Associations between Genetically Proxied Inhibition of Lipid-Lowering Drug Targets and Serum Micronutrients among Individuals of European Descent: A Mendelian Randomization Study.

BACKGROUND: Limited and inconclusive data exist concerning the associations between lipid-lowering drugs and serum micronutrient concentrations. METHODS: We conducted Mendelian randomization (MR) analyses to explore the associations between lipid-lowering drug targets and serum micronutrients. Single-nucleotide polymorphisms in genes encoding molecular targets of LDL cholesterol-lowering therapies were selected as instrumental variables for 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR; target of statins), proprotein convertase subtilisin/kexin type 9 (PCSK9; target of PCSK9 inhibitors), and Niemann-Pick C1-Like 1 (NPC1L1; target of ezetimibe). Exposure data were extracted from a published genome-wide association study (GWAS) of lipids in 188,577 European individuals, with outcome data obtained from the Integrative Epidemiology Unit (IEU) GWAS database (https://gwas.mrcieu.ac.uk). Overall, age and sex information were not calculable from the summary-level GWAS data. MR analyses were performed using the inverse-variance weighted method and MR sensitivity analysis methods. RESULTS: We found genetically proxied inhibition of HMGCR to lower iron (effect, -0.16; 95% CI: -0.27, -0.06; P value = 0.003), zinc (effect, -0.83; 95% CI: -1.36, -0.31; P value = 0.002), magnesium (effect, -0.17; 95% CI: -0.27, -0.06; P value = 0.003), potassium (effect, -0.17; 95% CI: -0.27, -0.06; P value = 0.002), genetically proxied inhibition of NPC1L1 to increase calcium (effect, 0.28; 95% CI: 0.10, 0.46; P value = 0.003), retinol (effect, 0.25; 95% CI: 0.07, 0.44; P value = 0.01), and genetically proxied inhibition of PCSK9 to increase vitamin D (effect, 0.10; 95% CI: 0.07, 0.12; P value = 1.8 × 10-19). These associations were robust in MR sensitivity analyses. However, the associations between genetically proxied inhibition of HMGCR and NPC1L1 and the micronutrients were not consistent in multiple comparisons. CONCLUSIONS: Our results provide evidence that statin use may lower serum concentrations of iron, zinc, magnesium, and potassium, PCSK9 inhibitors may increase serum vitamin D, and ezetimibe may increase serum calcium and retinol concentrations.

Improved detection of genetic loci in estimated glomerular filtration rate and type 2 diabetes using a pleiotropic cFDR method.

Genome-wide association studies (GWAS) have been shown to have the potential of explaining more of the "missing heritability" of complex human phenotypes by improving statistical approaches. Here, we applied a genetic-pleiotropy-informed conditional false discovery rate (cFDR) to capture additional polygenic effects associated with estimated glomerular filtration rate (creatinine) (eGFRcrea) and type 2 diabetes (T2D). The cFDR analysis improves the identification of pleiotropic variants by incorporating potentially shared genetic mechanisms between two related traits. The Q-Q and fold-enrichment plots were used to assess the enrichment of SNPs associated with eGFRcrea or T2D, and Manhattan plots were used for showing chromosomal locations of the significant loci detected. By applying the cFDR method, we newly identified 74 loci for eGFRcrea and 3 loci for T2D with the cFDR criterion of 0.05 compared with previous related GWAS studies. Four shared SNPs were detected to be associated with both eGFRcrea and T2D at the significant conjunction cFDR level of 0.05, and these shared SNPs had not been reported in previous studies. In addition, we used DAVID analysis to perform functional analysis of the shared SNPs' annotated genes and found their potential hidden associations with eGFRcrea and T2D. In this study, the cFDR method shows the feasibility to detect more genetic variants underlying the heritability of eGFRcrea and T2D, and the overlapping SNPs identified could be regarded as candidate loci that provide a thread of genetic mechanisms between eGFRcrea and T2D in future research.

Application of multiplex methylated-specific PCR with capillary electrophoresis to explore prognostic value of TSGs hypermethylation for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a malignant tumor that severely threatens human health. To date, early detection for HCC patients is particularly significant due to their poor survival rates even after liver resection. METHODS: Therefore, an efficient and sensitive detection method for monitoring liver cancer, multiplex methylation-specific PCR (MSP) coupled with capillary electrophoresis, is developed. RESULTS: Simulations demonstrated that the methylation status of RASSF1A, p16, SFRP1, and ELF could be detected even when DNA equaled or exceeded 12.5 ng simultaneously. Also, its accuracy for methylation detection outweighed polyacrylamide gel electrophoresis (87.5%) and agarose electrophoresis (84.3%), reaching 92.1%. Subsequently, we implemented multiplex MSP with capillary electrophoresis to investigate methylation status of the four tumor suppressor genes in tissue specimens and explore the prognostic value for HCC patients. As the data suggested, multivariate cox regression analysis revealed that the recurrence-free survival of 46 patients was greatly associated with portal vein tumor thrombus (PVTT) and p16 methylation and receiver operating characteristic (ROC) curves demonstrated that the predictive range of portal vein tumor thrombus (PVTT) combined with p16 hypermethylation was more sensitive than that of either PVTT or p16 hypermethylation alone with regard to disease recurrence in patients with HCC, which could be testified as a valuable biomarker in Clinical application. CONCLUSION: Multiplex MSP coupled with capillary electrophoresis has an excellent prospect of clinical application for monitoring early liver cancer and screening valuable biomarkers for prognosis of HCC patients.

[BORIS Regulates SOCS3 Expression Through Epigenetic Mechanisms in Human Hepatocellular Carcinoma Cells].

OBJECTIVE: To study the regulation of suppressor of cytokine signaling 3 (SOCS3) expression bythe brother of the regulator of the imprinted site (BORIS) in hepatocellular carcinoma cell. METHODS: The expression of SOCS3 mRNA in HCC cell lines was detected by real-time quantitative PCR (qRT-PCR). The expression of SOCS3 protein in knockdown and overexpression BORIS of HCC cell lines was tested by Western blot. The SOCS3 gene promoter methylation statusin the knockdown and overexpression BORIS of hepatocarcinoma cell lines was detected by using methylation specific PCR (MSP-PCR) method.The potential BORIS binding site of SOCS3promoter region was found by UCSC database analysis.The enrichment of BORIS in SOCS3 promoter region in endogenous high expression BORIS of HCC cells was evaluated by using chromatin immunoprecipitation (ChIP)-qPCR (ChIP-qPCR).The SOCS3 promoter region histone methylation status in the knockdown and overexpression BORIS of HCC was detected by ChIP-qPCR. RESULTS: The expression of SOCS3 mRNA in hepatocellular carcinoma cells was higher and SOCS3 protein expression was down-regulated or up-regulated in the knockdown or overexpression of BORIS mRNA hepatocarcinoma cells,so BORIS has a positive regulatory effect on SOCS3 protein expression in hepatocarcinoma cells. MSP-PCR experiments showed that the SOCS3 promoter in SMMC-7721 and HepG2 cells was unmethylated and knockdown of BORIS did not change the methylation status; the SOCS3 promoter region of Huh7 cells was methylated; after overexpression of BORIS,the SOCS3 promoter region was changed to an unmethylated state; the SOCS3 promoter was unmethylated in HCCLM3,overexpression of BORIS did not alter the methylation status. The ChIP-qPCR assay demonstrated that BORIS specifically binds to the SOCS3 promoter region in HCC cells with high expression of BORIS. Histone methylation assay indicated that knockdown of BORIS reduced BORIS enrichment in the SOCS3 promoter region, with decreasing H3K4 me2 and increasing H3K27 me3 in the region of histone,whereas the overexpress BORIS in HCC cells showed the opposite situation. CONCLUSION: BORIS plays a role of epigenetic regulationon SOCS3 gene promoter methylation and histone methylation,modulating the expression of SOCS3,and then involved in the development of hepatocellular carcinoma.

Novel common variants associated with body mass index and coronary artery disease detected using a pleiotropic cFDR method.

Genome-wide association studies (GWAS) have been successfully applied in identifying single nucleotide polymorphisms (SNPs) associated with body mass index (BMI) and coronary heart disease (CAD). However, the SNPs to date can only explain a small percentage of the genetic variances of traits. Here, we applied a genetic pleiotropic conditional false discovery rate (cFDR) method that combines summary statistic p values from different multi-center GWAS datasets, to detect common genetic variants associated with these two traits. The enrichment of SNPs associated with BMI and CAD was assessed by conditional Q-Q plots and the common variants were identified by the cFDR method. By applying the cFDR level of 0.05, 7 variants were identified to be associated with CAD (2 variants being novel), 34 variants associated with BMI (11 variants being novel), and 3 variants associated with both BMI and CAD (2 variants being novel). The SNP rs653178 (ATXN2) is noteworthy as this variant was replicated in an independent analysis. SNP rs12411886 (CNNM2) and rs794356 (HIP1) were of note as the annotated genes may be associated with processes that are functionally important in lipid metabolism. In conclusion, the cFDR method identified novel variants associated with BMI and/or CAD by effectively incorporating different GWAS datasets.

Aberrant promoter methylation of SOCS-1 gene may contribute to the pathogenesis of hepatocellular carcinoma: a meta-analysis.

PURPOSE: We conducted this meta-analysis of published case-control studies aiming to evaluate the relationship between abnormal suppression of cytokine signaling-1 (SOCS-1) promoter methylation and the risk of hepatocellular carcinoma (HCC). METHODS: Relevant studies were retrieved from PubMed, Embase, Web of Science, Cochrane Library, Chinese National Knowledge Infrastructure (CNKI) and China Biological Medicine (CBM) databases without language restrictions. Meta-analysis was conducted using the STATA 12.0 software. We calculated odds ratio (OR) and its 95 % confidence interval (95% CI) to estimate the correlations. RESULTS: Sixteen case-control studies with a total of 941 HCC patients and 114 individuals with benign liver diseases met our inclusion criteria. Our results demonstrated that the frequency of SOCS-1 promoter methylation in cancer tissues was significantly higher than in adjacent non-tumorous tissues and benign tissues (cancer tissue vs adjacent tissue: OR=3.05, 95%CI 1.62-5.77, p=0.001; cancer tissue vs benign tissue: OR=11.55, 95%CI 5.93-22.49, p=0.000). Subgroup analyses by ethnicity, detecting method and sample size also suggested that abnormal SOCS-1 promoter methylation was correlated to the risk of HCC in the majority of these subgroups. CONCLUSION: Our findings provide empirical evidence that abnormal SOCS-1 promoter methylation may contribute to the pathogenesis of HCC. Thus, detection of SOCS-1 promoter methylation may be a valuable diagnostic biomarker for HCC.

A Paal-Knorr agent for chemoproteomic profiling of targets of isoketals in cells.

Natural systems produce various γ-dicarbonyl-bearing compounds that can covalently modify lysine in protein targets via the classic Paal-Knorr reaction. Among them is a unique class of lipid-derived electrophiles - isoketals that exhibit high chemical reactivity and critical biological functions. However, their target selectivity and profiles in complex proteomes remain unknown. Here we report a Paal-Knorr agent, 4-oxonon-8-ynal (herein termed ONAyne), for surveying the reactivity and selectivity of the γ-dicarbonyl warhead in biological systems. Using an unbiased open-search strategy, we demonstrated the lysine specificity of ONAyne on a proteome-wide scale and characterized six probe-derived modifications, including the initial pyrrole adduct and its oxidative products (i.e., lactam and hydroxylactam adducts), an enlactam adduct from dehydration of hydroxylactam, and two chemotypes formed in the presence of endogenous formaldehyde (i.e., fulvene and aldehyde adducts). Furthermore, combined with quantitative chemoproteomics in a competitive format, ONAyne permitted global, in situ, and site-specific profiling of targeted lysine residues of two specific isomers of isoketals, levuglandin (LG) D2 and E2. The functional analyses reveal that LG-derived adduction drives inhibition of malate dehydrogenase MDH2 and exhibits a crosstalk with two epigenetic marks on histone H2B in macrophages. Our approach should be broadly useful for target profiling of bioactive γ-dicarbonyls in diverse biological contexts.

Additional common variants associated with type 2 diabetes and coronary artery disease detected using a pleiotropic cFDR method.

Genome-wide association studies (GWASs) have been performed extensively in diverse populations to identify single nucleotide polymorphisms (SNPs) associated with complex diseases or traits. However, to date, the SNPs identified fail to explain a large proportion of the variance of the traits/diseases. GWASs on type 2 diabetes (T2D) and coronary artery disease (CARD) are generally performed as single-trait studies, rather than analyzing the related traits simultaneously. Despite the extensive evidence suggesting that these two phenotypes share both genetic and environmental risk factors, the shared overlapping genetic biological mechanisms between these traits remain largely unexplored. Here, we adopted a recently developed genetic pleiotropic conditional false discovery rate (cFDR) approach to discover novel loci associated with T2D and CARD by incorporating the summary statistics from existing GWASs of these two traits. Applying the cFDR level of 0.05, 33 loci were identified for T2D and 34 loci for CARD, 9 of which for both. By incorporating pleiotropic effects into a conditional analysis framework, we observed that there is significant pleiotropic enrichment between T2D and CARD. These findings may provide novel insights into the etiology of T2D and CARD, as well as the processes that may influence disease development both individually and jointly.

Predicting hepatocellular carcinoma development for cirrhosis patients via methylation detection of heparocarcinogenesis-related genes.

Background: Most hepatocellular carcinoma (HCC) patients have undergone a progression from chronic hepatitis, then liver cirrhosis (LC), and finally to carcinoma. The objective of this study was to elucidate risk factors to predict HCC development for cirrhosis patients. Methods: Multiple methylated specific PCR (MSP) was applied to determine methylation status of heparocarcinogenesis-related genes in 396 tissue and plasma specimens and multivariate cox model was used to analyze the relationship between risk variables and HCC development among cirrhosis patients, followed up in a median period of 30 months. Results: Among 105 LC cases, HCC incidence rate at 30 months was 30.48% (32/105), which were statistically associated with patients' age and aberrant methylation of p16, SFRP, and LINE1 (p<0.05). Receiver operating characteristic (ROC) curve showed the overall predictive accuracy reached the highest (90.7%) if the four risk variables were concurrent to predict HCC development. Moreover, along with the growth of age from 0-40, 40-55, to 55-70 years or the increased number of aberrantly-methylated gene from 0-1 to 2-3, the HCC incidence rate of cirrhosis patients rised from 10.00%, 12.28% to 82.14% and 17.44% to 89.47%, separately. Thus, based on combined analysis with diverse age and number of aberrantly-methylated gene, 105 cases were divided into five groups and computed their respective HCC incidecne rate to categorize them into different risk groups. Of note, A significant lifting of HCC incidence rate in the high-risk group (40-55 years coupled with 2-3 aberrantly-methylated genes, 55-70 years coupled with 0-1 aberrantly-methylated gene, 55-70 years coupled with 2-3 aberrantly-methylated genes; n=33) was observed compared with the low-risk group (0-40 years coupled with 0-1 aberrantly-methylated gene, 40-55 years coupled with 0-1 aberrantly-methylated gene; (n=72) (p<0.01). Conclusions: Ultimately, high-risk cirrhosis patients with 55-over years or 2-3 aberrantly-methylated genes should be paid more attention to be regularly screened with HCC development.

Novel Common Variants Associated with Obesity and Type 2 Diabetes Detected Using a cFDR Method.

Genome-wide association studies (GWASs) have been performed extensively in diverse populations to identify single nucleotide polymorphisms (SNPs) associated with complex diseases or traits. However, to date, the SNPs identified fail to explain a large proportion of the variance of the traits/diseases. GWASs on type 2 diabetes (T2D) and obesity are generally focused on individual traits independently, and genetic intercommunity (common genetic contributions or the product of over correlated phenotypic world) between them are largely unknown, despite extensive data showing that these two phenotypes share both genetic and environmental risk factors. Here, we applied a recently developed genetic pleiotropic conditional false discovery rate (cFDR) approach to discover novel loci associated with BMI and T2D by incorporating the summary statistics from existing GWASs of these two traits. Conditional Q-Q and fold enrichment plots were used to visually demonstrate the strength of pleiotropic enrichment. Adopting a cFDR nominal significance level of 0.05, 287 loci were identified for BMI and 75 loci for T2D, 23 of which for both traits. By incorporating related traits into a conditional analysis framework, we observed significant pleiotropic enrichment between obesity and T2D. These findings may provide novel insights into the etiology of obesity and T2D, individually and jointly.

[Epidemiological characteristics of hepatitis C in Henan province].

OBJECTIVE: To understand the epidemiological characteristics of hepatitis C in Henan province. METHODS: Multistage sampling was used to investigate a group of 1-75 year-old general population, living in 30 districts of 18 cities, Henan province. Blood samples were collected and tested for anti-HCV and hepatitis C virus(HCV), from April to July 2012. RESULTS: 32 203 persons were investigated. Among the general population aged 1 to 75 years old, the overall prevalence rates of anti-HCV and HCV RNA were 0.64% and 0.35% respectively. The prevalence rates of anti-HCV among males and females were 0.60%, and 0.68% respectively, with the rates of HCV RNA as 0.37%, and 0.33% respectively. The prevalence rates of anti-HCV and HCV RNA were increasing with age. The prevalence rates of anti-HCV and HCV RNA in urban area were 0.58% and 0.32%, and in rural area as 0.41%, and 0.19% respectively. CONCLUSION: After the implementation of comprehensive measures for HCV control and prevention, Henan province became low prevalent for HCV infection. Our data revealed that the overall prevalence remained low, but the epidemic was severe in some geographical regions in Henan province.