Expression of the congenital heart disease 5/tryptophan rich basic protein homologue gene during heart development in Medaka fish,Oryzias latipes

The congenital heart disease 5 (CHD5)/tryptophan rich basic protein (WRB) is a protein containing a tryptophan‐rich carboxy‐terminal region, which was discovered in the human fetal heart. In humans, this CHD5/WRB is located between the markers ACTL5‐D21S268 within the Down syndrome (DS) Region‐2 at chromosome 21. Congenital heart disease is commonly linked to DS patients. The functions of this gene product are unknown. To identify the functions of CHD5/WRB in heart formation during embryogenesis, the medaka CHD5 cDNA (mCHD5) was isolated and its gene expression pattern and the localization of its gene product were investigated. The obtained mCHD5 belongs to the CHD5 superfamily, whose members include coiled‐coil proteins. The mCHD5 gene was found to be expressed in the developing heart after stage 28 at which the chamber (ventricle and atrium) differentiation in the heart tube is initiated in the embryo. Its gene product was also detected in the developing heart at embryonic stage 28 and 35. Knocking‐down of mCHD5 function caused severe cardiac disorder, including abnormal chamber differentiation, abnormal looping and ocular abnormality such as Cyclops. Our results provide the mCHD5 gene expression pattern as well as its physiological role during heart formation in a vertebrate model system.     

Chromosomal aberrations in tire plant workers and interaction with polymorphisms of biotransformation and DNA repair genes

We evaluated chromosomal aberrations in lymphocytes of 177 workers exposed to xenobiotics in a tire plant and in 172 controls, in relation to their genetic background. Nine polymorphisms in genes encoding biotransformation enzymes and nine polymorphisms in genes involved in main DNA repair pathways were investigated for possible modulation of chromosomal damage. Chromosomal aberration frequencies were the highest among exposed smokers and the lowest in non-smoking unexposed individuals (2.5 ± 1.8% vs. 1.7 ± 1.2%, respectively). The differences between groups (ANOVA) were borderline significant (F = 2.6, P = 0.055). Chromosomal aberrations were higher in subjects with GSTT1-null (2.4 ± 1.7%) than in those with GSTT1-plus genotype (1.8 ± 1.4%; F = 7.2, P = 0.008). Considering individual groups, this association was significant in smoking exposed workers (F = 4.4, P = 0.040). Individuals with low activity EPHX1 genotype exhibited significantly higher chromosomal aberrations (2.3 ± 1.6%) in comparison with those bearing medium (1.7 ± 1.2%) and high activity genotype (1.5 ± 1.2%; F = 4.7, P = 0.010). Both chromatid- and chromosome-type aberration frequencies were mainly affected by exposure and smoking status. Binary logistic regression analysis revealed that frequencies of chromatid-type aberrations were modulated by NBS1 Glu185Gln (OR 4.26, 95%CI 1.38–13.14, P = 0.012), and to a moderate extent, by XPD Lys751Gln (OR 0.16, 95%CI 0.02–1.25, P = 0.081) polymorphisms. Chromosome-type aberrations were lowest in individuals bearing the EPHX1 genotype conferring the high activity (OR 0.38, 95%CI 0.15–0.98, P = 0.045). Present results show that exposed individuals in the tire production, who smoke, exhibit higher chromosomal aberrations frequencies, and the extent of chromosomal damage may additionally be modified by relevant polymorphisms.     

Promoter methylation and functional variants in arachidonate 5-lipoxygenase and forkhead box protein O1 genes associated with coronary artery disease

Coronary artery disease (CAD) is a multifactorial chronic inflammatory disease, which is the most common form of heart disease. This is one of the main causes of death in the United States. Inflammation is one of the key drivers of atherosclerotic plaque development. Forkhead box protein O1 (FOXO1s) family and 5-lipoxygenase make an important contribution to atherosclerosis. The aim of this study was to investigate the methylation pattern and polymorphism analysis of FOXO1 and arachidonate 5-lipoxygenase (ALOX5) promoter genes. We studied 50 patients with CAD and 50 age- and sex-matched healthy controls by high resolution melt technique. Overall, we found significant differences between patients and controls in terms of the promoter methylation of ALOX5 (P > 0.05). But there was no significant difference in FOXO1 promoter methylation between patient and controls. Single nucleotide polymorphisms genotyping of rs12762303 and rs2297627, in ALOX5 and FOXO1 genes were demonstrated a significant correlation between mutant allele and the risk of CAD, respectively. Furthermore, there were significant associations between CT + CC genotype and ALOX5 expression. Our findings demonstrated functional effects of single nucleotide polymorphisms (SNPs) and DNA methylation in ALOX5 on mentioned genes expression and they resulted in CAD progression.     

Genome‐wide screen of genes imprinted in sorghum endosperm,and the roles of allelic differential cytosine methylation

Imprinting is an epigenetic phenomenon referring to allele‐biased expression of certain genes depending on their parent of origin. Accumulated evidence suggests that, while imprinting is a conserved mechanism across kingdoms, the identities of the imprinted genes are largely species‐specific. Using deep RNA sequencing of endosperm 14 days after pollination in sorghum, 5683 genes (29.27% of the total 19 418 expressed genes) were found to harbor diagnostic single nucleotide polymorphisms between two parental lines. The analysis of parent‐of‐origin expression patterns in the endosperm of a pair of reciprocal F₁ hybrids between the two sorghum lines led to identification of 101 genes with ≥ fivefold allelic expression difference in both hybrids, including 85 maternal expressed genes (MEGs) and 16 paternal expressed genes (PEGs). Thirty of these genes were previously identified as imprinted in endosperm of maize (Zea mays), rice (Oryza sativa) or Arabidopsis, while the remaining 71 genes are sorghum‐specific imprinted genes relative to these three plant species. Allele‐biased expression of virtually all of the 14 tested imprinted genes (nine MEGs and five PEGs) was validated by pyrosequencing using independent sources of RNA from various developmental stages and dissected parts of endosperm. Forty‐six imprinted genes (30 MEGs and 16 PEGs) were assayed by quantitative RT–PCR, and the majority of them showed endosperm‐specific or preferential expression relative to embryo and other tissues. DNA methylation analysis of the 5’ upstream region and gene body for seven imprinted genes indicated that, while three of the four PEGs were associated with hypomethylation of maternal alleles, no MEG was associated with allele‐differential methylation.     

Conservation of imprinting of MKRN3 and NAP1L5 in rabbits

Maternally imprinted genes of makorin ring finger protein 3 (MKRN3) and nucleosome assembly protein 1‐like 5 (NAP1L5) have been identified in many species but have not yet been investigated in rabbits. In this study, a polymorphism‐based approach and bisulfite‐sequencing PCR (BSP) were used to determine the imprinting status of MKRN3 and NAP1L5 in rabbits. The single nucleotide polymorphism (SNP)‐based sequencing results demonstrated that MKRN3 and NAP1L5 were expressed preferentially from the paternal allele. Furthermore, the BSP results showed the gamete‐specific methylation patterns and hemimethylation in brain and full methylation in liver were observed in MKRN3 and NAP1L5 respectively. Thus, we provide the first evidence that MKRN3 and NAP1L5 are paternally expressed genes and that the CpG islands located in the promoter region may be the putative differentially methylated region of these two genes in rabbits.     

Different effects of PPARA,PPARG and ApoE SNPs on serum lipids in patients with coronary heart disease based on the presence of diabetes

Background

The aim of this study was to investigate the individual or combined effects of PPARA-L162V, PPARG-C161T and APOE polymorphisms on hyperlipidemia in coronary heart disease (CHD) patients.

Methods

Our study included 223 patients with CHD (103 with type 2 diabetes (T2DM), 120 without diabetes) and 101 controls. All genotypes were determined by PCR–RFLP technique.

Results

Genotypic and allelic distributions of PPARA-L162V polymorphism were similar between study and control groups (p > 0.05). The serum total-cholesterol (TC) and LDL-cholesterol (LDL-C) levels were higher in PPARA-V162 allele carriers in non-diabetic CHD patients (p = 0.007 and p = 0.038, respectively). The increasing effect of the PPARA-V162 allele on serum TC and LDL-C levels was weakened with the presence of PPARG-161T allele in the non-diabetic CHD patients. The ApoE4–PPARA-V162 allelic combination of the ApoE/PPARA genes was found to be more frequent in diabetic CHD patients independent of serum lipids (p = 0.035).

Conclusions

The PPARA V162 allele has an increasing effect on TC and LDL-C levels and this effect was reduced by carrying PPARG T161 allele in non-diabetic CHD patients. On the other hand, the V162 allele may be associated with an increased risk of CHD in diabetic CHD patients due to the presence of ApoE4 allele independent of serum lipids. We suggest that the PPARA L162V polymorphism may have diverse effects on serum lipids and CHD risk depends on the presence of T2DM.     

Mutations of the GATA4 and NKX2.5 genes in Chinese pediatric patients with non-familial congenital heart disease

A number of mutations in GATA4 and NKX2.5 have been identified to be causative for a subset of familial congenital heart defects (CHDs) and a small number of sporadic CHDs. In this study, we evaluated common GATA4 and NKX2.5 mutations in 135 Chinese pediatric patients with non-familial congenital heart defects. Two novel mutations in the coding region of GATA4 were identified, namely, 487C >T (Pro163Ser) in exon 1 in a child with tetralogy of Fallot and 1220C >A (Pro407Gln) in exon 6 in a pediatric patient with outlet membranous ventricular septal defect. We also found 848C >A (Pro283Gln) in exon 2 of the NKX2.5 gene in a pediatric patient with ventricular septal defect, patent ductus arteriosus and aortic isthmus stenosis. None of the mutations was detected in healthy control subjects (n = 114). This study suggests that GATA4 and NKX2.5 missense mutations may be associated with congenital heart defects in pediatric Chinese patients. Further clinical studies with large samples are warranted.     

DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is a highly lethal malignancy due to a lack of early detection approaches coupled with poor outcomes for patients with clinically advanced disease. Cancer-testis (CT) or cancer-germline genes encode antigens known to generate spontaneous anti-tumor immunity in cancer patients. CT45 genes are a recently discovered 6-member family of X-linked CT genes with oncogenic function. Here, we determined CT45 expression in EOC and fully defined its epigenetic regulation by DNA methylation. CT45 was silent and hypermethylated in normal control tissues, but a large subset of EOC samples showed increased CT45 expression in conjunction with promoter DNA hypomethylation. In contrast, copy number status did not correlate with CT45 expression in the TCGA database for EOC. CT45 promoter methylation inversely correlated with both CT45 mRNA and protein expression, the latter determined using IHC staining of an EOC TMA. CT45 expression was increased and CT45 promoter methylation was decreased in late-stage and high-grade EOC, and both measures were associated with poor survival. CT45 hypomethylation was directly associated with LINE-1 hypomethylation, and CT45 was frequently co-expressed with other CT antigen genes in EOC. Decitabine treatment induced CT45 mRNA and protein expression in EOC cells, and promoter transgene analyses indicated that DNA methylation directly represses CT45 promoter activity. These data verify CT45 expression and promoter hypomethylation as possible prognostic biomarkers, and suggest CT45 as an immunological or therapeutic target in EOC. Treatment with decitabine or other epigenetic modulators could provide a means for more effective immunological targeting of CT45.     

Expression and promoter methylation status of hMLH1, MGMT,APC, and CDH1 genes in patients with colon adenocarcinoma

Colorectal cancer (CRC) is the third most common cancer in men and the second in women worldwide. CRC development is the result of genetic and epigenetic alterations accumulation in the epithelial cells of colon mucosa. In the present study, DNA methylation, an epigenetic event, was evaluated in tumoral and matching normal epithelium in a cohort of 61 CRC patients. The results confirmed and expanded knowledge for the tumor suppressor genes hMLH1, MGMT, APC, and CDH1. Promoter methylation was observed for all the examined genes in different percentage. A total of 71% and 10% of the examined cases were found to be methylated in two or more and in all genes, respectively. mRNA and protein levels were also evaluated. Promoter methylation of hMLH1, MGMT, APC, and CDH1 genes was present at the early stages of tumor’s formation and it could also be detected in the normal mucosa. Correlations of the methylated genes with patient’s age and tumor’s clinicopathological characteristics were also observed. Our findings suggest that DNA methylation is a useful marker for tumor progression monitoring and that promoter methylation in certain genes is associated with more advanced tumor stage, poor differentiation, and metastasis.     

重金属镉胁迫对果蝇dDnmt2、dMBD2/3表达量的影响

【目的】探明重金属镉(Cadmium,Cd)对黑腹果蝇Drosophila melanogaster DNA甲基化修饰相关基因表达的影响,初步分析镉胁迫可能导致果蝇的表观遗传变异及其可遗传性。【方法】收集8 h内羽化未交配的雌、雄果蝇,在添加不同质量浓度(0、0.9375、1.875、3.75、7.5、15.0、30.0、60.0 mg/kg)Cd的培养基中培养,以Real-time PCR定量检测亲代(F0)果蝇生殖系统、去生殖系统体细胞、整体表达量变化趋势及解除胁迫的子代(F1)果蝇DNA甲基化修饰系统相关基因(dDnmt2、dMBD2/3)在mRNA水平的表达变化。【结果】重金属镉胁迫诱导了果蝇卵巢、精巢、去卵巢雌果蝇、去精巢雄果蝇、完整雌果蝇、完整雄果蝇的dDnmt2、dMBD2/3在mRNA水平的表达上调,呈现一定剂量依赖性及雌、雄组织差异性,且这种表达变化持续至子一代。【结论】研究结果揭示了重金属镉胁迫可诱导果蝇dDnmt2、dMBD2/3表达量上调,其可能与果蝇的DNA甲基化修饰过程相关联,导致表观遗传变异并可能向子代传递。     

Association of maternal and nutrient supply line factors with DNA methylation at the imprinted IGF2/H19 locus in multiple tissues of newborn twins

Epigenetic events are crucial for early development, but can be influenced by environmental factors, potentially programming the genome for later adverse health outcomes. The insulin-like growth factor 2 (IGF2)/H19 locus is crucial for prenatal growth and the epigenetic state at this locus is environmentally labile. Recent studies have implicated maternal factors, including folate intake and smoking, in the regulation of DNA methylation at this locus, although data are often conflicting in the direction and magnitude of effect. Most studies have focused on single tissues and on one or two differentially-methylated regions (DMRs) regulating IGF2/H19 expression. In this study, we investigated the relationship between multiple shared and non-shared gestational/maternal factors and DNA methylation at four IGF2/H19 DMRs in five newborn cell types from 67 pairs of monozygotic and 49 pairs of dizygotic twins. Data on maternal and non-shared supply line factors were collected during the second and third trimesters of pregnancy and DNA methylation was measured via mass spectrometry using Sequenom MassArray EpiTyper analysis. Our exploratory approach showed that the site of umbilical cord insertion into the placenta in monochorionic twins has the strongest positive association with methylation in all IGF2/H19 DMRs (p < 0.05). Further, evidence for tissue- and locus-specific effects were observed, emphasizing that responsiveness to environmental exposures in utero cannot be generalized across genes and tissues, potentially accounting for the lack of consistency in previous findings. Such complexity in responsiveness to environmental exposures in utero has implications for all epigenetic studies investigating the developmental origins of health and disease.     

The anti-infection resistance system and the pathogenesis of bacterial endocarditis in patients with congenital heart defects

The clinico-immuno-microbiological study of 24 patients with congenital heart diseases was made. Clinically, the patients were subdivided into two groups: 8 patients with diagnosed bacterial endocarditis (BE) prior to the operation and 16 patients without BE. The analysis of the results of investigation carried out prior to the operation showed that age, the form of the disease and its duration did not affect the level of anti-infectious resistance factors (AIRF): in both groups a decrease in the level of AIRF characteristics (cell-mediated and humoral) was noted and no characteristic differences were determined. The results of the study of microflora from the nasal and laryngeal mucosa showed no difference in both groups of the patients. At the same time the detailed analysis of the results made it possible to suggest that BE caused by Streptococcus epidermidis and Streptococcus aureus inhabiting the mucous membrane of the anterior sections of the nasal cavity, alpha-hemolytic streptococci and S. epidermidis inhibiting the laryngeal mucosa. The results of the analysis made 3-6 months after the operation were also indicative of the absence of essential differences between AIRF characteristics observed in patients with confirmed and clinically unconfirmed BE. The clinical manifestation of this fact was an increase in the percentage of diagnosed BE cases up to 70% as compared with that before the operation (33%). This clinico-immuno-microbiological study made it possible to come to the conclusion that any form of congenital heart disease develops in combination with BE and/or is its prodrome.     

Key genes and functional coexpression modules involved in the pathogenesis of systemic lupus erythematosus

We performed a systematic review of genome‐wide gene expression datasets to identify key genes and functional modules involved in the pathogenesis of systemic lupus erythematosus (SLE) at a systems level. Genome‐wide gene expression datasets involving SLE patients were searched in Gene Expression Omnibus and ArrayExpress databases. Robust rank aggregation (RRA) analysis was used to integrate those public datasets and identify key genes associated with SLE. The weighted gene coexpression network analysis (WGCNA) was adapted to identify functional modules involved in SLE pathogenesis, and the gene ontology enrichment analysis was utilized to explore their functions. The aberrant expressions of several randomly selected key genes were further validated in SLE patients through quantitative real‐time polymerase chain reaction. Fifteen genome‐wide gene expression datasets were finally included, which involved a total of 1,778 SLE patients and 408 healthy controls. A large number of significantly upregulated or downregulated genes were identified through RRA analysis, and some of those genes were novel SLE gene signatures and their molecular roles in etiology of SLE remained vague. WGCNA further successfully identified six main functional modules involved in the pathogenesis of SLE. The most important functional module involved in SLE included 182 genes and mainly enriched in biological processes, including defense response to virus, interferon signaling pathway, and cytokine‐mediated signaling pathway. This study identifies a number of key genes and functional coexpression modules involved in SLE, which provides deepening insights into the molecular mechanism of SLE at a systems level and also provides some promising therapeutic targets.     

Degree of methylation of ZAC1 (PLAGL1) is associated with prenatal and post-natal growth in healthy infants of the EDEN mother child cohort

The ZAC1 gene, mapped to the 6q24 region, is part of a network of co-regulated imprinted genes involved in the control of embryonic growth. Loss of methylation at the ZAC1 differentially methylated region (DMR) is associated with transient neonatal diabetes mellitus, a developmental disorder involving growth retardation and diabetes in the first weeks of post-natal life. We assessed whether the degree of methylation of the ZAC1 DMR in leukocytes DNA extracted from cord blood is associated with fetal, birth and post-natal anthropometric measures or with C-peptide concentrations in cord serum. We also searched for an influence of dietary intake and maternal parameters on ZAC1 DMR methylation. We found positive correlations between the ZAC1 DMR methylation index (MI) and estimated fetal weight (EFW) at 32 weeks of gestation, weight at birth and weight at one year of age (respectively, r = 0.15, 0.09, 0.14; P values = 0.01, 0.15, 0.03). However, there were no significant correlations between the ZAC1 DMR MI and cord blood C-peptide levels. Maternal intakes of alcohol and of vitamins B2 were positively correlated with ZAC1 DMR methylation (respectively, r = 0.2 and 0.14; P = 0.004 and 0.04). The influence of ZAC1 seems to start in the second half of pregnancy and continue at least until the first year of life. The maternal environment also appears to contribute to the regulation of DNA methylation.     

Constitutive and light-induced DNAseI hypersensitive sites in the rbcS genes of pea (Pisum sativum)

The chromatin structure of pea (Pisum sativum) rbcS genes in inactive (root), potentially active (dark-grown leaf), and active states (light-grown leaf) was analysed using (a) pancreatic DNAseI to detect general DNAseI sensitivity and DNAseI-hypersensitive sites, and (b) methyl-sensitive restriction endonucleases to probe for cytosine methylation within the promoter region. We showed that within the same organ individual members of the pea rbcS multigene family are differentially sensitive to DNAseI suggesting differential protection in nuclei. During light activation general DNAseI sensitivity increases in some genes, especially their 5 upstream regulatory sequences. DNAseI-hypersensitive sites are constitutively present in 5 upstream regulatory sequences around positions –335, –465, –650, and –945 (5 constitutive domain) and in the coding region around position +340, +450, +530, +640, and +810 (3 constitutive domain). One additional hypersensitive site appears after light induction (inducible site). This region is centred around position –190 and flanked by light-responsive elements (LREs). In spite of changes in the chromatin structure of rbcS genes during their transition from an inactive to an active state, their cytosine methylation at Alu I, Fnu 4HI, Hae III, Sau 3AI and Sau 96I sites in the promoter region remains uniform.     

Epidemiology of congenital heart disease in Louisiana: an association between race and sex and the prevalence of specific cardiac malformations.

We hypothesized that susceptibility to the genetic and environmental factors that disrupt cardiac development is associated with race and sex. To evaluate this hypothesis, we asked whether the prevalence of specific cardiac malformations differs by race and sex. We attempted to include all infants born alive in the State of Louisiana from January 1, 1988, through December 31, 1989, and diagnosed by echocardiography, catheterization and/or autopsy within a year of birth as having one of ten specific cardiac malformations. The prevalence of atrioventricular canal defects (AVCD) per 1,000 live births was significantly higher for black females (.744) compared to black males (.198) and for white females (.414) compared to white males (.116). Complete transposition of the great arteries (TGA) was significantly higher for white males (.559) compared to white females (.122); in contrast, TGA was not significantly different for black males (.198) and black females (.169). Obstructive left heart syndrome (OLHS)--aortic stenosis and/or coarctation of the aorta--was significantly higher for white males (.652) compared to white females (.317); in contrast, OLHS was not significantly different for black males (.264) and black females (.169). Single ventricle (SV) was significantly higher for whites (.202) compared to blacks (.067). We did not find that race and sex were associated with differences in the prevalence of tetralogy of Fallot and hypoplastic left heart syndrome. The numbers of infants with anomalous pulmonary venous return, tricuspid atresia, double outlet right ventricle, or truncus arteriosus were too small to measure an association with race and sex. These results demonstrate that the prevalence of a subset of cardiac malformations differs by race and sex.(ABSTRACT TRUNCATED AT 250 WORDS)