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Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function (LoF) and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. LoF variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2, and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

Supplemental Digital Content is available in the text., Background: De novo genic and copy number variants are enriched in patients with congenital heart disease, particularly those with extra-cardiac anomalies. The impact of de novo damaging variants on outcomes following cardiac repair is unknown. Methods: We studied 2517 patients with congenital heart disease who had undergone whole-exome sequencing as part of the CHD GENES study (Congenital Heart Disease Genetic Network). Results: Two hundred ninety-four patients (11.7%) had clinically significant de novo variants. Patients with de novo damaging variants were 2.4 times more likely to have extra-cardiac anomalies (P=5.63×10−12). In 1268 patients (50.4%) who had surgical data available and underwent open-heart surgery exclusive of heart transplantation as their first operation, we analyzed transplant-free survival following the first operation. Median follow-up was 2.65 years. De novo variants were associated with worse transplant-free survival (hazard ratio, 3.51; P=5.33×10−04) and longer times to final extubation (hazard ratio, 0.74; P=0.005). As de novo variants had a significant interaction with extra-cardiac anomalies for transplant-free survival (P=0.003), de novo variants conveyed no additional risk for transplant-free survival for patients with these anomalies (adjusted hazard ratio, 1.96; P=0.06). By contrast, de novo variants in patients without extra-cardiac anomalies were associated with worse transplant-free survival during follow-up (hazard ratio, 11.21; P=1.61×10−05) than that of patients with no de novo variants. Using agnostic machine-learning algorithms, we identified de novo copy number variants at 15q25.2 and 15q11.2 as being associated with worse transplant-free survival and 15q25.2, 22q11.21, and 3p25.2 as being associated with prolonged time to final extubation. Conclusions: In patients with congenital heart disease undergoing open-heart surgery, de novo variants were associated with worse transplant-free survival and longer times on the ventilator. De novo variants were most strongly associated with adverse outcomes among patients without extra-cardiac anomalies, suggesting a benefit for preoperative genetic testing even when genetic abnormalities are not suspected during routine clinical practice. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT01196182.

BackgroundThe contribution of somatic mosaicism, or genetic mutations arising after oocyte fertilization, to congenital heart disease (CHD) is not well understood. Further, the relationship between mosaicism in blood and cardiovascular tissue has not been determined.ResultsWe developed a computational method, Expectation-Maximization-based detection of Mosaicism (EM-mosaic), to analyze mosaicism in exome sequences of 2530 CHD proband-parent trios. EM-mosaic detected 326 mosaic mutations in blood and/or cardiac tissue DNA. Of the 309 detected in blood DNA, 85/97 (88%) tested were independently confirmed, while 7/17 (41%) candidates of 17 detected in cardiac tissue were confirmed. MosaicHunter detected an additional 64 mosaics, of which 23/46 (50%) among 58 candidates from blood and 4/6 (67%) of 6 candidates from cardiac tissue confirmed. Twenty-five mosaic variants altered CHD-risk genes, affecting 1% of our cohort. Of these 25, 22/22 candidates tested were confirmed. Variants predicted as damaging had higher variant allele fraction than benign variants, suggesting a role in CHD. The frequency of mosaic variants above 10% mosaicism was 0.13/person in blood and 0.14/person in cardiac tissue. Analysis of 66 individuals with matched cardiac tissue available revealed both tissue-specific and shared mosaicism, with shared mosaics generally having higher allele fraction.ConclusionsWe estimate that ~1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants expressed at higher allele fraction compared to benign variants. Although blood is a readily-available DNA source, cardiac tissues analyzed contributed ~5% of somatic mosaic variants identified, indicating the value of tissue mosaicism analyses.

The discovery of damaging gene mutations in congenital heart disease (CHD) patients enables identification of regulators of cardiac development. Exome sequencing identified de novo heterozygous loss-of-function (LoF) and missense variants in GATA6 among CHD probands, most with outflow tract malformations. Other subjects with GATA6 LoF mutations developed pancreatic agenesis. To elucidate the molecular basis for the predominance of this heart defect, we modeled GATA6 mutations in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs). GATA6 variants were introduced into isogenic hiPSCs using CRISPR/Cas9 genome editing. Genome-wide molecular profiles including chromatin accessibility (ATAC-Seq) and gene expression (single cell and bulk RNA-Seq) were evaluated during hiPSC-CM differentiation. Analyses of GATA6 mutant hiPSC-CMs showed deficits in hiPSC-CM differentiation, chromatin accessibility and transcriptional profiles. Heterozygous GATA6 LoF hiPSCs made hiPSC-CMs but exhibited reduced expression of second heart field genes. Homozygous GATA6 LoF hiPSCs failed to differentiate and adopted fibroblast expression profiles. hiPSCs carrying a homozygous GATA6 missense variant, R456G, which altered a DNA-binding domain residue, showed enhanced capacity to differentiate into neuroepithelial-like cells. Chromatin-accessibility studies confirmed that GATA6 normally binds to genes in the promoter region and other genes at distal enhancers. Human GATA6 haploinsufficiency disrupts developmental transcriptional responses driving cardiac morphogenesis. The HAND2 -dependent genetic program, operant during outflow tract development, is particularly sensitive to GATA6 dosage. The mixed differentiation patterns observed in mutation-carrying hiPSCs likely contributes to vascular phenotypes observed in CHD patients. GATA6 haploinsufficiency preferentially alters binding of distal enhancers to promoters in genes where GATA6 normally binds the enhancer rather than the promoter. We speculate that pathogenicity of GATA6 haploinsufficiency is mediated by weaker binding of GATA6 to distal enhancers than to promoter elements, altering expression of these genes in GATA6 haploinsufficient patients.

Dilated cardiomyopathy (DCM) is a leading cause for heart failure and is associated with a rate of mortality of 20% within 5 years of diagnosis. The most common genetic causes for DCM are mutations of the sarcomere protein titin (encoded by TTN ), which occurs in 10-20% of DCM cases. Dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency. Thus, strategies to increase the expression of the wild type TTN allele could attenuate damaging effects of TTNtv. Utilizing bioinformatic tools, we identified a putative enhancer for TTN in its intron 1. We deleted a 658 bp region from intron 1 which encompasses the region of interest in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing to validate its function. Utilizing RNA sequencing and qPCR of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs), we demonstrated that a homozygous deletion in this region leads to a drop in TTN expression compared to the wild type (WT) control (0.344-fold change, p < 0.001). To further characterize this region, we subdivided it into three parts which we called E1 (296 bp), E2 (206 bp), and E3 (139 bp). E1 includes a highly conserved region and a region of open chromatin as identified by the Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) performed on hiPSC-CMs. A homozygous E1 deletion resulted in a decreased TTN expression of 0.63-fold compared to the WT control (p < 0.001) when performing RNA sequencing on hiPSC-CMs. Both homozygous E2 and E3 deletions resulted in an increased TTN expression (1.56-fold change, p < 0.001; 1.19 fold change, p < 0.001). Utilizing a published sarcomere tracking platform, SarcTrack, to investigate hiPSC-CM physiology, we saw a decreased contractility of 6.6% in hiPSC-CMs carrying a homozygous E1 deletion compared to 10.1% in the WT control (p < 0.001). Cells carrying homozygous E2 or E3 deletions were hypercontractile (13.8%, p < 0.001; 13.7%, p < 0.001). Given our results, we hypothesize that TTN expression depends on the E1 region. If confirmed, we expect that increasing the activity of this enhancer using small molecules may provide a novel therapeutic target for DCM caused by TTNtv.

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin binding protein-C; cMyBPC) or myosin missense mutations cause hyper-contractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches we explored how depletion of cMyBPC altered sarcomere function. We demonstrate that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM) normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation - enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across all phases of the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in DCM patients, while inhibiting myosin by MYK-461 should benefit the substantial proportion of HCM patients with MYBPC3 mutations.

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin binding protein-C; cMyBPC) or myosin missense mutations cause hyper-contractility and poor relaxation in hypertrophic cardiomyopathy (HCM) are incompletely understood. Using genetic and biochemical approaches we explored how depletion of cMyBPC altered sarcomere function. We demonstrate that stepwise loss of cMyBPC resulted in reciprocal augmentation of myosin contractility. Direct attenuation of myosin function, via a damaging missense variant (F764L) that causes dilated cardiomyopathy (DCM) normalized the increased contractility from cMyBPC depletion. Depletion of cMyBPC also altered dynamic myosin conformations during relaxation - enhancing the myosin state that enables ATP hydrolysis and thin filament interactions while reducing the super relaxed conformation associated with energy conservation. MYK-461, a pharmacologic inhibitor of myosin ATPase, rescued relaxation deficits and restored normal contractility in mouse and human cardiomyocytes with MYBPC3 mutations. These data define dosage-dependent effects of cMyBPC on myosin that occur across all phases of the cardiac cycle as the pathophysiologic mechanisms by which MYBPC3 truncations cause HCM. Therapeutic strategies to attenuate cMyBPC activity may rescue depressed cardiac contractility in DCM patients, while inhibiting myosin by MYK-461 should benefit the substantial proportion of HCM patients with MYBPC3 mutations.One Sentence SummaryAnalyses of cardiomyocytes with hypertrophic cardiomyopathy mutations in MYBPC3 reveal that these directly activate myosin contraction by disrupting myosin states of relaxation, and that genetic or pharmacological manipulation of myosin therapeutically abates the effects of MYBPC3 mutations.