Data on cardiac lncRNA STX18-AS1 expression in developing human hearts and function during in vitro hESC-cardiomyocyte differentiation.

This article presents data concerning STX18-AS1, a long noncoding RNA gene identified from a Genome-wide association study of Atrial Septal Defect (ASD). The data describes its expression patterns in human tissues and functions in regulating cardiomyocyte differentiation in vitro. STX18-AS1 is a lncRNA with a higher abundance in developing tissues, including hearts. Its transcription distribution within the embryonic hearts during key heart septation stages supports STX18-AS1's association with risk SNPs for ASD. The CRISPR stem cell pool in which STX18-AS1 was knocked down, showed reduced CM differentiation efficiency and lower expression of key cardiac transcriptional factors. This indicated its regulative role in supporting the lineage specification from cardiac mesoderm into cardiac progenitors and cardiomyocytes. These data can benefit the understanding of human embryonic heart developmental biology, and the time-course changes of cardiac transcriptional factors during in vitro cardiomyocyte differentiation from human embryonic stem cells.

A novel RNA-mediated mechanism causing down-regulation of insulating promoter interactions in human embryonic stem cells.

The genome-wide promoter interactome is primarily maintained and regulated by architectural proteins such as CTCF and cohesin. However, some studies suggest a role for non-coding RNAs (ncRNAs) in this process. We aimed to characterise the regulatory role of RNA-mediated promoter interactions in the control of gene expression. We integrated genome-wide datasets of RNA-chromatin and promoter-genome interactions in human embryonic stem cells (hESCs) to identify putative RNA-mediated promoter interactions. We discovered that CTCF sites were enriched in RNA-PIRs (promoter interacting regions co-localising with RNA-chromatin interaction sites) and genes interacting with RNA-PIRs containing CTCF sites showed higher expression levels. One of the long noncoding RNAs (lncRNAs) expressed in hESCs, Syntaxin 18-Antisense 1 (STX18-AS1), appeared to be involved in an insulating promoter interaction with the neighbouring gene, MSX1. By knocking down STX18-AS1, the MSX1 promoter-PIR interaction was intensified and the target gene (MSX1) expression was down-regulated. Conversely, reduced MSX1 promoter-PIR interactions, resulting from CRISPR-Cas9 deletion of the PIR, increased the expression of MSX1. We conclude that STX18-AS1 RNA antagonised local CTCF-mediated insulating promoter interactions to augment gene expression. Such down-regulation of the insulating promoter interactions by this novel mechanism may explain the higher expression of genes interacting with RNA-PIRs linked to CTCF sites.

Global prevalence of congenital heart disease in school-age children: a meta-analysis and systematic review.

BACKGROUND: Congenital heart disease (CHD) is the commonest birth defect. Studies estimating the prevalence of CHD in school-age children could therefore contribute to quantifying unmet health needs for diagnosis and treatment, particularly in lower-income countries. Data at school age are considerably sparser, and individual studies have generally been of small size. We conducted a literature-based meta-analysis to investigate global trends over a 40-year period. METHODS AND RESULTS: Studies reporting on CHD prevalence in school-age children (4-18 years old) from 1970 to 2017 were identified from PubMed, EMBASE, Web of Science and Google Scholar. According to the inclusion criteria, 42 studies including 2,638,475 children, reporting the prevalence of unrepaired CHDs (both pre-school diagnoses and first-time school-age diagnoses), and nine studies including 395,571 children, specifically reporting the prevalence of CHD first diagnosed at school ages, were included. Data were combined using random-effects models. The prevalence of unrepaired CHD in school children during the entire period of study was 3.809 (95% confidence intervals 3.075-4.621)/1000. A lower proportion of male than female school children had unrepaired CHD (OR = 0.84 [95% CI 0.74-0.95]; p = 0.001). Between 1970-1974 and 1995-1999, there was no significant change in the prevalence of unrepaired CHD at school age; subsequently there was an approximately 2.5-fold increase from 1.985 (95% CI 1.074-3.173)/1000 in 1995-1999 to 4.832 (95% CI 3.425-6.480)/1000 in 2010-2014, (p = 0.009). Among all CHD conditions, atrial septal defects and ventricular septal defects chiefly accounted for this increasing trend. The summarised prevalence (1970-2017) of CHD diagnoses first made in childhood was 1.384 (0.955, 1.891)/1000; during this time there was a fall from 2.050 [1.362, 2.877]/1000 pre-1995 to 0.848 [0.626, 1.104]/1000 in 1995-2014 (p = 0.04). CONCLUSIONS: Globally, these data show an increased prevalence of CHD (mainly mild CHD conditions) recognised at birth/infancy or early childhood, but remaining unrepaired at school-age. In parallel there has been a decrease of first-time CHD diagnoses in school-age children. These together imply a favourable shift of CHD recognition time to earlier in the life course. Despite this, substantial inequalities between higher and lower income countries remain. Increased healthcare resources for people born with CHD, particularly in poorer countries, are required.

Global birth prevalence of congenital heart defects 1970-2017: updated systematic review and meta-analysis of 260 studies.

BACKGROUND: Globally, access to healthcare and diagnostic technologies are known to substantially impact the reported birth prevalence of congenital heart disease (CHD). Previous studies have shown marked heterogeneity between different regions, with a suggestion that CHD prevalence is rising globally, but the degree to which this reflects differences due to environmental or genetic risk factors, as opposed to improved detection, is uncertain. We performed an updated systematic review to address these issues. METHODS: Studies reporting the birth prevalence of CHD between the years 1970-2017 were identified from searches of PubMed, EMBASE, Web of Science and Google Scholar. Data on the prevalence of total CHD and 27 anatomical subtypes of CHD were collected. Data were combined using random-effect models. Subgroup and meta-regression analyses were conducted, focused on geographical regions and levels of national income. RESULTS: Two hundred and sixty studies met the inclusion criteria, encompassing 130 758 851 live births. The birth prevalence of CHD from 1970-2017 progressively increased to a maximum in the period 2010-17 of 9.410/1000 [95% CI (confidence interval) 8.602-10.253]. This represented a significant increase over the fifteen prior years (P = 0.031). The change in prevalence of mild CHD lesions (ventricular septal defect, atrial septal defect and patent ductus arteriosus) together explained 93.4% of the increased overall prevalence, consistent with a major role of improved postnatal detection of less severe lesions. In contrast the prevalence of lesions grouped together as left ventricular outflow tract obstruction (which includes hypoplastic left heart syndrome) decreased from 0.689/1000 (95% CI 0.607-0.776) in 1995-99, to 0.475/1000 (95% CI 0.392-0.565; P = 0.004) in 2010-17, which would be consistent with improved prenatal detection and consequent termination of pregnancy when these very severe lesions are discovered. There was marked heterogeneity among geographical regions, with Africa reporting the lowest prevalence [2.315/1000 (95% CI 0.429-5.696)] and Asia the highest [9.342/1000 (95% CI 8.072-10.704)]. CONCLUSIONS: The reported prevalence of CHD globally continues to increase, with evidence of severe unmet diagnostic need in Africa. The recent prevalence of CHD in Asia for the first time appears higher than in Europe and America, where disease ascertainment is likely to be near-complete, suggesting higher genetic or environmental susceptibility to CHD among Asian people.

Author Correction: Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks.

In the original version of the Article, the gene symbol for tissue factor pathway inhibitor was inadvertently given as 'TFP1' instead of 'TFPI'. This has now been corrected in both the PDF and HTML versions of the Article.

Promoter interactome of human embryonic stem cell-derived cardiomyocytes connects GWAS regions to cardiac gene networks.

Long-range chromosomal interactions bring distal regulatory elements and promoters together to regulate gene expression in biological processes. By performing promoter capture Hi-C (PCHi-C) on human embryonic stem cell-derived cardiomyocytes (hESC-CMs), we show that such promoter interactions are a key mechanism by which enhancers contact their target genes after hESC-CM differentiation from hESCs. We also show that the promoter interactome of hESC-CMs is associated with expression quantitative trait loci (eQTLs) in cardiac left ventricular tissue; captures the dynamic process of genome reorganisation after hESC-CM differentiation; overlaps genome-wide association study (GWAS) regions associated with heart rate; and identifies new candidate genes in such regions. These findings indicate that regulatory elements in hESC-CMs identified by our approach control gene expression involved in ventricular conduction and rhythm of the heart. The study of promoter interactions in other hESC-derived cell types may be of utility in functional investigation of GWAS-associated regions.

Genetic variants associated with risk of atrial fibrillation regulate expression of PITX2, CAV1, MYOZ1, C9orf3 and FANCC.

Genome-wide association studies (GWAS) have identified genetic variants in a number of chromosomal regions that are associated with atrial fibrillation (AF). The mechanisms underlying these associations are unknown, but are likely to involve effects of the risk haplotypes on expression of neighbouring genes. To investigate the association between genetic variants at AF-associated loci and expression of nearby candidate genes in human atrial tissue and peripheral blood. Right atrial appendage (RAA) samples were collected from 122 patients undergoing cardiac surgery, of these, 12 patients also had left atrial appendage samples taken. 22 patients had a history of AF. Peripheral blood samples were collected from 405 patients undergoing diagnostic cardiac catheterisation. In order to tag genetic variation at each of nine loci, a total of 367 single nucleotide polymorphisms (SNPs) were genotyped using the Sequenom platform. Total expression of 16 candidate genes in the nine AF-associated regions was measured by quantitative PCR. The relative expression of each allele of the candidate genes was measured on the Sequenom platform using one or more transcribed SNPs to distinguish between alleles in heterozygotes. We tested association between the SNPs of interest and gene expression using total gene expression (integrating cis and trans acting sources of variation), and allelic expression ratios (specific for cis acting influences), in atrial tissue and peripheral blood. We adjusted for multiple comparisons using a Bonferroni approach. In subsidiary analyses, we compared the expression of candidate genes between patients with and without a history of AF. Total expression of 15 transcripts of 14 genes and allelic expression ratio of 14 transcripts of 14 genes in genomic regions associated with AF were measured in right atrial appendage tissue. 8 of these transcripts were also expressed in peripheral blood. Risk alleles at AF-associated SNPs were associated in cis with an increased expression of PITX2a (2.01-fold, p=6.5×10(-4)); and with decreased expression of MYOZ1 (0.39 fold; p=5.5×10(-15)), CAV1 (0.89 fold; p=5.9×10(-8)), C9orf3 (0.91 fold; 1.5×10(-5)), and FANCC (0.94-fold; p=8.9×10(-8)) in right atrial appendage. Of these five genes, only CAV1 was expressed in peripheral blood; association between the same AF risk alleles and lower expression of CAV1 was confirmed (0.91 fold decrease; p=4.2×10(-5)). A history of AF was also associated with a decrease in expression of CAV1 in both right and left atria (0.84 and 0.85 fold, respectively; p=0.03), congruent with the magnitude of the effect of the risk SNP on expression, and independent of genotype. The analyses in peripheral blood showed association between AF risk SNPs and decreased expression of KCNN3 (0.85-fold; p=2.1×10(-4)); and increased expression of SYNE2 (1.12-fold; p=7.5×10(-24)); however, these associations were not detectable in atrial tissue. We identified novel cis-acting associations in atrial tissue between AF risk SNPs and increased expression of PITX2a/b; and decreased expression of CAV1 (an association also seen in peripheral blood), C9orf3 and FANCC. We also confirmed a previously described association between AF risk variants and MYOZ1 expression. Analyses of peripheral blood illustrated tissue-specificity of cardiac eQTLs and highlight the need for larger-scale genome-wide eQTL studies in cardiac tissue. Our results suggest novel aetiological roles for genes in four AF-associated genomic regions.

The landscape of DNA repeat elements in human heart failure.

BACKGROUND: The epigenomes of healthy and diseased human hearts were recently examined by genome-wide DNA methylation profiling. Repetitive elements, heavily methylated in post-natal tissue, have variable methylation profiles in cancer but methylation of repetitive elements in the heart has never been examined. RESULTS: We analyzed repetitive elements from all repeat families in human myocardial samples, and found that satellite repeat elements were significantly hypomethylated in end-stage cardiomyopathic hearts relative to healthy normal controls. Satellite repeat elements are almost always centromeric or juxtacentromeric, and their overexpression correlates with disease aggressiveness in cancer. Similarly, we found that hypomethylation of satellite repeat elements correlated with up to 27-fold upregulation of the corresponding transcripts in end-stage cardiomyopathic hearts. No other repeat family exhibited differential methylation between healthy and cardiomyopathic hearts, with the exception of the Alu element SINE1/7SL, for which a modestly consistent trend of increased methylation was observed. CONCLUSIONS: Satellite repeat element transcripts, a form of non-coding RNA, have putative functions in maintaining genomic stability and chromosomal integrity. Further studies will be needed to establish the functional significance of these non-coding RNAs in the context of heart failure.

Distinct epigenomic features in end-stage failing human hearts.

BACKGROUND: The epigenome refers to marks on the genome, including DNA methylation and histone modifications, that regulate the expression of underlying genes. A consistent profile of gene expression changes in end-stage cardiomyopathy led us to hypothesize that distinct global patterns of the epigenome may also exist. METHODS AND RESULTS: We constructed genome-wide maps of DNA methylation and histone-3 lysine-36 trimethylation (H3K36me3) enrichment for cardiomyopathic and normal human hearts. More than 506 Mb sequences per library were generated by high-throughput sequencing, allowing us to assign methylation scores to ≈28 million CG dinucleotides in the human genome. DNA methylation was significantly different in promoter CpG islands, intragenic CpG islands, gene bodies, and H3K36me3-enriched regions of the genome. DNA methylation differences were present in promoters of upregulated genes but not downregulated genes. H3K36me3 enrichment itself was also significantly different in coding regions of the genome. Specifically, abundance of RNA transcripts encoded by the DUX4 locus correlated to differential DNA methylation and H3K36me3 enrichment. In vitro, Dux gene expression was responsive to a specific inhibitor of DNA methyltransferase, and Dux siRNA knockdown led to reduced cell viability. CONCLUSIONS: Distinct epigenomic patterns exist in important DNA elements of the cardiac genome in human end-stage cardiomyopathy. The epigenome may control the expression of local or distal genes with critical functions in myocardial stress response. If epigenomic patterns track with disease progression, assays for the epigenome may be useful for assessing prognosis in heart failure. Further studies are needed to determine whether and how the epigenome contributes to the development of cardiomyopathy.

Genome-wide conserved consensus transcription factor binding motifs are hyper-methylated.

BACKGROUND: DNA methylation can regulate gene expression by modulating the interaction between DNA and proteins or protein complexes. Conserved consensus motifs exist across the human genome ("predicted transcription factor binding sites": "predicted TFBS") but the large majority of these are proven by chromatin immunoprecipitation and high throughput sequencing (ChIP-seq) not to be biological transcription factor binding sites ("empirical TFBS"). We hypothesize that DNA methylation at conserved consensus motifs prevents promiscuous or disorderly transcription factor binding. RESULTS: Using genome-wide methylation maps of the human heart and sperm, we found that all conserved consensus motifs as well as the subset of those that reside outside CpG islands have an aggregate profile of hyper-methylation. In contrast, empirical TFBS with conserved consensus motifs have a profile of hypo-methylation. 40% of empirical TFBS with conserved consensus motifs resided in CpG islands whereas only 7% of all conserved consensus motifs were in CpG islands. Finally we further identified a minority subset of TF whose profiles are either hypo-methylated or neutral at their respective conserved consensus motifs implicating that these TF may be responsible for establishing or maintaining an un-methylated DNA state, or whose binding is not regulated by DNA methylation. CONCLUSIONS: Our analysis supports the hypothesis that at least for a subset of TF, empirical binding to conserved consensus motifs genome-wide may be controlled by DNA methylation.

High-throughput sequencing identifies STAT3 as the DNA-associated factor for p53-NF-kappaB-complex-dependent gene expression in human heart failure.

BACKGROUND: Genome-wide maps of DNA regulatory elements and their interaction with transcription factors may form a framework for understanding regulatory circuits and gene expression control in human disease, but how these networks, comprising transcription factors and DNA-binding proteins, form complexes, interact with DNA and modulate gene expression remains largely unknown. METHODS: Using microRNA-21 (mir-21), which is an example of genes that are regulated in heart failure, we performed chromatin immunoprecipitation (ChIP) assays to determine the occupancy of transcription factors at this genetic locus. Tissue ChIP was further performed using human hearts and genome-wide occupancies of these transcription factors were analyzed by high-throughput sequencing. RESULTS: We show that the transcription factor p53 piggy-backs onto NF-kappaB/RELA and utilizes the kappaB-motif at a cis-regulatory region to control mir-21 expression. p53 behaves as a co-factor in this complex because despite a mutation in its DNA binding domain, mutant p53 was still capable of binding RELA and the cis-element, and inducing mir-21 expression. In dilated human hearts where mir-21 upregulation was previously demonstrated, the p53-RELA complex was also associated with this cis-element. Using high-throughput sequencing, we analyzed genome-wide binding sites for the p53-RELA complex in diseased and control human hearts and found a significant overrepresentation of the STAT3 motif. We further determined that STAT3 was necessary for the p53-RELA complex to associate with this cis-element and for mir-21 expression. CONCLUSIONS: Our results uncover a mechanism by which transcription factors cooperate in a multi-molecular complex at a cis-regulatory element to control gene expression.

MICA polymorphism: biology and importance in immunity and disease.

The human major histocompatibility complex class I chain-related gene A (MICA) is one of the genes in the HLA class I region of chromosome 6. Unlike HLA classical class I gene products, MICA does not present any antigen but acts as a ligand for several immune cells including natural killer (NK) cells bearing NKG2D receptors. MICA is the member of the non-classical class I family that displays the greatest degree of polymorphism. MICA alleles can be divided into two large groups with the polymorphisms found in alpha3 domains. This division could be explained by a possible polyphyletic origin that is in line with recent findings from evolutionary, population and functional studies of this gene. MICA polymorphisms are associated with a number of diseases related to NK activity, such as viral infection, cancer and allograft rejection or graft-versus-host disease (GVHD). The mechanisms underlying these associations include NK cell-mediated cytotoxicity and MICA shedding to produce immunosuppressive soluble MICA particles. The MICA-induced humoral response has attracted interest recently because of its possible role in graft rejection in solid organ transplantation. Here, we discuss the genetics and biology of the MICA gene and its products, and their importance in disease.

Differential DNA methylation correlates with differential expression of angiogenic factors in human heart failure.

Epigenetic mechanisms such as microRNA and histone modification are crucially responsible for dysregulated gene expression in heart failure. In contrast, the role of DNA methylation, another well-characterized epigenetic mark, is unknown. In order to examine whether human cardiomyopathy of different etiologies are connected by a unifying pattern of DNA methylation pattern, we undertook profiling with ischaemic and idiopathic end-stage cardiomyopathic left ventricular (LV) explants from patients who had undergone cardiac transplantation compared to normal control. We performed a preliminary analysis using methylated-DNA immunoprecipitation-chip (MeDIP-chip), validated differential methylation loci by bisulfite-(BS) PCR and high throughput sequencing, and identified 3 angiogenesis-related genetic loci that were differentially methylated. Using quantitative RT-PCR, we found that the expression of these genes differed significantly between CM hearts and normal control (p<0.01). Moreover, for each individual LV tissue, differential methylation showed a predicted correlation to differential expression of the corresponding gene. Thus, differential DNA methylation exists in human cardiomyopathy. In this series of heterogeneous cardiomyopathic LV explants, differential DNA methylation was found in at least 3 angiogenesis-related genes. While in other systems, changes in DNA methylation at specific genomic loci usually precede changes in the expression of corresponding genes, our current findings in cardiomyopathy merit further investigation to determine whether DNA methylation changes play a causative role in the progression of heart failure.

PKB/Akt activation inhibits p53-mediated HIF1A degradation that is independent of MDM2.

Cross-talk between the two transcription factors, p53 and hypoxia inducible factor 1alpha (HIF1A), is important in different pathophysiological conditions (Hammond and Giaccia, 2006, Clin Cancer Res 12:5007-5009) such as in the transition from myocardial hypertrophy to cardiac dilatation and heart failure. In that context, p53 induces HIF1A degradation which in turn provokes the transition from compensatory hypertrophy to myocardial thinning and chamber dilatation (Sano et al., 2007, Nature 446:444-448). In order to investigate the mechanism of p53-induced HIF1A degradation, we used the established in vitro model of deferroxamine (DFX)-induced HIF1A accumulation in H9c2 cardiac cells (Sano et al., 2007, Nature 446:444-448). Here, we report that opposite to HIF1A accumulation following exposure to DFX, prolonged DFX-induced p53 activation and HIF1A protein decrease, without any change in Hif1a mRNA. HIF1A protein decrease accompanied upregulated HIF1A ubiquitination. MDM2, an ubiquitin E3 ligase target gene of p53, was upregulated following prolonged DFX, but using p53/Mdm2 double-null mouse embryonic fibroblasts, we found that p53 upregulated HIF1A ubiquitination and degradation independently of MDM2. Moreover, with prolonged DFX treatment, an enhanced interaction between MDM2 and HIF1A was lacking. Instead, phospho-Akt(ser473) was decreased during the phase coinciding with HIF1A degradation, and inhibition of PKB/Akt phosphorylation using PI3K inhibitor (LY294002) upregulated HIF1A ubiquitination. In summary, we propose that p53-induced HIF1A degradation is not exclusively MDM2-mediated, but reversible by PKB/Akt phosphorylation.

An Arabidopsis mutant able to green after extended dark periods shows decreased transcripts of seed protein genes and altered sensitivity to abscisic acid.

An Arabidopsis mutant showing an altered ability to green on illumination after extended periods of darkness has been isolated in a screen for genomes uncoupled (gun) mutants. Following illumination for 24 h, 10-day-old dark-grown mutant seedlings accumulated five times more chlorophyll than wild-type seedlings and this was correlated with differences in plastid morphology observed by transmission electron microscopy. The mutant has been named greening after extended darkness 1 (ged1). Microarray analysis showed much lower amounts of transcripts of genes encoding seed storage proteins, oleosins, and late embryogenesis abundant (LEA) proteins in 7-day-old seedlings of ged1 compared with the wild type. RNA gel-blot analyses confirmed very low levels of transcripts of seed protein genes in ged1 seedlings grown for 2-10 d in the dark, and showed higher amounts of transcripts of photosynthesis-related genes in illuminated 10-day-old dark-grown ged1 seedlings compared with the wild type. Consensus elements similar to abscisic acid (ABA) response elements (ABREs) were detected in the upstream regions of all genes highly affected in ged1. Germination of ged1 seeds was hypersensitive to ABA, although no differences in ABA content were detected in 7-day-old seedlings. This suggests the mutant may have an altered responsiveness to ABA, affecting expression of ABA-responsive genes and plastid development during extended darkness.

Possible polyphyletic origin of major histocompatibility complex class I chain-related gene A (MICA) alleles.

Phylogenetic relationships among 23 nonhuman primate (NHP) major histocompatibility complex class I chain-related gene (MIC) sequences, 54 confirmed human MICA alleles, and 16 human MICE alleles were constructed with methods of sequence analysis. Topology of the phylogenetic tree showed separation between NHP MICs and human MICs. For human MICs, the topology indicated monophyly for the MICB alleles, while MICA alleles were separated into two lineages, LI and LII. Of these, LI MICA alleles shared a common ancestry with gorilla (Ggo) MIC. One conservative amino acid difference and two nonconservative amino acid differences in the alpha3 domain were found between the MICA lineages. The nonconservative amino acid differences might imply structural and functional differences. Transmembrane (TM) trinucleotide-repeat variants were found to be specific to the MICA lineages such as A4, A9, and A10 to LI and A5 to LII. Variants such as A5.1 and A6 were commonly found in both MICA lineages. Based on these analyses, we postulate a polyphyletic origin for MICA alleles and their division into two lineages, LI and LII. As such, there would be 30 alleles in LI and 24 alleles in LII, thereby reducing the current level of polymorphism that exists, based on a presumed monophyletic origin. The lower degree of polymorphism in MICA would then be in line with the rest of the human major histocompatibility complex nonclassical class I genes.