Discrimination of SARS-CoV-2 omicron variant and its lineages by rapid detection of immune-escape mutations in spike protein RBD using asymmetric PCR-based melting curve analysis.

BACKGROUND: The SARS-CoV-2 Omicron strain has multiple immune-escape mutations in the spike protein receptor-binding domain (RBD). Rapid detection of these mutations to identify Omicron and its lineages is essential for guiding public health strategies and patient treatments. We developed a two-tube, four-color assay employing asymmetric polymerase chain reaction (PCR)-based melting curve analysis to detect Omicron mutations and discriminate the BA.1, BA.2, BA.4/5, and BA.2.75 lineages. METHODS: The presented technique involves combinatory analysis of the detection of six fluorescent probes targeting the immune-escape mutations L452R, N460K, E484A, F486V, Q493R, Q498R, and Y505H within one amplicon in the spike RBD and probes targeting the ORF1ab and N genes. After protocol optimization, the analytical performance of the technique was evaluated using plasmid templates. Sensitivity was assessed based on the limit of detection (LOD), and reliability was assessed by calculating the intra- and inter-run precision of melting temperatures (Tms). Specificity was assessed using pseudotyped lentivirus of common human respiratory pathogens and human genomic DNA. The assay was used to analyze 40 SARS-CoV-2-positive clinical samples (including 36 BA.2 and 4 BA.4/5 samples) and pseudotyped lentiviruses of wild-type and BA.1 viral RNA control materials, as well as 20 SARS-CoV-2-negative clinical samples, and its accuracy was evaluated by comparing the results with those of sequencing. RESULTS: All genotypes were sensitively identified using the developed method with a LOD of 39.1 copies per reaction. The intra- and inter-run coefficients of variation for the Tms were ≤ 0.69% and ≤ 0.84%, with standard deviations ≤ 0.38 °C and ≤ 0.41 °C, respectively. Validation of the assay using known SARS-CoV-2-positive samples demonstrated its ability to correctly identify the targeted mutations and preliminarily characterize the Omicron lineages. CONCLUSION: The developed assay can provide accurate, reliable, rapid, simple and low-cost detection of the immune-escape mutations located in the spike RBD to detect the Omicron variant and discriminate its lineages, and its use can be easily generalized in clinical laboratories with a fluorescent PCR platform.

Role of HLA class I and II alleles in susceptibility to ankylosing spondylitis in Chinese Han.

OBJECTIVE: The objective of the study was to clarify the associations of HLA class I and II alleles with ankylosing spondylitis (AS) among Chinese Han. METHODS: We performed HLA genotyping and Sanger sequencing for 68 HLA-B*27(-), 62 HLA-B*27(+) AS patients, and 70 controls. Case-control analyses and separate analyses of HLA-B*27(-) patients were performed. One-way ANOVA and Kruskal-Wallis multiple comparisons test were used to analyze the effects of HLA-A\B\C\DRB1\DQB1 alleles on clinical characteristics of HLA-B*27(-) and HLA-B*27(+) patients. RESULTS: In the HLA-B*27(+) group, positive associations were seen with A*11:02, B*27:04, B*27:05, C*02:02, C*12:02, and DRB1*04:01 and negative associations were seen with A*33:03, B*07:02, B*57:01, and C*07:02. The age at onset was greater in HLA-B*27(-) patients than in HLA-B*27(+) patients (30.03 ± 15.15 vs. 23.08 ± 7.79 years). In the HLA-B*27(-) group, those with A*01:01, B*13:01, B*13:02, C*01:02, C*04:01, DQB1*02:01, DQB1*06:01, and DRB1*03:01 had an earlier onset than those without these alleles, while patients carrying B*40:02, C*07:02, C*12:02, C*15:02, DQB1*05:02, and DQB1*05:03 had a delayed onset. In the HLA-B*27(-) group, A*32:01(+), C*08:01(+), and DRB1*04:05(-) women were likely to develop AS. In the HLA-B*27(+) group, DQB1*03:02(+) women may be more likely to develop AS. DRB1*12:02 and HLA-B*27 interacted with the distribution of AS-affected sites. In the HLA-B*27(+) group, DRB1*12:02(+) patients were likely to have peripheral joint involvement. CONCLUSION: HLA class I and II alleles other than HLA-B*27 contribute to AS predisposition and characteristics among Chinese Han patients.

Causal effect between total cholesterol and HDL cholesterol as risk factors for chronic kidney disease: a mendelian randomization study.

BACKGROUND: While observational studies show an association between serum lipid levels and cardiovascular disease (CVD), intervention studies that examine the preventive effects of serum lipid levels on the development of CKD are lacking. METHODS: To estimate the role of serum lipid levels in the etiology of CKD, we conducted a two-sample mendelian randomization (MR) study on serum lipid levels. Single nucleotide polymorphisms (SNPs), which were significantly associated genome-wide with serum lipid levels from the GLGC and CKDGen consortium genome-wide association study (GWAS), including total cholesterol (TC, n = 187,365), triglyceride (TG, n = 177,861), HDL cholesterol (HDL-C, n = 187,167), LDL cholesterol (LDL-C, n = 173,082), apolipoprotein A1 (ApoA1, n = 20,687), apolipoprotein B (ApoB, n = 20,690) and CKD (n = 117,165), were used as instrumental variables. None of the lipid-related SNPs was associated with CKD (all P > 0.05). RESULTS: MR analysis genetically predicted the causal effect between TC/HDL-C and CKD. The odds ratio (OR) and 95% confidence interval (CI) of TC within CKD was 0.756 (0.579 to 0.933) (P = 0.002), and HDL-C was 0.85 (0.687 to 1.012) (P = 0.049). No causal effects between TG, LDL-C- ApoA1, ApoB and CKD were observed. Sensitivity analyses confirmed that TC and HDL-C were significantly associated with CKD. CONCLUSIONS: The findings from this MR study indicate causal effects between TC, HDL-C and CKD. Decreased TC and elevated HDL-C may reduce the incidence of CKD but need to be further confirmed by using a genetic and environmental approach.

SYNE1-QK1 SNPs, G × G and G × E interactions on the risk of hyperlipidaemia.

This study aimed to assess the relationship of 3 spectrin repeat containing nuclear envelope protein 1 (SYNE1) and 4 KH domain containing RNA binding (QK1) single nucleotide polymorphisms (SNPs), their haplotypes, gene-gene (G × G), gene-environment (G × E) interactions and hypercholesterolaemia (HCH) and hypertriglyceridaemia (HTG) in the Chinese Maonan minority. The genetic make-up of the SYNE1-QK1 SNPs in 1932 unrelated subjects (normal, 641; HCH, 649; and HTG, 642) was obtained by next-generation sequencing technologies. The genotypic frequencies of following SNPs were suggestively distinctive between the control and HCH groups (rs2623963, rs7745725, rs9459317, rs16897566), or between the control and HTG groups (rs2623963, rs1358317, rs7745725, rs1923608, rs16897566 SNPs; P < .05, respectively). Multiple-locus linkage disequilibrium analysis indicated that the identified SNPs were not inherited independently. Several haplotypes and gene-gene interaction haplotypes among the detected SNPs may be related with an increased morbidity of HCH (C-G-A, C-G-G and C-G-G-T-C-A-T) and HTG (C-G-G, G-T-G-C, C-G-G-G-T-G-C and C-G-G-T-C-A-T), whereas others may be related with an decreased risk of HCH (G-A-A, G-C-A-T, C-A-A-T-C-A-T and G-A-A-G-C-A-T) and HTG (G-A-A, G-C-A-T, C-A-A-T-C-A-T and G-A-A-G-C-A-T). The association evaluation based on haplotypes and gene-gene interactions could improve the power of detecting the risk of dyslipidaemia than anyone of SNP alone. There was significant three-locus model involving SNP-SNP, haplotype-haplotype/environment and G × G interactions (P < .05-0.001) that were detected by GMDR in HCH and HTG groups. Different interactions between genetic and environmental factors would produce different redundancy or synergy effects on the morbidity of HCH and/or HTG.

The MC4R SNPs, their haplotypes and gene-environment interactions on the risk of obesity.

BACKGROUND: Little is known about the correlation between the melanocortin 4 receptor gene (MC4R) single nucleotide polymorphisms (SNPs) and the risk of obesity. This research sought to test the MC4R rs17782313, rs476828 and rs12970134 SNPs, their haplotypes and gene-environment interactions on the risk of obesity in the Maonan ethnic group, an isolated minority in China. METHODS: A case-control study comprised of 1836 participants (obesity group, 858; and control group, 978) was conducted. Genotypes of the three SNPs were determined by the next-generation sequencing (NGS) technology. RESULTS: The genotypic frequencies of the three SNPs were different between the obesity and control groups (P <  0.05 for all). The minor allelic frequency of the MC4R rs17782313C, rs476828C and rs12970134A was higher in obesity than in control groups (13.8% vs. 8.3%, P <  0.001, 17.1% vs. 10.9%, P <  0.001; and 15.5% vs. 11.5%, P <  0.001; respectively). Additionally, the dominant model of rs17782313 and rs476828 SNPs revealed an increased morbidity function on the risk of obesity (P <  0.05). A correlation between SNP-environment and the risk of obesity was also observed. The rs17782313C-rs476828C-rs12970134A haplotype was associated with high risk of obesity (OR = 1.796, 95% CI = 1.447-2.229), whereas the rs17782313T-rs476828T-rs12970134G and rs17782313T-rs476828T-rs12970134A haplotypes were associated with low risk of obesity (OR = 0.699, 95% CI = 0.586-0.834 and OR = 0.620, 95% CI = 0.416-0.925; respectively). The interactions between haplotype and waist circumference on the risk of obesity were also noted. CONCLUSIONS: We discovered that the MC4R rs17782313, rs476828 and rs12970134 SNPs and their haplotypes were associated with the risk of obesity in the Chinese Maonan population.

Association between the XKR6 rs7819412 SNP and serum lipid levels and the risk of coronary artery disease and ischemic stroke.

BACKGROUND: The present study aimed to expound the association between the XK related 6 gene (XKR6) rs7819412 single nucleotide polymorphism (SNP) and serum lipid profiles and the risk of coronary artery disease (CAD) and ischemic stroke. METHODS: The genetic makeup of the XKR6 rs7819412 SNP in 1783 unrelated participants (controls, 643; CAD, 588 and ischemic stroke, 552) of Han Chinese was obtained by the Snapshot technology. RESULTS: The genotypic frequencies of the SNP were disparate between CAD (GG, 81.0%; GA/AA, 19.0%) or ischemic stroke (GG, 81.2%; GA/AA, 18.8%) patients and healthy controls (GG, 85.7%, GA/AA, 14.3%; P < 0.05 vs. CAD or ischemic stroke; respectively). The A allele frequency was also diverse between CAD (10.1%) or ischemic stroke (10.0%) and control groups (7.5%; P < 0.05 vs. CAD or ischemic stroke; respectively). The GA/AA genotypes and A allele were associated with high risk of CAD and ischemic stroke (CAD: P = 0.026 for GA/AA vs. GG, P = 0.024 for A vs. G; Ischemic stroke: P = 0.029 for GA/AA vs. GG, P = 0.036 for A vs. G). The GA/AA genotypes were also associated with increased serum triglyceride (TG) concentration in CAD and total cholesterol (TC) concentration in ischemic stroke patients. CONCLUSIONS: These data revealed that the XKR6 rs7819412 A allele was related to increased serum TG levels in CAD, TC levels in ischemic stroke patients and high risk of CAD and ischemic stroke.

Potential molecular mechanism of ACE gene at different time points in STEMI patients based on genome-wide microarray dataset.

BACKGROUND: This study aimed to investigate the angiotensin converting enzyme (ACE) co-expression genes and their pathways involved in ST-segment elevation myocardial infarction (STEMI) at different time points. METHODS: The array data set of GSE59867 was examined for the ACE co-expression genes in peripheral blood samples from 111 patients with STEMI at four time points (admission, discharge, and 1 and 6 months after MI). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, Gene Ontology (GO) annotation and protein-protein interaction (PPI) of the co-expression genes were determined using online analytical tools. The Cytoscape software was used to create modules and hub genes. RESULTS: The number of biological processes (BP), cellular components (CC) and molecular functions (MF) was 43, 22 and 24 at admission; 18, 19 and 11 at discharge; 30, 37 and 21 at 1 month after MI; and 12, 19 and 14 at 6 months after MI; respectively. There were 6 BP, 8 CC and 4 MF enriched at every time point. The co-expression genes were substantially enriched in 12, 5, 6 and 14 KEGG pathways at the four time points, respectively, but no KEGG pathway was found to be common in all time points. We identified 132 intersectional co-expression genes (90 positive and 42 negative) from the four time points and 17 BP, 13 CC, 11 MF and 7 KEGG pathways were enriched. In addition, the PPI network contained 129 nodes and 570 edges, and only 1 module was identified to be significantly enriched in just 1 BP (chromatin-mediated maintenance of transcription). CONCLUSIONS: The results of the present study showed that the ACE co-expression genes and their pathways involved in STEMI were significantly different at four different time points. These findings may be helpful for further understanding the functions and roles of ACE in different stages of STEMI, and providing reference for the treatment of STEMI.

Development of a Novel Multiplex PCR Method for the Rapid Detection of SARS-CoV-2, Influenza A Virus, and Influenza B Virus.

Objective: A sensitive and specific multiplex fluorescence rapid detection method was established for simultaneous detection of SARS-CoV-2, influenza A virus, and influenza B virus in a self-made device within 30 min, with a minimum detection limit of 200 copies/mL. Methods: Based on the genome sequences of SARS-CoV-2, influenza A virus (FluA), and influenza B virus (FluB) with reference to the Chinese Center for Disease Control and Prevention and related literature, specific primers were designed, and a multiplex fluorescent PCR system was established. The simultaneous and rapid detection of SARS-CoV-2, FluA, and FluB was achieved by optimizing the concentrations of Taq DNA polymerase as well as primers, probes, and Mg2+. The minimum detection limits of the nucleic acid rapid detection system for SARS-CoV-2, FluA, and FluB were evaluated. Results: By optimizing the amplification system, the N enzyme with the best amplification performance was selected, and the optimal concentration of Mg2+ in the multiamplification system was 3 mmol/L; the final concentrations of SARS-CoV-2 NP probe and primer were 0.15 µmol/L and 0.2 µmol/L, respectively; the final concentrations of SARS-CoV-2 ORF probe and primer were both 0.15 µmol/L; the final concentrations of FluA probe and primer were 0.2 µmol/L and 0.3 µmol/L, respectively; the final concentrations of FluB probe and primer were 0.15 µmol/L and 0.25 µmol/L, respectively. Conclusion: A multiplex real-time quantitative fluorescence RT-PCR system for three respiratory viruses of SARS-CoV-2, FluA, and FluB was established with a high amplification efficiency and sensitivity reaching 200 copies/mL for all samples. Combined with the automated microfluidic nucleic acid detection system, the system can achieve rapid detection in 30 minutes.

Tangshen formula improves diabetic nephropathy in STZ-induced diabetes rats fed with hyper-methionine by regulating the methylation status of kidney.

BACKGROUND: The objective of this study was to examine and analyze differential methylation profiles in order to investigate the influence of hyper-methioninemia (HM) on the development of diabetic nephropathy (DN). Male Wistar rats, aged eight weeks and weighing 250-300 g, were randomly assigned into four groups: a control group (Healthy, n = 8), streptozocin-induced rats (STZ group, n = 8), HM + STZ group (n = 8), and the Tangshen Formula (TSF) treatment group (TSF group, n = 8). Blood glucose levels and other metabolic indicators were monitored before treatment and at four-week intervals until 12 weeks. Total DNA was extracted from the aforementioned groups, and DNA methylation landscapes were analyzed via reduced representative bisulfite sequencing. RESULTS: Both the STZ group and HM + STZ group exhibited increased blood glucose levels and urinary albumin/creatinine ratios in comparison with the control group. Notably, the HM + STZ group exhibited a markedly elevated urinary albumin/creatinine ratio (411.90 ± 88.86 mg/g) compared to the STZ group (238.41 ± 62.52 mg/g). TSF-treated rats demonstrated substantial reductions in both blood glucose levels and urinary albumin/creatinine ratios in comparison with the HM + STZ group. In-depth analysis of DNA methylation profiles revealed 797 genes with potential therapeutic effects related to TSF, among which approximately 2.3% had been previously reported as homologous genes. CONCLUSION: While HM exacerbates DN through altered methylation patterns at specific CpG sites, TSF holds promise as a viable treatment for DN by restoring abnormal methylation levels. The identification of specific genes provides valuable insights into the underlying mechanisms of DN pathogenesis and offers potential therapeutic targets for further investigation.

A novel assay based on DNA melting temperature for multiplexed identification of SARS-CoV-2 and influenza A/B viruses.

Introduction: The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and influenza viruses can cause respiratory illnesses with similar clinical symptoms, making their differential diagnoses challenging. Additionally, in critically ill SARS-CoV-2-infected patients, co-infections with other respiratory pathogens can lead to severe cytokine storm and serious complications. Therefore, a method for simultaneous detection of SARS-CoV-2 and influenza A and B viruses will be clinically beneficial. Methods: We designed an assay to detect five gene targets simultaneously via asymmetric PCR-mediated melting curve analysis in a single tube. We used specific probes that hybridize to corresponding single-stranded amplicons at low temperature and dissociate at high temperature, creating different detection peaks representing the targets. The entire reaction was conducted in a closed tube, which minimizes the risk of contamination. The limit of detection, specificity, precision, and accuracy were determined. Results: The assay exhibited a limit of detection of <20 copies/µL for SARS-CoV-2 and influenza A and <30 copies/µL for influenza B, with high reliability as demonstrated by a coefficient of variation for melting temperature of <1.16% across three virus concentrations. The performance of our developed assay and the pre-determined assay showed excellent agreement for clinical samples, with kappa coefficients ranging from 0.98 (for influenza A) to 1.00 (for SARS-CoV-2 and influenza B). No false-positive, and no cross-reactivity was observed with six common non-influenza respiratory viruses. Conclusion: The newly developed assay offers a straightforward, cost-effective and nucleic acid contamination-free approach for simultaneous detection of the SARS-CoV-2, influenza A, and influenza B viruses. The method offers high analytical sensitivity, reliability, specificity, and accuracy. Its use will streamline testing for co-infections, increase testing throughput, and improve laboratory efficacy.

RUNX1 and CCL3 in Diabetes Mellitus-Related Coronary Artery Disease: A Bioinformatics Analysis.

BACKGROUND: Cardiovascular complications are a major cause of death and disability in patients with diabetes mellitus, but how such complications arise is unclear. METHODS: Weighted gene correlation network analysis (WGCNA) was performed on gene expression profiles from healthy controls, individuals with diabetes mellitus, and individuals with diabetes mellitus-associated coronary artery disease (DMCAD). Phenotypically related module genes were analyzed for enrichment in Gene Ontology (GO) terms and Kyoto Gene and Genome Encyclopedia (KEGG) pathways. Predicted biological functions were validated using gene set enrichment analysis (GSEA) and ClueGo analysis. Based on the TRRUST v2 database and hypergeometric tests, a global network was built to identify transcription factors (TFs) and downstream target genes potentially involved in DMCAD. RESULTS: WGCNA identified three modules associated with progression from diabetes mellitus to DMCAD. The module genes were significantly involved in biological processes related to interferon and viral infection, while GSEA of DMCAD samples suggested involvement in viral myocarditis, chemokine signaling and phagosomes. RUNX1 was identified as a potential TF regulating these module genes. Analysis of the global regulatory network of TFs and their targets suggested that CCL3 may be a key regulator in DMCAD. CONCLUSION: We found bioinformatic evidence that CCL3 may be a key regulator and RUNX1 a key TF in DMCAD.

Association of RDW, NLR, and PLR with Atrial Fibrillation in Critical Care Patients: A Retrospective Study Based on Propensity Score Matching.

Objective: Previous studies have shown inconsistent results in relation to the red cell distribution width (RDW), neutrophil to lymphocyte ratio (NLR), and platelet to lymphocyte ratio (PLR) of atrial fibrillation (AF). This retrospective study is aimed at detecting the association of RDW, NLR, and PLR with AF. Methods: A total of 4717 critical care patients were screened from the Medical Information Mart for Intensive Care- (MIMIC-) III database. The patients were separated into the non-AF and AF groups. The imbalances between the groups were reduced using propensity score matching (PSM). ROC curves were generated to detect the diagnostic value of RDW, NLR, and PLR. Logistic regression analysis was used to detect the risk factors for AF. Results: A total of 991 non-AF patients paired with 991 AF patients were included after PSM in this study. The RDW level in the AF group was significantly higher than that in the non-AF group (15.09 ± 1.93vs. 14.89 ± 1.91, P = 0.017). Neither NLR nor PLR showed any significant difference between the two groups (P > 0.05 for each). According to ROC curve, RDW showed a very low diagnostic value of AF (AUC = 0.5341), and the best cutoff of RDW was 14.1 (ACU = 0.5257, sensitivity = 0.658, specificity = 0.395). Logistic regression analysis showed that an elevated RDW level increased 1.308-fold (95%CI = 1.077-1.588, P = 0.007) risk of AF. Neither elevated NLR nor elevated PLR was a significant risk factor for AF (OR = 0.993, 95%CI = 0.802-1.228, P = 0.945 for NLR; OR = 0.945, 95%CI = 0.763-1.170, P = 0.603 for PLR). Conclusions: Elevated RDW level but not NLR or PLR levels is associated with AF. RDW > 14.1 is a risk factor for AF, but its diagnostic capacity for AF is not of great value.

EHBP1, TUBB, and WWOX SNPs, Gene-Gene and Gene-Environment Interactions on Coronary Artery Disease and Ischemic Stroke.

The associations among the EH domain-binding protein 1 (EHBP1), tubulin beta class I (TUBB), and WW domain-containing oxidoreductase (WWOX) single nucleotide polymorphisms (SNPs) and coronary artery disease (CAD) and ischemic stroke (IS) are not yet understood. This study aimed to detect the associations of these SNPs, gene-gene and gene-environment interactions and CAD and IS in the Guangxi Han population. A total of 1853 unrelated subjects were recruited into normal control (n = 638), CAD (n = 622), and IS (n = 593) groups. Related genotypes were determined by high-throughput sequencing. The genotypic and minor allelic frequencies of rs2278075 were different between the CAD and control groups, and those of rs2710642, rs3130685, and rs2278075 were also different between the IS and control groups. The rs2278075T allele, rs3130685-rs2222896-rs2278075, rs3130685-rs2222896-diabetes, rs3130685-rs2222896-drinking, and haplotype rs2710642A-rs10496099C-diabetes interactions were associated with increased risk, while G-T-G-C-G-A and G-T-T-T-G-T-drinking were associated with reduced risk of CAD. The rs2278075T and rs2710642G alleles, rs2710642G-rs10496099C haplotype, rs3130685-rs2278075-rs2222896, and rs2710642-rs2278075-hypertension interactions aggravated the association with IS, whereas the rs3130685T allele, rs2710642A-rs10496099C haplotype and the interactions of H1 (s2710642A-rs10496099C)-H2 (rs2710642G-rs10496099C)-drinking and I1 (A-C-G-C-A-A)-I3 (A-C-G-T-A-A)-I4 (A-C-G-T-G-A)-I5 (G-T-G-C-G-A) diminished the association with IS. Carrying WWOX rs2278075T was strongly associated with CAD or IS, while EHBP1 rs2710642 and TUBB rs3130685 might alter the association of IS by modifying the serum lipid profile. This study demonstrates that the EHBP1, TUBB, and WWOX SNPs, gene-gene and gene-environment interactions are associated with the risk of CAD and IS in the Guangxi Han population.

Classifiers for Predicting Coronary Artery Disease Based on Gene Expression Profiles in Peripheral Blood Mononuclear Cells.

OBJECTIVE: Coronary artery disease (CAD) is a serious global health concern. Current diagnostic methods for CAD involve risk to the patient and are costly, so better diagnostic tools are needed. We defined four classifiers based on gene expression profiles in peripheral blood mononuclear cells and determined their potential for CAD detection. METHODS: We downloaded a CAD-related data set (GSE113079) from the Gene Expression Omnibus (GEO) database. We identified differentially expressed genes (DEGs) in peripheral blood mononuclear cells between CAD samples and healthy controls. DEGs were analyzed for functional enrichment. To create a robust CAD classifier, DEGs were identified by feature selection using the principal component analysis. Then, least absolute shrinkage and selection operator (LASSO) logistic regression, random forest, and support vector machine (SVM) models were created. Gene set variation analysis (GSVA) score and gene set enrichment analysis (GSEA) were also conducted. The performance of the models was evaluated in terms of the area under receiver operating characteristic curves (AUC). RESULTS: In the training set, we found 135 up-regulated genes and 104 down-regulated genes in CAD patients compared with controls. The DEGs were involved in some pathways associated with CAD, such as pathways involving calcium and interleukin-17 signaling. Twenty genes were identified as optimal features and used to generate the logistic classifier based on LASSO. The AUC for the classifier was 1.00 in the training set and 0.997 in the test set. Using the 20 DEGs, SVM and random forest classifiers were also generated and showed high diagnostic efficacy, with respective AUCs of 0.997 and 1.00 against the training set. A GSVA score was also established using the top 20 significant DEGs, which showed an AUC of 0.971 in the training set and 0.989 in the test set. Furthermore, GSEA showed autophagy and the proteasome to be major pathways involving the DEGs. CONCLUSION: We identified a set of genes specific for CAD whose expression can be measured non-invasively. Using these genes, we defined four diagnostic classifiers using multiple methods.

SYTL3-SLC22A3 Single-Nucleotide Polymorphisms and Gene-Gene/Environment Interactions on the Risk of Hyperlipidemia.

The current study aims to further delineate the associations between the synaptotagmin-like 3 (SYTL3) and solute carrier family 22 member 3 (SLC22A3) single-nucleotide polymorphisms (SNPs) and their haplotypes and gene-gene (G × G)/environment (G × E) interactions on the risk of hyperlipidemia (HLP) in the Maonan and Han ethnic groups. Genotype distribution among the SYTL3-SLC22A3 SNPs in 2,829 individual patients bearing no relationship to each other (Han, 1,436; Maonan, 1,393) was analyzed utilizing next-generation sequencing techniques. The genotype frequencies of the rs6455600, rs2129209, and rs446809 SNPs were varied between the two ethnic groups (P < 0.05-0.001). Various SNPs were correlated with serum levels of triglyceride (TG; rs446809), total cholesterol (TC; rs6455600, rs2129209, and rs539298), and low-density lipoprotein cholesterol (LDL-C; rs446809) among the Han population, whereas various SNPs were also correlated with TC (rs6455600 and rs539298), TG (rs446809), and LDL-C (rs446809) levels in the Maonan ethnic group (P < 0.008-0.001). One part of haplotypes resulted in worsened HLP-related morbidity in the Han (SYTL3 A-C-A-A; SLC22A3 A-A and A-G; and SYTL3-SLC22A3 A-C-A-A-A-A and A-C-A-A-A-G) and Maonan (SYTL3 A-C-A-A; SLC22A3 A-A and A-G; and SYTL3-SLC22A3 A-C-A-A-A-A, G-T-C-A-A-A, and G-T-C-A-C-A) ethnic groups, whereas another part of haplotypes lowered HLP-related health risks in the Han (SLC22A3 C-A and C-G and SYTL3-SLC22A3 A-C-A-A-C-A, A-C-A-A-C-G, and G-T-C-A-C-A) and Maonan (SLC22A3 C-G and SYTL3-SLC22A3 A-C-A-A-C-G) ethnic groups. We discovered that the SYTL3-SLC22A3 SNPs and their haplotypes were associated with serum lipid levels and the risk of HLP in our studied populations.

Associations between TUBB-WWOX SNPs, their haplotypes, gene-gene, and gene-environment interactions and dyslipidemia.

In this study, we investigated associations between single nucleotide polymorphisms (SNPs) in the tubulin beta class I (TUBB) and WW domain-containing oxidoreductase (WWOX) genes, gene-gene interactions, and gene-environment interactions and dyslipidemia in the Chinese Maonan ethnic group. Four SNPs (rs3132584, rs3130685, rs2222896, and rs2548861) were genotyped in unrelated subjects with normal lipid levels (864) or dyslipidemia (1129). While 5.0% of Maonan subjects carried the rs3132584TT genotype, none of the Chinese Han in Beijing subjects did. Allele and genotype frequencies differed between the normal and dyslipidemia groups for three SNPs (rs3132584, rs3130685, and rs2222896). rs2222896G allele carriers in the normal group had higher low-density lipoprotein cholesterol and lower high-density lipoprotein cholesterol levels. The rs3132584GG, rs3130685CC+TT, and rs2222896GG genotypes as well as the rs2222896G-rs2548861G and rs2222896G-rs2548861T haplotypes were associated with an elevated risk of dyslipidemia; the rs2222896A-rs2548861T and rs2222896A-rs2548861G haplotypes were associated with a reduced risk of dyslipidemia. Among the thirteen TUBB-WWOX interaction types identified, rs3132584T-rs3130685T-rs2222896G-rs2548861T increased the risk of dyslipidemia 1.371-fold. Fourteen two- to four-locus optimal interactive models for SNP-SNP, haplotype-haplotype, gene-gene, and gene-environment interactions exhibited synergistic or contrasting effects on dyslipidemia. Finally, the interaction between rs3132584 and rs2222896 increased the risk of dyslipidemia 2.548-fold and predicted hypertension.

No causal effects of plasma homocysteine levels on the risk of coronary heart disease or acute myocardial infarction: A Mendelian randomization study.

BACKGROUND: Although many observational studies have shown an association between plasma homocysteine levels and cardiovascular diseases, controversy remains. In this study, we estimated the role of increased plasma homocysteine levels on the etiology of coronary heart disease and acute myocardial infarction. METHODS: A two-sample Mendelian randomization study on disease was conducted, i.e. "coronary heart disease" (n = 184,305) and "acute myocardial infarction" (n = 181,875). Nine single nucleotide polymorphisms, which were genome-wide significantly associated with plasma homocysteine levels in 57,644 subjects from the Coronary ARtery DIsease Genome wide Replication and Meta-analysis (CARDIoGRAM) plus The Coronary Artery Disease (C4D) Genetics (CARDIoGRAMplusC4D) consortium genome-wide association study and were known to be associated at p < 5×10-8, were used as an instrumental variable. RESULTS: None of the nine single nucleotide polymorphisms were associated with coronary heart disease or acute myocardial infarction (p > 0.05 for all). Mendelian randomization analysis revealed no causal effects of plasma homocysteine levels, either on coronary heart disease (inverse variance weighted; odds ratio = 1.015, 95% confidence interval = 0.923-1.106, p = 0.752) or on acute myocardial infarction (inverse variance weighted; odds ratio = 1.037, 95% confidence interval = 0.932-1.142, p = 0.499). The results were consistent in sensitivity analyses using the weighted median and Mendelian randomization-Egger methods, and no directional pleiotropy (p = 0.213 for coronary heart disease and p = 0.343 for acute myocardial infarction) was observed. Sensitivity analyses confirmed that plasma homocysteine levels were not significantly associated with coronary heart disease or acute myocardial infarction. CONCLUSIONS: The findings from this Mendelian randomization study indicate no causal relationship between plasma homocysteine levels and coronary heart disease or acute myocardial infarction. Conflicting findings from observational studies might have resulted from residual confounding or reverse causation.

CYP17A1-ATP2B1 SNPs and Gene-Gene and Gene-Environment Interactions on Essential Hypertension.

Background: The association between the CYP17A1 and ATP2B1 SNPs and essential hypertension (referred to as hypertension) is far from being consistent. In addition to the heterogeneity of hypertension resulting in inconsistent results, gene-gene and gene-environment interactions may play a major role in the pathogenesis of hypertension rather than a single gene or environmental factor. Methods: A case-control study consisting of 1,652 individuals (hypertension, 816; control, 836) was conducted in Maonan ethnic minority of China. Genotyping of the four SNPs was performed by the next-generation sequencing technology. Results: The frequencies of minor alleles and genotypes of four SNPs were different between the two groups (p < 0.001). According to genetic dominance model analysis, three (rs1004467, rs11191548, and rs17249754) SNPs and two haplotypes (CYP17A1 rs1004467G-rs11191548C and ATP2B1 rs1401982G-rs17249754A) were negatively correlated, whereas rs1401982 SNP and the other two haplotypes (CYP17A1 rs1004467A-rs11191548T and ATP2B1 rs1401982A-rs17249754G) were positively associated with hypertension risk (p ≤ 0.002 for all). Two best significant two-locus models were screened out by GMDR software involving SNP-environment (rs11191548 and BMI ≥ 24 kg/m2) and haplotype-environment (CYP17A1 rs1004467G-rs11191548C and BMI ≥ 24 kg/m2) interactions (p ≤ 0.01). The subjects carrying some genotypes increased the hypertension risk. Conclusions: Our outcomes implied that the rs1004467, rs11191548, and rs17249754 SNPs and CYP17A1 rs1004467G-rs11191548C and ATP2B1 rs1401982G-rs17249754A haplotypes have protective effects, whereas the rs1401982 SNP and CYP17A1 rs1004467A-rs11191548T and ATP2B1 rs1401982A-rs17249754G haplotypes showed adverse effect on the prevalence of hypertension. Several SNP-environment interactions were also detected.

Comprehensive Identification of Key Genes Involved in Development of Diabetes Mellitus-Related Atherogenesis Using Weighted Gene Correlation Network Analysis.

Coronary heart disease (CHD) is common in patients with diabetes mellitus (DM), however, the relevant mechanism remains elusive. The whole blood gene expression profiles of healthy control, patients with DM, patients with DM and CHD (DMCHD) were used to performed weight gene correlation network analysis (WGCNA) to identify the gene modules associated with DM-related atherogenesis. The candidate module was significantly involved in immune- and T cell activity-related biological process. GSEA results suggested that lysosome and apoptosis were enriched in DM and DMCHD samples. The protein-protein-KEGG pathway network may reveal the potential transcriptional regulatory network involving in DM-related atherosclerosis. Nineteen genes (RTKN, DCP1B, PDZD4, CACNA2D2, TSEN54, PVRIG, PLEKHF1, NKG7, ZAP70, NUDCD3, SLAMF6, CCDC107, NAG6, ZDHHC14, EOMES, VIL2, WDR54, DMAP1, and PMPCA) were considered as DM-related atherogenesis genes (DRAGs). The Gene Set Variation Analysis (GSVA) score of the DRAG set gradually increased in the control, DM and DMCHD. ROC curve analysis showed that ZAP70, TSEN54, and PLEKHF1 may be potential blood circulation biomarkers for DMCHD in patients with DM. In conclusion, we identified nineteen hallmark genes involving in DM-related atherogenesis and constructed a potential transcriptional regulatory network involving in DM-related atherosclerosis.

Potential Molecular Mechanism of the NPPB Gene in Postischemic Heart Failure with and without T2DM.

BACKGROUND: This study is aimed at investigating natriuretic peptide B (NPPB) coexpression genes and their pathways involved in heart failure (HF) among patients both with and without type 2 diabetes mellitus (T2DM). METHODS: The microarray dataset GSE26887, containing 19 postischemic HF patients' peripheral blood samples (7 with T2DM and 12 without T2DM), was examined to detect the genes coexpressed with NPPB using the corr.test function in the R packet. Furthermore, using online analytical tools, we determined the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, Gene Ontology (GO) annotation, and protein-protein interaction (PPI) network of the coexpression genes. The modules and hub genes of the PPI network were then identified using the Cytoscape software. RESULTS: In patients with T2DM, a total of 41 biological processes (BP), 20 cellular components (CC), 13 molecular functions (MF), and 41 pathways were identified. Furthermore, a total of 61 BPs, 16 CCs, 13 MFs, and 22 pathways in patients without T2DM were identified. In both groups of patients, 17 BPs, 10 CCs, 6 MFs, and 13 pathways were enriched. We also identified 173 intersectional coexpression genes (63 positively, 106 negatively, and 4 differently coexpressed in patients with and without T2DM, respectively) in both types of patients, which were enriched in 16 BPs, 8 CCs, 3 MFs, and 8 KEGG pathways. Moreover, the PPI network (containing 237 edges and 170 nodes) with the top module significantly enriched in 4 BPs (tricarboxylic acid metabolic process, citrate metabolic process, tricarboxylic acid cycle, and aerobic respiration) and 3 pathways (citrate cycle, malaria parasite metabolic pathway, and AGE-RAGE signaling pathway in diabetic complications) was constructed. DECR1, BGN, TIMP1, VCAN, and CTCF are the top hub genes. CONCLUSIONS: Our findings may elucidate the functions and roles of the NPPB gene in patients with postischemic HF and facilitate HF management.