Transcriptional characteristics and functional validation of three monocyte subsets during aging.

BACKGROUND: Age-associated changes in immunity are inextricably linked to chronic inflammation and age-related diseases, the impact of aging on monocyte subsets is poorly understood. METHODS: Flow cytometry was applied to distinguish three monocyte subsets between 120 young and 103 aged individuals. We then analyzed the expression profiles of three monocyte subsets from 9 young and 9 older donors and CD14+ monocytes from 1202 individuals between 44 and 83 years old. Flow cytometry was used to measure ß-galactosidase activities, ROS levels, mitochondrial contents, mitochondrial membrane potentials (MMPs) and intracellular IL-6 levels in three monocyte subsets of young and elderly individuals, and plasma IL-6 levels were detected by electrochemiluminescence immunoassay. Mitochondrial stress and glycolytic rate of CD14+ monocytes from young and aged individuals were measured by Seahorse XFe24 Analyzer. RESULTS: Compared with young individuals, the percentage of classical subset in aged persons significantly decreased, while the proportion of nonclassical subset increased. Age-related differential genes were obviously enriched in cellular senescence, ROS, oxidative phosphorylation, mitochondrial respiratory chain, IL-6 and ribosome-related pathways. Compared with young individuals, the ß-galactosidase activities, ROS contents, intracellular IL-6 levels of three monocyte subsets, and plasma IL-6 levels in aged individuals were significantly elevated, while the MMPs apparently declined with age and the mitochondrial contents were only increased in intermediate and nonclassical subsets. CD14+ monocytes from elderly adults had conspicuously lower basal and spare respiratory capacity and higher basal glycolysis than those from young individuals. CONCLUSIONS: During aging, monocytes exhibited senescence-associated secretory phenotype, mitochondrial dysfunction, decreased oxidative phosphorylation and increased glycolysis and the nonclassical subset displayed the clearest features of aging. Our study comprehensively investigated age-related transcriptional alterations of three monocyte subsets and identified the pivotal pathways of monocyte senescence, which may have significant implications for tactics to alleviate age-related conditions.

[Analysis of a child with X-linked intellectual disability due to a maternal de novo splicing variant of the PAK3 gene].

OBJECTIVE: To explore the genetic etiology for a child with profound intellectual disabilities and obvious behavioral abnormalities. METHODS: A male child who had presented at the Zhongnan Hospital of Wuhan University on December 2, 2020 was selected as the study subject. Peripheral blood samples of the child and his parents were collected and subjected to whole exome sequencing (WES). Candidate variant was verified by Sanger sequencing. Short tandem repeat (STR) analysis was carried out to determine its parental origin. The splicing variant was also validated in vitro with a minigene assay. RESULTS: WES results revealed that the child had harbored a novel splicing variant of c.176-2A>G in the PAK3 gene, which was inherited from his mother. The results of minigene assay have confirmed aberrant splicing of exon 2. According to the guidelines from the American College of Medical Genetics and Genomics, it was classified as a pathogenic variant (PVS1+PM2_Supporting+PP3). CONCLUSION: The novel splicing variant c.176-2A>G of the PAK3 gene probably underlay the disorder in this child. Above finding has expanded the variation spectrum of the PAK3 gene and provided a basis for genetic counseling and prenatal diagnosis for this family.

A Tool for Early Prediction of Severe Coronavirus Disease 2019 (COVID-19): A Multicenter Study Using the Risk Nomogram in Wuhan and Guangdong, China.

BACKGROUND: Because there is no reliable risk stratification tool for severe coronavirus disease 2019 (COVID-19) patients at admission, we aimed to construct an effective model for early identification of cases at high risk of progression to severe COVID-19. METHODS: In this retrospective multicenter study, 372 hospitalized patients with nonsevere COVID-19 were followed for > 15 days after admission. Patients who deteriorated to severe or critical COVID-19 and those who maintained a nonsevere state were assigned to the severe and nonsevere groups, respectively. Based on baseline data of the 2 groups, we constructed a risk prediction nomogram for severe COVID-19 and evaluated its performance. RESULTS: The training cohort consisted of 189 patients, and the 2 independent validation cohorts consisted of 165 and 18 patients. Among all cases, 72 (19.4%) patients developed severe COVID-19. Older age; higher serum lactate dehydrogenase, C-reactive protein, coefficient of variation of red blood cell distribution width, blood urea nitrogen, and direct bilirubin; and lower albumin were associated with severe COVID-19. We generated the nomogram for early identifying severe COVID-19 in the training cohort (area under the curve [AUC], 0.912 [95% confidence interval {CI}, .846-.978]; sensitivity 85.7%, specificity 87.6%) and the validation cohort (AUC, 0.853 [95% CI, .790-.916]; sensitivity 77.5%, specificity 78.4%). The calibration curve for probability of severe COVID-19 showed optimal agreement between prediction by nomogram and actual observation. Decision curve and clinical impact curve analyses indicated that nomogram conferred high clinical net benefit. CONCLUSIONS: Our nomogram could help clinicians with early identification of patients who will progress to severe COVID-19, which will enable better centralized management and early treatment of severe disease.

Luminescence Sensing for Qualitative and Quantitative Detection of 5-Methylcytosine.

5-Methylcytosine (5mC) is revealed as a heritable epigenetic modification in genomic DNA. It has been reported that cytosine/guanine dinucleotides (CpG) hypermethylation in the promoter regions of tumor suppressor genes is related with inappropriate gene silencing, so the determination of 5mC in the CpG islands of mammals has attracted much attention. In this paper, a luminescence sensing strategy based on bisulfite treatment, asymmetric polymerase chain reaction (PCR), and adenosine triphosphate (ATP)-releasing nucleotide is proposed. With little background, this method can provide accurate quantitative information about methylation changes at CpG sites, even at a specific site. The proposed method can be successfully employed to determine the methylation status of three hepatocellular carcinomas (HCC) related genes in clinical tissues.

Hypermethylation of ACP1, BMP4, and TSPYL5 in Hepatocellular Carcinoma and Their Potential Clinical Significance.

BACKGROUND AND AIM: Aberrant methylation of specific genes is frequent event in hepatocellular carcinoma (HCC). Our present study aims to explore the methylation levels of acid phosphatase locus 1 (ACP1), bone morphogenetic protein 4 (BMP4), and testis-specific protein, Y-encoded-like 5 (TSPYL5) and their potential clinical applications in HCC. METHODS: The methylation levels of ACP1, BMP4 and TSPYL5 were analyzed in 188 HCC tissues, 163 matched adjacent non-tumor tissues, and 29 normal liver tissues using a method of methylation-sensitive restriction enzyme-based quantitative PCR, and their associations with clinicopathological features and prognosis were evaluated. RESULTS: Compared with adjacent non-tumor tissues and normal liver tissues, the methylation levels of ACP1, BMP4, and TSPYL5 were significantly increased in HCC tissues (All p < 0.0001). The methylation of each individual gene could distinguish HCC tissues well from adjacent non-tumor tissues with the area under the receiver operating characteristic curves (AUC) of 0.753, 0.785 and 0.917, respectively. Furthermore, a higher methylation of BMP4 was statistically associated with worse disease-free survival (p = 0.006) and might be an independent unfavorable factor for disease-free survival by univariate and multivariate analysis (p = 0.011, HR 3.431, 95 % CI 1.333-8.833). CONCLUSIONS: Our findings suggest that hypermethylation of ACP1, BMP4, and TSPYL5 are common events in HCC and could be used as potentially detectable biomarkers in HCC tissues. Moreover, BMP4 could be potentially served as a methylated biomarker to predict recurrence and metastasis after hepatectomy for HCC patients. However, their potential clinical application value need to be further clarified.

Deletion of Pr130 Interrupts Cardiac Development in Zebrafish.

Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A), a regulatory subunit of protein phosphatase 2A (PP2A), is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR), plays an important role in the excitation-contraction (EC) coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system. Pr130-knockout zebrafish exhibited cardiac looping defects and decreased cardiac function (decreased fractional area and fractional shortening). Hematoxylin and eosin (H&E) staining demonstrated reduced cardiomyocytes. Subsequent transmission electron microscopy revealed that the bright and dark bands were narrowed and blurred, the Z- and M-lines were fogged, and the gaps between longitudinal myocardial fibers were increased. Additionally, increased apoptosis was observed in cardiomyocyte in pr130-knockout zebrafish compared to wild-type (WT). Taken together, our results suggest that pr130 is required for normal myocardium formation and efficient cardiac contractile function.

Angiotensin-converting enzyme gene polymorphisms and risk for sporadic Alzheimer's disease: a meta-analysis.

Numerous studies have tested for associations between common polymorphisms of the angiotensin-converting enzyme gene and sporadic Alzheimer disease (SAD), but results have been inconclusive. Using meta-analysis, our study aimed to clarify the nature of the genetic risks contributed by the three polymorphisms (rs4291, rs4343, rs1800764) for developing SAD. Through searching of Pubmed, Embase, Alzgene and manually searching relevant references, a total of 14 articles with 26 independent studies were included. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of the association studies. The heterogeneity across the studies was tested, as was publication bias. We observed significant association between SNP rs4291 and SAD using allelic comparison (OR = 1.08, 95% CI 1.03-1.14), homozygote comparison (OR = 1.16, 95% CI 1.04-1.30) and the recessive model (OR = 1.10, 95% CI 1.02-1.18). Association with SNP rs1800764 was revealed but it was not sufficiently robust to withstand the Benjamini-Hochberg method and stepdown Bonferroni correction. Significant association was not identified in the analysis for SNP rs4343. In subgroup analyses, the risk of SAD associated with SNP rs4291 appeared to be significant among Caucasians and in older cases (mean age ≥75 years). Our results confirmed a significant but modest association between SNP rs4291 and SAD susceptibility. Further study of the pathogenetic characteristics of this polymorphism and independent confirmation of the association in larger studies are warranted.

Common rs7138803 variant of FAIM2 and obesity in Han Chinese.

BACKGROUND: Obesity causes severe healthcare problem worldwide leading to numerous diseases, such as cardiovascular diseases and diabetes mellitus. Previous Genome-Wide Association Study (GWAS) identified an association between a single nucleotide polymorphism (SNP) rs7138803, on chromosome 12q13 and obesity in European Caucasians. Since the genetic architecture governing the obesity may vary among different populations, we investigate the variant rs7138803 in Chinese population to find out whether it is associated with obesity. METHODS: A population-based cohort association study was carried out using the High Resolution Melt (HRM) method with 1851 participants. The association between rs7138803 genotypes and body mass index (BMI) was modeled with a general linear model, and a case-control study for the association between rs7138803 genotypes and obesity was performed using Pearson's χ2 test. There was no indication of a deviation from Hardy-Weinberg equilibrium (HWE p value = 0.51) in our sample. RESULTS: No association was detected between SNP rs7138803 and BMI in our Chinese Han population with a P value of 0.51. SNP rs7138803 was found to be not associated with common forms of obesity after adjusting for age and sex in the Chinese population. SNP rs7138803 was not associated with other obesity related traits, including T2DM, hypertension, lipid profiles, and ischemic stroke. CONCLUSION: Our data suggest that the rs7138803 exerts no significant effect on obesity in Chinese Han population. Larger cohorts may be more appropriate to detect an effect of this SNP on common obesity.

Atheroprotective mechanism by which folic acid regulates monocyte subsets and function through DNA methylation.

BACKGROUND: Recent studies have suggested that folic acid can restore abnormal DNA methylation and monocyte subset shifts caused by hyperhomocysteinemia (HHcy) and hyperlipidemia (HL). However, the exact mechanism of action is still not fully understood. In this study, we further investigated the reversal effect and underlying mechanism of folic acid on the shift in monocyte subsets induced by aberrant lipids and Hcy metabolism via DNA methylation in vitro and in vivo. RESULTS: Our results showed that intermediate monocytes were significantly increased but had the lowest global 5-methylcytosine (5-mC) levels in coronary artery disease (CAD) patients, which might lead to a decrease in the global 5-mC levels of peripheral blood leukocytes (PBLs). We also discovered that ARID5B might mediate the increased proportion of intermediate monocytes, as this factor was related to the proportion of monocyte subsets and the expression of CCR2. The expression of ARID5B was inversely associated with the hypermethylated cg25953130 CpG site, which was induced by HL and HHcy. ARID5B could also regulate monocyte CCR2, MCP-1, and TNF-α expression, adhesion and migration, macrophage polarization, and monocyte/macrophage apoptosis, which might explain the regulatory effect of ARID5B on monocyte subset shifting. Folic acid reversed HL- and HHcy-mediated aberrant global and cg25953130 DNA methylation, reduced the proportion of intermediate monocytes, and inhibited the formation of atherosclerotic plaques. CONCLUSION: Folic acid plays a protective role against atherosclerosis through the regulation of DNA methylation, ARID5B expression, and monocyte subsets.

Development and Validation of a Nomogram to Assist Monitoring Nosocomial SARS-CoV-2 Infection of Hospitalized Patients.

PURPOSE: SARS-CoV-2 is extremely infectious, and the incidence of nosocomial infection is conceivably high. We aimed to develop and validate a nomogram to assist monitoring nosocomial SARS-CoV-2 infection in hospitalized patients. PATIENTS AND METHODS: There were 437 COVID-19 hospitalized cases and 420 negative inpatients enrolled from two hospitals in Hubei province, China. We compared the demographic and clinical characteristics of participants between the two groups. Then, LASSO regression and logistic regression were applied to build a nomogram for SARS-CoV-2 infection prediction in the development cohort. Our nomogram was assessed by area under the curve (AUC), calibration curve, decision curve (DCA) and clinical impact curve analysis (CICA). RESULTS: After LASSO regression filtration, eleven laboratory indicators were correlated with SARS-CoV-2 infection. Then, we integrated these features and constructed a nomogram, which showed a high AUC 0.863 (95% CI: 0.834-0.892) in the development cohort with a sensitivity of 80.41% and specificity of 77.38% and 0.813 (95% CI: 0.760-0.866) in validation cohort with a sensitivity of 82.98% and specificity of 70.43%. The calibration plot displayed that the predicted outcomes were in good concordance with the actual observations. DCA and CICA further showed a larger clinical net benefit. CONCLUSION: We constructed and validated a nomogram that integrated eleven laboratory indexes to assist monitoring of nosocomial SARS-CoV-2 infection in hospitalized patients. Our nomogram is remarkably informative for clinical practice, which will be helpful for preventing SARS-CoV-2 further transmission in hospital and avoiding nosocomial infection.

Dynamic changes of throat swabs RNA and serum antibodies for SARS-CoV-2 and their diagnostic performances in patients with COVID-19.

Dynamic changes of RNA and antibodies in SARS-CoV-2 infected patients remain largely unknown, and influence factors for antibody production have not been fully clarified. In this study, consecutive throat swabs specimens (n = 1875) from 187 patients were collected to analyse the dynamic changes of RNA. Moreover, 162 serial serum samples from 31 patients were tested for seroconversion of IgM and IgG. Meanwhile, IgM and IgG were also detected in 409 COVID-19 patients and 389 controls. Additionally, the logistic regression analysis was executed to identify the possible influence factors for antibody production. The median positive conversion time for RNA was day 7 (IQR, 3-11), and the positive rate was highest in day 1-5 (74.59 %) and then gradually decreased. The median time of seroconversion for IgM and IgG were both day 12 (IQR, 10-15). The sensitivity and specificity for IgM (or IgG) was 87.04% and 96.92%, respectively. Multivariate logistic regression indicated that reduced lymphocytes and short positive conversion time for SARS-CoV-2 RNA were independent factors for negative results of IgM and IgG. In conclusion, RNA and antibodies should be combined for COVID-19 diagnosis, and delayed seroconversion was influenced by the decreased lymphocytes and short positive conversion time for RNA.

Clinical significance of aberrant cyclin-dependent kinase-like 2 methylation in hepatocellular carcinoma.

BACKGROUND AND AIMS: Aberrant DNA methylation of cyclin-dependent kinase-like 2 (CDKL2) had been observed in several types of tumors. Herein, the present study was aimed to explore the epigenetic and expression status of CDKL2 and evaluate the diagnostic potential of CDKL2 methylation in hepatocellular carcinoma (HCC). METHODS: The methylation status of CDKL2 was detected by methylation-sensitive restriction enzyme based quantitative PCR (MSRE-qPCR) and bisulfite genomic sequencing (BGS). The mRNA expression of CDKL2 was measured using real-time quantitative PCR (qPCR). The correlations between the methylation of CDKL2 and mRNA expression, clinicopathological features were evaluated. RESULTS: Compared with normal liver tissues, the methylation levels of CDKL2 were significantly increased in the HCC tissues and cell lines (All p < 0.05). And the receiver operating characteristic (ROC) analysis showed that the hypermethylation of CDKL2 had a high specificity and sensitivity to distinguish adjacent non-tumor tissues from HCC tissues. Additionally, the mRNA expression levels of CDKL2 were decreased both in HCC tissues and cell lines than those in normal liver tissues (All p < 0.05), and the expression could be upregulated by 5-aza-2'-deoxycytidine treatment in HCC cell lines. Furthermore, the methylation of CDKL2 was negatively correlated with its mRNA expression (p < 0.001, rs = -0.513), and was associated with gender (p = 0.023), age (p = 0.001) and tumor size (p = 0.016). CONCLUSIONS: Our results showed that CDKL2 promoter hypermethylation played an important role in hepatocarcinogenesis and might be a valuable biomarker for HCC diagnosis.

Using circular RNA SMARCA5 as a potential novel biomarker for hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common malignant tumor worldwide. Circular RNAs (circRNAs), a new class of endogenous non-coding RNAs, are widespread and abundant in mammalian cells. Cumulative evidence showed that circRNAs play significant roles in the process of cancer. However, the expression and function of circRNAs in HCC remain to be investigated. METHODS: The expression of circular RNA SMARCA5 (circSMARCA5) in tissues and plasma samples was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The role of circSMARCA5 in HCC progression was assessed by in vitro experiments. A receiver operating characteristic (ROC) curve was established to evaluate the value of circSMARCA5 as a biomarker in HCC. RESULTS: The expression of circSMARCA5 was significantly downregulated in HCC tissues compared with para-carcinoma tissues. CircSMARCA5 levels were correlated with tumor differentiation (p = 0.023), Tumor-node-metastasis (TNM) stage (p = 0.001), cancer invasion (p = 0.004), as well as cancer diameter (p = 0.018). In vitro cell experiments revealed that overexpression of circSMARCA5 resulted in inhibited proliferation, increased apoptosis and suppressed invasion. Moreover, we found that circSMARCA5 expression was down-regulated in plasma samples of patients with HCC. The ROC curve analyses revealed that plasma circSMARCA5 showed a high accuracy (AUC = 0.938, 0.853, 0.711) for diagnosing HCC from healthy controls, hepatitis and cirrhosis. The area under the ROC curve of plasma circSMARCA5 in combination with AFP in diagnosing HCC from hepatitis and cirrhosis was 0.903 and 0.858. Especially, plasma circSMARCA5 presented a high accuracy (AUC = 0.847, 0.706) for detecting HCC with serum AFP below 200 ng/ml from those hepatitis and cirrhosis with AFP below 200 ng/ml. CONCLUSION: Our study revealed that circSMARCA5 may promote apoptosis, inhibit proliferation, invasion and metastasis of HCC cells. CircSMARCA5 may serve as a potential prediction and monitor biomarker for HCC, especially in HCC patients with AFP blow the cutoff value.

CDKN2A promoter methylation and hepatocellular carcinoma risk: A meta-analysis.

AIM: Lots of studies have explored cyclin-dependent kinase inhibitor 2A (CDKN2A) promoter methylation in hepatocellular carcinoma (HCC), but the established results were controversial. Hence, we conducted the meta-analysis to comprehensively investigate the association between CDKN2A promoter methylation and HCC risk. METHODS: A comprehensive search was implemented through searching PubMed, Web of Science and Embase. Associations of CDKN2A promoter methylation with HCC risk, clinicopathological features, and CDKN2A expression were assessed by the pooled odds ratios (ORs) with corresponding 95% confidence intervals (CIs). Subgroup analyses and meta-regression were served for exploring the potential sources of heterogeneity. RESULTS: A total of 59 articles including 3067 cases and 2951 controls were incorporated in this meta-analysis. Overall, we observed a high CDKN2A promoter methylation rate (58.18%) in HCC and a significant association between the methylation and HCC risk (OR, 7.07; 95% CI, 5.67-8.80). Furthermore, CDKN2A promoter methylation was robustly associated with decreased mRNA (OR, 13.89; 95% CI, 5.44-35.45) and protein (OR, 48.19; 95% CI, 5.56-417.29). In addition, we found the methylation was related with HBV infection (OR, 3.31; 95% CI, 1.47-7.47), HCV infection (OR, 2.76; 95% CI, 1.80-4.23), cirrhosis status (OR, 1.57; 95% CI, 1.01-2.44) and older age (OR, 1.83; 95% CI, 1.14-2.94). CONCLUSIONS: Our results indicated that CDKN2A promoter methylation was associated with an enhancive HCC risk and played a crucial role in the process of HCC with a potential value to being a triage marker for HCC.

Genomic 5-mC contents in peripheral blood leukocytes were independent protective factors for coronary artery disease with a specific profile in different leukocyte subtypes.

Background: Alterations in DNA methylation are demonstrated in atherosclerosis pathogenesis. However, changing rules of global DNA methylation and hydroxymethylation in peripheral blood leukocytes (PBLs) and different blood cell subtypes of coronary artery disease (CAD) patients are still inconclusive, and much less is known about mechanisms underlying. Results: We recruited 265 CAD patients and 270 healthy controls with genomic DNA from PBLs, of which 50 patients and 50 controls were randomly chosen with DNA from isolated neutrophils, lymphocytes and monocytes, and RNA from PBLs. Genomic 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) contents were quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay. Genomic 5-mC contents were negatively associated with the serum total cholesterol (TC) level (P = 0.010), age (P = 0.016), and PBL classifications (P = 0.023), explaining 6.8% individual variation in controls. Furthermore, genomic 5-mC contents were inversely associated with an increased risk of CAD (odds ratio (OR) = 0.325, 95% confidence interval (CI) = 0.237~0.445, P = 2.62 × 10- 12), independent of PBL counts and classifications, age, sex, histories of hyperlipidemia, hypertension, and diabetes. Within-individual analysis showed a general 5-mC decrease in PBL subtypes, but significant difference was found in monocytes only (P = 0.001), accompanied by increased 5-hmC (P = 3.212 × 10- 4). In addition, coincident to the reduced DNMT1 expression in patients' PBLs, the expression level of DNMT1 was significantly lower (P = 0.022) in oxidized low-density lipoprotein (ox-LDL) stimulated THP-1-derived foam cells compared to THP-1 monocytes, with decreased genomic 5-mdC content (P = 0.038). Conclusions: Global hypomethylation of blood cells defined dominantly by the monocyte DNA hypomethylation is independently associated with the risk of CAD in Chinese Han population. The importance of monocytes in atherosclerosis pathophysiology may demonstrate via an epigenetic pathway, but prospective studies are still needed to test the causality.

Novel compound heterozygous mutations in low density lipoprotein receptor gene causes a severe phenotype in a Chinese hypercholesterolemia family.

Mutations in the low density lipoprotein receptor (LDLR) gene serve a causative role in the pathophysiology of familial hypercholesterolemia (FH), a common autosomal inherited disorder characterized by abnormal lipid metabolism. The aim of the present study was to investigate genetic defects in a Chinese family with FH. Clinical features and family histories were collected, as were the results of various laboratory tests, including determinations of serum lipid concentrations, ultrasonography and angiography results. Potential mutations in LDLR were screened using direct polymerase chain reaction (PCR) sequencing. Multiple sequence alignments, structure and hydrophobicity predictions were performed in silico. Novel compound heterozygote mutations in LDLR of the proband were identified, with a Trp577Term-bearing maternal allele and a Pro685Leu-bearing paternal allele. The proband, a 27-year-old male, had severe and diffuse coronary stenosis and non-ST segment elevation myocardial infarction, as well as multiple skin xanthomas and high serum lipid levels. The allele-dosage-dependent clinical features, including hypercholesterolemia and peripheral arterial atherosclerosis, were observed in the proband and the other heterozygous patients. Therefore, the coexistence of Pro685Leu and Trp577Term mutations in LDLR is a novel compound heterozygosis in Chinese patients and may lead to a severe FH phenotype. The explanation for the existence of compound heterozygous mutations instead of homozygous mutations in this particular family requires further study.

A novel mutation in nuclear prelamin a recognition factor-like causes diffuse pulmonary arteriovenous malformations.

Two daughters in a Chinese consanguineous family were diagnosed as diffuse pulmonary arteriovenous malformations (PAVMs) and screened using whole exome sequencing (WES) and copy number variations (CNVs) chips. Though no mutation was found in the established causative genes of capillary malformation-AVMs (CM-AVMs) or PAVMs, Ser161Ile (hg19 NM_022493 c.482G>T) mutation in nuclear prelamin A recognition factor-like (NARFL) was identified. Ser161Ile mutation in NARFL conservation region was predicted to be deleterious and absent in 500 population controls and Exome Aggregation Consortium (ExAC) Database. And there was a dosage effect of the mutation on mRNA levels among family members and population controls, consistent with the instability of mutant mRNA in vitro. Accordingly, in lung tissue of the proband, NARFL protein expression was reduced but Fe3+ was overloaded with vascular endothelial growth factor (VEGF) overexpression. Furthermore, NARFL-knockdown cell lines demonstrated decreased activity of cytosolic aconitase, while NARFL-knockout zebrafish presented ectopic subintestinal vessels sprouts and upregulated VEGF. So we concluded that the Ser161Ile mutant induced NARFL deficiency and eventually diffuse PAVMs probably through VEGF pathway. In a word, we detected a functional mutation in NARFL, which might be the pathogenic gene in this pedigree.

Aberrant methylation of TRIM58 in hepatocellular carcinoma and its potential clinical implication.

TRIM58 (tripartite motif containing 58) has been reported as a novel methylated gene in hepatocellular carcinoma (HCC) by methylation microarrays. However, its associations with mRNA expression and clinicopathological characteristics have not been evaluated. In this study, we explored the potential clinical implications of TRIM58 methylation in HCC. We analyzed the methylation level of TRIM58 in 181 HCC tissues, 172 matched adjacent non-tumor tissues and 13 normal liver tissues using methylation-sensitive restriction enzyme based quantitative PCR and bisulfite genomic sequencing. Further, the mRNA expression level of TRIM58 was measured in 46 paired HCC and adjacent non-tumor tissues by quantitative real-time PCR. Moreover, the relationship between TRIM58 methylation and mRNA expression, the clinicopathological features, as well as prognostic value were evaluated. The results showed that TRIM58 methylation was significantly higher in HCC tissues compared with adjacent non-tumor tissues and normal liver tissues (both p<0.0001). Using 10% as the cut-off value, hypermethylation of TRIM58 was specific in HCC tissues (28.18%, 51/181), with a tendency to correlate with unfavorable disease-free survival (p=0.047). Moreover, TRIM58 expression was significantly decreased in HCC tissues compared with adjacent non-tumor tissues (p<0.0001), and showed a negative association with DNA methylation (p=0.015, rs= -0.260). Our data indicate that TRIM58 methylation is a common event in HCC and may contribute to downregulation of its mRNA expression. Furthermore, hypermethylation of TRIM58 tends to be associated with worse DFS after hepatectomy. However, the potential clinical application of TRIM58 need to be further investigated.

Associations of ChREBP and Global DNA Methylation with Genetic and Environmental Factors in Chinese Healthy Adults.

Age, gender, diet, gene and lifestyle have been reported to affect metabolic status and disease susceptibility through epigenetic pathway. But it remains indistinct that which factors account for certain epigenetic modifications. Our aim was to identify the influencing factors on inter-individual DNA methylation variations of carbohydrate response element binding protein (ChREBP) and global genome in peripheral blood leucocytes (PBLs). ChREBP DNA methylation was determined by bisulfite sequencing, and genomic 5mdC contents were quantified by capillary hydrophilic-interaction liquid chromatography/ in-source fragmentation/ tandem mass spectrometry system in about 300 healthy individuals. Eleven single nucleotide polymorphisms (SNPs) spanning ChREBP and DNA methyltransferase 1 (DNMT1) were genotyped by high resolution melting or PCR-restriction fragment length polymorphism. DNMT1 mRNA expression was analyzed by quantitative PCR. We found ChREBP DNA methylation levels were statistically associated with age (Beta (B) = 0.028, p = 0.006) and serum total cholesterol concentrations (TC) (B = 0.815, p = 0.010), independent of sex, concentrations of triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol (LDL-C), fasting blood glucose and systolic blood pressure, diastolic blood pressure, PBLs counts and classifications. The DNMT1 haplotypes were related to ChREBP (odds ratio (OR) = 0.668, p = 0.029) and global (OR = 0.450, p = 0.015) DNA methylation as well as LDL-C, but not DNMT1 expression. However, only the relation to LDL-C was robust to correction for multiple testing (ORFDR = 1.593, pFDR = 0.013). These results indicated that the age and TC were independent influential factors of ChREBP methylation and DNMT1 variants could probably influence LDL-C to further modify ChREBP DNA methylation. Certainly, sequential comprehensive analysis of the interactions between genetic variants and blood lipid levels on ChREBP and global DNA methylation was required.

Novel keratin 5 mutation in a family with epidermolysis bullosa simplex.

The aim of the present study was to identify the causative gene defects associated with epidermolysis bullosa simplex (EBS) in a pedigree. The diagnosis of EBS was confirmed in two patients from that pedigree based on the clinical manifestations, histopathological examination of the skin and family history. Blood samples were collected from 6 family members and 100 heathy controls, and genomic DNA and RNA were extracted. Mutation analysis of the keratin 5 gene (KRT5) was conducted using polymerase chain reaction (PCR) direct sequencing and PCR-restriction fragment length polymorphism. In the pedigree, the results of PCR direct sequencing revealed a heterozygous missense mutation in codon 202 of exon 2 of KRT5 (c.605T>A), which led to an amino acid change (p.L202Q) in the patients with EBS but was absent from the unaffected family members and 100 population controls. In conclusion, a novel missense mutation in the KRT5 gene was identified that had a pathogenic role in EBS in the population studied, which enriches the germline mutation spectrum of the KRT5 gene.