Cells of the adult human heart.

Cardiovascular disease is the leading cause of death worldwide. Advanced insights into disease mechanisms and therapeutic strategies require a deeper understanding of the molecular processes involved in the healthy heart. Knowledge of the full repertoire of cardiac cells and their gene expression profiles is a fundamental first step in this endeavour. Here, using state-of-the-art analyses of large-scale single-cell and single-nucleus transcriptomes, we characterize six anatomical adult heart regions. Our results highlight the cellular heterogeneity of cardiomyocytes, pericytes and fibroblasts, and reveal distinct atrial and ventricular subsets of cells with diverse developmental origins and specialized properties. We define the complexity of the cardiac vasculature and its changes along the arterio-venous axis. In the immune compartment, we identify cardiac-resident macrophages with inflammatory and protective transcriptional signatures. Furthermore, analyses of cell-to-cell interactions highlight different networks of macrophages, fibroblasts and cardiomyocytes between atria and ventricles that are distinct from those of skeletal muscle. Our human cardiac cell atlas improves our understanding of the human heart and provides a valuable reference for future studies.

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk.

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.

An ancient founder mutation located between ROBO1 and ROBO2 is responsible for increased microtia risk in Amerindigenous populations.

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 cell­derived 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.

Damaging variants in FOXI3 cause microtia and craniofacial microsomia.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Deciphering the super relaxed state of human ß-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.

Mutations in ß-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 ß-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.

Cardiovascular homeostasis dependence on MICU2, a regulatory subunit of the mitochondrial calcium uniporter.

Comparative analyses of transcriptional profiles from humans and mice with cardiovascular pathologies revealed consistently elevated expression of MICU2, a regulatory subunit of the mitochondrial calcium uniporter complex. To determine if MICU2 expression was cardioprotective, we produced and characterized Micu2-/- mice. Mutant mice had left atrial enlargement and Micu2-/- cardiomyocytes had delayed sarcomere relaxation and cytosolic calcium reuptake kinetics, indicating diastolic dysfunction. RNA sequencing (RNA-seq) of Micu2-/- ventricular tissues revealed markedly reduced transcripts encoding the apelin receptor (Micu2-/- vs. wild type, P = 7.8 × 10-40), which suppresses angiotensin II receptor signaling via allosteric transinhibition. We found that Micu2-/- and wild-type mice had comparable basal blood pressures and elevated responses to angiotensin II infusion, but that Micu2-/- mice exhibited systolic dysfunction and 30% lethality from abdominal aortic rupture. Aneurysms and rupture did not occur with norepinephrine-induced hypertension. Aortic tissue from Micu2-/- mice had increased expression of extracellular matrix remodeling genes, while single-cell RNA-seq analyses showed increased expression of genes related to reactive oxygen species, inflammation, and proliferation in fibroblast and smooth muscle cells. We concluded that Micu2-/- mice recapitulate features of diastolic heart disease and define previously unappreciated roles for Micu2 in regulating angiotensin II-mediated hypertensive responses that are critical in protecting the abdominal aorta from injury.

Identification of pathogenic gene mutations in LMNA and MYBPC3 that alter RNA splicing.

Genetic variants that cause haploinsufficiency account for many autosomal dominant (AD) disorders. Gene-based diagnosis classifies variants that alter canonical splice signals as pathogenic, but due to imperfect understanding of RNA splice signals other variants that may create or eliminate splice sites are often clinically classified as variants of unknown significance (VUS). To improve recognition of pathogenic splice-altering variants in AD disorders, we used computational tools to prioritize VUS and developed a cell-based minigene splicing assay to confirm aberrant splicing. Using this two-step procedure we evaluated all rare variants in two AD cardiomyopathy genes, lamin A/C (LMNA) and myosin binding protein C (MYBPC3). We demonstrate that 13 LMNA and 35 MYBPC3 variants identified in cardiomyopathy patients alter RNA splicing, representing a 50% increase in the numbers of established damaging splice variants in these genes. Over half of these variants are annotated as VUS by clinical diagnostic laboratories. Familial analyses of one variant, a synonymous LMNA VUS, demonstrated segregation with cardiomyopathy affection status and altered cardiac LMNA splicing. Application of this strategy should improve diagnostic accuracy and variant classification in other haploinsufficient AD disorders.

Cardiac myosin binding protein C regulates postnatal myocyte cytokinesis.

Homozygous cardiac myosin binding protein C-deficient (Mybpc(t/t)) mice develop dramatic cardiac dilation shortly after birth; heart size increases almost twofold. We have investigated the mechanism of cardiac enlargement in these hearts. Throughout embryogenesis myocytes undergo cell division while maintaining the capacity to pump blood by rapidly disassembling and reforming myofibrillar components of the sarcomere throughout cell cycle progression. Shortly after birth, myocyte cell division ceases. Cardiac MYBPC is a thick filament protein that regulates sarcomere organization and rigidity. We demonstrate that many Mybpc(t/t) myocytes undergo an additional round of cell division within 10 d postbirth compared with their wild-type counterparts, leading to increased numbers of mononuclear myocytes. Short-hairpin RNA knockdown of Mybpc3 mRNA in wild-type mice similarly extended the postnatal window of myocyte proliferation. However, adult Mybpc(t/t) myocytes are unable to fully regenerate the myocardium after injury. MYBPC has unexpected inhibitory functions during postnatal myocyte cytokinesis and cell cycle progression. We suggest that human patients with homozygous MYBPC3-null mutations develop dilated cardiomyopathy, coupled with myocyte hyperplasia (increased cell number), as observed in Mybpc(t/t) mice. Human patients, with heterozygous truncating MYBPC3 mutations, like mice with similar mutations, have hypertrophic cardiomyopathy. However, the mechanism leading to hypertrophic cardiomyopathy in heterozygous MYBPC3(+/-) individuals is myocyte hypertrophy (increased cell size), whereas the mechanism leading to cardiac dilation in homozygous Mybpc3(-/-) mice is primarily myocyte hyperplasia.

Loss of FHL1 induces an age-dependent skeletal muscle myopathy associated with myofibrillar and intermyofibrillar disorganization in mice.

Recent human genetic studies have provided evidences that sporadic or inherited missense mutations in four-and-a-half LIM domain protein 1 (FHL1), resulting in alterations in FHL1 protein expression, are associated with rare congenital myopathies, including reducing body myopathy and Emery-Dreifuss muscular dystrophy. However, it remains to be clarified whether mutations in FHL1 cause skeletal muscle remodeling owing to gain- or loss of FHL1 function. In this study, we used FHL1-null mice lacking global FHL1 expression to evaluate loss-of-function effects on skeletal muscle homeostasis. Histological and functional analyses of soleus, tibialis anterior and sternohyoideus muscles demonstrated that FHL1-null mice develop an age-dependent myopathy associated with myofibrillar and intermyofibrillar (mitochondrial and sarcoplasmic reticulum) disorganization, impaired muscle oxidative capacity and increased autophagic activity. A longitudinal study established decreased survival rates in FHL1-null mice, associated with age-dependent impairment of muscle contractile function and a significantly lower exercise capacity. Analysis of primary myoblasts isolated from FHL1-null muscles demonstrated early muscle fiber differentiation and maturation defects, which could be rescued by re-expression of the FHL1A isoform, highlighting that FHL1A is necessary for proper muscle fiber differentiation and maturation in vitro. Overall, our data show that loss of FHL1 function leads to myopathy in vivo and suggest that loss of function of FHL1 may be one of the mechanisms underlying muscle dystrophy in patients with FHL1 mutations.

Genetic and environmental risk factors in congenital heart disease functionally converge in protein networks driving heart development.

Congenital heart disease (CHD) occurs in ∼1% of newborns. CHD arises from many distinct etiologies, ranging from genetic or genomic variation to exposure to teratogens, which elicit diverse cell and molecular responses during cardiac development. To systematically explore the relationships between CHD risk factors and responses, we compiled and integrated comprehensive datasets from studies of CHD in humans and model organisms. We examined two alternative models of potential functional relationships between genes in these datasets: direct convergence, in which CHD risk factors significantly and directly impact the same genes and molecules and functional convergence, in which risk factors significantly impact different molecules that participate in a discrete heart development network. We observed no evidence for direct convergence. In contrast, we show that CHD risk factors functionally converge in protein networks driving the development of specific anatomical structures (e.g., outflow tract, ventricular septum, and atrial septum) that are malformed by CHD. This integrative analysis of CHD risk factors and responses suggests a complex pattern of functional interactions between genomic variation and environmental exposures that modulate critical biological systems during heart development.

Functional dissection of human cardiac enhancers and noncoding de novo variants in congenital heart disease.

Rare coding mutations cause ∼45% of congenital heart disease (CHD). Noncoding mutations that perturb cis-regulatory elements (CREs) likely contribute to the remaining cases, but their identification has been problematic. Using a lentiviral massively parallel reporter assay (lentiMPRA) in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we functionally evaluated 6,590 noncoding de novo variants (ncDNVs) prioritized from the whole-genome sequencing of 750 CHD trios. A total of 403 ncDNVs substantially affected cardiac CRE activity. A majority increased enhancer activity, often at regions with undetectable reference sequence activity. Of ten DNVs tested by introduction into their native genomic context, four altered the expression of neighboring genes and iPSC-CM transcriptional state. To prioritize future DNVs for functional testing, we used the MPRA data to develop a regression model, EpiCard. Analysis of an independent CHD cohort by EpiCard found enrichment of DNVs. Together, we developed a scalable system to measure the effect of ncDNVs on CRE activity and deployed it to systematically assess the contribution of ncDNVs to CHD.

Direct reprogramming of non-limb fibroblasts to cells with properties of limb progenitors.

The early limb bud consists of mesenchymal limb progenitors derived from the lateral plate mesoderm (LPM). The LPM also gives rise to the mesodermal components of the flank and neck. However, the cells at these other levels cannot produce the variety of cell types found in the limb. Taking advantage of a direct reprogramming approach, we find a set of factors (Prdm16, Zbtb16, and Lin28a) normally expressed in the early limb bud and capable of imparting limb progenitor-like properties to mouse non-limb fibroblasts. The reprogrammed cells show similar gene expression profiles and can differentiate into similar cell types as endogenous limb progenitors. The further addition of Lin41 potentiates the proliferation of the reprogrammed cells. These results suggest that these same four factors may play pivotal roles in the specification of endogenous limb progenitors.

Increased endothelial sclerostin caused by elevated DSCAM mediates multiple trisomy 21 phenotypes.

Trisomy 21 (T21), a recurrent aneuploidy occurring in 1:800 births, predisposes to congenital heart disease (CHD) and multiple extracardiac phenotypes. Despite a definitive genetic etiology, the mechanisms by which T21 perturbs development and homeostasis remain poorly understood. We compared the transcriptome of CHD tissues from 49 patients with T21 and 226 with euploid CHD (eCHD). We resolved cell lineages that misexpressed T21 transcripts by cardiac single-nucleus RNA sequencing and RNA in situ hybridization. Compared with eCHD samples, T21 samples had increased chr21 gene expression; 11-fold-greater levels (P = 1.2 × 10-8) of SOST (chr17), encoding the Wnt inhibitor sclerostin; and 1.4-fold-higher levels (P = 8.7 × 10-8) of the SOST transcriptional activator ZNF467 (chr7). Euploid and T21 cardiac endothelial cells coexpressed SOST and ZNF467; however, T21 endothelial cells expressed 6.9-fold more SOST than euploid endothelial cells (P = 2.7 × 10-27). Wnt pathway genes were downregulated in T21 endothelial cells. Expression of DSCAM, residing within the chr21 CHD critical region, correlated with SOST (P = 1.9 × 10-5) and ZNF467 (P = 2.9 × 10-4). Deletion of DSCAM from T21 endothelial cells derived from human induced pluripotent stem cells diminished sclerostin secretion. As Wnt signaling is critical for atrioventricular canal formation, bone health, and pulmonary vascular homeostasis, we concluded that T21-mediated increased sclerostin levels would inappropriately inhibit Wnt activities and promote Down syndrome phenotypes. These findings imply therapeutic potential for anti-sclerostin antibodies in T21.

Heterogeneous myocyte enhancer factor-2 (Mef2) activation in myocytes predicts focal scarring in hypertrophic cardiomyopathy.

Unknown molecular responses to sarcomere protein gene mutations account for pathologic remodeling in hypertrophic cardiomyopathy (HCM), producing myocyte growth and increased cardiac fibrosis. To determine if hypertrophic signals activated myocyte enhancer factor-2 (Mef2), we studied mice carrying the HCM mutation, myosin heavy-chain Arg403Gln, (MHC(403/+)) and an Mef2-dependent ß-galactosidase reporter transgene. In young, prehypertrophic MHC(403/+) mice the reporter was not activated. In hypertrophic hearts, activation of the Mef2-dependent reporter was remarkably heterogeneous and was observed consistently in myocytes that bordered fibrotic foci with necrotic cells, MHC(403/+) myocytes with Mef2-dependent reporter activation reexpressed the fetal myosin isoform (ßMHC), a molecular marker of hypertrophy, although MHC(403/+) myocytes with or without ßMHC expression were comparably enlarged over WT myocytes. To consider Mef2 roles in severe HCM, we studied homozygous MHC(403/403) mice, which have accelerated remodeling, widespread myocyte necrosis, and neonatal lethality. Levels of phosphorylated class II histone deacetylases that activate Mef2 were substantially increased in MHC(403/403) hearts, but Mef2-dependent reporter activation was patchy. Sequential analyses showed myocytes increased Mef2-dependent reporter activity before death. Our data dissociate myocyte hypertrophy, a consistent response in HCM, from heterogeneous Mef2 activation and reexpression of a fetal gene program. The temporal and spatial relationship of Mef2-dependent gene activation with myocyte necrosis and fibrosis in MHC(403/+) and MHC(403/403) hearts defines Mef2 activation as a molecular signature of stressed HCM myocytes that are poised to die.

Cardiomyocyte infection by Trypanosoma cruzi promotes innate immune response and glycolysis activation.

Introduction: Chagas cardiomyopathy, a disease caused by Trypanosoma cruzi (T. cruzi) infection, is a major contributor to heart failure in Latin America. There are significant gaps in our understanding of the mechanism for infection of human cardiomyocytes, the pathways activated during the acute phase of the disease, and the molecular changes that lead to the progression of cardiomyopathy. Methods: To investigate the effects of T. cruzi on human cardiomyocytes during infection, we infected induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) with the parasite and analyzed cellular, molecular, and metabolic responses at 3 hours, 24 hours, and 48 hours post infection (hpi) using transcriptomics (RNAseq), proteomics (LC-MS), and metabolomics (GC-MS and Seahorse) analyses. Results: Analyses of multiomic data revealed that cardiomyocyte infection caused a rapid increase in genes and proteins related to activation innate and adaptive immune systems and pathways, including alpha and gamma interferons, HIF-1α signaling, and glycolysis. These responses resemble prototypic responses observed in pathogen-activated immune cells. Infection also caused an activation of glycolysis that was dependent on HIF-1α signaling. Using gene editing and pharmacological inhibitors, we found that T. cruzi uptake was mediated in part by the glucose-facilitated transporter GLUT4 and that the attenuation of glycolysis, HIF-1α activation, or GLUT4 expression decreased T. cruzi infection. In contrast, pre-activation of pro-inflammatory immune responses with LPS resulted in increased infection rates. Conclusion: These findings suggest that T. cruzi exploits a HIF-1α-dependent, cardiomyocyte-intrinsic stress-response activation of glycolysis to promote intracellular infection and replication. These chronic immuno-metabolic responses by cardiomyocytes promote dysfunction, cell death, and the emergence of cardiomyopathy.

Efficient in vivo genome editing prevents hypertrophic cardiomyopathy in mice.

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.

Tbx5 maintains atrial identity by regulating an atrial enhancer network.

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 factor Tbx5 in the atrial working myocardium of the neonatal mouse heart to show that it is required to maintain atrial identity. Atrial Tbx5 inactivation downregulated highly chamber specific genes such as Myl7 and Nppa , and conversely, increased the expression of ventricular identity genes including Myl2 . 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.

Tbx5 maintains atrial identity in post-natal cardiomyocytes by regulating an atrial-specific enhancer network.

Understanding how the atrial and ventricular heart chambers maintain distinct identities is a prerequisite for treating chamber-specific diseases. Here, we selectively knocked out (KO) the transcription factor Tbx5 in the atrial working myocardium to evaluate its requirement for atrial identity. Atrial Tbx5 inactivation downregulated atrial cardiomyocyte (aCM) selective gene expression. Using concurrent single nucleus transcriptome and open chromatin profiling, genomic accessibility differences were identified between control and Tbx5 KO aCMs, revealing that 69% of the control-enriched ATAC regions were bound by TBX5. Genes associated with these regions were downregulated in KO aCMs, suggesting they function as TBX5-dependent enhancers. Comparing enhancer chromatin looping using H3K27ac HiChIP identified 510 chromatin loops sensitive to TBX5 dosage, and 74.8% of control-enriched loops contained anchors in control-enriched ATAC regions. Together, these data demonstrate TBX5 maintains the atrial gene expression program by binding to and preserving the tissue-specific chromatin architecture of atrial enhancers.

Contribution of Previously Unrecognized RNA Splice-Altering Variants to Congenital Heart Disease.

BACKGROUND: Known genetic causes of congenital heart disease (CHD) explain <40% of CHD cases, and interpreting the clinical significance of variants with uncertain functional impact remains challenging. We aim to improve diagnostic classification of variants in patients with CHD by assessing the impact of noncanonical splice region variants on RNA splicing. METHODS: We tested de novo variants from trio studies of 2649 CHD probands and their parents, as well as rare (allele frequency, <2×10-6) 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.

Multiplexed Single-Nucleus RNA Sequencing Using Lipid-Oligo Barcodes.

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.