Role In Congenital Heart Defects Research Articles

The Role of SHROOM3 in Congenital Heart Disease

Background and Hypothesis: Congenital heart defects (CHD) are the most common, and most frequently fatal birth defects, but most etiology remains unknown. We identified a patient with CHD and implicated a gene called SHROOM3. SHROOM3 binds Dishevelled2 which is the central cytoplasmic component of both canonical and noncanonical Wnt/planar cell polarity (PCP) signaling pathways. PCP drives cell movement and is important to embryogenesis and disruption causes CHD. We hypothesize CHD can result from SHROOM3-loss-of-function due to PCP disruption.
 Project Methods: To interrogate SHROOM3’s role in CHD and PCP we utilized an established in vivo SHROOM3-loss-of-function model, Shroom3 gene trap mice (Shroom3gt). We also utilized a loss-of-function model for PCP membrane component VANGL2, (Vangl2+/-). We assayed genetic interaction between Shroom3 and Vangl2 during cardiac development by crossing singly heterozygous null mice to produce compound heterozygous embryos, harvested embryos, and performed histologic analysis for cardiac defects. We also utilized a human in vitro SHROOM3-loss-of-function model, a CRISPR-Cas9 edited SHROOM3 knockout HELA cell line. We assayed cell movement using a scratch assay.
 Results: Compound heterozygous Shroom3+/gt;Vangl2+/- embryos had a three fold increase in heart defects compared to singly heterozygous Shroom3+/gt;Vangl2+/+ or Shroom3+/+;Vangl2+/- embryos (3 of 19 or 15.7%, versus 1 of 17 or 5.2%, and 1 of 19 or 4.8%, respectively), demonstrating a trend towards genetic interaction between SHROOM3 and VANGL2/PCP during cardiac development. The scratch assays demonstrated cell movement defects due to SHROOM3-loss-of-function consistent with increased cell movement.
 Conclusion and Potential Impact: We demonstrate SHROOM3 interacts with Wnt/PCP during cardiac development. Further interrogation of SHROOM3’s role in Wnt signaling will provide insight into the mechanisms by which a novel CHD candidate participates in cardiogenesis and will improve CHD diagnosis, management, and therapeutic development.

Transcriptional associations between maternal pregestational diabetes, congenital heart defects, and the human placenta

A critical role has been recognized for the placenta in the development of congenital heart defects (CHD), for which infants of pregestationally diabetic (PGDM) mothers are at pronounced risk. PGDM induces stark histological and gross anatomical changes in the placenta, yet little is known about the PGDM placenta’s role in CHD. High-throughput transcriptomic processes have enabled large-scale association between the gene expression signatures of the heart, placenta, and diabetic state. However, limited integrative data exists, necessitating a comparative appraisal of existing literature to identify placental and cardiac signaling pathways that may be altered in the PGDM state.

Axenfeld-Rieger syndrome-associated mutants of the transcription factor FOXC1 abnormally regulate NKX2-5 in model zebrafish embryos

FOXC1 is a member of the forkhead family of transcription factors, and whose function is poorly understood. A variety of FOXC1 mutants have been identified in patients diagnosed with the autosomal dominant disease Axenfeld-Rieger syndrome, which is mainly characterized by abnormal development of the eyes, particularly those who also have accompanying congenital heart defects (CHD). However, the role of FOXC1 in CHD, and how these mutations might impact FOXC1 function, remains elusive. Our previous work provided one clue to possible function, demonstrating that zebrafish foxc1a, an orthologue of human FOXC1 essential for heart development, directly regulates the expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells. Abnormal expression of Nkx2-5 leads to CHD in mice and is also associated with CHD patients. Whether this link extends to the human system, however, requires investigation. In this study, we demonstrate that FOXC1 does regulate human NKX2-5 expression in a dose-dependent manner via direct binding to its proximal promoter. A comparison of FOXC1 mutant function in the rat cardiac cell line H9c2 and zebrafish embryos suggested that the zebrafish embryos might serve as a more representative model system than the H9c2 cells. Finally, we noted that three of the Axenfeld-Rieger syndrome FOXC1 mutations tested increased, whereas a fourth repressed the expression of NKX2-5 These results imply that mutant FOXC1s might play etiological roles in CHD by abnormally regulating NKX2-5 in the patients. And zebrafish embryos can serve as a useful in vivo platform for rapidly evaluating disease-causing roles of mutated genes.

Functional testing of a human PBX3 variant in zebrafish reveals a potential modifier role in congenital heart defects.

ABSTRACTWhole-genome and exome sequencing efforts are increasingly identifying candidate genetic variants associated with human disease. However, predicting and testing the pathogenicity of a genetic variant remains challenging. Genome editing allows for the rigorous functional testing of human genetic variants in animal models. Congenital heart defects (CHDs) are a prominent example of a human disorder with complex genetics. An inherited sequence variant in the human PBX3 gene (PBX3 p.A136V) has previously been shown to be enriched in a CHD patient cohort, indicating that the PBX3 p.A136V variant could be a modifier allele for CHDs. Pbx genes encode three-amino-acid loop extension (TALE)-class homeodomain-containing DNA-binding proteins with diverse roles in development and disease, and are required for heart development in mouse and zebrafish. Here, we used CRISPR-Cas9 genome editing to directly test whether this Pbx gene variant acts as a genetic modifier in zebrafish heart development. We used a single-stranded oligodeoxynucleotide to precisely introduce the human PBX3 p.A136V variant in the homologous zebrafish pbx4 gene (pbx4 p.A131V). We observed that zebrafish that are homozygous for pbx4 p.A131V are viable as adults. However, the pbx4 p.A131V variant enhances the embryonic cardiac morphogenesis phenotype caused by loss of the known cardiac specification factor, Hand2. Our study is the first example of using precision genome editing in zebrafish to demonstrate a function for a human disease-associated single nucleotide variant of unknown significance. Our work underscores the importance of testing the roles of inherited variants, not just de novo variants, as genetic modifiers of CHDs. Our study provides a novel approach toward advancing our understanding of the complex genetics of CHDs.

MEF2C regulates outflow tract alignment and transcriptional control of Tdgf1.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHF-restricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

Dosage changes of MED13L further delineate its role in congenital heart defects and intellectual disability

A chromosomal balanced translocation disrupting the MED13L (Mediator complex subunit13-like) gene, encoding a subunit of the Mediator complex, was previously associated with transposition of the great arteries (TGA) and intellectual disability (ID), and led to the identification of missense mutations in three patients with isolated TGA. Recently, a homozygous missense mutation in MED13L was found in two siblings with non-syndromic ID from a consanguineous family. Here, we describe for the first time, three patients with copy number changes affecting MED13L and delineate a recognizable MED13L haploinsufficiency syndrome. Using high resolution molecular karyotyping, we identified two intragenic de novo frameshift deletions, likely resulting in haploinsufficiency, in two patients with a similar phenotype of hypotonia, moderate ID, conotruncal heart defect and facial anomalies. In both, Sanger sequencing of MED13L did not reveal any pathogenic mutation and exome sequencing in one patient showed no evidence for a non-allelic second hit. A further patient with hypotonia, learning difficulties and perimembranous VSD showed a 1 Mb de novo triplication in 12q24.2, including MED13L and MAP1LC3B2. Our findings show that MED13L haploinsufficiency in contrast to the previously observed missense mutations cause a distinct syndromic phenotype. Additionally, a MED13L copy number gain results in a milder phenotype. The clinical features suggesting a neurocristopathy may be explained by animal model studies indicating involvement of the Mediator complex subunit 13 in neural crest induction.

Analysis of MTHFR and MTRR Gene Polymorphisms in Iranian Ventricular Septal Defect Subjects.

Ventricular septal defect (VSD) is one of the most common types of congenital heart defects (CHD). There are vivid multifactorial causes for VSD in which both genetic and environmental risk factors are consequential in the development of CHD. Methionine synthase reductase (MTRR) and methylenetetrahydrofolate reductase (MTHFR) are two of the key regulatory enzymes involved in the metabolic pathway of homocysteine. Genes involved in homocysteine/folate metabolism may play an important role in CHDs. In this study; we determined the association of A66G and C524T polymorphisms of the MTRR gene and C677T polymorphism of the MTHFR gene in Iranian VSD subjects. A total of 123 children with VSDs and 125 healthy children were included in this study. Genomic DNA was extracted from the buccal cells of all the subjects. The restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) method was carried out to amplify the A66G and C524T polymorphism of MTRR and C677T polymorphism of MTHFR genes digested with Hinf1, Xho1 and Nde1 enzymes, respectively. The genotype frequencies of CC, CT and TT of MTRR gene among the studied cases were 43.1%, 40.7% and 16.3%, respectively, compared to 52.8%, 43.2% and 4.0%, respectively among the controls. For the MTRR A66G gene polymorphism, the genotypes frequencies of AA, AG and GG among the cases were 33.3%, 43.9% and 22.8%, respectively, while the frequencies were 49.6%, 42.4% and 8.0%, respectively, among control subjects. The frequencies for CC and CT genotypes of the MTHFR gene were 51.2% and 48.8%, respectively, in VSD patients compared to 56.8% and 43.2% respectively, in control subjects. Apart from MTHFR C677T polymorphism, significant differences were noticed (p < 0.05) in C524T and A66G polymorphisms of the MTRR gene between cases and control subjects.

A66G and C524T polymorphisms of the methionine synthase reductase gene are associated with congenital heart defects in the Chinese Han population

Congenital heart defects (CHDs) are the most common birth defects; genes involved in homocysteine/folate metabolism may play important roles in CHDs. Methionine synthase reductase (MTRR) is one of the key regulatory enzymes involved in the metabolic pathway of homocysteine. We investigated whether two polymorphisms (A66G and C524T) of the MTRR gene are associated with CHDs. A total of 599 children with CHDs and 672 healthy children were included; the polymorphisms were detected by PCR and RFLP analysis. Significant differences in the distributions of A66G and C524T alleles were observed between CHD cases and controls, and slightly increased risks of CHD were associated with 66GG and 524CT genotypes (odds ratios = 1.545 and 1.419, respectively). The genotype frequencies of 524CT in the VSD subgroup, 66GG and 524CT in the PDA subgroup were significantly different from those of controls. In addition, the combined 66AA/524CT, 66AG/524CT and 66GG/524CT in CHDs had odds ratios = 1.589, 1.422 and 1.934, respectively. Increased risks were also observed in 66AA/524CT and 66GG/524CT for ASD, 66AG/524CT for VSD, as well as 66GG/524CT for PDA. In conclusion, MTRR A66G and C524T polymorphisms are associated with increased risk of CHDs.

Haploinsufficiency of TAB2 Causes Congenital Heart Defects in Humans

Congenital heart defects (CHDs) are the most common major developmental anomalies and the most frequent cause for perinatal mortality, but their etiology remains often obscure. We identified a locus for CHDs on 6q24-q25. Genotype-phenotype correlations in 12 patients carrying a chromosomal deletion on 6q delineated a critical 850 kb region on 6q25.1 harboring five genes. Bioinformatics prioritization of candidate genes in this locus for a role in CHDs identified the TGF-beta-activated kinase 1/MAP3K7 binding protein 2 gene (TAB2) as the top-ranking candidate gene. A role for this candidate gene in cardiac development was further supported by its conserved expression in the developing human and zebrafish heart. Moreover, a critical, dosage-sensitive role during development was demonstrated by the cardiac defects observed upon titrated knockdown of tab2 expression in zebrafish embryos. To definitively confirm the role of this candidate gene in CHDs, we performed mutation analysis of TAB2 in 402 patients with a CHD, which revealed two evolutionarily conserved missense mutations. Finally, a balanced translocation was identified, cosegregating with familial CHD. Mapping of the breakpoints demonstrated that this translocation disrupts TAB2. Taken together, these data clearly demonstrate a role for TAB2 in human cardiac development.

Defective Decapentaplegic Signaling Results in Heart Overgrowth and Reduced Cardiac Output in Drosophila

During germ-band extension, Decapentaplegic (Dpp) signals from the dorsal ectoderm to maintain Tinman (Tin) expression in the underlying mesoderm. This signal specifies the cardiac field, and homologous genes (BMP2/4 and Nkx2.5) perform this function in mammals. We showed previously that a second Dpp signal from the dorsal ectoderm restricts the number of pericardial cells expressing the transcription factor Zfh1. Here we report that, via Zfh1, the second Dpp signal restricts the number of Odd-skipped-expressing and the number of Tin-expressing pericardial cells. Dpp also represses Tin expression independently of Zfh1, implicating a feed-forward mechanism in the regulation of Tin pericardial cell number. In the adjacent dorsal muscles, Dpp has the opposite effect. Dpp maintains Krüppel and Even-skipped expression required for muscle development. Our data show that Dpp refines the cardiac field by limiting the number of pericardial cells. This maintains the boundary between pericardial and dorsal muscle cells and defines the size of the heart. In the absence of the second Dpp signal, pericardial cells overgrow and this significantly reduces larval cardiac output. Our study suggests the existence of a second round of BMP signaling in mammalian heart development and that perhaps defects in this signal play a role in congenital heart defects.