine the relative expression of 18 key inflammatory genes associated with MetS and incident CAD in a representative group of patients. METHODS: A total of 178 male patients, including 57 with CAD and 121 without CAD, were enrolled in the study. The participants without CAD were characterized for the presence of MetS using modified criteria specific for Asian Indians, which included a lower cut-off for waist circumference (≥90 cm for men). The expression of 18 inflammatory genes was evaluated in peripheral whole blood by quantitative polymerase chain reaction method. RESULTS: Of the 121 participants without CAD, 53 (43.8%) had three or more risk factors (MetS group), 50 (41.3%) had one or two risk factors (non-MetS group), while 18 (14.8%) did not have any risk factors (control group). High nuclear factor-kappa B (NF-κB) expression levels and low interleukin-10 (IL-10) levels were observed in MetS patients. Linear association was seen between NF-κB and vascular endothelial growth factor A (VEGFA) expression and with increase in MetS components. Comparison of gene expression pattern between CAD and MetS revealed significantly higher expression of leukotriene genes - arachidonate 5-lipoxygenase (ALOX5), arachidonate 5-lipoxygenase activating protein (ALOX5 AP), leukotriene A4 hydrolase (LTA4H) and leukotriene C4 synthase (LTC4S), and lower expression of NF-κB, interleukin 1 beta (IL-1ß), monocyte chemoattractant protein-1 (MCP-1/CCL2) and signal transducer and activator of transcription 3 (STAT3) genes in CAD. There was linear increase in expression of LTA4H, LTC4S, IL-8 and VEGFA genes across the four groups, namely from controls, non-MetS, MetS and CAD. INTERPRETATION & CONCLUSIONS: A distinct gene expression pattern was seen in MetS and CAD implying a well-orchestrated inflammatory and immune activity. Specifically, NF-κB might be playing an active role in MetS, allowing further expansion of the inflammatory process with resolution of inflammation in full-blown CAD, wherein other gene players such as leukotrienes may dominate.

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.

Network analysis of inflammatory genes and their transcriptional regulators in coronary artery disease.

Network analysis is a novel method to understand the complex pathogenesis of inflammation-driven atherosclerosis. Using this approach, we attempted to identify key inflammatory genes and their core transcriptional regulators in coronary artery disease (CAD). Initially, we obtained 124 candidate genes associated with inflammation and CAD using Polysearch and CADgene database for which protein-protein interaction network was generated using STRING 9.0 (Search Tool for the Retrieval of Interacting Genes) and visualized using Cytoscape v 2.8.3. Based on betweenness centrality (BC) and node degree as key topological parameters, we identified interleukin-6 (IL-6), vascular endothelial growth factor A (VEGFA), interleukin-1 beta (IL-1B), tumor necrosis factor (TNF) and prostaglandin-endoperoxide synthase 2 (PTGS2) as hub nodes. The backbone network constructed with these five hub genes showed 111 nodes connected via 348 edges, with IL-6 having the largest degree and highest BC. Nuclear factor kappa B1 (NFKB1), signal transducer and activator of transcription 3 (STAT3) and JUN were identified as the three core transcription factors from the regulatory network derived using MatInspector. For the purpose of validation of the hub genes, 97 test networks were constructed, which revealed the accuracy of the backbone network to be 0.7763 while the frequency of the hub nodes remained largely unaltered. Pathway enrichment analysis with ClueGO, KEGG and REACTOME showed significant enrichment of six validated CAD pathways - smooth muscle cell proliferation, acute-phase response, calcidiol 1-monooxygenase activity, toll-like receptor signaling, NOD-like receptor signaling and adipocytokine signaling pathways. Experimental verification of the above findings in 64 cases and 64 controls showed increased expression of the five candidate genes and the three transcription factors in the cases relative to the controls (p<0.05). Thus, analysis of complex networks aid in the prioritization of genes and their transcriptional regulators in complex diseases.

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.

Improved detection of gastrointestinal pathogens using generalised sample processing and amplification panels.

We aimed to streamline the diagnosis of gastrointestinal disease by producing multiplexed real time polymerase chain reaction (PCR) panels employing universal sample processing for DNA and RNA containing pathogens. A total of 487 stored, previously characterised stool samples comprising bacterial, viral, protozoan and Clostridium difficile positive samples were tested using four multiplexed real time PCR panels. A further 81 pre-selected clinical samples from a teaching hospital were included to provide an independent validation of assay performance. Improved sensitivity was achieved using the protozoan panels and 16 more mixed infections were observed compared to tests with conventional methods. Using the C. difficile panels, 100% sensitivity was achieved when compared to the gold standard of toxigenic culture. In addition, hypervirulent strains including ribotype 027 could be identified directly from primary sample without the need for ribotyping methods. Bacterial and viral panels detecting Salmonella, Shigella, Campylobacter, Yersinia enterocolitica, Listeria monocytogenes, norovirus groups I and II, rotavirus A, astrovirus, sapovirus, rotavirus B, adenovirus and adenovirus 40/41 performed as well as conventional methods, whilst allowing detection in 3 hours from processing to result. Multiplex real time PCR panels with universal sample preparation allow streamlined, rapid diagnosis of gastrointestinal pathogens whilst extending the characterisation of pathogens present in stool samples from affected patients.

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.

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.