Pathogenesis Of Congenital Heart Disease Research Articles

Model system identification of novel congenital heart disease gene candidates: focus on RPL13.

Genetics is a significant factor contributing to congenital heart disease (CHD), but our understanding of the genetic players and networks involved in CHD pathogenesis is limited. Here, we searched for de novo copy number variations (CNVs) in a cohort of 167 CHD patients to identify DNA segments containing potential pathogenic genes. Our search focused on new candidate disease genes within 19 deleted de novo CNVs, which did not cover known CHD genes. For this study, we developed an integrated high-throughput phenotypical platform to probe for defects in cardiogenesis and cardiac output in human induced pluripotent stem cell (iPSC)-derived multipotent cardiac progenitor (MCPs) cells and, in parallel, in the Drosophila in vivo heart model. Notably, knockdown (KD) in MCPs of RPL13, a ribosomal gene and SON, an RNA splicing cofactor, reduced proliferation and differentiation of cardiomyocytes, while increasing fibroblasts. In the fly, heart-specific RpL13 KD, predominantly at embryonic stages, resulted in a striking 'no heart' phenotype. KD of Son and Pdss2, among others, caused structural and functional defects, including reduced or abolished contractility, respectively. In summary, using a combination of human genetics and cardiac model systems, we identified new genes as candidates for causing human CHD, with particular emphasis on ribosomal genes, such as RPL13. This powerful, novel approach of combining cardiac phenotyping in human MCPs and in the in vivo Drosophila heart at high throughput will allow for testing large numbers of CHD candidates, based on patient genomic data, and for building upon existing genetic networks involved in heart development and disease.

Epigenetics and Mechanobiology in Heart Development and Congenital Heart Disease.

Congenital heart disease (CHD) is the most common birth defect worldwide and the number one killer of live-born infants in the United States. Heart development occurs early in embryogenesis and involves complex interactions between multiple cell populations, limiting the understanding and consequent treatment of CHD. Furthermore, genome sequencing has largely failed to predict or yield therapeutics for CHD. In addition to the underlying genome, epigenetics and mechanobiology both drive heart development. A growing body of evidence implicates the aberrant regulation of these two extra-genomic systems in the pathogenesis of CHD. In this review, we describe the stages of human heart development and the heart defects known to manifest at each stage. Next, we discuss the distinct and overlapping roles of epigenetics and mechanobiology in normal development and in the pathogenesis of CHD. Finally, we highlight recent advances in the identification of novel epigenetic biomarkers and environmental risk factors that may be useful for improved diagnosis and further elucidation of CHD etiology.

Functional analysis of the congenital heart disease‑associated GATA4 H436Y mutation invitro.

Congenital heart disease (CHD) is the most common type of developmental defect, with high rates of morbidity in infants. The transcription factor GATA-binding factor 4 (GATA4) has been reported to serve a critical role in embryogenesis and cardiac development. Our previous study reported a heterozygous GATA4 c.1306C>T (p.H436Y) mutation in four Chinese infants with congenital heart defects. In the present study, functional analysis of the GATA4 H436Y mutation was performed in vitro. The functional effect of GATA4 mutation was compared with GATA4 wild-type using a dual-luciferase reporter assay system and immunofluorescence. Electrophoretic mobility-shift assays were performed to explore the binding affinity of the mutated GATA4 to the heart and neural crest derivatives expressed 2 (HAND2) gene. The results revealed that the mutation had no effect on normal nuclear localization, but resulted in diminished GATA-binding affinity to HAND2 and significantly decreased gene transcriptional activation. These results indicated that this GATA4 mutation may not influence cellular localization in transfected cells, but may affect the affinity of the GATA-binding site on HAND2 and decrease transcriptional activity, thus suggesting that the GATA4 mutation may be associated with the pathogenesis of CHD.

Research progress in genetic mechanism for congenital heart disease

Congenital heart disease (CHD) is the most common birth defect and is also the leading cause of death in infants and young children.Studying the pathogenesis of CHD and preventing the development of CHD are major scientific problems that need to be solved urgently.Now, it is known that the occurrence of CHD is caused by multiple factors such as genetic factors and environmental factors.In this paper, the genetic mechanisms in the progress of CHD were summarized. Key words: Congenital heart disease; Genetics; Chromosome; Gene

Single cell expression analysis reveals anatomical and cell cycle-dependent transcriptional shifts during heart development.

The heart is a complex organ composed of multiple cell and tissue types. Cardiac cells from different regions of the growing embryonic heart exhibit distinct patterns of gene expression, which are thought to contribute to heart development and morphogenesis. Single cell RNA sequencing allows genome-wide analysis of gene expression at the single cell level. Here, we have analyzed cardiac cells derived from early stage developing hearts by single cell RNA-seq and identified cell cycle gene expression as a major determinant of transcriptional variation. Within cell cycle stage-matched CMs from a given heart chamber, we found that CMs in the G2/M phase downregulated sarcomeric and cytoskeletal markers. We also identified cell location-specific signaling molecules that may influence the proliferation of other nearby cell types. Our data highlight how variations in cell cycle activity selectively promote cardiac chamber growth during development, reveal profound chamber-specific cell cycle-linked transcriptional shifts, and open the way to deeper understanding of pathogenesis of congenital heart disease.

Copy number variants detection by microarray and multiplex ligation-dependent probe amplification in congenital heart diseases

Congenital heart diseases (CHDs) are the most common birth defects among life births, which could be presented as isolated or syndromic with other congenital malformations. The etiology of CHD largely unknown, genetic and environmental factors contribute to the disease. Recurrent copy number variants (CNVs) have been reported in the pathogenesis of CHD. The aim of this study was to evaluate the clinical utility of multiplex ligation-dependent probe amplification (MLPA) and microarray analyses on isolated and syndromic CHD cases and to explore the relationship between identified CNVs and CHD. Eighteen prenatal samples, 16 isolated and 33 syndromic patients with mild to severe CHD phenotype were tested. Prenatal and isolated CHD cases did not show pathogenic CNVs. Clinically significant CNVs were detected in 7/33 (21%) syndromic CHD patients: del 22q11.2 (n = 2), 8p23.1 duplication (n = 2), deletion 5p (n = 1), deletion 6q21q22 (n = 1), unbalanced translocation causing partial deletion of 4q34.3 and duplication of 6q25.1 (n = 1). These genomic imbalances contain genes that has been associated with human CHD before. The present study demonstrates that using microarray and MLPA analysis increase the detection rate of causal CNVs in individuals with syndromic CHD.

Association of NKX2-5, GATA4, and TBX5 polymorphisms with congenital heart disease in Egyptian children.

BackgroundSeveral genes encoding transcription factors are known to be the primary cause of congenital heart disease. NKX2‐5 and GATA4 were the first congenital heart disease–causing genes identified by linkage analysis. This study designed to study the association of five single–nucleotide variants of NKX2‐5, GATA4, and TBX5 genes with sporadic nonsyndromic cases of a congenital cardiac septal defect in Egyptian children.MethodsVenous blood samples from 150 congenital heart disease children (including a ventricular septal defect, atrial septal defect, tetralogy of Fallot, and patent ductus arteriosus) and 90 apparently healthy of matched age and sex were studied by polymerase chain reaction followed by direct sequencing in order to study two single–nucleotide variants of NKX2‐5 (rs2277923, rs28936670), two single–nucleotide variants of GATA4 (rs368418329, rs56166237) and one single–nucleotide variant TBX5 (rs6489957). The distribution of genotype and allele frequency in the congenital heart diseases (CHD) group and control group were analyzed.ResultsWe found different genotype frequencies of the two variants of NKX2‐5, as CT genotype of rs2277923 was present in 58% and 36% in cases and control respectively, and TT genotype present in 6% of the cases. Also regarding missense variant rs28936670, heterozygous AG presented in 82% of the cases. Also, we observed a five prime UTR variant rs368418329, GT (42% of the cases) and GG (46% of the cases) genotypes showed the most frequent presentation in cases. While regarding a synonymous variant rs56166237, GT and GG were the most presented in cases (41.4%, 56% respectively) in contrast to control group (20%, 1.7% respectively). Also, a synonymous variant in TBX5, the distribution of genotype frequency was significantly different between the CHD group and control group. CT genotype of TBX5 ‐rs6489957 was found in 12 ASD, 24 VSD, six PDA, three aortic coarctation and nine fallot that represent 42% of the cases.ConclusionsSignificantly higher frequency of different allelle of five variants was observed in cases when compared to the control group, with significant risky effect for the development of septal defect. In addition to two polymorphisms of NKX2‐5 (rs2277923, rs28936670) variant in the cardiac septal defect, two variants in GATA4 (rs368418329, rs56166237) and one variant in TBX5 (rs6489957) seem to have a role in the pathogenesis of congenital heart disease.

Maternal zinc metabolism and changes of metallothionein-1 and zinc transporter-1 in fetal congenital heart diseases

Objective To investigate maternal zinc metabolism and the changes of zinc-related factors as metallothionein-1(MT1) and zinc transporter-1(ZnT1) in certain types of congenital heart diseases (CHD). Methods Fifteen infants with interventricular septal defect, 12 infants with atrial septal defect and 7 infants with tetralogy of Fallot, together with their mothers were enrolled, and normal infants and their mothers were enrolled by a ratio of 1∶1 with the above three types of CHD diseases. General conditions of the mothers, along with their diets and zinc-containing drug supplementation during the pregnancy, were surveyed. Maternal blood zinc levels and serum alkaline phosphatase activities at gestation week 32 and delivery or induced abortion, and the protein and mRNA expressions of MT1 and ZnT1 in maternal serum and placental tissue at delivery or induced abortion were assayed. Results The general conditions were comparable between the CHD group and control group. The ratio of the mothers taking more zinc-rich food was significantly lower in the CHD group than in the control group. Circulating zinc levels in interventricular septal defect (73.55±5.79 μmol/L), atrial septal defect (72.66±5.82 μmol/L) and tetralogy of Fallot (68.72±6.72 μmol/L) groups were significantly lower than those in the control groups (82.77±7.88, 84.58±7.55 and 85.66±7.30 μmol/L) at delivery (P all <0.05). Similar change patterns were seen for serum alkaline phosphatase activities. The relative quantities of serum MT1 and ZnT1 proteins in interventricular septal defect (73.22±36.54 and 68.55±27.82), atrial septal defect (64.29±38.26 and 74.55±29.67) and tetralogy of Fallot (67.88±30.50 and 70.13±29.65) groups were significantly lower than those in their corresponding control groups (166.31±67.43 and 97.67±30.22, 182.56±71.40 and 111.65±32.70, and 173.81±62.36 and 108.27±28.52, P<0.01 or P<0.05). The relative quantities of placental MT1 and ZnT1 proteins and mRNA expressions in interventricular septal defect (protein quantities 0.438±0.096 and 0.384±0.061, mRNA expressions 1.23±0.82 and 0.96±0.39), atrial septal defect (0.427±0.093 and 0.377±0.059, 1.17±0.70 and 0.85±0.40) and tetralogy of Fallot (0.414±0.111 and 0.336±0.066, 1.31±0.97 and 0.90±0.38) groups were significantly lower than those in their corresponding control groups (protein quantities 0.565±0.083 and 0.541±0.090, mRNA expressions 2.78±1.06 and 1.67±0.33; protein quantities 0.622±0.136 and 0.493±0.079, mRNA expressions 2.85±0.89 and 1.72±0.38; protein quantities 0.637±0.125 and 0.521±0.089, mRNA expressions 3.21±0.99 and 1.61±0.29; P<0.01 or P<0.05). Conclusion Mothers with their fetus of certain types of CHD are found zinc deficiency, and down-regulation of MT1 and ZnT1 expressions in the serum and placenta may involve in the pathogenesis of CHD when maternal zinc deficiency. Key words: Zinc deficiency; Congenital heart disease; Pregnancy; Metallothionein-1; Zinc transporter-1

Akt3 is a target of miR-29c-3p and serves an important function in the pathogenesis of congenital heart disease.

Our previous studies identified that the expression of microRNA‑29c (miR‑29c‑3p) was significantly increased in the serum of pregnant women carrying fetuses with congenital heart disease (CHD) compared with in that of normal pregnant women. However, the mechanism by which miR‑29c‑3p affects development of the embryonic heart remained unclear. The aim of the present study was to investigate the effect and potential molecular mechanism of miR‑29c‑3p overexpression on P19 cell proliferation, apoptosis and differentiation. miR‑29c‑3p‑overexpression and protein kinase Bγ (Akt3)‑knockdown cell lines were constructed using transfection technology. The function of miR‑29c‑3p and Akt3 in cardiomyocyte development was investigated by determining the proliferation, apoptosis and differentiation of P19 cells, which can differentiate into cardiomyocytes induced by dimethylsulfoxide. Bioinformatic analysis and luciferase assays were performed to explore the association between Akt3 and miR‑29c‑3p. The results of the present study revealed that miR‑29c‑3p overexpression and Akt3 knockdown suppressed proliferation, and promoted apoptosis and differentiation in P19 cells. Akt3 was also demonstrated to be a target of miR‑29c‑3p. Therefore, overexpression of miR‑29c‑3p may inhibit proliferation, and promote apoptosis and differentiation in P19 cells by inhibiting the expression of Akt3. miR‑29c‑3p may be a potential therapeutic target for the treatment of CHD.

Genetic Basis for Congenital Heart Disease: Revisited: A Scientific Statement From the American Heart Association.

This review provides an updated summary of the state of our knowledge of the genetic contributions to the pathogenesis of congenital heart disease. Since 2007, when the initial American Heart Association scientific statement on the genetic basis of congenital heart disease was published, new genomic techniques have become widely available that have dramatically changed our understanding of the causes of congenital heart disease and, clinically, have allowed more accurate definition of the pathogeneses of congenital heart disease in patients of all ages and even prenatally. Information is presented on new molecular testing techniques and their application to congenital heart disease, both isolated and associated with other congenital anomalies or syndromes. Recent advances in the understanding of copy number variants, syndromes, RASopathies, and heterotaxy/ciliopathies are provided. Insights into new research with congenital heart disease models, including genetically manipulated animals such as mice, chicks, and zebrafish, as well as human induced pluripotent stem cell-based approaches are provided to allow an understanding of how future research breakthroughs for congenital heart disease are likely to happen. It is anticipated that this review will provide a large range of health care-related personnel, including pediatric cardiologists, pediatricians, adult cardiologists, thoracic surgeons, obstetricians, geneticists, genetic counselors, and other related clinicians, timely information on the genetic aspects of congenital heart disease. The objective is to provide a comprehensive basis for interdisciplinary care for those with congenital heart disease.

Abstract 16756: Coxsackievirus Infection During Early Pregnancy Leads to Congenital Heart Defects

Introduction: Coxsackievirus B (CVB), the most common cause of myocarditis, targets cardiomyocytes through the Coxsackie and Adenovirus Receptor which is highly expressed in the fetal heart. Association between maternal CVB infection and fetal cardiac malformations has not been studied. Hypothesis: CVB infection during early pregnancy may play a role in the pathogenesis of congenital heart disease (CHD). Methods: Pregnant C57Bl/6J mice were infected between embryonic days (E) 5-11 with CVB3 (Nancy strain) at doses 1x10 6 , 2.5x10 6 , and 5x10 6 TCID50. Presence of the virus was confirmed by RT-PCR and infectivity assay and quantified by qPCR. Fetal heart phenotype was assessed by histology and to determine changes at the molecular level after infection, RNA was collected from fetal hearts using laser microdissection and analyzed by RNA-Seq. The results were confirmed by immunohistochemistry and qPCR. To assess the potential clinical relevance of our observations in mice, we analyzed the sera from pregnant women for presence of antibody titers against of CVB. Results: Offspring of pregnant mice infected with CVB3 at different time points developed a spectrum of CHD (38.2%, n=78/204): ventricular septal defect, double outlet right ventricle, and non-compaction of the ventricular myocardium, along with fetal death. Infection at E9 led to the highest incidence of VSD compared to infection earlier (E5, E7 or E8) or later (E11) in gestation. Transcripts found to be differentially expressed between infected fetal hearts with VSDs compared with controls were mediators of the Transforming growth factor β and Extracellular matrix signaling pathways. Consistent with the former, expression levels of BMP2 and Smad1/5/9 mRNA were elevated. Simultaneously suppressed proliferation of cardiomyocytes in these fetuses was observed. Serum analysis from 66 pregnant women who had babies with heart defects showed elevated titers of antibodies against CVB underlining the clinical significance of CVB infection during pregnancy. Conclusions: In mice, there is a critical window during pregnancy where CVB infection leads to abnormal cardiac development, likely through reduction of cardiomyocyte proliferation mediated by elevated levels of BMP2 and Smad1/5/9. Collectively, the data confirm a link between CVB infection and CHD development in mouse and human.

Abstract 16596: Dominant Mouse Mutagenesis Screen Unveils a Complex Genetic Architecture for Congenital Heart Disease

Introduction: Human studies show congenital heart disease (CHD) has a complex non-Mendelian genetic etiology. We previously conducted a recessive N-ethyl-N-nitrosourea (ENU) mouse mutagenesis screen using fetal ultrasound and recovered over 150 recessive mutations causing CHD. In the present study, we conducted a sensitized screen with driver mutations to recover dominant mutations causing CHD. We hypothesize this dominant screen can help to elucidate the complex genetic architecture of CHD. Methods: G1 males with heterozygous ENU induced mutations were mated to females harboring heterozygous “driver” mutation in Kif7, Anks6, or Sap130 that can cause CHD when homozygous. The resulting G2 offspring were screened for CHD by fetal ultrasound. Mutations in G1 males were identified by whole exome sequencing and the G2 offspring were genotyped using Ion Torrent sequencing. Linkage analysis was conducted to identify the CHD causing mutation. Results: We found 9.3% of the G1 pedigrees (n=551) yielded CHD mutants, similar to the 8.2% observed in the previous recessive screen. However, only 2.5 mutants were recovered per line vs. 4.5 in the recessive screen despite having screened similar number of embryos, supporting non-Mendelian genetics in the dominant screen. Genotype-phenotype analysis revealed only 32%, 45%, and 55% of the CHD mutants recovered carried the Kif7, Anks6, or Sap130 driver mutations, respectively, suggesting the driver mutations are not required for CHD pathogenesis. For mouse line 4142 with DORV/OA, we identified a heterozygous p21/Rac-activated kinase 2 ( Pak2) mutation as disease causing (p=4.8e-12). CHD penetrance was 83% among Pak2 +/m embryos. Among surviving 4142 G2 offspring, only 14% have the Pak2 mutation vs. 50% expected. We observed 75% of the surviving Pak2 mutant mice also carry a mutation in Zbtb2, a gene identified to regulate p21, suggesting this may act as a protective modifier. Conclusion: We showed the feasibility of using mouse mutagenesis to recover dominant mutations causing CHD, and showed for the first time a role for Pak2 in CHD. These findings demonstrate that the genetic diversity created by ENU mutagenesis can provide the genomic context to investigate the complex genetics and genetic architecture of CHD.

Search of Somatic Mutations of NKX2-5 and GATA4 Genes in Chinese Patients with Sporadic Congenital Heart Disease.

Congenital heart disease (CHD) usually occurs sporadically, with only a minority of cases associated with a known genetic mechanism. Cardiac-specific transcription factors NKX2-5 and GATA4 play key roles in the mammalian heart development, and the affected cardiac tissues of CHD patients are prone to somatic mutations which thus participate in the pathogenesis of CHD. We collected 98 patients with sporadic CHD, extracted genomic DNA from cardiac tissues and blood, and then screened NKX2-5 and GATA4 genes using PCR-direct sequence analysis. A novel heterozygous missense mutation (c.907G>A, p.V303I) of NKX2-5 gene was identified in a patient with tetralogy of Fallots. Functional assay revealed that this mutant was associated with significantly reduced transcriptional activity. In addition, we found two known single-nucleotide polymorphisms (SNPs) (rs2277923, rs3729753) in NKX2-5 and two known SNPs (rs56166237, rs3729856) in GATA4. All variations identified in cardiac tissues were consistent with those of peripheral blood, and no somatic mutations were found in cardiac tissues. Our study shows no evidence of NKX2-5 and GATA4 somatic mutations playing a role in the pathogenesis of sporadic CHD.

Abstract 565: Coxsackievirus Infection During the First Trimester of Pregnancy Suppresses Cardiomyocyte Proliferation Leading to Congenital Heart Defects

Purpose: Coxsackievirus B (CVB), known to be the most common cause of myocarditis, target cardiomyocytes through Coxsackie and Adenovirus Receptor, which is highly expressed in the fetal heart. We hypothesized that CVB infection during early pregnancy may play a role in the pathogenesis of congenital heart disease (CHD). Methods: Pregnant C57Bl/6J mice were infected with varying doses of CVB3 (Nancy strain), 1x10 6 , 2.5x10 6 , and 5x10 6 TCID50, between embryonic days (E) 5-11. Presence of the virus was confirmed by RT-PCR and infectivity assay. Fetal heart phenotype was assessed by histology. RNA collected from fetal hearts using laser microdissection was analyzed by RNA-Seq to understand changes at the molecular level after infection. Results were confirmed by immunohistochemistry and qPCR. To determine if virus serotype alters the outcome, CVB1, CVB4, and a combination of CVB3 and CVB4 were used for infection. To assess the clinical significance serum from pregnant human subjects, grouped based on outcome of pregnancy (healthy baby vs babies with CHD), was checked for the presence of CVB serotypes. Results: Fetuses developed spectrum of CHD (38.2%, n=78/204): ventricular septal defect, double outlet right ventricle, and non-compaction of ventricular myocardium after CVB3 infection. Approximately one third of the fetuses were resorbed (38.7%, n=129/333). Infection at E9 led to the highest incidence of VSD with female fetuses being more susceptible to the phenotype. Transcripts found to be differentially expressed in hearts with VSDs were mediators of the Transforming growth factor β and Extracellular matrix signaling pathways. Consistent with the former, expression levels of BMP2 and Smad1/5/9 mRNA were elevated in fetal hearts with VSDs. This correlated with suppressed proliferation of cardiomyocytes in these mice. In pregnant human subjects who had babies with CHD elevated titers of antibodies against CVB3, CVB4, and CVB3+CVB4 were seen. In mice, infection with CVB1, CVB4, or CVB3+4 showed that CVB3 in combination with CVB4 induces more VSDs than infection with any one serotype. Conclusion: There is a critical window during pregnancy where CVB infection leads to abnormal cardiac development likely through reduction of cardiomyocyte proliferation.

Alteration in the expression of the renin-angiotensin system in the myocardium of mice conceived by in vitro fertilization.

Epidemiological studies have revealed that offspring conceived by in vitro fertilization (IVF) have an elevated risk of cardiovascular malformations at birth, and are more predisposed to cardiovascular diseases. The renin-angiotensin system (RAS) plays an essential role in both the pathogenesis of congenital heart disease in fetuses and cardiovascular dysfunction in adults. This study aimed to assess the relative expression levels of genes in the RAS pathway in mice conceived using IVF, compared to natural mating with superovulation. Results demonstrated that expression of the angiotensin II receptor type 1 (AGTR1), connective tissue growth factor (CTGF), and collagen 3 (COL3), in the myocardial tissue of IVF-conceived mice, was elevated at 3 weeks, 10 weeks, and 1.5 years of age, when compared to their non-IVF counterparts. These data were supported by microRNA microarray analysis of the myocardial tissue of aged IVF-conceived mice, where miR-100, miR-297, and miR-758, which interact with COL3, AGTR1, and COL1 respectively, were upregulated when compared to naturally mated mice of the same age. Interestingly, bisulfite sequencing data indicated that IVF-conceived mice exhibited decreased methylation of CpG sites in Col1. In support of our in vivo investigations, miR-297 overexpression was shown to upregulate AGTR1 and CTGF, and increased cell proliferation in cultured H9c2 cardiomyocytes. These findings indicate that the altered expression of RAS in myocardial tissue might contribute to cardiovascular malformation and/or dysfunction in IVF-conceived offspring. Furthermore, these cardiovascular abnormalities might be the result of altered DNA methylation and abnormal regulation of microRNAs.

Rs4759314 polymorphism located in HOTAIR is associated with the risk of congenital heart disease by alternating downstream signaling via reducing its expression.

Our study was aimed to investigate the mechanism of rs4759314 located in HOTAIR involved in congenital heart disease. Luciferase assay was performed to evaluate whether rs4759314 affected the transcription efficiency of HOTAIR promoter, and confirm miR-545 target gene. Real-time PCR, Western-blot and IHC were carried out to investigate the relationship among HOTAIR, EGFR, p-ERK, P-P38 MAPK. MTT assay and flow cytometry analysis were performed to detect the effect of HOTAIR on cell viability and apoptosis. The presence of G allele of the polymorphism located in HOTAIR promoted transcription activity of HOTAIR promoter. Furthermore, HOTAIR inhibited miR-545 expression. EGFR was identified as a virtual target gene of miR-545 using bioinformatics analysis, and miR-545 apparently decreased luciferase activity of wild-type EGFR 3'UTR, while miR-545 had no effect on luciferase activity of mutant EGFR 3'UTR. HOTAIR and EGFR were lowly expressed, while miR-545 was highly expressed in VSD group. Higher levels of HOTAIR and ECFR, while a lower level of miR-545 were observed in AG than AA groups. Regulatory relationships between HOTAIR and miR-545, as well as EGFR and miR-545 were found to be negatively correlated, with the negative correlation coefficient being -0.49 and -0.46, respectively. HOTAIR evidently increased EGFR p-ERK and P-P38 MAPK expression levels, moreover HOTAIR substantially promoted cell viability, and inhibited cell apoptosis. In this study, we suggested the possible relation between the rs4759314 polymorphism and the risk of congenital heart disease, and explored the deregulation of HOTAIR/miR-545/EGFR/MAPK signaling pathway in the pathogenesis of congenital heart disease.

A Screening Approach to Identify Clinically Actionable Variants Causing Congenital Heart Disease in Exome Data

Congenital heart disease (CHD)-structural abnormalities of the heart that arise during embryonic development-is the most common inborn malformation, affecting ≤1% of the population. However, currently, only a minority of cases can be explained by genetic abnormalities. The goal of this study was to identify disease-causal genetic variants in 30 families affected by CHD. Whole-exome sequencing was performed with the DNA of multiple family members. We utilized a 2-tiered whole-exome variant screening and interpretation procedure. First, we manually curated a high-confidence list of 90 genes known to cause CHD in humans, identified predicted damaging variants in genes on this list, and rated their pathogenicity using American College of Medical Genetics and Genomics-Association for Molecular Pathology guidelines. In 3 families (10%), we found pathogenic variants in known CHD genes TBX5, TFAP2B, and PTPN11, explaining the cardiac lesions. Second, exomes were comprehensively analyzed to identify additional predicted damaging variants that segregate with disease in CHD candidate genes. In 10 additional families (33%), likely disease-causal variants were uncovered in PBX1, CNOT1, ZFP36L2, TEK, USP34, UPF2, KDM5A, KMT2C, TIE1, TEAD2, and FLT4. The pathogenesis of CHD could be explained using our high-confidence CHD gene list for variant filtering in a subset of cases. Furthermore, our unbiased screening procedure of family exomes implicates additional genes and variants in the pathogenesis of CHD, which suggest themselves for functional validation. This 2-tiered approach provides a means of (1) identifying clinically actionable variants and (2) identifying additional disease-causal genes, both of which are essential for improving the molecular diagnosis of CHD.

MicroRNA-34a modulates the Notch signaling pathway in mice with congenital heart disease and its role in heart development

The objective of the study was to elucidate the mechanism by which microRNA-34a (miR-34a) influences heart development and participates in the pathogenesis of congenital heart disease (CHD) by targeting NOTCH-1 through the Notch signaling pathway. Forty D7 pregnant mice were recruited for the purposes of the study and served as the CHD (n=20, successfully established as CHD model) and normal (n=20) groups. The positive expression of the NOTCH-1 protein was evaluated by means of immunohistochemistry. Embryonic endocardial cells (ECCs) were assigned into the normal, blank, negative control (NC), miR-34a mimics, miR-34a inhibitors, miR-34a inhibitors+siRNA-NOTCH-1, siRNA-NOTCH-1, miR-34a mimics+NOTCH-1 OE and miR-34a mimics+crispr/cas9 (mutant NOTCH-1) groups. The expressions of miR-34a, NOTCH-1, Jagged1, Hes1, Hey2 and Csx in cardiac tissues and ECCs were determined by both RT-qPCR and western blotting methods. MTT assay and flow cytometry were conducted for cell proliferation and apoptosis measurement. A dual luciferase reporter assay was applied to demonstrate that NOTCH-1 was the target gene of miR-34a. In comparison to the normal group, the expressions of miR-34a, Jagged1, Hes1 and Hey2 displayed up-regulated levels, while the expressions of NOTCH-1 and Csx were down-regulated in the CHD group. Compared with the blank and NC groups, the miR-34a mimics and siRNA-NOTCH-1 groups displayed reduced expressions of NOTCH-1 and Csx as well as a decreased proliferation rate, higher miR-34a, Jagged1, Hes1 and Hey2 expressions and an increased rate of apoptosis; while an reverse trend was observed in the miR-34a inhibitors group. The expressions of MiR-34a recorded increased levels in the miR-34a mimics+NOTCH-1 OE and miR-34a mimics+crispr/cas9 (mutant NOTCH-1) groups, however no changes in the expressions of NOTCH-1, Jagged1, Hes1, Hey2, Csx, as well as cell proliferation and apoptosis were observed when compared to the blank and NC groups. The results of our study demonstrated that miR-34a increases the risk of CHD through its downregulation of NOTCH-1 by modulating the Notch signaling pathway.

Identification of Novel Congenital Heart Disease Candidate Genes Using Chromosome Microarray.

While the majority of patients have isolated heart disease, congenital heart disease (CHD) may be associated with other congenital anomalies or syndromes. Our institution utilizes chromosomal microarray (CMA) to identify chromosomal abnormalities, specifically copy number variations (CNVs). While CNVs have been associated with CHD, their direct impact on cardiac development remains unclear. This study sought to identify potential novel CHD candidate genes by comparing CNVs present in our institution's CHD population with those already recognized in the literature. A list of candidate genes was compiled from recent medical literature that utilized CMA. Records from neonatal cases at our institution over 10years were reviewed. Genes identified from CMAs were compared with those reported in the literature and cross-referenced with the Online Mendelian Inheritance in Man catalog. We identified 375 CNVs reported in patients with CHD. At our institution between 2005 and 2015, 307 neonates with CHD had CMA. Of these, 77 patients (25%) had CNVs containing 832 unique candidate genes. 49 patients (16%) had isolated CHD with 353 candidate genes expressed within the CNVs, many of which were previously reported. However, there were 16 unique candidate genes identified that have been expressed with heart structure of the mouse knock-out models. Our findings demonstrate a high incidence of abnormal genes identified by CMA in CHD patients, including many CNVs of "unknown clinical significance". We conclude that a portion of these CNVs (including 16 genes expressed in the heart of the mouse knock-out models) could be candidate genes involved in CHD pathogenesis.

Hypomethylation and decreased expression of BRG1 in the myocardium of patients with congenital heart disease.

BRG1, an ATPase subunit of the SWItch/Sucrose Non-Fermentable complex, is tightly associated with cardiac development. However, little is known about the association between the pathogenesis of CHD and BRG1. The methylation of a BRG1 promoter and a novel CpG island in the second intron was analyzed in the myocardium of congenital heart disease (CHD) patients (n = 24) and normal controls (n = 11) using pyrosequencing and the MassARRAY platform. BRG1 expression was sketched in the normal fetal and postnatal heart using real-time PCR. BRG1 mRNA and protein expression was detected by means of real-time PCR and immunohistochemistry. The expression of GATA4 was analyzed with real-time PCR. The CpG shore in the second intron of BRG1 was hypomethylated in the myocardium of patients (p < 0.05). BRG1 showed a high level of expression in the normal fetal heart in the second trimester (p < 0.01). Compared with that of the normal subjects, BRG1 expression was decreased by 70% in the myocardium of patients (n = 92; p < 0.05). Of note, the expression of GATA4 was significantly correlated with BRG1 expression (r = 0.7475; p = 0.0082) in the myocardium, and it was also decreased by 70% in these patients (n = 92; p < 0.05). These results suggested that the early high expression of BRG1 in fetal hearts maintained normal cardiac development and that the abnormal hypomethylation and decreased expression of BRG1 in human hearts probably affect the expression of GATA4, which affects the pathogenesis of CHD. Birth Defects Research 109:1183-1195, 2017. © 2017 Wiley Periodicals, Inc.