Biology subjects

Huang, Y.

Publications and source records attributed to Huang, Y..

At least 991 records · Page 55Linked to original sources

bioRxiv2018

Base pair editing of goat embryos: nonsense codon introgression into FGF5 to improve cashmere yield

The ability to alter single bases without DNA double strand breaks provides a potential solution for multiplex editing of livestock genomes for quantitative traits. Here, we report using a single base editing system, Base Editor 3 (BE3), to induce nonsense codons (C-to-T transitions) at four target sites in caprine FGF5. All five progenies produced from microinjected single-cell embryos had alleles with a targeted nonsense mutation and yielded expected phenotypes. The effectiveness of BE3 to make single base changes varied considerably based on sgRNA design. Also, the rate of mosaicism differed between animals, target sites, and tissue type. PCR amplicon and whole genome resequencing analyses for off-target changes caused by BE3 were low at a genome-wide scale. This study provides first evidence of base editing in livestock, thus presenting a potentially better method to introgress complex human disease alleles into large animal models and provide genetic improvement of complex health and production traits in a single generation.

genetics↗

bioRxiv2018

New insights into human nostril microbiome from the expanded Human Oral Microbiome Database (eHOMD): a resource for species-level identification of microbiome data from the aerodigestive tract

The expanded Human Oral Microbiome Database (eHOMD) is a comprehensive microbiome database for sites along the human aerodigestive tract that revealed new insights into the nostril microbiome. The eHOMD provides well-curated 16S rRNA gene reference sequences linked to available genomes and enables assignment of species-level taxonomy to most NextGeneration sequences derived from diverse aerodigestive tract sites, including the nasal passages, sinuses, throat, esophagus and mouth. Using Minimum Entropy Decomposition coupled with the RDP Classifier and our eHOMD V1-V3 training set, we reanalyzed 16S rRNA V1-V3 sequences from the nostrils of 210 Human Microbiome Project participants at the species level revealing four key insights. First, we discovered that Lawsonella clevelandensis, a recently named bacterium, and Neisseriaceae [G-1] HMT-174, a previously unrecognized bacterium, are common in adult nostrils. Second, just 19 species accounted for 90% of the total sequences from all participants. Third, one of these 19 belonged to a currently uncultivated genus. Fourth, for 94% of the participants, two to ten species constituted 90% of their sequences, indicating nostril microbiome may be represented by limited consortia. These insights highlight the strengths of the nostril microbiome as a model system for studying interspecies interactions and microbiome function. Also, in this cohort, three common nasal species (Dolosigranulum pigrum and two Corynebacterium species) showed positive differential abundance when the pathobiont Staphylococcus aureus was absent, generating hypotheses regarding colonization resistance. By facilitating species-level taxonomic assignment to microbes from the human aerodigestive tract, the eHOMD is a vital resource enhancing clinical relevance of microbiome studies.\n\nIMPORTANCEThe eHOMD (ehomd.org) is a valuable resource for researchers, from basic to clinical, who study the microbiomes, and the individual microbes, in health and disease of body sites in the human aerodigestive tract, which includes the nasal passages, sinuses, throat, esophagus and mouth, and the lower respiratory tract. The eHOMD is an actively curated, web-based, open-access resource. eHOMD provides the following: (1) species-level taxonomy based on grouping 16S rRNA gene sequences at 98.5% identity, (2) a systematic naming scheme for unnamed and/or uncultivated microbial taxa, (3) reference genomes to facilitate metagenomic, metatranscriptomic and proteomic studies and (4) convenient cross-links to other databases (e.g., PubMed and Entrez). By facilitating the assignment of species names to sequences, the eHOMD is a vital resource for enhancing the clinical relevance of 16S rRNA gene-based microbiome studies, as well as metagenomic studies.

microbiology↗

bioRxiv2018

Sublethal β-lactam antibiotics induce PhpP phosphatase expression and StkP kinase phosphorylation in PBP-independent β-lactam antibiotic resistance of Streptococcus pneumoniae

StkP and PhpP of Streptococcus pneumoniae have been confirmed to compose a signaling couple, in which the former is a serine/threonine (Ser/Thr) kinase while the latter was annotated as a phosphotase. StkP has been reported to be involved in penicillin-binding protein (PBP)-independent penicillin resistance of S. pneumoniae. However, the enzymatic characterization of PhpP and the role of PhpP in StkP-PhpP couple remain poorly understood. Here we showed that 1/4 minimal inhibitory concentration (MIC) of penicillin (PCN) or cefotaxime (CTX), the representatives of {beta}-lactam antibiotics, could induce the expression of stkP and phpP genes and phosphorylation of StkP in PCN/CTX-sensitive strain ATCC6306 and three isolates of S. pneumoniae (MICs: 0.02-0.5 g/ml). The product of phpP gene hydrolyzed PP2C type Ser/Thr phosphotase-specific RRA(pT)VA phosphopeptide substrate with the Km and Kcat values of 277.35 mol/L and 0.71 S-1, and the hydrolytic activity was blocked by sodium fluoride, a PP2C type Ser/Thr phosphatase inhibitor. The phosphorylation levels of StkP in the four phpP gene-knockout ({Delta}phpP) mutants were significantly higher than that in the wild-type strains. In particular, the MICs of PCN and CTX against the {Delta}phpP mutants were significantly elevated as 4-16 g/ml. Therefore, our findings confirmed that sublethal PCN and CTX act as environmental inducers to cause the increase of phpP and stkP gene expression and StkP phosphorylation. PhpP is a PP2C type Ser/Thr protein phosphatase responsible for dephosphorylation of StkP. Knockout of the phpP gene results in a high level of StkP phosphorylation and PBP-independent PCN/CTX resistance of S. pneumoniae.\n\nImportanceStreptococcus pneumoniae is a common pathogen in human populations in many countries and areas due to the prevalence of {beta}-lactam antibiotic-resistant pneumococcal strains. Production of {beta}-lactamases and mutation of penicillin-binding proteins (PBP) have been considered as the major {beta}-lactam antibiotic-resistant mechanisms in bacteria, but S. pneumoniae has not been confirmed to produce any {beta}-lactamases and many pneumococcal strains present PBP mutation-independent {beta}-lactam antibiotic resistance. StkP is a Ser/Thr kinase of S. pneumoniae to compose a signal-couple with PhpP protein. The present study demonstrated that the PhpP is a PP2C-type phosphotase for dephosphorylation of StkP and the sublethal penicillin (PCN) or cefotaxime (CTX) acted as environmental signal molecules to induce the expression of PhpP. The knockout of PhpP-encoding gene caused the PCN/CTX resistance generation of PCN/CTX-sensitive pneumococcal strains. All the data indicate that StkP-PhpP couple of S. pneumoniae is involved in PBP mutation-independent {beta}-lactam antibiotic resistance by phosphorylation of StkP.

microbiology↗

bioRxiv2018

C1 CAGE detects transcription start sites and enhancer activity at single-cell resolution

Single-cell transcriptomic profiling is a powerful tool to explore cellular heterogeneity. However, most of these methods focus on the 3-end of polyadenylated transcripts and provide only a partial view of the transcriptome. We introduce C1 CAGE, a method for the detection of transcript 5-ends with an original sample multiplexing strategy in the C1 microfluidic system. We first quantified the performance of C1 CAGE and found it as accurate and sensitive as other methods in C1 system. We then used it to profile promoter and enhancer activities in the cellular response to TGF-{beta} of lung cancer cells and discovered subpopulations of cells differing in their response. We also describe enhancer RNA dynamics revealing transcriptional bursts in subsets of cells with transcripts arising from either strand within a single-cell in a mutually exclusive manner, which was validated using single molecule fluorescence in-situ hybridization.

genomics↗

bioRxiv2018

phastWeb: a web interface for evolutionary conservation scoring of multiple sequence alignments using phastCons and phyloP

The Phylogenetic Analysis with Space/Time models (PHAST) package is a widely used software package for comparative genomics that has been freely available for download since 2002. Here we introduce a web interface (phastWeb) that makes it possible to use two of the most popular programs in PHAST, phastCons and phyloP, without downloading and installing the PHAST software. This interface allows users to upload a sequence alignment and either upload a corresponding phylogeny or have one estimated from the alignment. After processing, users can visualize alignments and conservation scores as genome browser tracks, and download estimated tree models and raw scores for further analysis. Altogether, this resource makes key features of the PHAST package conveniently available to a broad audience.\n\nAVAILABILITYphastWeb is freely available on the web at http://compgen.cshl.edu/phastweb/. The website provides instructions as well as examples.\n\nCONTACTphasthelp@cshl.edu

evolutionary biology↗

bioRxiv2018

High-throughput identification and marker development of perfect SSR for cultivated genus of passion fruit (Passiflora edulis)

Simple sequence repeat (SSR) markers are characterized by high polymorphism, good reproducibility and co-dominance etc. They can be easily applied to develop efficient, simple and practical molecular markers. In the present study, bioinformatics methods were applied to identify high-throughput perfect SSRs of cultivar Passiflora genome. A total of 13104 perfect SSRs were obtained. SSR core sequence structure is mainly 2-4 bases, the maximum numbers are TA, AT, TC and AG. The maximum numbers of repetitions were up to 20 times. A total of 12934 pairs of SSR markers were developed by using bioinformatics software, and 20 pairs of markers were selected for amplification specificity assessment of MTX and WJ10, and the polymorphism rate was as high as 60%. The large-scale development of the SSR markers of Passiflora cultivar has paved a foundation for the efficient utilization of the germplasm resources of passion fruit, genetic improvement of the varieties and molecular breeding.

genomics↗

bioRxiv2018

DeepEM: Deep 3D ConvNets With EM For Weakly Supervised Pulmonary Nodule Detection

Recently deep learning has been witnessing widespread adoption in various medical image applications. However, training complex deep neural nets requires large-scale datasets labeled with ground truth, which are often unavailable in many medical image domains. For instance, to train a deep neural net to detect pulmonary nodules in lung computed tomography (CT) images, current practice is to manually label nodule locations and sizes in many CT images to construct a sufficiently large training dataset, which is costly and difficult to scale. On the other hand, electronic medical records (EMR) contain plenty of partial information on the content of each medical image. In this work, we explore how to tap this vast, but currently unexplored data source to improve pulmonary nodule detection. We propose DeepEM, a novel deep 3D ConvNet framework augmented with expectation-maximization (EM), to mine weakly supervised labels in EMRs for pulmonary nodule detection. Experimental results show that DeepEM can lead to 1.5% and 3.9% average improvement in free-response receiver operating characteristic (FROC) scores on LUNA16 and Tianchi datasets, respectively, demonstrating the utility of incomplete information in EMRs for improving deep learning algorithms.1

bioinformatics↗

bioRxiv2018

Extracellular HBV RNAs are heterogeneous in length and circulate as virions and capsid-antibody-complexes in chronic hepatitis B patients

Extracellular HBV RNA has been detected in both HBV-replicating cell culture media and sera from chronic hepatitis B (CHB) patients, but its exact origin and composition remain controversial. Here, we demonstrated that extracellular HBV RNA species were of heterogeneous lengths, ranging from the length of pregenomic RNA to a few hundred nucleotides. In cell models, these RNAs were predominantly associated with naked capsids although virions also harbored a minority of them. Moreover, HBV RNAs in hepatitis B patients blood circulation were localized in unenveloped capsids in the form of capsid-antibody-complexes (CACs) and in virions. Furthermore, we showed that extracellular HBV RNAs could serve as template for viral DNA synthesis. In conclusion, extracellular HBV RNAs mainly consist of pgRNA or the pgRNA species degraded by the RNase H domain of the polymerase in the process of viral DNA synthesis and circulate as CACs and virions. Their presence in the blood circulation of CHB patients may be exploited to develop novel biomarkers for HBV persistence.

microbiology↗

bioRxiv2018

Comparative analysis of droplet-based ultra-high-throughput single-cell RNA-seq systems

Since its establishment in 2009, single-cell RNA-seq has been a major driver behind progress in biomedical research. In developmental biology and stem cell studies, the ability to profile single cells confers particular benefits. While most studies still focus on individual tissues or organs, the recent development of ultra-high-throughput single-cell RNA-seq has demonstrated potential power in characterizing more complex systems or even the entire body. However, although multiple ultra-high-throughput single-cell RNA-seq systems have attracted attention, no systematic comparison of these systems has been performed. Here, we focus on three widely used droplet-based ultra-high-throughput single-cell RNA-seq systems, inDrop, Drop-seq, and 10X Genomics Chromium. While each system is capable of profiling single-cell transcriptomes, their detailed comparison revealed the distinguishing features and suitable applications for each system.

genomics↗

bioRxiv2018

Single Cell Transcriptomics of Pancreatic Cancer Precursors Demonstrates Epithelial and Microenvironmental Heterogeneity as an Early Event in Neoplastic Progression

BackgroundEarly detection of pancreatic ductal adenocarcinoma (PDAC) remains elusive. Precursor lesions of PDAC, specifically, intraductal papillary mucinous neoplasms (IPMNs) represent a bona fide pathway to invasive neoplasia, although the molecular correlates of progression remain to be fully elucidated. Single cell transcriptomics provides a unique avenue for dissecting both the epithelial and microenvironmental heterogeneity that accompany multistep progression from non-invasive IPMNs to PDAC.\n\nMethodsSingle cell RNA-sequencing was performed through droplet-based sequencing on 5,403 cells from two low-grade IPMNs (LGD-IPMN), two high-grade IPMNs (HGD-IPMN), and two PDACs (all surgically resected).\n\nResultsAnalysis of single cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of non-invasive dysplasia to invasive cancer. While HGD-IPMNs expressed many core-signaling pathways described in PDAC, LGD-IPMNs harbored subsets of single cells with a transcriptomic profile that overlapped with invasive cancer. Notably, a pro-inflammatory immune component was readily seen in low-grade IPMNs, comprised of cytotoxic T-cells, activated T-helper cells, and dendritic cells, which was progressively depleted during neoplastic progression, accompanied by infiltration of myeloid-derived suppressor cells. Finally, stromal myofibroblast populations were heterogeneous, and acquired a previously described tumor-promoting and immune-evading phenotype during invasive carcinogenesis.\n\nConclusionsThis study demonstrates the ability to perform high resolution profiling of the transcriptomic changes that occur during multistep progression of cystic PDAC precursors to cancer. Notably, single cell analysis provides an unparalleled insight into both the epithelial and microenvironmental heterogeneity that accompany early cancer pathogenesis, and might be a useful substrate to identify targets for cancer interception.

pathology↗

bioRxiv2018

Distinctive types of postzygotic single-nucleotide mosaicisms in healthy individuals revealed by genome-wide profiling of multiple organs

Postzygotic single-nucleotide mosaicisms (pSNMs) have been extensively studied in tumors and are known to play critical roles in tumorigenesis. However, the patterns and origin of pSNMs in normal organs of healthy humans remain largely unknown. Using whole-genome sequencing and ultra-deep amplicon re-sequencing, we identified and validated 164 pSNMs from 27 postmortem organ samples obtained from five healthy donors. The mutant allele fractions ranged from 1.0% to 29.7%. Inter- and intra-organ comparison revealed two distinctive types of pSNMs, with about half originating during early embryogenesis (embryonic pSNMs) and the remaining more likely to result from clonal expansion events that had occurred more recently (clonal expansion pSNMs). Compared to clonal expansion pSNMs, embryonic pSNMs had higher proportion of C>T mutations with elevated mutation rate at CpG sites. We observed differences in replication timing between these two types of pSNMs, with embryonic and clonal expansion pSNMs enriched in early- and late-replicating regions, respectively. An increased number of embryonic pSNMs were located in open chromatin states and topologically associating domains that transcribed embryonically. Our findings provide new insights into the origin and spatial distribution of postzygotic mosaicism during normal human development.\n\nAuthor SummaryGenomic mosaicism led by postzygotic mutation is the major cause of cancers and many non-cancer developmental disorders. Theoretically, postzygotic mutations should be accumulated during the developmental process of healthy individuals, but the genome-wide characterization of postzygotic mosaicisms across many organ types of the same individual remained limited. In this study, we identified and validated two types of postzygotic mosaicism from the whole-genomes of 27 organs obtained from five healthy donors. We further found that the postzygotic mosaicisms arising during early embryogenesis and later clonal expansion events show distinct genomic patterns in mutation spectrum, replication timing, and chromatin status.

genomics↗

bioRxiv2018

Isonitrile Formation by a Non-heme Iron(II)-dependent Oxidase/Decarboxylase

The electron-rich isonitrile is an important functionality in bioactive natural products, but its biosynthesis has been restricted to the IsnA family of isonitrile synthases. We here provide the first structural and biochemical evidence of an alternative mechanism for isonitrile formation. ScoE, a putative non-heme iron(II)-dependent enzyme from Streptomyces coeruleorubidus, was shown to catalyze the conversion of (R)-3-((carboxymethyl)amino)butanoic acid to (R)-3-isocyanobutanoic acid through an oxidative decarboxylation mechanism. This work further provides a revised scheme for the biosynthesis of a unique class of isonitrile lipopeptides, members of which are critical for the virulence of pathogenic mycobacteria.

biochemistry↗

bioRxiv2018

Branch-recombinant Gaussian processes for analysis of perturbations in biological time series

MotivationA common class of behaviour encountered in the biological sciences involves branching and recombination. During branching, a statistical process bifurcates resulting in two or more potentially correlated processes that may under-go further branching; the contrary is true during recombination, where two or more statistical processes converge into one. A key objective is to identify the time of this bifurcation (branch time) from time series measurements e.g., comparing a control time series with a perturbed time series. Whilst statistical treatments for the two branch (control versus treatment) case exists, the ability to infer more complex branching structure from time series data remains open. Gaussian processes (GPs) represents an ideal framework for such analysis, allowing for nonlinear regression that includes a rigorous treatment of uncertainty. Currently, however, GP models only exist for two-branch systems. Here we highlight how arbitrarily complex branching processes can be built using the correct composition of covariance functions within a GP framework, thus outlining a general framework for the treatment of branching and recombination in the form of branch-recombinant Gaussian processes (B-RGPs). We first demonstrate the performance of B-RGPs compared to a variety of existing regression approaches, and demonstrate robustness to model misspecification. B-RGPs are then used to investigate the branching patterns of Arabidopsis thaliana gene expression following inoculation with the hemibotrophic bacteria, Pseudomonas syringae DC3000, and a disarmed mutant strain, hrpA. By grouping genes according to the number of branches, we could naturally separate out genes involved in basal immune response from those subverted by the virulent strain, and show enrichment for targets of pathogen protein effectors. Finally, we identify two early branching genes WRKY11 and WRKY17, and showed that groups of genes that branched at similar times to WRKY11/17 were enriched for W-box binding motifs, and overrepresented for genes differentially expressed in WRKY11/17 knockouts, suggesting that branch time could be used for identifying direct and indirect binding targets of key transcription factors. Software is available from: https://github.com/cap76/BranchingGPs.

bioinformatics↗

bioRxiv2018

Single-molecule optical mapping enables accurate molecular diagnosis of facioscapulohumeral muscular dystrophy (FSHD)

Facioscapulohumeral Muscular Dystrophy (FSHD) is a common adult muscular dystrophy in which the muscles of the face, shoulder blades and upper arms are among the most affected. FSHD is the only disease in which \"junk\" DNA is reactivated to cause disease, and the only known repeat array-related disease where fewer repeats cause disease. More than 95% of FSHD cases are associated with copy number loss of a 3.3kb tandem repeat (D4Z4 repeat) at the subtelomeric chromosomal region 4q35, of which the pathogenic allele contains less than 10 repeats and has a specific genomic configuration called 4qA. Currently, genetic diagnosis of FSHD requires pulsed-field gel electrophoresis followed by Southern blot, which is labor-intensive, semi-quantitative and requires long turnaround time. Here, we developed a novel approach for genetic diagnosis of FSHD, by leveraging Bionano Saphyr single-molecule optical mapping platform. Using a bioinformatics pipeline developed for this assay, we found that the method gives direct quantitative measurement of repeat numbers, can differentiate 4q35 and the highly paralogous 10q26 regions, can determine the 4qA/4qB allelic configuration, and can quantitate levels of post-zygotic mosaicism. We evaluated this approach on 5 patients (including two with post-zygotic mosaicism) and 2 patients (including one with post-zygotic mosaicism) from two separate cohorts, and had complete concordance with Southern blots, but with improved quantification of repeat numbers resolved between haplotypes. We concluded that single-molecule optical mapping is a viable approach for molecular diagnosis of FSHD and may be applied in clinical diagnostic settings once more validations are performed.

genomics↗

bioRxiv2018

Human Puf-A, a Novel Component of 90S Pre-ribosome, Links Ribosome Biogenesis to Cancer Progression

We describe a novel biogenesis factor of the 90S pre-ribosome, Puf-A, which is a negative transcriptional target of p53. The expression of Puf-A is not only upregulated in advanced human lung cancer and tumors of patients especially with TP53 mutation, but also is highly prognostic for stage I lung cancer. Loss of Puf-A expression prevents KrasG12D/p53-/--induced tumor progression in the lungs and induces apoptosis in TP53- mutated cancers and c-Myc/p53-/--transformed cells as well. Overexpression of Puf-A enhances proliferation of normal cells after c-Myc induction and overcomes the cell-cycle checkpoints incurred by p53 expression. Mechanistically, Puf-A interacts with double-stranded structures of the 5.8S sequence within pre-rRNA and maintains the integrity of 90S pre-ribosomes, thereby impacting early ribosome assembly and export of ribosomes from nuclei. Silencing of Puf-A disrupts the assembly of 90S pre-ribosomes and induces the translocation of its associated nucleophosmin (NPM1) from nucleoli to the nucleoplasm, resulting in impairment of ribosome synthesis. Thus, Puf-A is crucial for over-activation of ribosome biogenesis and contributes to tumor progression and cancer growth.

cancer biology↗

bioRxiv2018

Mitochondrial Fusion Suppresses Pancreatic Cancer Growth via Reduced Oxidative Metabolism

Pancreatic cancer is a highly lethal disease whose aggressive biology that is driven by mitochondrial oxidative metabolism. Mitochondria normally form a network of fused organelles, but we find that patient-derived and genetically engineered murine pancreatic cancer cells exhibit highly fragmented mitochondria with robust oxygen consumption rates (OCR). When mitochondrial fusion was activated by the genetic or pharmacological inhibition Drp1, the morphology and metabolism of human and murine pancreatic cancer cells more closely resembled that of normal pancreatic epithelial cells. This reduced metabolism was correlated with slower tumor growth, fewer metastases, and enhanced survival in a syngeneic orthotopic model. Similarly, directly activating mitochondrial fusion by overexpression of Mfn2 also reduced tumor growth and metastases. Mitochondrial fusion in pancreatic cancer cells was associated with reduced mitochondrial mass and Complex I expression and function. Thus, these data suggest that enhancing mitochondrial fusion through Drp1 inhibition or enhanced Mfn2 expression or function has strong tumor suppressive activity against pancreatic cancer and may thus represent a highly novel and efficacious therapeutic target.

cancer biology↗

bioRxiv2018

Tracing the Transitions from Pluripotency to Germ Cell Fate with CRISPR Screening

Early mammalian development entails a series of cell fate transitions that includes transit through naive pluripotency to post-implantation epiblast. This subsequently gives rise to primordial germ cells (PGC), the founding population of the germline lineage. To investigate the gene regulatory networks that control these critical cell fate decisions, we developed a compound-reporter system to track cellular identity in a model of PGC specification (PGC-like cells; PGCLC), and coupled it with unbiased genome-wide CRISPR screening. This enabled identification of key genes both for exit from pluripotency and for acquisition of PGC fate, with further characterisation revealing a central role for the transcription factors Nr5a2 and Zfp296 in germline ontogeny. Abrogation of these genes results in significantly impaired PGCLC development due to widespread activation (Nr5a2-/-) or inhibition (Zfp296-/-) of WNT pathway components. This leads to aberrant upregulation of the somatic programme or failure to appropriately activate germline genes in PGCLC, respectively, and consequently loss of germ cell identity. Overall our study places Zfp296 and Nr5a2 as key components of an expanded PGC gene regulatory network, and outlines a transferable strategy for identifying critical regulators of complex cell fate transitions.

developmental biology↗

bioRxiv2018

Oligogenic inheritance of congenital heart disease involving a NKX2-5 modifier

Complex genetic inheritance is thought to underlie many human diseases, yet experimental proof of this model has been elusive. Here, we show that a human congenital heart defect, left ventricular non-compaction (LVNC), can be caused by a combination of rare, inherited heterozygous missense single nucleotide variants. Whole exome sequencing of a nuclear family revealed novel single nucleotide variants of MYH7 and MKL2 in an asymptomatic father while the offspring with severe childhood-onset LVNC harbored an additional missense variant in the cardiac transcription factor, NKX2-5, inherited from an unaffected mother. Mice bred to compound heterozygosity for the orthologous missense variants in Myh7 and Mkl2 had mild cardiac pathology; the additional inheritance of the Nkx2-5 variant yielded a more severe LVNC-like phenotype in triple compound heterozygotes. RNA sequencing identified genes associated with endothelial and myocardial development that were dysregulated in hearts from triple heterozygote mice and human induced pluripotent stem cell-derived cardiomyocytes harboring the three variants, with evidence for NKX2-5s contribution as a modifier on the molecular level. These studies demonstrate that the deployment of efficient gene editing tools can provide experimental evidence for complex inheritance of human disease.\n\nOne sentence summaryA combination of three inherited heterozygous missense single nucleotide variants underlying a familial congenital heart defect.

genetics↗