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This protocol describes a robust pipeline for simultaneously analyzing multiple samples by single-nucleus (sn)RNA-seq. cDNA obtained from each single sample are labeled with the same lipid-coupled oligonucleotide barcode (10X Genomics). Nuclei from as many as 12 individual samples can be pooled together and simultaneously processed for cDNA library construction and subsequent DNA sequencing. While previous protocols using lipid-coupled oligonucleotide barcodes were optimized for analysis of samples consisting of viable cells, this protocol is optimized for analyses of quick-frozen cell samples. The protocol ensures efficient recovery of nuclei both by incorporating high sucrose buffered solutions and by including a tracking dye (trypan blue) during nuclei isolation. The protocol also describes a procedure for removing single nuclei 'artifacts' by removing cell debris prior to single nuclear fractionation. This protocol informs the use of computational tools for filtering poorly labeled nuclei and assigning sample identity using barcode unique molecular identifier (UMI) read counts percentages. The computational pipeline is applicable to either cultured or primary, fresh or frozen cells, regardless of their cell types and species. Overall, this protocol reduces batch effects and experimental costs while enhancing sample comparison. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Labeling cells with lipid oligo barcodes and generating multiplexed single-nucleus RNA-seq libraries Basic Protocol 2: Bioinformatic deconvolution of the multiplexed snRNAseq libraries.

Dominant missense pathogenic variants in cardiac myosin heavy chain cause hypertrophic cardiomyopathy (HCM), a currently incurable disorder that increases risk for stroke, heart failure and sudden cardiac death. In this study, we assessed two different genetic therapies—an adenine base editor (ABE8e) and a potent Cas9 nuclease delivered by AAV9—to prevent disease in mice carrying the heterozygous HCM pathogenic variant myosin R403Q. One dose of dual-AAV9 vectors, each carrying one half of RNA-guided ABE8e, corrected the pathogenic variant in ≥70% of ventricular cardiomyocytes and maintained durable, normal cardiac structure and function. An additional dose provided more editing in the atria but also increased bystander editing. AAV9 delivery of RNA-guided Cas9 nuclease effectively inactivated the pathogenic allele, albeit with dose-dependent toxicities, necessitating a narrow therapeutic window to maintain health. These preclinical studies demonstrate considerable potential for single-dose genetic therapies to correct or silence pathogenic variants and prevent the development of HCM.

BACKGROUND: Known genetic causes of congenital heart disease (CHD) explain METHODS: We tested de novo variants from trio studies of 2649 CHD probands and their parents, as well as rare (allele frequency, − 6 ) variants from 4472 CHD probands in the Pediatric Cardiac Genetics Consortium through a combined computational and in vitro approach. RESULTS: We identified 53 de novo and 74 rare variants in CHD cases that alter splicing and thus are loss of function. Of these, 77 variants are in known dominant, recessive, and candidate CHD genes, including KMT2D and RBFOX2 . In 1 case, we confirmed the variant’s predicted impact on RNA splicing in RNA transcripts from the proband’s cardiac tissue. Two probands were found to have 2 loss-of-function variants for recessive CHD genes HECTD1 and DYNC2H1 . In addition, SpliceAI—a predictive algorithm for altered RNA splicing—has a positive predictive value of ≈93% in our cohort. CONCLUSIONS: Through assessment of RNA splicing, we identified a new loss-of-function variant within a CHD gene in 78 probands, of whom 69 (1.5%; n=4472) did not have a previously established genetic explanation for CHD. Identification of splice-altering variants improves diagnostic classification and genetic diagnoses for CHD. REGISTRATION: URL: https://clinicaltrials.gov ; Unique identifier: NCT01196182.

Understanding how the atrial and ventricular chambers of the heart maintain their distinct identity is a prerequisite for treating chamber-specific diseases. Here, we selectively inactivated the transcription factorTbx5in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. AtrialTbx5inactivation downregulated highly chamber specific genes such asMyl7andNppa, and conversely, increased the expression of ventricular identity genes includingMyl2. Using combined single nucleus transcriptome and open chromatin profiling, we assessed genomic accessibility changes underlying the altered atrial identity expression program, identifying 1846 genomic loci with greater accessibility in control atrial cardiomyocytes compared to KO aCMs. 69% of the control-enriched ATAC regions were bound by TBX5, demonstrating a role for TBX5 in maintaining atrial genomic accessibility. These regions were associated with genes that had higher expression in control aCMs compared to KO aCMs, suggesting they act as TBX5-dependent enhancers. We tested this hypothesis by analyzing enhancer chromatin looping using HiChIP and found 510 chromatin loops that were sensitive to TBX5 dosage. Of the loops enriched in control aCMs, 73.7% contained anchors in control-enriched ATAC regions. Together, these data demonstrate a genomic role for TBX5 in maintaining the atrial gene expression program by binding to atrial enhancers and preserving tissue-specific chromatin architecture of atrial enhancers.

Rationale: Dominant heterozygous variants in filamin C ( FLNC ) cause diverse cardiomyopathies, although the underlying molecular mechanisms remain poorly understood. Objective: We aimed to define the molecular mechanisms by which FLNC variants altered human cardiomyocyte gene and protein expression, sarcomere structure, and contractile performance. Methods and Results: Using CRISPR/Cas9, we introduced FLNC variants into human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). We compared isogenic hiPSC-CMs with normal (wild-type), ablated expression ( FLNC −/− ), or haploinsufficiency ( FLNC +/− ) that causes dilated cardiomyopathy. We also studied a heterozygous in-frame deletion ( FLNC +/Δ7aa ) which did not affect FLNC expression but caused aggregate formation, similar to FLNC variants associated with hypertrophic cardiomyopathy. FLNC −/− hiPSC-CMs demonstrated profound sarcomere misassembly and reduced contractility. Although sarcomere formation and function were unaffected in FLNC +/ − and FLNC +/Δ7aa hiPSC-CMs, these heterozygous variants caused increases in lysosome content, enhancement of autophagic flux, and accumulation of FLNC-binding partners and Z-disc proteins. Conclusions: FLNC expression is required for sarcomere organization and physiological function. Variants that produce misfolded FLNC proteins cause the accumulation of FLNC and FLNC-binding partners which leads to increased lysosome expression and activation of autophagic pathways. Surprisingly, similar pathways were activated in FLNC haploinsufficient hiPSC-CMs, likely initiated by the loss of stoichiometric FLNC protein interactions and impaired turnover of proteins at the Z-disc. These results indicate that both FLNC haploinsufficient variants and variants that produce misfolded FLNC protein cause disease by similar proteotoxic mechanisms and indicate the therapeutic potential for augmenting protein degradative pathways to treat a wide range of FLNC -related cardiomyopathies.

INTRODUCTION Human heart failure is a highly morbid condition that affects 23 million individuals worldwide. It emerges in the setting of an array of different cardiovascular disorders, which has propelled the notion that diverse stimuli converge on a common final pathway. Consistent with this, initiating etiologies do not direct heart failure treatments, which are often inadequate and necessitate mechanical interventions and cardiac transplantation. The recent application of single-nucleus RNA sequencing (snRNAseq) transcriptional analyses to characterize the cellular composition and molecular states in the healthy adult human heart provides an emerging benchmark by which disease-related changes can be assessed. Moreover, the discovery of human pathogenic variants that cause dilated cardiomyopathy (DCM) and arrhythmogenic cardiomyopathy (ACM), disorders associated with high rates of heart failure, provides direct opportunities to evaluate whether genotype influences heart failure pathways. RATIONALE A systematic identification of shared and distinct molecules and pathways involved in heart failure is lacking, and knowledge of these fundamental data could propel the development of more effective treatments. To enable these discoveries, we performed snRNAseq of explanted ventricular tissues from 18 healthy donors and 61 heart failure patients. By focusing analyses on multiple samples with pathogenic variants in DCM genes (LMNA, RBM20, and TTN), ACM genes (PKP2), or pathogenic variant–negative (PV negative) samples, we characterized genotype-stratified and common heart failure responses. RESULTS From 881,081 nuclei isolated from left and right diseased and healthy ventricles, we identified 10 major cell types and 71 distinct transcriptional states. DCM and ACM tissues showed significant depletion of cardiomyocytes and increased endothelial and immune cells. Fibrosis was expanded in disease hearts, but, unexpectedly, fibroblasts were not increased, and instead showed altered transcriptional states that indicated activated remodeling of the extracellular matrix. Genotype-stratified analyses identified multiple transcriptional changes shared only among the hearts harboring pathogenic variants or distinctive for individual and subsets of DCM and ACM genotypes. We validated many of these by single-molecule fluorescent in situ hybridization. Through analyses of receptor and ligand expression across all cells, we observed changes in intercellular signaling and communications, such as increased endothelin signaling in LMNA hearts, tumor necrosis factor in PKP2 hearts, and others. We also identified specific cardiac cell lineages expressing genes with common polymorphisms that were identified in validated association studies of DCM. Because our findings indicated genotype-enriched transcripts and cell states, we harnessed machine learning to develop a graph attention network for the multinomial classification of genotypes. This network showed remarkably high prediction of the genotypes for each cardiac sample, thereby reinforcing our conclusion that genotypes activate very specific heart failure pathways. CONCLUSION snRNAseq of human ventricular samples illuminated cell types and states, molecular signals, and intercellular communications that characterize DCM and ACM. The cellular and molecular architectures that induce heart failure are both shared and distinct across genotypes. These data provide candidate therapeutic targets for future research and interventional opportunities to improve and personalize treatments for cardiomyopathies and heart failure.

Craniofacial microsomia (CFM) represents a spectrum of craniofacial malformations, ranging from isolated microtia with or without aural atresia to underdevelopment of the mandible, maxilla, orbit, facial soft tissue, and/or facial nerve. The genetic causes of CFM remain largely unknown.We performed genome sequencing and linkage analysis in patients and families with microtia and CFM of unknown genetic etiology. The functional consequences of damaging missense variants were evaluated through expression of wild-type and mutant proteins in vitro.We studied a 5-generation kindred with microtia, identifying a missense variant in FOXI3 (p.Arg236Trp) as the cause of disease (logarithm of the odds = 3.33). We subsequently identified 6 individuals from 3 additional kindreds with microtia-CFM spectrum phenotypes harboring damaging variants in FOXI3, a regulator of ectodermal and neural crest development. Missense variants in the nuclear localization sequence were identified in cases with isolated microtia with aural atresia and found to affect subcellular localization of FOXI3. Loss of function variants were found in patients with microtia and mandibular hypoplasia (CFM), suggesting dosage sensitivity of FOXI3.Damaging variants in FOXI3 are the second most frequent genetic cause of CFM, causing 1% of all cases, including 13% of familial cases in our cohort.

Microtia is a congenital malformation that encompasses mild hypoplasia to complete loss of the external ear, or pinna. Although the contribution of genetic variation and environmental factors to microtia remains elusive, Amerindigenous populations have the highest reported incidence. Here, using both transmission disequilibrium tests and association studies in microtia trios (parents and affected child) and microtia cohorts enrolled in Latin America, we map an ∼10-kb microtia locus (odds ratio = 4.7; P = 6.78e-18) to the intergenic region between Roundabout 1 (ROBO1) and Roundabout 2 (ROBO2) (chr3: 78546526 to 78555137). While alleles at the microtia locus significantly increase the risk of microtia, their penetrance is low (1%). We demonstrate that the microtia locus contains a polymorphic complex repeat element that is expanded in affected individuals. The locus is located near a chromatin loop region that regulates ROBO1 and ROBO2 expression in induced pluripotent stem cell–derived neural crest cells. Furthermore, we use single nuclear RNA sequencing to demonstrate ROBO1 and ROBO2 expression in both fibroblasts and chondrocytes of the mature human pinna. Because the microtia allele is enriched in Amerindigenous populations and is shared by some East Asian subjects with craniofacial malformations, we propose that both populations share a mutation that arose in a common ancestor prior to the ancient migration of Eurasian populations into the Americas and that the high incidence of microtia among Amerindigenous populations reflects the population bottleneck that occurred during the migration out of Eurasia.

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p= 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p= 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean= 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p= 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r= 0.975) and RNA VAF (r= 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.

A genetic etiology is identified for one-third of patients with congenital heart disease (CHD), with 8% of cases attributable to coding de novo variants (DNVs). To assess the contribution of noncoding DNVs to CHD, we compared genome sequences from 749 CHD probands and their parents with those from 1,611 unaffected trios. Neural network prediction of noncoding DNV transcriptional impact identified a burden of DNVs in individuals with CHD (n = 2,238 DNVs) compared to controls (n = 4,177; P = 8.7 × 10-4). Independent analyses of enhancers showed an excess of DNVs in associated genes (27 genes versus 3.7 expected, P = 1 × 10-5). We observed significant overlap between these transcription-based approaches (odds ratio (OR) = 2.5, 95% confidence interval (CI) 1.1-5.0, P = 5.4 × 10-3). CHD DNVs altered transcription levels in 5 of 31 enhancers assayed. Finally, we observed a DNV burden in RNA-binding-protein regulatory sites (OR = 1.13, 95% CI 1.1-1.2, P = 8.8 × 10-5). Our findings demonstrate an enrichment of potentially disruptive regulatory noncoding DNVs in a fraction of CHD at least as high as that observed for damaging coding DNVs.

Description, PKP2 mutations lead to instability in cell-cell junctions and sarcomeres that impairs cardiac tissue contractility in ACM., Progressive loss of cardiac systolic function in arrhythmogenic cardiomyopathy (ACM) has recently gained attention as an important clinical consideration in managing the disease. However, the mechanisms leading to reduction in cardiac contractility are poorly defined. Here, we use CRISPR gene editing to generate human induced pluripotent stem cells (iPSCs) that harbor plakophilin-2 truncating variants (PKP2tv), the most prevalent ACM-linked mutations. The PKP2tv iPSC–derived cardiomyocytes are shown to have aberrant action potentials and reduced systolic function in cardiac microtissues, recapitulating both the electrical and mechanical pathologies reported in ACM. By combining cell micropatterning with traction force microscopy and live imaging, we found that PKP2tvs impair cardiac tissue contractility by destabilizing cell-cell junctions and in turn disrupting sarcomere stability and organization. These findings highlight the interplay between cell-cell adhesions and sarcomeres required for stabilizing cardiomyocyte structure and function and suggest fundamental pathogenic mechanisms that may be shared among different types of cardiomyopathies.

Background:HeterozygousTTNtruncating variants cause 10% to 20% of idiopathic dilated cardiomyopathy (DCM). Although variants which disrupt canonical splice signals (ie, invariant dinucleotide of the splice donor site, invariant dinucleotide of the splice acceptor site) at exon-intron junctions are readily recognized asTTNtruncating variants, the effects of other nearby sequence variations on splicing and their contribution to disease is uncertain.Methods:Rare variants of unknown significance located in the splice regions of highly expressedTTNexons from 203 DCM cases, 3329 normal subjects, and clinical variant databases were identified. The effects of these variants on splicing were assessed using an in vitro splice assay.Results:Splice-altering variants of unknown significance were enriched in DCM cases over controls and present in 2% of DCM patients (P=0.002). Application of this method to clinical variant databases demonstrated 20% of similar variants of unknown significance inTTNsplice regions affect splicing. Noncanonical splice-altering variants were most frequently located at position +5 of the donor site (P=4.4×107) and position -3 of the acceptor site (P=0.002). SpliceAI, an emerging in silico prediction tool, had a high positive predictive value (86%–95%) but poor sensitivity (15%–50%) for the detection of splice-altering variants. Alternate exons spliced out of mostTTNtranscripts frequently lacked the consensus base at +5 donor and −3 acceptor positions.Conclusions:Noncanonical splice-altering variants inTTNexplain 1-2% of DCM and offer a 10-20% increase in the diagnostic power ofTTNsequencing in this disease. These data suggest rules that may improve efforts to detect splice-altering variants in other genes and may explain the low percent splicing observed for many alternateTTNexons.

SUMMARYThe early limb bud consists of mesenchymal progenitors (limb progenitors) derived from the lateral plate mesoderm (LPM) that produce most of the tissues of the mature limb bud. The LPM also gives rise to the mesodermal components of the trunk, flank and neck. However, the mesenchymal cells generated at these other axial levels cannot produce the variety of cell types found in the limb bud, nor can they be directed to form a patterned appendage-like structure, even when placed in the context of the signals responsible for organizing the limb bud. Here, by taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28) normally expressed in the early limb bud, that are capable of imparting limb progenitor-like properties to non-limb fibroblasts. Cells reprogrammed by these factors show similar gene expression profiles, and can differentiate into similar cell types, as endogenous limb progenitors. The further addition of Lin41 potentiates proliferation of the reprogrammed cells while suppressing differentiation. These results suggest that these same four key factors may play pivotal roles in the specification of endogenous limb progenitors.

Background Human mutations in the X‐linked lysosome‐associated membrane protein‐2 ( LAMP2 ) gene can cause a multisystem Danon disease or a primary cardiomyopathy characterized by massive hypertrophy, conduction system abnormalities, and malignant ventricular arrhythmias. We introduced an in‐frame LAMP2 gene exon 6 deletion mutation (denoted L2 Δ6 ) causing human cardiomyopathy, into mouse LAMP2 gene, to elucidate its consequences on cardiomyocyte biology. This mutation results in in‐frame deletion of 41 amino acids, compatible with presence of some defective LAMP2 protein. Methods and Results Left ventricular tissues from L2 Δ6 and wild‐type mice had equivalent amounts of LAMP2 RNA, but a significantly lower level of LAMP2 protein. By 20 weeks of age male mutant mice developed left ventricular hypertrophy which was followed by left ventricular dilatation and reduced systolic function. Cardiac electrophysiology and isolated cardiomyocyte studies demonstrated ventricular arrhythmia, conduction disturbances, abnormal calcium transients and increased sensitivity to catecholamines. Myocardial fibrosis was strikingly increased in 40‐week‐old L2 Δ6 mice, recapitulating findings of human LAMP2 cardiomyopathy. Immunofluorescence and transmission electron microscopy identified mislocalization of lysosomes and accumulation of autophagosomes between sarcomeres, causing profound morphological changes disrupting the cellular ultrastructure. Transcription profile and protein expression analyses of L2 Δ6 hearts showed significantly increased expression of genes encoding activators and protein components of autophagy, hypertrophy, and apoptosis. Conclusions We suggest that impaired autophagy results in cardiac hypertrophy and profound transcriptional reactions that impacted metabolism, calcium homeostasis, and cell survival. These responses define the molecular pathways that underlie the pathology and aberrant electrophysiology in cardiomyopathy of Danon disease.

BACKGROUND: Heterozygous truncating variants in titin (TTNtv) cause 10–20% of idiopathic dilated cardiomyopathy (DCM). Though variants which disrupt canonical splice signals (i.e. GT, AG) at exon-intron junctions are readily recognized as TTNtv, the effects of other nearby sequence variations on splicing and their contribution to disease is uncertain. METHODS: Rare variants of unknown significance (VUS) located in the splice regions of highly expressed TTN exons from 203 DCM cases, 3329 normal subjects, and clinical variant databases were identified. The effects of these variants on splicing were assessed using an in vitro splice assay. RESULTS: Splice-altering VUS were enriched in DCM cases over controls and present in 2% of DCM patients (p=0.002). Application of this method to clinical variant databases demonstrated 20% of similar VUS in TTN splice regions affect splicing. Non-canonical splice-altering variants were most frequently located at position +5 of the donor site (p=4.4e-7) and position −3 of the acceptor site (p=0.002). SpliceAI, an emerging in-silico prediction tool, had a high positive predictive value (86–95%) but poor sensitivity (15–50%) for the detection of splice-altering variants. Alternate exons spliced out of most TTN transcripts frequently lacked the consensus base at +5 donor and −3 acceptor positions. CONCLUSION: Non-canonical splice-altering variants in TTN explain 1–2% of DCM and offer a 10–20% increase in the diagnostic power of TTN sequencing in this disease. These data suggest rules that may improve efforts to detect splice-altering variants in other genes and may explain the low percent splicing observed for many alternate TTN exons.

Motivation Non-coding rare variants (RVs) may contribute to Mendelian disorders but have been challenging to study due to small sample sizes, genetic heterogeneity and uncertainty about relevant non-coding features. Previous studies identified RVs associated with expression outliers, but varying outlier definitions were employed and no comprehensive open-source software was developed. Results We developed Outlier-RV Enrichment (ORE) to identify biologically-meaningful non-coding RVs. We implemented ORE combining whole-genome sequencing and cardiac RNAseq from congenital heart defect patients from the Pediatric Cardiac Genomics Consortium and deceased adults from Genotype-Tissue Expression. Use of rank-based outliers maximized sensitivity while a most extreme outlier approach maximized specificity. Rarer variants had stronger associations, suggesting they are under negative selective pressure and providing a basis for investigating their contribution to Mendelian disorders. Availability and implementation ORE, source code, and documentation are available at https://pypi.python.org/pypi/ore under the MIT license. Supplementary information Supplementary data are available at Bioinformatics online.

Rationale: NAA15 (N-alpha-acetyltransferase 15) is a component of the NatA (N-terminal acetyltransferase complex). The mechanism by which NAA15 haploinsufficiency causes congenital heart disease remains unknown. To better understand molecular processes by which NAA15 haploinsufficiency perturbs cardiac development, we introduced NAA15 variants into human induced pluripotent stem cells (iPSCs) and assessed the consequences of these mutations on RNA and protein expression. Objective: We aim to understand the role of NAA15 haploinsufficiency in cardiac development by investigating proteomic effects on NatA complex activity and identifying proteins dependent upon a full amount of NAA15. Methods and Results: We introduced heterozygous loss of function, compound heterozygous, and missense residues (R276W) in iPSCs using CRISPR/Cas9. Haploinsufficient NAA15 iPSCs differentiate into cardiomyocytes, unlike NAA15 -null iPSCs, presumably due to altered composition of NatA. Mass spectrometry analyses reveal ≈80% of identified iPSC NatA targeted proteins displayed partial or complete N-terminal acetylation. Between null and haploinsufficient NAA15 cells, N-terminal acetylation levels of 32 and 9 NatA-specific targeted proteins were reduced, respectively. Similar acetylation loss in few proteins occurred in NAA15 R276W induced pluripotent stem cells. In addition, steady-state protein levels of 562 proteins were altered in both null and haploinsufficient NAA15 cells; 18 were ribosomal-associated proteins. At least 4 proteins were encoded by genes known to cause autosomal dominant congenital heart disease. Conclusions: These studies define a set of human proteins that requires a full NAA15 complement for normal synthesis and development. A 50% reduction in the amount of NAA15 alters levels of at least 562 proteins and N-terminal acetylation of only 9 proteins. One or more modulated proteins are likely responsible for NAA15-haploinsufficiency mediated congenital heart disease. Additionally, genetically engineered induced pluripotent stem cells provide a platform for evaluating the consequences of amino acid sequence variants of unknown significance on NAA15 function.

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.

The bromodomain and extraterminal (BET) family comprises epigenetic reader proteins that are important regulators of inflammatory and hypertrophic gene expression in the heart. We previously identified the activation of proinflammatory gene networks as a key early driver of dilated cardiomyopathy (DCM) in transgenic mice expressing a mutant form of phospholamban (PLN(R9C)) — a genetic cause of DCM in humans. We hypothesized that BETs coactivate this inflammatory process, representing a critical node in the progression of DCM. To test this hypothesis, we treated PLN(R9C) or age-matched WT mice longitudinally with the small molecule BET bromodomain inhibitor JQ1 or vehicle. BET inhibition abrogated adverse cardiac remodeling, reduced cardiac fibrosis, and prolonged survival in PLN(R9C) mice by inhibiting expression of proinflammatory gene networks at all stages of disease. Specifically, JQ1 had profound effects on proinflammatory gene network expression in cardiac fibroblasts, while having little effect on gene expression in cardiomyocytes. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Mechanistically, we demonstrated that BRD4 serves as a direct and essential regulator of NF-κB–mediated proinflammatory gene expression in cardiac fibroblasts. Suppressing proinflammatory gene expression via BET bromodomain inhibition could be a novel therapeutic strategy for chronic DCM in humans.

Background The 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. Methods We developed a new computational method, EM-mosaic (Expectation-Maximization-based detection of mosaicism), to analyze mosaicism in exome sequences derived primarily from blood DNA of 2530 CHD proband-parent trios. To optimize this method, we measured mosaic detection power as a function of sequencing depth. In parallel, we analyzed our cohort using MosaicHunter, a Bayesian genotyping algorithm-based mosaic detection tool, and compared the two methods. The accuracy of these mosaic variant detection algorithms was assessed using an independent resequencing method. We then applied both methods to detect mosaicism in cardiac tissue-derived exome sequences of 66 participants for which matched blood and heart tissue was available. Results 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 estimated true frequency of mosaic variants above 10% mosaicism was 0.14/person in blood and 0.21/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. Conclusions We estimate that ~ 1% of CHD probands have a mosaic variant detectable in blood that could contribute to cardiac malformations, particularly those damaging variants with relatively higher allele fraction. 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.

SummaryCardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and strategies to improve therapeutic opportunities require deeper understanding of the molecular processes of the normal heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavor. Here, using large-scale single cell and nuclei transcriptomic profiling together with state-of-the-art analytical techniques, we characterise the adult human heart cellular landscape covering six anatomical cardiac regions (left and right atria and ventricles, apex and interventricular septum). Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, revealing distinct subsets in the atria and ventricles indicative of diverse developmental origins and specialized properties. Further we define the complexity of the cardiac vascular network which includes clusters of arterial, capillary, venous, lymphatic endothelial cells and an atrial-enriched population. By comparing cardiac cells to skeletal muscle and kidney, we identify cardiac tissue resident macrophage subsets with transcriptional signatures indicative of both inflammatory and reparative phenotypes. Further, inference of cell-cell interactions highlight a macrophage-fibroblast-cardiomyocyte network that differs between atria and ventricles, and compared to skeletal muscle. We expect this reference human cardiac cell atlas to advance mechanistic studies of heart homeostasis and disease.

The bromodomain and extraterminal (BET) family of epigenetic reader proteins are key regulators of pathologic gene expression in the heart. Using mice carrying a human mutation in phospholamban (PLNR9C) that develop progressive dilated cardiomyopathy (DCM), we previously identified the activation of inflammatory gene networks as a key early driver of DCM. We reasoned that BETs control this inflammatory process, representing a key node in the progression of genetic DCM. Using a chemical genetic strategy, PLNR9C or age-matched wild type mice were treated longitudinally with the BET inhibitor JQ1 or vehicle. JQ1 abrogated DCM, reduced cardiac fibrosis, and prolonged survival in PLNR9C mice by inhibiting inflammatory gene network expression at all disease stages. Cardiac fibroblast proliferation was also substantially reduced by JQ1. Interestingly, JQ1 had profound effects on pathologic gene network expression in cardiac fibroblasts, while having little effect on transcription in cardiomyocytes. Using co-immunoprecipitation, we identified BRD4 as a direct and essential regulator of NFκB-mediated inflammatory gene transcription in cardiac fibroblasts. In this this model of chronic, heritable DCM, BETs activate inflammatory gene networks in cardiac fibroblasts via an NFκB-dependent mechanism, marking them as critical effectors of pathologic gene expression.

Summary Sirtuin 3 (SIRT3) is a NAD+-dependent deacetylase downregulated in aging and age-associated diseases such as cancer and neurodegeneration and in high-fat diet (HFD)-induced metabolic disorders. Here, we performed a small-molecule screen and identified an unexpected metabolic vulnerability associated with SIRT3 loss. Azaserine, a glutamine analog, was the top compound that inhibited growth and proliferation of cells lacking SIRT3. Using stable isotope tracing of glutamine, we observed its increased incorporation into de novo nucleotide synthesis in SIRT3 knockout (KO) cells. Furthermore, we found that SIRT3 KO cells upregulated the diversion of glutamine into de novo nucleotide synthesis through hyperactive mTORC1 signaling. Overexpression of SIRT3 suppressed mTORC1 and growth in vivo in a xenograft tumor model of breast cancer. Thus, we have uncovered a metabolic vulnerability of cells with SIRT3 loss by using an unbiased small-molecule screen., Graphical abstract SIRT3 is lost or downregulated in numerous pathologies. Loss of SIRT3 results in increased cell proliferation. Gonzalez Herrera et al. identify glutamine incorporation into nucleotides to be a driving force behind increased proliferation of cells lacking SIRT3.

Mosaicism due to somatic mutations can cause multiple diseases including cancer, developmental and overgrowth syndromes, neurodevelopmental disorders, autoinflammatory diseases, and atrial fibrillation. With the increased use of next generation sequencing technology, multiple tools have been developed to identify low-frequency variants, specifically from matched tumor-normal tissues in cancer studies. To investigate whether mosaic variants are implicated in congenital heart disease (CHD), we developed a pipeline using the cancer somatic variant caller MuTect to identify mosaic variants in whole-exome sequencing (WES) data from a cohort of parent/affected child trios (n = 715) and a cohort of healthy individuals (n = 416). This is a novel application of the somatic variant caller designed for cancer to WES trio data. We identified two cases with mosaic KMT2D mutations that are likely pathogenic for CHD, but conclude that, overall, mosaicism detectable in peripheral blood or saliva does not account for a significant portion of CHD etiology.

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.

Rationale: Congenital heart disease (CHD) occurs in about 1 in 100 live births, yet known genetic causes explain less than 20% of CHD cases. While variants that cause frameshift, nonsense, start/stop site gain or loss, and canonical splice site alterations are readily categorized as being pathogenic or loss-of-function (LOF), interpreting the clinical significance of variants without obvious functional consequences remains challenging. Here, we aim to improve classification of variants of unknown significance (VUS) in non-canonical RNA splice sites that may be pathogenic for CHD. Methods: We tested candidate LOF de novo (DNV) and rare (p < 2E-5) inherited variants from whole exome sequencing of 4474 CHD probands and their parents in the NHLBI Pediatric Cardiac Genetics Consortium. Briefly, variants underwent computational selection to prioritize VUS in splice regions that are likely to alter splicing (“high-likelihood VUS”). These variants then underwent in vitro analysis including Minigene construction, transfection, RNA isolation, and sequencing to confirm splicing outcomes. Results: Preliminary results limited to DNV variants showed that 163 of 2678 (6.08%) were high-likelihood VUS. Subsequent analysis in vitro assay of high-likelihood VUS yielded 53 as splice-altering (p < 0.05) and thus LOF. Combined with previously identified 366 DNV LOF variants, the addition of these splice-altering variants represents a 15.3% increase in total LOF DNV variant calls. This includes new pathogenic mutations in known CHD genes such as KMT2D . In one case, a CHD proband with features of Kabuki Syndrome without a definitive diagnosis was found to have a splice-altering variant in KMT2D . We have extended this assay to 34518 rare, inherited variants in the same cohort, of which 868 (2.54%) are in genes previously associated with CHD. Conclusion: Consideration of non-canonical RNA splice sites in this assay increased the yield of LOF mutations from traditional sequencing methods by 15.3% in the CHD cohort. Further analysis of splice-altering variants in both known and unknown pathogenic genes will improve diagnostic classification of VUS and gene-based diagnosis of CHD.

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.

Rationale: Neural crest cells (NCCs) play a critical role in normal cardiac development, and defects in NCCs likely cause congenital heart disease (CHD). NCCs are transient and multipotent migratory stem cells that give rise to diverse tissues, including cardiac structures such as the smooth muscles of the great arteries and semilunar valves. Induced pluripotent stem cells (iPSC) can be differentiated into NCCs, as demonstrated by expression of several marker proteins including NGFR and HNK1. To better define iPSC-NCCs and to better understand the progression of iPSCs to NCCs, we have compared the transcriptomes of iPSC and iPSC-NCCs. We have also begun to investigate the consequences of loss-of-function mutations in genes implicated in CHD on the differentiation of iPSC-NCCs. Methods and Results: PGP1 iPSCs were differentiated to NCCs and RNA was collected at 0, 5, 10, and 15 days of differentiation. RNAseq analysis showed that by day 15, 6483 genes were upregulated in NCC vs iPSCs, 6406 downregulated, and 6715 unchanged (FDR 5%). Enrichment analysis for the top 500 upregulated genes showed 4 cardiac gene ontology (GO) terms in the top 10, including ‘endocardial cushion morphogenesis’ (fold enrichment 11.85, p = 0.012). Notably, of 45 genes under GO term ‘endocardial cushion development’, 38 (84%) differentially expressed in NCCs by day 15 (FDR 5%). Next, we compared this RNAseq data with that of iPSC-derived cardiomyocyte (CM) differentiation in a subset of 253 genes previously implicated in CHD. Of these, 143 genes were differentially expressed in both iPSC-CMs and iPSC-NCCs. However, 27 genes (10.6%) including MYH6, PITX2, and TBX5 were uniquely upregulated during CM differentiation, while 65 genes (25.7%) including CHD4 and NOTCH1 were uniquely upregulated during NCC differentiation. Conclusion: Transcriptomic changes during iPSC-NCC differentiation, assessed by both bulk and single cell RNAseq, reveal an upregulation of genes involved in cardiac development, particularly endocardial cushions and the outflow tract. Importantly, a subset of genes implicated in CHD are altered during iPSC-NCC differentiation but not in iPSC-CM differentiation. Thus, iPSC-NCCs offer a new model with which to investigate the pathogenesis and mechanisms of CHD.

Left ventricular (LV) dilatation is a key step in transition to heart failure (HF) in response to pressure overload. Cardiac extracellular matrix (ECM) contains fibrillar collagens and proteoglycans, important for maintaining tissue integrity. Alterations in collagen production and cross-linking are associated with cardiac LV dilatation and HF. Lumican (LUM) is a collagen binding proteoglycan with increased expression in hearts of patients and mice with HF, however, its role in cardiac function remains poorly understood. To examine the role of LUM in pressure overload induced cardiac remodeling, we subjected LUM knock-out (LUMKO) mice to aortic banding (AB) and treated cultured cardiac fibroblasts (CFB) with LUM. LUMKO mice exhibited increased mortality 1–14 days post-AB. Echocardiography revealed increased LV dilatation, altered hypertrophic remodeling and exacerbated contractile dysfunction in surviving LUMKO 1–10w post-AB. LUMKO hearts showed reduced collagen expression and cross-linking post-AB. Transcriptional profiling of LUMKO hearts by RNA sequencing revealed 714 differentially expressed transcripts, with enrichment of cardiotoxicity, ECM and inflammatory pathways. CFB treated with LUM showed increased mRNAs for markers of myofibroblast differentiation, proliferation and expression of ECM molecules important for fibrosis, including collagens and collagen cross-linking enzyme lysyl oxidase. In conclusion, we report the novel finding that lack of LUM attenuates collagen cross-linking in the pressure-overloaded heart, leading to increased mortality, dilatation and contractile dysfunction in mice.

The mechanisms by which truncating mutations in MYBPC3 (encoding cardiac myosin-binding protein C; cMyBPC) or myosin missense mutations cause hypercontractility 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 demonstrated 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 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 patients with DCM, whereas inhibiting myosin by MYK-461 should benefit the substantial proportion of patients with HCM with MYBPC3 mutations.

Immune checkpoint inhibitors have improved clinical outcomes associated with numerous cancers, but high-grade, immune-related adverse events can occur, particularly with combination immunotherapy. We report the cases of two patients with melanoma in whom fatal myocarditis developed after treatment with ipilimumab and nivolumab. In both patients, there was development of myositis with rhabdomyolysis, early progressive and refractory cardiac electrical instability, and myocarditis with a robust presence of T-cell and macrophage infiltrates. Selective clonal T-cell populations infiltrating the myocardium were identical to those present in tumors and skeletal muscle. Pharmacovigilance studies show that myocarditis occurred in 0.27% of patients treated with a combination of ipilimumab and nivolumab, which suggests that our patients were having a rare, potentially fatal, T-cell-driven drug reaction. (Funded by Vanderbilt-Ingram Cancer Center Ambassadors and others.).

Powering down yields a healthier heart In hypertrophic cardiomyopathy (HCM), the heart muscle enlarges and becomes progressively less efficient at pumping blood. HCM can be caused by mutations in components of the sarcomere (the heart's contractile unit), most notably myosin. Hypercontractility is among the earliest heart disturbances seen in mice carrying these myosin mutations, implying that the mutations inflict their damage by increasing myosin's power production. Green et al. identified a small molecule that binds to myosin and inhibits its activity (see the Perspective by Warshaw). When orally administered to young mice, the molecule prevented the development of several hallmark features of HCM without adversely affecting skeletal muscle. Science , this issue p. 617 ; see also p. 556

Heterozygous truncating variants in titin (TTNtv) are the major genetic cause of dilated cardiomyopathy (DCM). Though variants which disrupt essential splicing dinucleotides (GT/AG) are readily recognized as TTNtv, the effects of other nearby sequence variations on splicing is uncertain. We

Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.

Dilated cardiomyopathy (DCM), a disorder that occurs in 1:250 individuals, is associated with rates of mortality of 20% within 5 years of diagnosis and is a leading cause for heart failure and cardiac transplantation. Mutations in the massive sarcomere protein titin (encoded by TTN ) are the most common genetic cause of DCM, occurring in 10-20% of cases. As dominant DCM mutations truncate titin (TTNtv) and result in haploinsufficiency, we predict that strategies to increase the expression of the wild type (WT) TTN allele might attenuate the damaging effects of TTNtv. We used bioinformatic analyses to identify a putative TTN enhancer within intron 1. To confirm its function, we deleted 658 bp from intron 1 that encompasses the putative TTN enhancer in human induced pluripotent stem cells (hiPSCs) using CRISPR/Cas9 genome editing. We used qPCR and RNA sequencing of RNA harvested from hiPSC-derived cardiomyocytes (hiPSC-CMs) and demonstrated that a homozygous deletion in this region leads to decreased TTN gene expression compared to the WT control (0.344 fold change, p < 0.001), and also to decreased expression of other sarcomeric genes such as TNNT2 (0.074 fold change, p < 0.001), MYH6 (0.18 fold change, p < 0.001), MYH7 (0.008 fold change, p < 0.001), and ACTN2 (0.118 fold change, p < 0.001). The expression of transcription factors (TF) that have binding sites in this region is also affected, such as MEIS2 (0.4 fold change, p < 0.001) and KLF6 (0.33 fold change, p < 0.001), both of which are known to be involved in cardiogenesis. These TF may act as a link between the deletion in TTN Intron 1 and a decreased transcription of other cardiac-relevant genes. We also utilized Assay for Transposase-Accessible Chromatin Sequencing (ATAC-Seq) on WT hiPSC-CMs to identify open regions of chromatin that are accessible for TF binding. This provided additional evidence that this 658 bp region in TTN intron 1 has enhancer activity. Ongoing studies are aiming to refine the TTN Intron 1 enhancer by further studies using CRISPR/Cas9, CRISPR droplet sequencing (CROP-Seq), and luciferase-based enhancer activity assays. If confirmed, we expect that increasing the activity of this 658 bp region with small molecules may provide a novel therapeutic target for DCM caused by TTNtv.

We describe a variation of droplet digital PCR (ddPCR) for DNA structural variant analysis, where expensive fluorescent probes are replaced by low-cost EvaGreen, a DNA binding dye, hence reducing cost without reducing sensitivity. ddPCR is a method that employs water-oil microfluidics and fluorescence technology to quantify target nucleic acids with extreme precision and sensitivity. Traditional ddPCR uses standard Taqman probe assays to determine target DNA concentrations, however these two-color fluorescent oligonucleotide probes are expensive and require extensive optimization. EvaGreen’s DNA binding properties allow target products to be effectively quantified at a significantly lower cost. This protocol employs the EvaGreen ddPCR assay to confirm structural variants on a ddPCR system and produces results comparable to the standard, far more expensive Taqman approach.

Identification of sequence variants that create or eliminate splice sites has proven to be a significant challenge and represents one of many roadblocks in the clinical interpretation of rare genetic variation. Current methods of identifying splice altering sequence variants exist, however, these are limited by an imperfect understanding of splice signals and cumbersome functional assays. We have recently developed a computational tool that prioritizes putative splice-altering sequence variants, and a moderate-throughput minigene assay that confirms the variants which alter splicing. This bioinformatic strategy represents a substantial increase in accuracy and efficiency of historical in vitro splicing assays. In this unit we give detailed instructions on how to organize, run, and interpret various features of this protocol. We expect that splice-altering variants revealed through this protocol can be reliably carried forward for further clinical and biological analyses.

and KeywordsBackgroundSex differences exist in the prevalence, presentation, and outcomes of ischemic heart disease. Females have higher risk of heart failure post myocardial infarction relative to males and the female sex is an independent risk factor for hospital and operative mortality after cardiac surgery. However, the mechanisms underlying this sexual dimorphism remain unclear. We examined sex differences in human myocardial gene expression in response to ischemia.MethodsLeft ventricular biopsies from 68 male and 46 female patients undergoing aortic valve replacement surgery were obtained at baseline and after a median 74 minutes of cold cardioplegic arrest/ischemia and respective transcriptomes were quantified by RNA-Seq. Sex-specific responses to ischemia were quantified by differential gene expression, expression quantitative trait loci (eQTL) and pathway and functional analysis. Cell-type enrichment analysis. was used to obtain an estimate of the identity and relative proportions of different cell types present in each sample.ResultsA sex-specific response to ischemia was observed for 271 genes. Functional annotation analysis revealed sex-specific modulation of the oxytocin signaling pathway and common pathway of fibrin clot formation. The eQTL analysis identified variant-by-sex interaction eQTLs at baseline and post-ischemia, indicative of sex differences in the genotypic effects on gene expression, and cell-type enrichment analysis showed sex-bias in proportion of specific cell types.ConclusionIn response to myocardial ischemia, the human left ventricle demonstrates changes in gene expression that differ between the sexes. These differences provide insight into the sexual dimorphism of ischemic heart disease and may aid in the development of sex-specific therapies that reduce myocardial injury.

Multiple tools have been developed to identify copy number variants (CNVs) from whole exome (WES) and whole genome sequencing (WGS) data. Current tools such as XHMM for WES and CNVnator for WGS identify CNVs based on changes in read depth. For WGS, other methods to identify CNVs include utilizing discordant read pairs and split reads and genome-wide local assembly with tools such as Lumpy and SvABA, respectively. Here, we introduce a new method to identify deletion CNVs from WES and WGS trio data based on the clustering of Mendelian errors (MEs). Using our Mendelian Error Method (MEM), we identified 127 deletions (inherited and de novo) in 2,601 WES trios from the Pediatric Cardiac Genomics Consortium, with a validation rate of 88% by digital droplet PCR. MEM identified additional de novo deletions compared to XHMM, and also identified sample switches, DNA contamination, a significant enrichment of 15q11.2 deletions compared to controls and eight cases of uniparental disomy. We applied MEM to WGS data from the Genome In A Bottle Ashkenazi trio and identified deletions with 97% specificity. MEM provides a robust, computationally inexpensive method for identifying deletions, and an orthogonal approach for verifying deletions called by other tools.

Background In the second trimester of human fetal development, a tenfold increase in fetal size occurs while cardiac valves grow and retain their function. Patterns of transcription in normally growing human aortic valves are unknown. Methods Discarded human aortic valve samples were collected from the second trimester, 6 from early (14, 15, 17 weeks) and 6 from late (20, 21, 22 weeks) gestation. Network analysis of RNA sequencing data identified subnetworks of significantly increasing and decreasing transcripts. Subsequent cluster analysis identified patterns of transcription through the time course. Pathway enrichment analysis determined the predominant biological processes at each interval. Results We observed phasic transcription over the time course, including an early decrease in cell proliferation and developmental genes (14 to 15 weeks). Pattern specification, shear stress, and adaptive immune genes were induced early. Cell adhesion genes were increased from 14 to 20 weeks. A phase involving cell differentiation and apoptosis (17 to 20 weeks) was followed by downregulation of endothelial-to-mesenchymal transformation genes and then by increased extracellular matrix organization and stabilization (20 to 22 weeks). Conclusions We present a unique data set, comprehensively characterizing human valve development after valve primordia are formed, focusing on key processes displayed by normal aortic valves undergoing significant growth. We build a time course of genes and processes in second trimester fetal valve growth and observe the sequential regulation of gene clusters over time. Critical valve growth genes are potential targets for therapeutic intervention in congenital heart disease and have implications for regenerative medicine and tissue engineering.

The mechanisms by which truncating mutations in

Deep nucleotide sequencing enables the unbiased, broad-spectrum detection of viruses in clinical samples without requiring an a priori hypothesis for the source of infection. However, its use in clinical research applications is limited by low cost-effectiveness given that most of the sequencing information from clinical samples is related to the human genome, which renders the analysis of viral genomes challenging. To overcome this limitation we developed ViroFind, an in-solution target-enrichment platform for virus detection and discovery in clinical samples. ViroFind comprises 165,433 viral probes that cover the genomes of 535 selected DNA and RNA viruses that infect humans or could cause zoonosis. The ViroFind probes are used in a hybridization reaction to enrich viral sequences and therefore enhance the detection of viral genomes via deep sequencing. We used ViroFind to detect and analyze all viral populations in the brain of 5 patients with progressive multifocal leukoencephalopathy (PML) and of 18 control subjects with no known neurological disease. Compared to direct deep sequencing, by using ViroFind we enriched viral sequences present in the clinical samples up to 127-fold. We discovered highly complex polyoma virus JC populations in the PML brain samples with a remarkable degree of genetic divergence among the JC virus variants of each PML brain sample. Specifically for the viral capsid protein VP1 gene, we identified 24 single nucleotide substitutions, 12 of which were associated with amino acid changes. The most frequent (4 of 5 samples, 80%) amino acid change was D66H, which is associated with enhanced tissue tropism, and hence likely a viral fitness advantage, compared to other variants. Lastly, we also detected sparse JC virus sequences in 10 of 18 (55.5%) of control samples and sparse human herpes virus 6B (HHV6B) sequences in the brain of 11 of 18 (61.1%) control subjects. In sum, ViroFind enabled the in-depth analysis of all viral genomes in PML and control brain samples and allowed us to demonstrate a high degree of JC virus genetic divergence in vivo that has been previously underappreciated. ViroFind can be used to investigate the structure of the virome with unprecedented depth in health and disease state.