Research Article

Oligogenic inheritance of a human heart disease involving a genetic modifier

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Science  31 May 2019:
Vol. 364, Issue 6443, pp. 865-870
DOI: 10.1126/science.aat5056

Three rights can make a wrong

Many diseases are thought to arise from co-inheritance of rare genetic variants that are benign on their own but harmful in combination. This hypothesis has been difficult to validate by functional experiments. Gifford et al. sequenced the genomes of two parents who were asymptomatic and their three children, all of whom had early-onset heart disease. They identified three likely culprit genetic variants, two in transcription factor genes linked to heart development and one in a gene encoding a muscle structural protein. When they introduced these three variants together into mice by gene editing, the mice developed heart disease resembling that seen in the children.

Science, this issue p. 865

Abstract

Complex genetic mechanisms are thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human cardiac anomaly can be caused by a combination of rare, inherited heterozygous mutations. Whole-exome sequencing of a nuclear family revealed that three offspring with childhood-onset cardiomyopathy had inherited three missense single-nucleotide variants in the MKL2, MYH7, and NKX2-5 genes. The MYH7 and MKL2 variants were inherited from the affected, asymptomatic father and the rare NKX2-5 variant (minor allele frequency, 0.0012) from the unaffected mother. We used CRISPR-Cas9 to generate mice encoding the orthologous variants and found that compound heterozygosity for all three variants recapitulated the human disease phenotype. Analysis of murine hearts and human induced pluripotent stem cell–derived cardiomyocytes provided histologic and molecular evidence for the NKX2-5 variant’s contribution as a genetic modifier.

The genetic etiologies of complex phenotypes or diseases such as type 2 diabetes, Parkinson’s disease, and cardiovascular disease are not fully understood (14). High-throughput DNA sequencing is establishing the landscape of genetic variation in the absence of disease, enhancing identification of the pathogenic variants involved in Mendelian disorders (5). However, the lack of experimental approaches to determine the involvement of multiple genetic variants and their epistatic relationships has hampered mechanistic dissection of complex phenotypes, especially those involving oligogenic or polygenic inheritance and genetic modifiers (3, 6, 7). Definitive genetic causes of congenital heart disease (CHD), the most common congenital malformation, have been particularly elusive (8, 9). Rare inherited and de novo monogenic aberrations account for ~10% of cases, on the basis of a recent exome-sequencing study of CHD trios, whereas copy number variants have been identified in ~25% of cases (10, 11). Oligogenic inheritance and the involvement of genetic modifiers may contribute to CHD and cardiomyopathies; however, experimental confirmation of this model is lacking (12, 13).

Recent improvements in gene editing facilitated by CRISPR-Cas technology provide the opportunity to test hypotheses involving the potential for oligogenic inheritance of disease (14). In parallel, the establishment of human induced pluripotent stem cell (hiPSC) models of differentiation has fostered the ability to study aberrant regulatory events that occur during embryonic development in a human cellular context (15). Here, we used these advances to dissect a complex familial case of heart disease and identified a rare missense NKX2-5 variant that acts as a modifier, on both the phenotypic and the molecular level, in conjunction with previously unknown missense variants in the myocardin-related transcription factor MKL2 and the sarcomeric protein MYH7.

Familial left ventricular noncompaction

Upon presentation of a 2-month-old infant with congestive heart failure requiring mechanical ventilation and inotropic support, echocardiography revealed severely depressed left ventricular (LV) function and deep LV trabeculations, characteristic of a type of cardiomyopathy known as left ventricular noncompaction (LVNC) (Fig. 1A, right). LVNC is thought to represent a failure of cardiomyocyte maturation during embryonic development and accounts for almost 10% of all cardiomyopathies, though its incidence may be underestimated (16). This defect exhibits variability in presentation from neonates to adulthood but appears to have a congenital etiology.

Fig. 1 Presentation of a familial case of LVNC.

(A) Four-chamber echocardiography view showing the left atrium (LA), right atrium (RA), left ventricle (LV), and right ventricle (RV). The unaffected individual is shown on the left (43) and the index patient (a 2-month-old infant with LVNC) is shown on the right. Yellow arrowheads indicate abnormal hypertrabeculation of the dilated LV. (B) Transverse section of heart from the sibling of the index patient who suffered fetal demise. Higher magnification of the LV is shown on the right. Scale bar, 6 mm. (C) Pedigree showing inheritance pattern of LVNC in this family. Light gray indicates individuals whose cardiac status was not determined (ND), and white indicates unaffected individuals assessed by imaging. Orange indicates the asymptomatic adult individual, and purple indicates the individuals with childhood-onset LVNC.

A family medical history disclosed a sibling who suffered fetal demise at 24 weeks of gestation. Initial autopsy results concluded that death was due to pulmonary hypoplasia. However, our examination of histologic sections revealed that the fetus suffered from biventricular noncompaction, based on the deep recesses in the myocardial walls of both ventricles, right ventricular dilation, and widespread fibrosis (Fig. 1B). Cardiac imaging of living immediate family members exposed previously undetected evidence of LVNC in a 4-year-old sibling and subtle signs of LVNC in the father (fig. S1, A and B). The proband’s paternal grandfather had a history of arrhythmia but, similar to the extended family, no cardiac functional or structural abnormalities were detected (Fig. 1C). Collectively, these findings suggested vertical transmission of LVNC from the father with a markedly increased severity of disease and age of onset in offspring.

Multiple genetic variants segregate with familial LVNC

To investigate potential genetic causes of LVNC in this family, we performed whole-exome sequencing on the immediate family of the proband. We reasoned that individual de novo events were unlikely to be the cause of disease because of the penetrance in all three offspring and instead pursued an inherited variant hypothesis. We initially focused on inherited private and/or rare (minor allele frequency, MAF < 0.005) exonic, nonsynonymous variants inherited from the father and identified 30 single-nucleotide variants (SNVs) of interest (Fig. 2A, left). Eight of these variants were predicted to be damaging, but only two were enriched in heart tissue transcripts (table S1).

Fig. 2 Genotypic analysis of LVNC family by exome sequencing.

(A) Workflow for analysis of sequence variants inherited from the father is shown in blue on the left and from the unaffected mother in red on the right. Inheritance pattern for variants of interest is shown in pedigree on lower right. (B) Conservation of each amino acid residue across species. The variant of interest is highlighted in yellow. Predicted effects of variants on protein function based on the Combined Annotation Dependent Depletion (CADD), Sift, Polyphen-2, and FATHMM programs. D, damaging; B, benign; T, tolerated; ND, not determined.

The first variant of interest present only in the four family members with image-based evidence of LVNC was a previously unknown heterozygous missense variant in myosin heavy chain 7 (MYH7) involving a leucine-to-phenylalanine substitution at position 387 (L387F) (Fig. 2A, left). This amino acid residue is highly conserved, predicted to be damaging by multiple algorithms, and resides within the adenosine triphosphatase domain of the protein (Fig. 2B and fig. S2A). SNVs within this gene have previously been associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, and LVNC (16, 17). One interpretation of the phenotypic variability associated with variants in MYH7 is that genetic modifiers may be involved, although this has not been demonstrated (17, 18).

The second variant that met our filtering criteria in individuals with phenotypic evidence of LVNC resulted in a previously undescribed heterozygous glutamine-to-histidine substitution at position 670 (Q670H) in the transcription factor MKL2 (also known as myocardin-related transcription factor B, MRTF-B) (19). This residue is adjacent to the leucine zipper domain, is highly conserved, and is also predicted to be damaging by multiple algorithms (Fig. 2, A and B, and fig. S2A). Myocardin activity at paired MEF2 sites requires leucine zipper domain–dependent homodimizeration (20). The myocardin family of regulators are potent transcriptional coactivators of SRF and Mef2c, both essential for cardiogenesis. Consistent with a detrimental effect of the variant, we found that MKL2 Q670H had reduced transcriptional activity in vitro compared with wild-type (WT) MKL2 (fig. S2B). Homozygous Mkl2 deletion leads to defects in the cardiovascular epithelial-to-mesenchymal transition during mouse embryonic development and disrupts vascular development (21). Although this gene has not previously been associated with cardiac disease, loss-of-function variants are underrepresented in the human population (observed/expected = 0.07), suggesting that the gene is important for human viability (22).

Given the marked increase in severity of disease in the three children compared with the father, we investigated whether variants inherited from the unaffected mother might serve as genetic modifiers of the LVNC phenotype. We applied the same filtering criteria described earlier, but in this case, we did not require variants of interest to be damaging based on traditional metrics (e.g., Sift or Polyphen), as we reasoned that genetic modifiers inherited from an unaffected parent could have a relatively subtle alteration of function. We identified 34 previously unknown and/or rare variants (MAF < 0.005) inherited by all three affected children from the unaffected mother (Fig. 2A, right). After filtering for those that were cardiac enriched (23), we focused on a rare heterozygous missense variant in NKX2-5 that produced an alanine-to-serine substitution at position 119 (A119S, MAF = 0.0012). NKX2-5 is an essential “core” transcriptional regulator of cardiac development, and heterozygous mutations have been associated with CHD (24, 25). The alanine residue at position 119 is not conserved in rhesus macaques, although the hydrophobicity at this site is maintained by the valine substitution in these animals (Fig. 2B). The NKX2-5 A119S variant is not predicted to be damaging (Fig. 2B) and is outside the critical DNA-binding homeodomain (fig. S2A), but it has been reported to slightly reduce DNA binding in vitro and has been noted in the setting of CHD and cardiomyopathies, although with incomplete penetrance (2628).

Sequence analysis validated all three variants (fig. S2C and table S2), and sequencing of all paternal family members exposed the presence of the MKL2 Q670H variant in an unaffected uncle of the proband and a grandfather, both phenotyped by echocardiography, indicating that this variant is not sufficient to cause cardiac dysfunction (fig. S2D). The SNV in MYH7 arose de novo in the father (fig. S2D). The observation of isolated MKL2 Q670H or NKX2-5 A119S variants in unaffected individuals raised the possibility that these alterations have subtle effects on protein function and either have no consequence or may act as modifiers of the phenotype in the presence of the MYH7 L387F variant. Although it is possible that other genetic variants and environmental factors may contribute to the severity of the disease, the inheritance pattern of the heterozygous missense variants in MKL2, MYH7, and NKX2-5 led us to hypothesize that their collective inheritance was sufficient to cause LVNC.

Functional significance of MKL2, MYH7, and NKX2-5 missense variants in vivo

To evaluate the in vivo functional consequence of the MKL2, MYH7, and NKX2-5 SNVs identified in this family, we generated mice (C57BL/6J) harboring the orthologous missense variants by CRISPR-Cas9 gene editing (fig. S3, A to C). Animals were bred to homozygosity to test the effects of each individual missense variant in vivo. Whereas Myh7+/L387F animals were observed at the expected Mendelian ratio, homozygous animals died by embryonic day 9.5 (E9.5) to E10.0 with evidence of heart failure (Fig. 3A). Thus, the MYH7 L387F substitution is damaging.

Fig. 3 Functional evaluation of disease-associated SNVs in mice.

(A) Left lateral images of E9.5 embryos from a representative Myh7L387F/+ × Myh7L387F/+ cross. h, head; lv, left ventricle; a, atrium. Scale bar, 5 mm. (B) Hematoxylin and eosin–stained transverse histologic sections of E13.5 embryos with the indicated Nkx2-5 genotypes. Width of the compact layer is indicated by black bars in higher-magnification views of boxed areas (bottom). Top scale bar, 500 μm; bottom scale bar, 50 μm. (C and D) Quantification (μm) of LV free wall compact layer thickness (C) and apical wall thickness (D) from E13.5 mice collected from three litters of mice. P value was calculated using a t test. (E) Short-axis view of LV in systole viewed by echocardiography of P4 mice with indicated Mkl2 genotypes. m, myocardium signified by dark area; e, endocardium indicated by arrowheads. (F and G) Average pixel intensity (PI) (F) and minimum PI (G) within the LV of P4 mouse hearts calculated from three litters of mice. P value was calculated using a t test.

Mendelian ratios were observed for Nkx2-5+/A118S or Mkl2+/Q664H mice and, although animals homozygous for each did not exhibit evidence of cardiac dysfunction by echocardiography, they had subtle abnormalities in the ventricular wall before the first week of life. At embryonic time points, we noted a thin apical myocardial wall in Nkx2-5A118S homozygous mice (Fig. 3, B to D). In Mkl2Q664H-homozygous mice, marked echogenic foci in the left ventricular cavity of postnatal day 4 (P4) homozygous mice were present and were similar to those in human patients with LVNC (Fig. 3, E to G, and movies S1 to S3). Previous work did not observe a similar phenotype in mice with cardiomyocyte-specific deletion of Mkl2, suggesting that the echogenicity results from MKL2’s involvement in the development of another cell type within the heart (29). These results indicate that the SNVs identified in MYH7, MKL2, and NKX2-5 adversely affect the ability of the encoded protein to promote timely ventricular development in a homozygous mouse model.

Triple–compound heterozygous mice exhibit LVNC

We next investigated whether compound heterozygosity of the MKL2, MYH7, and NKX2-5 variants produced an LVNC-like phenotype in mice. Immunohistochemistry with an antibody to the endocardial marker endomucin at P3 revealed mild hypertrabeculation and apical recesses in Mkl2Q664H/+Myh7L387F/+ and Nkx2-5A118S/+ mice (Fig. 4A). By contrast, triple-heterozygous mice (Mkl2Q664H/+Myh7L387F/+Nkx2-5A118S/+) exhibited deep trabeculations in the left ventricular wall that were similar to those seen in patients with LVNC and to those observed in the autopsy of the affected child in the family described here (Fig. 4A). Quantification of P3 sections confirmed a decrease in the apical wall thickness and a statistically significant difference in trabecular complexity but revealed no changes in LV free wall thickness (Fig. 4, B to D). Few differences were noted between Myh7L387F/+Nkx2-5A118S/+ and Nkx2-5A118S/+ mice, illustrating the contribution of Mkl2Q664H/+ to the LV phenotype (Fig. 4, A to D). There were no major phenotypic differences between WT and Myh7L387F/+ mice, consistent with the hypothesis that this variant does not independently lead to severe disease (fig. S4A).

Fig. 4 Mkl2Q664H/+Myh7L387F/+Nkx2-5A118S/+ compound-heterozygous mice recapitulate features of human LVNC.

(A) Left, Representative histologic sections in four-chambered views of hearts from P3 mice. Scale bar, 200 mm. Right, Higher magnification of the apical region of the same hearts shown on the left. Scale bar, 50 mm. Shown is immunohistochemistry with green indicating wheat germ agglutinin (cell membrane) and red indicating endomucin (endocardium). (B to D) Quantification of LV free wall thickness (B), apical wall thickness (C), and trabeculation (D) based on fractal dimension analysis. *P < 0.05, **P < 0.01; ns, not significant. P values were calculated by one-way analysis of variance (ANOVA) with Tukey’s multiple-comparisons test. Means are indicated by horizontal bars. Data were collected from 10 litters of mice. (E) Heatmap of all genes differentially expressed between WT and Mkl2Q664H/+Myh7L387F/+ or Mkl2Q664H/+Myh7L387F/+Nkx2-5A118S/+ mice. Key at bottom indicates log2-fold change. (F) Heatmap of selected genes chosen from and generated as in (E).

Despite the ventricular hypertrabeculation, cardiac function by echocardiography was normal at baseline in adult Mkl2Q664H/+Myh7L387F/+Nkx2-5A118S/+ mice (fig. S3, B and C). Transverse aortic constriction was used to increase the pressure load on the left ventricle and caused a statistically significant reduction in cardiac function in triple-heterozygous mice compared with WT mice (fig. S4, D and E). This pathological response exhibited incomplete penetrance, suggesting that the triple-heterozygous mice were on the threshold of functional abnormality.

RNA sequencing of tissue from the apex of P7 hearts revealed subtle yet statistically significant up-regulation of genes associated with metabolism in triple–compound heterozygous mice, whereas genes associated with vasculogenesis were down-regulated (fig. S4, F and G, and table S3). Of 378 protein-coding genes differentially expressed between WT and triple-heterozygous mice, 158 were also differentially expressed between WT and Mkl2Q664H/+Myh7L387F/+ mice, more than expected by chance (Fig. 4E and table S4) (Fisher’s test P value = 8.8e-155, odds ratio = 37.6). However, 58% (224 out of 378) of genes were uniquely differentially expressed in triple-heterozygous mice, including many essential for cardiovascular development and function, highlighting the contribution of the Nkx2-5 A118S variant.

Increased expression of genes involved in the cell cycle and mitosis was observed in triple-heterozygous mice, supporting previous evidence that the noncompaction phenotype is associated with dysregulation of proliferation (fig. S4G) (30, 31). Genes expressed at higher levels in the myocardial trabeculae during embryonic development were also up-regulated in triple-mutant mice, supporting the histological observation of hypertrabeculation (Fig. 4F and table S4) (32). Genes associated with earlier stages of development and bound by Nkx2-5 were additionally expressed at higher levels in triple–compound heterozygous mice, consistent with a less-mature state compared with WT (Fig. 4F) (3336). Conversely, genes associated with endothelial cell development and the coronary vasculature were down-regulated (Fig. 4F and table S4). Disruption of Notch signaling contributes to improper endothelial cell development and subsequent LVNC through reciprocal interactions with the myocardium, suggesting that poor endothelial cell function may be associated with the LVNC phenotype (30, 37). Consistent with our observations, RNA sequencing after cardiomyocyte-specific deletion of Mkl1/2 hearts similarly demonstrated dysregulation of epithelial cell–related pathways, but no disruption in cardiomyocyte function (29). Although we cannot exclude the possibility that additional variants influence the disease phenotype in humans, these results suggest that inheritance of the SNVs in Mkl2, Myh7, and Nkx2-5 is sufficient to mimic the pathology of LVNC in a mouse model.

Human iPSC-derived cardiomyocytes exhibit disease-related alterations

To determine whether the phenotype exhibited by triple-heterozygous mice reflected the effects of the genetic mutations in human cardiomyocytes, we generated patient-specific hiPSC lines from multiple family members and differentiated them to cardiomyocytes using WNT pathway modulation (Fig. 5, A and B) (38). Differentiation efficiency was similar in all lines, as assessed by cardiac troponin T expression (fig. S5A). However, discrepancies in adherence between patient lines became apparent by day 7 of differentiation as cells derived from an individual with symptomatic LVNC formed aggregates that were less apparent in the unaffected individual (Fig. 5, B and C), consistent with previous work showing that Mkl2 regulates genes associated with cell adhesion (21).

Fig. 5 Human iPSC-derived cardiomyocytes exhibit features of LVNC.

(A) Pedigree of family members included in hiPSC studies. (B) Bright-field images of hiPSC-derived cardiomyocytes from three family members as indicated. Scale bars, 100 mm. Orange arrowheads indicate abnormal aggregation. (C) Quantification of cellular aggregation. y axis is log scale. n = 2. *P < 0.05, **P < 0.01. (D) Heatmap depicting log2-fold change of cardiomyocyte maturation–related genes in hiPSC-derived cardiomyocytes from asymptomatic (2) and symptomatic (3) LVNC individuals compared with those from an unaffected individual (1). Key at bottom indicates log2-fold change. A full list of genes can be found in table S6. (E) Box plot illustrating the distance from genes expressed at higher levels in the individual labeled in red compared with black to the closest NKX2-5 ChIP-sequencing peak. Peak distances > 2 megabases (Mb) were excluded from the graph. *P = 0.005, ns = not significant. P values were calculated using Wilcoxon’s rank-sum test with Bonferroni correction.

RNA sequencing on day 8 of cardiomyocyte differentiation revealed down-regulation of gene sets associated with cell adhesion and extracellular matrix deposition in the symptomatic case (MKL2Q670H/+MYH7L387F/+NKX2-5A119S/+), supporting the visual observation of decreased adhesion of these cells (fig. S5B). Gene ontology analysis revealed up-regulation of cell cycle and cardiac developmental genes in cells derived from the symptomatic LVNC case, which was similar to that observed in triple-heterozygous mice (fig. S5B and table S5). Evaluation of genes associated with the cardiac progenitor state and trabecular myocardium exhibited higher expression in the LVNC lines compared with the unaffected line (Fig. 5D and table S6). There was no temporal change in T (Brachyury) induction and repression, suggesting normal mesendoderm specification (fig. S5C); however, BMP10, a marker of trabeculated myocardium, exhibited delayed activation (fig. S5D).

A statistically significant overlap was observed between differentially expressed genes shared by the asymptomatic and symptomatic individuals’ cell lines compared with those from unaffected individuals (fig. S5E). However, in agreement with the mouse transcriptome data, the fold change of many key genes was often greater in the cell line derived from the individual diagnosed with symptomatic LVNC and harboring all three genetic variants (Fig. 5D). Despite hiPSC-derived cardiomyocytes representing an earlier stage of development than the mouse heart tissue included in this study (39), we also found that 43 genes were differentially expressed in both model systems (table S7).

To infer gene dysregulation that may be related to disruption of NKX2-5 function due to the A119S SNV, we used published NKX2-5 chromatin immunoprecipitation (ChIP)–sequencing data collected on day 10 of cardiomyocyte differentiation (table S8) (40). We found that genes expressed at higher levels in the triple-heterozygous childhood-onset individual (Fig. 5A, no. 3) compared with her father (Fig. 5A, no. 2) were significantly closer to NKX2-5–binding events compared with gene sets identified in alternative differential expression scenarios and randomly permuted data (Fig. 5E; 3 versus 2 compared with permuted peak set, P = 7.12 × 10−13; 3 versus 2 compared with randomly sampled expressed genes, P = 0.002). The lack of association between genes expressed at lower levels may suggest that the populations from the unaffected and asymptomatic individuals have progressed to a developmental state whose gene signature is not associated with NKX2-5 binding (fig. S5G). Collectively, these results suggest that, whereas disruption of proper endothelial cell development and function may contribute to LVNC, there is a cardiomyocyte cell-autonomous component.

Discussion

The development of assays to test variants of unknown significance is essential for the advancement of precision medicine initiatives. Although deletions, insertions, and frameshift variants have predictable consequences, the effect of millions of SNVs identified in each individual’s genome is difficult to assess computationally. Recent advances in gene-editing technologies have created an avenue to interrogate the contribution of these variants to phenotypes and disease (14). Our data suggest that traditional metrics used to identify phenotype-associated variants are likely not designed to identify the subtle effects of genetic modifiers (41) and that such genetic modifiers may explain the wide spectrum of cardiomyopathies observed among individuals with mutations in the same sarcomeric gene (42). Accordingly, additional experimentation and analysis will ultimately expose the prevalence and context in which NKX2-5 A119S, or other missense variants, can function as a modifier of CHD or cardiomyopathies.

Using human genetic variation to investigate biological processes experimentally will undoubtedly reveal new insights regarding disease mechanisms. As we refine our understanding of the regulatory mechanisms that govern cell-autonomous and non–cell-autonomous cellular states using advances in both gene-editing and single-cell next-generation sequencing approaches, our ability to correlate genetic variation with phenotypic outcome will improve, bringing precision medicine closer to reality. Although various genetic mechanisms, such as noncoding variation and omnigenics, have advanced our understanding of the genetics underlying complex phenotypes, the work presented here suggests that experimental exploration of SNV-associated phenotypes is a worthwhile endeavor and can shed light on the mechanisms of complex diseases.

Supplementary Materials

science.sciencemag.org/content/364/6443/865/suppl/DC1

Materials and Methods

Figs. S1 to S5

Tables S1 to S8

References (4458)

Movies S1 to S3

References and Notes

Acknowledgments: We thank K. Samse-Knapp and the members of the Srivastava laboratory and Gladstone Institutes for helpful discussion and feedback; the Gladstone Histology and Microscopy, Stem Cell, Transgenic, and Genomics Core for making this work possible; the family who generously offered to be part of this study; and B. Taylor and K. Claiborn for editorial assistance. Funding: C.A.G. is an HHMI fellow of the Damon Runyon Cancer Research Foundation (DRG-2206-14). Y.K.B. was a Gladstone CIRM scholar (TG2-01160). S.S.R. is a Winslow Family Fellow. D.S. is supported by NHLBI/NIH grants (R01 HL057181, U01 HL098179, U01 HL100406, and UM1 HL128671), the Roddenberry Foundation, the L.K. Whittier Foundation, and the Younger Family Fund. This work was also supported by NIH/NCRR grant C06 RR018928 to Gladstone Institutes. Author contributions: C.A.G. and D.S. conceived the study, interpreted the data, and wrote the manuscript. S.K.W. and C.A.G. analyzed exome-sequencing data. C.A.G., S.S.R., T.Y.D.S., and H.T.S. collected embryos, sectioned tissue, and stained slides. C.A.G. and R.S. performed genotyping of mice. C.A.G. and R.S. cultured and differentiated hiPSCs. C.A.G. made RNA-sequencing libraries and analyzed data. C.A.G. analyzed NKX2-5 ChIP-sequencing data. K.R.C.M., K.N.I., and Y.K.B. collected DNA for exomes and tissue to generate hiPSCs. C.A.G. and P.Z. cloned vectors and performed luciferase assays. Y.H. and H.T.S. performed echocardiography and analyzed data. A.E. imaged heart sections. P.U. examined histological sections from human heart. Competing interests: D.S. is a co-founder and member of the board of directors of Tenaya Therapeutics. D.S. and K.I. have equity in Tenaya Therapeutics. Data and materials availability: All raw data are available in the manuscript or have been deposited to the Gene Expression Omnibus GSE131323 or the Sequence Read Archive (PRJNA531964).
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