Associations of Impulsivity and Risky Decision-Making with E-Cigarette-Related Outcomes Among Adolescents with Congenital Heart Disease: Variable- and Person-Oriented Approaches.

Adolescents with congenital heart disease (CHD) have elevated risk for acquired cardiovascular complications, increasing their vulnerability to e-cigarette-related health harms. Impulsivity and risky decision-making have been associated with adolescent substance use, but the relationships between these factors and e-cigarette-related outcomes among cardiovascular at-risk adolescents with CHD are unknown. This cross-sectional study aimed to (a) determine the associations of impulsivity and risky decision-making with e-cigarette-related outcomes (i.e. susceptibility, ever use, perceptions of harm and addictiveness) via variable-oriented analysis (logistic regression), (b) identify groups of adolescents with similar profiles of impulsivity and risky decision-making via exploratory person-oriented analysis (latent profile analysis; LPA), and (c) examine differences on e-cigarette-related outcomes between profile groups. Adolescents aged 12 to 18 years with CHD (N = 98) completed a survey assessing impulsivity facets (Short UPPS-P) and e-cigarette-related outcomes and were administered a risky decision-making task (Iowa Gambling Task, Version 2; IGT2). In variable-oriented analyses, impulsivity facets (negative urgency, positive urgency, lack of premeditation) but not risky decision-making were associated with e-cigarette susceptibility and ever use. The exploratory LPA identified two groups with similar patterns of responding on the Short UPPS-P and IGT2 labeled "Low Impulsivity" and "High Impulsivity," which were primarily characterized by significant differences in negative and positive urgency. Adolescents in the High Impulsivity group had increased odds of e-cigarette susceptibility but not ever use compared to the Low Impulsivity group. This work indicates that strategies to prevent e-cigarette use among adolescents with CHD may be enhanced by addressing impulsivity, particularly negative and positive urgency.

Endocardial HDAC3 is required for myocardial trabeculation.

Failure of proper ventricular trabeculation is often associated with congenital heart disease. Support from endocardial cells, including the secretion of extracellular matrix and growth factors is critical for trabeculation. However, it is poorly understood how the secretion of extracellular matrix and growth factors is initiated and regulated by endocardial cells. We find that genetic knockout of histone deacetylase 3 in the endocardium in mice results in early embryo lethality and ventricular hypotrabeculation. Single cell RNA sequencing identifies significant downregulation of extracellular matrix components in histone deacetylase 3 knockout endocardial cells. Secretome from cultured histone deacetylase 3 knockout mouse cardiac endothelial cells lacks transforming growth factor ß3 and shows significantly reduced capacity in stimulating cultured cardiomyocyte proliferation, which is remarkably rescued by transforming growth factor ß3 supplementation. Mechanistically, we identify that histone deacetylase 3 knockout induces transforming growth factor ß3 expression through repressing microRNA-129-5p. Our findings provide insights into the pathogenesis of congenital heart disease and conceptual strategies to promote myocardial regeneration.

Novel pathogenic GATA6 variant associated with congenital heart disease, diabetes mellitus and necrotizing enterocolitis.

BACKGROUND: Pathogenic GATA6 variants have been associated with congenital heart disease (CHD) and a spectrum of extracardiac abnormalities, including pancreatic agenesis, congenital diaphragmatic hernia, and developmental delay. However, the comprehensive genotype-phenotype correlation of pathogenic GATA6 variation in humans remains to be fully understood. METHODS: Exome sequencing was performed in a family where four members had CHD. In vitro functional analysis of the GATA6 variant was performed using immunofluorescence, western blot, and dual-luciferase reporter assay. RESULTS: A novel, heterozygous missense variant in GATA6 (c.1403 G > A; p.Cys468Tyr) segregated with affected members in a family with CHD, including three with persistent truncus arteriosus. In addition, one member had childhood onset diabetes mellitus (DM), and another had necrotizing enterocolitis (NEC) with intestinal perforation. The p.Cys468Tyr variant was located in the c-terminal zinc finger domain encoded by exon 4. The mutant protein demonstrated an abnormal nuclear localization pattern with protein aggregation and decreased transcriptional activity. CONCLUSIONS: We report a novel, familial GATA6 likely pathogenic variant associated with CHD, DM, and NEC with intestinal perforation. These findings expand the phenotypic spectrum of pathologic GATA6 variation to include intestinal abnormalities. IMPACT: Exome sequencing identified a novel heterozygous GATA6 variant (p.Cys468Tyr) that segregated in a family with CHD including persistent truncus arteriosus, atrial septal defects and bicuspid aortic valve. Additionally, affected members displayed extracardiac findings including childhood-onset diabetes mellitus, and uniquely, necrotizing enterocolitis with intestinal perforation in the first four days of life. In vitro functional assays demonstrated that GATA6 p.Cys468Tyr variant leads to cellular localization defects and decreased transactivation activity. This work supports the importance of GATA6 as a causative gene for CHD and expands the phenotypic spectrum of pathogenic GATA6 variation, highlighting neonatal intestinal perforation as a novel extracardiac phenotype.

Endocardial HDAC3 is required for myocardial trabeculation.

BACKGROUND: Trabeculation, a key process in early heart development, is the formation of myocardial trabecular meshwork. The failure of trabeculation often leads to embryonic lethality. Support from endocardial cells, including the secretion of extracellular matrix (ECM) and growth factors is critical for trabeculation; however, it is unknown how the secretion of ECM and growth factors is initiated and regulated by endocardial cells. METHODS: Various cellular and mouse models in conjunction with biochemical and molecular tools were employed to study the role of histone deacetylase 3 (HDAC3) in the developing endocardium. RESULTS: We found that genetic deletion of Hdac3 in endocardial cells in mice resulted in early embryo lethality presenting as a hypotrabeculation cardiac phenotype. Single cell RNA sequencing identified several ECM components including collagens that were significantly downregulated in Hdac3 knockout (KO) endocardial cells. When cultured with supernatant from Hdac3 KO mouse cardiac endothelial cells (MCECs), wild-type mouse embryonic cardiomyocytes showed decreased proliferation, suggesting that growth signaling from Hdac3 KO MCECs is disrupted. Subsequent transcriptomic analysis revealed that transforming growth factor ß3 (TGFß3) was significantly downregulated in Hdac3 KO MCECs and Hdac3 cardiac endothelial KO hearts. Mechanistically, we identified that microRNA (miR)-129-5p was significantly upregulated in Hdac3 KO MCECs and Hdac3 cardiac endothelial KO hearts. Overexpression of miR-129-5p repressed Tgfß3 expression in wild-type MCECs, whereas knockdown of miR-129-5p restored Tgfß3 expression in Hdac3 KO MCECs. CONCLUSION: Our findings reveal a critical signaling pathway in which endocardial HDAC3 promotes trabecular myocardium growth by stimulating TGFß signaling through repressing miR-129-5p, providing novel insights into the etiology of congenital heart disease and conceptual strategies to promote myocardial regeneration.

Generation and characterization of a human induced pluripotent stem cell line heterozygous for a NOTCH1 mutation (NCHi014-A).

NOTCH1 signaling is crucial for cardiovascular development. Numerous studies have identified heterozygous NOTCH1 loss of function and missense variants associated with a spectrum of congenital heart diseases (CHD). We generated induced pluripotent stem cells (iPSC) from a healthy individual to develop a model for NOTCH1+/- iPSC to study the molecular pathogenesis of CHD. NOTCH1+/-iPSC (NCHi014-A) have normal morphology and karyotype, are identical to the parental cell line, express pluripotency markers and have the ability to differentiate to the three germ layers. NOTCH1+/- iPSC can be used as a tool to study the cellular and molecular mechanisms underlying NOTCH1-associated human CHD.

Rare variants in CAPN2 increase risk for isolated hypoplastic left heart syndrome.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) characterized by hypoplasia of the left ventricle and aorta along with stenosis or atresia of the aortic and mitral valves. HLHS represents only ∼4%-8% of all CHDs but accounts for ∼25% of deaths. HLHS is an isolated defect (i.e., iHLHS) in 70% of families, the vast majority of which are simplex. Despite intense investigation, the genetic basis of iHLHS remains largely unknown. We performed exome sequencing on 331 families with iHLHS aggregated from four independent cohorts. A Mendelian-model-based analysis demonstrated that iHLHS was not due to single, large-effect alleles in genes previously reported to underlie iHLHS or CHD in >90% of families in this cohort. Gene-based association testing identified increased risk for iHLHS associated with variation in CAPN2 (p = 1.8 × 10-5), encoding a protein involved in functional adhesion. Functional validation studies in a vertebrate animal model (Xenopus laevis) confirmed CAPN2 is essential for cardiac ventricle morphogenesis and that in vivo loss of calpain function causes hypoplastic ventricle phenotypes and suggest that human CAPN2707C>T and CAPN21112C>T variants, each found in multiple individuals with iHLHS, are hypomorphic alleles. Collectively, our findings show that iHLHS is typically not a Mendelian condition, demonstrate that CAPN2 variants increase risk of iHLHS, and identify a novel pathway involved in HLHS pathogenesis.

Characterization of an induced pluripotent stem cell line NCHi011-A from a 23-year-old female with Alagille Syndrome harboring a heterozygous JAG1 pathogenic variant.

Alagille syndrome (ALGS) is a multisystem disease with high variability in clinical features. ALGS is predominantly caused by pathogenic variants in the Notch ligand JAG1. An iPSC line, NCHi011-A, was generated from a ALGS patient with complex cardiac phenotypes consisting of pulmonic valve and branch pulmonary artery stenosis. NCHi011-A is heterozygous for a single base duplication causing a frameshift in the JAG1 gene. This iPSC line demonstrates normal cellular morphology, expression of pluripotency markers, trilineage differentiation potential, and identity to the source patient. NCHi011-A provides a resource for modeling ALGS and investigating the role of Notch signaling in the disease.

A Multicenter Analysis of Abnormal Chromosomal Microarray Findings in Congenital Heart Disease.

Background Chromosomal microarray analysis (CMA) provides an opportunity to understand genetic causes of congenital heart disease (CHD). The methods for describing cardiac phenotypes in patients with CMA abnormalities have been inconsistent, which may complicate clinical interpretation of abnormal testing results and hinder a more complete understanding of genotype-phenotype relationships. Methods and Results Patients with CHD and abnormal clinical CMA were accrued from 9 pediatric cardiac centers. Highly detailed cardiac phenotypes were systematically classified and analyzed for their association with CMA abnormality. Hierarchical classification of each patient into 1 CHD category facilitated broad analyses. Inclusive classification allowing multiple CHD types per patient provided sensitive descriptions. In 1363 registry patients, 28% had genomic disorders with well-recognized CHD association, 67% had clinically reported copy number variants (CNVs) with rare or no prior CHD association, and 5% had regions of homozygosity without CNV. Hierarchical classification identified expected CHD categories in genomic disorders, as well as uncharacteristic CHDs. Inclusive phenotyping provided sensitive descriptions of patients with multiple CHD types, which occurred commonly. Among CNVs with rare or no prior CHD association, submicroscopic CNVs were enriched for more complex types of CHD compared with large CNVs. The submicroscopic CNVs that contained a curated CHD gene were enriched for left ventricular obstruction or septal defects, whereas CNVs containing a single gene were enriched for conotruncal defects. Neuronal-related pathways were over-represented in single-gene CNVs, including top candidate causative genes NRXN3, ADCY2, and HCN1. Conclusions Intensive cardiac phenotyping in multisite registry data identifies genotype-phenotype associations in CHD patients with abnormal CMA.

Generation of iPSC line NCHi012-A from a patient with Alagille syndrome and heterozygous pathogenic variant in the JAG1 gene.

Alagille syndrome (ALGS) is an autosomal dominant disease affecting the liver, heart and other organs with high variability. About 95% of ALGS cases are associated with pathogenic variants in JAG1, encoding the Jagged1 ligand that binds to Notch receptors. The iPSC line NCHi012-A was derived from an ALGS patient with cholestatic liver disease and mild pulmonary stenosis, who is heterozygous for a 2 bp deletion in the JAG1 coding sequence. We report here an initial characterization of NCHi012-A to evaluate its morphology, pluripotency, differentiation potential, genotype, karyotype and identity to the source patient.

Generation of an induced pluripotent stem cell line (NCHi010-A) from a 6-year-old female with Down syndrome and without congenital heart disease.

Down syndrome is a genetic anomaly that manifests when there is a mistake during cell division, resulting in an additional chromosome 21. Down syndrome can impact cognitive capabilities and physical development, giving rise to diverse developmental disparities and an elevated likelihood of certain health issues. The iPSC line NCHi010-A was generated from peripheral blood mononuclear cells of a 6-year-old female with Down syndrome and without congenital heart disease using Sendai virus reprogramming. NCHi010-A displayed a morphology of pluripotent stem cells, expressed pluripotency markers, retained trisomy 21 karyotype, and demonstrated potential to differentiate into cells representative of the three germ layers.

Creation of iPSC line NCHi004-A from a patient with down syndrome and congenital heart defects.

Down syndrome is a congenital disorder resulting from an extra full or partial chromosome 21, which is characterized by a spectrum of systemic developmental abnormalities, including those affecting the cardiovascular system. Here, we generated an iPSC line from peripheral blood mononuclear cells of a male adolescent with Down syndrome-associated congenital heart defects through Sendai virus-mediated transfection of 4 Yamanaka factors. This line exhibited normal morphology, expressed pluripotency markers, trisomy 21 karyotype, and could be differentiated into three germ layers. This iPSC line can be used for studying cellular and developmental etiologies of congenital heart defects induced by aneuploidy of chromosome 21.

Congenital aortic valve stenosis: from pathophysiology to molecular genetics and the need for novel therapeutics.

Congenital aortic valve stenosis (AVS) is one of the most common valve anomalies and accounts for 3%-6% of cardiac malformations. As congenital AVS is often progressive, many patients, both children and adults, require transcatheter or surgical intervention throughout their lives. While the mechanisms of degenerative aortic valve disease in the adult population are partially described, the pathophysiology of adult AVS is different from congenital AVS in children as epigenetic and environmental risk factors play a significant role in manifestations of aortic valve disease in adults. Despite increased understanding of genetic basis of congenital aortic valve disease such as bicuspid aortic valve, the etiology and underlying mechanisms of congenital AVS in infants and children remain unknown. Herein, we review the pathophysiology of congenitally stenotic aortic valves and their natural history and disease course along with current management strategies. With the rapid expansion of knowledge of genetic origins of congenital heart defects, we also summarize the literature on the genetic contributors to congenital AVS. Further, this increased molecular understanding has led to the expansion of animal models with congenital aortic valve anomalies. Finally, we discuss the potential to develop novel therapeutics for congenital AVS that expand on integration of these molecular and genetic advances.

Genetics of aortic valve disease.

PURPOSE OF REVIEW: Aortic valve disease is a leading global cause of morbidity and mortality, posing an increasing burden on society. Advances in next-generation technologies and disease models over the last decade have further delineated the genetic and molecular factors that might be exploited in development of therapeutics for affected patients. This review describes several advances in the molecular and genetic understanding of AVD, focusing on bicuspid aortic valve (BAV) and calcific aortic valve disease (CAVD). RECENT FINDINGS: Genomic studies have identified a myriad of genes implicated in the development of BAV, including NOTCH1 , SMAD6 and ADAMTS19 , along with members of the GATA and ROBO gene families. Similarly, several genes associated with the initiation and progression of CAVD, including NOTCH1 , LPA , PALMD , IL6 and FADS1/2 , serve as the launching point for emerging clinical trials. SUMMARY: These new insights into the genetic contributors of AVD have offered new avenues for translational disease investigation, bridging molecular discoveries to emergent pharmacotherapeutic options. Future studies aimed at uncovering new genetic associations and further defining implicated molecular pathways are fuelling the new wave of drug discovery.

The Association of Global and Disease-Related Stress With Susceptibility to and Use of E-Cigarettes and Marijuana Among Adolescents With Congenital Heart Disease.

OBJECTIVE: Adolescents with congenital heart disease (CHD) are exposed to disease-related stressors and have elevated risk for cardiovascular and cognitive complications that are exacerbated by e-cigarettes and marijuana. The aims of this cross-sectional study are to: (1) identify the association between perceived global and disease-related stress and susceptibility to e-cigarettes and marijuana, (2) determine if the association between stress and susceptibility differs by gender, and (3) explore the association between stress and ever use of e-cigarettes and marijuana among adolescents with CHD. METHODS: Adolescents with CHD (N = 98; aged 12-18 years) completed self-report measures of susceptibility to/ever use of e-cigarettes and marijuana and global and disease-related stress. RESULTS: Susceptibility to e-cigarettes and marijuana was reported by 31.3% and 40.2% of adolescents, respectively. Ever use of e-cigarettes and marijuana was reported by 15.3% and 14.3% of adolescents, respectively. Global stress was associated with susceptibility to and ever use of e-cigarettes and marijuana. Disease-related stress was associated with susceptibility to marijuana. Females reported more global and disease-related stress than males, but the association of stress with susceptibility to e-cigarettes and marijuana did not differ by gender. CONCLUSIONS: Susceptibility to e-cigarettes and marijuana is common among adolescents with CHD and is associated with stress. Future work to examine the longitudinal associations between susceptibility, stress, and use of e-cigarettes and marijuana is warranted. Global stress may be an important consideration in the development of strategies to prevent these risky health behaviors among adolescents with CHD.

Establishment of NCHi009-A, an iPSC line from a patient with hypoplastic left heart syndrome (HLHS) carrying a heterozygous NOTCH1 mutation.

Hypoplastic left heart syndrome (HLHS) is a congenital heart malformation clinically characterized by an underdeveloped left ventricle, mitral or aortic valve stenosis or atresia, and narrowed ascending aorta. Although genetic etiology of HLHS is heterogenous, recurrent NOTCH1 variants have been associated with this defect. We report generation of an iPSC line derived from a female with HLHS with a heterozygous missense NOTCH1 (c.2058G > A; p.Gly661Ser) mutation within the conserved EGF-like repeat 17. This iPSC line exhibited typical cellular morphology, normal karyotype, high expression of pluripotent markers, and trilineage differentiation potential; and can be leveraged to dissect the complex NOTCH1-mediated HLHS disease mechanism.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

Single-cell RNA-sequencing analysis of aortic valve interstitial cells demonstrates the regulation of integrin signaling by nitric oxide.

Calcific aortic valve disease (CAVD) is an increasingly prevalent condition among the elderly population that is associated with significant morbidity and mortality. Insufficient understanding of the underlying disease mechanisms has hindered the development of pharmacologic therapies for CAVD. Recently, we described nitric oxide (NO) mediated S-nitrosylation as a novel mechanism for preventing the calcific process. We demonstrated that NO donor or an S-nitrosylating agent, S-nitrosoglutathione (GSNO), inhibits spontaneous calcification in porcine aortic valve interstitial cells (pAVICs) and this was supported by single-cell RNA sequencing (scRNAseq) that demonstrated NO donor and GSNO inhibited myofibroblast activation of pAVICs. Here, we investigated novel signaling pathways that are critical for the calcification of pAVICs that are altered by NO and GSNO by performing an in-depth analysis of the scRNA-seq dataset. Transcriptomic analysis revealed 1,247 differentially expressed genes in pAVICs after NO donor or GSNO treatment compared to untreated cells. Pathway-based analysis of the differentially expressed genes revealed an overrepresentation of the integrin signaling pathway, along with the Rho GTPase, Wnt, TGF-ß, and p53 signaling pathways. We demonstrate that ITGA8 and VCL, two of the identified genes from the integrin signaling pathway, which are known to regulate cell-extracellular matrix (ECM) communication and focal adhesion, were upregulated in both in vitro and in vivo calcific conditions. Reduced expression of these genes after treatment with NO donor suggests that NO inhibits calcification by targeting myofibroblast adhesion and ECM remodeling. In addition, withdrawal of NO donor after 3 days of exposure revealed that NO-mediated transcriptional and translational regulation is a transient event and requires continuous NO exposure to inhibit calcification. Overall, our data suggest that NO and S-nitrosylation regulate the integrin signaling pathway to maintain healthy cell-ECM interaction and prevent CAVD.

Patent Ductus Arteriosus: A Contemporary Perspective for the Pediatric and Adult Cardiac Care Provider.

The burden of patent ductus arteriosus (PDA) continues to be significant. In view of marked differences in preterm infants versus more mature, term counterparts (viewed on a continuum with adolescent and adult patients), mechanisms regulating ductal patency, genetic contributions, clinical consequences, and diagnostic and treatment thresholds are discussed separately, when appropriate. Among both preterm infants and older children and adults, a range of hemodynamic profiles highlighting the markedly variable consequences of the PDA are provided. In most contemporary settings, transcatheter closure is preferable over surgical ligation, but data on longer-term outcomes, particularly among preterm infants, are lacking. The present review provides recommendations to identify gaps in PDA diagnosis, management, and treatment on which subsequent research can be developed. Ultimately, the combination of refined diagnostic thresholds and expanded treatment options provides the best opportunities to address the burden of PDA. Although fundamental gaps remain unanswered, the present review provides pediatric and adult cardiac care providers with a contemporary framework in PDA care to support the practice of evidence-based medicine.

Single-cell transcriptomic profiling unveils dysregulation of cardiac progenitor cells and cardiomyocytes in a mouse model of maternal hyperglycemia.

Congenital heart disease (CHD) is the most prevalent birth defect, often linked to genetic variations, environmental exposures, or combination of both. Epidemiological studies reveal that maternal pregestational diabetes is associated with ~5-fold higher risk of CHD in the offspring; however, the causal mechanisms affecting cardiac gene-regulatory-network (GRN) during early embryonic development remain poorly understood. In this study, we utilize an established murine model of pregestational diabetes to uncover the transcriptional responses in key cell-types of the developing heart exposed to maternal hyperglycemia (matHG). Here we show that matHG elicits diverse cellular responses in E9.5 and E11.5 embryonic hearts compared to non-diabetic hearts by single-cell RNA-sequencing. Through differential-gene-expression and cellular trajectory analyses, we identify perturbations in genes, predominantly affecting Isl1+ second heart field progenitors and Tnnt2+ cardiomyocytes with matHG. Using cell-fate mapping analysis in Isl1-lineage descendants, we demonstrate that matHG impairs cardiomyocyte differentiation and alters the expression of lineage-specifying cardiac genes. Finally, our work reveals matHG-mediated transcriptional changes in second heart field lineage that elevate CHD risk by perturbing Isl1-GRN during cardiomyocyte differentiation. Gene-environment interaction studies targeting the Isl1-GRN in cardiac progenitor cells will have a broader impact on understanding the mechanisms of matHG-induced risk of CHD associated with diabetic pregnancies.