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Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease

Ameen, Mohamed and Sundaram, Laksshman and Banerjee, Abhimanyu and Shen, Mengcheng and Kundu, Soumya and Nair, Surag and Shcherbina, Anna and Gu, Mingxia and Wilson, Kitchener D. and Varadarajan, Avyay and Vadgama, Nirmal and Balsubramani, Akshay and Wu, Joseph C. and Engreitz, Jesse and Farh, Kyle and Karakikes, Ioannis and Wang, Kevin C. and Quertermous, Thomas and Greenleaf, William J. and Kundaje, Anshul (2022) Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease. . (Unpublished)

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Congenital heart defects, the most common birth disorders, are the clinical manifestation of anomalies in fetal heart development - a complex process involving dynamic spatiotemporal coordination among various precursor cell lineages. This complexity underlies the incomplete understanding of the genetic architecture of congenital heart diseases (CHDs). To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We identified similarities and differences of regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts. We interpreted deep learning models that predict cell-type resolved, base-resolution chromatin accessibility profiles from DNA sequence to decipher underlying TF motif lexicons and infer the regulatory impact of non-coding variants. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in CHD cases versus controls. We used CRISPR-based perturbations to validate an enhancer harboring a nominated regulatory CHD mutation, linking it to effects on the expression of a known CHD gene JARID2. Together, this work defines the cell-type resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements as a component of the genetic etiology of CHD.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Discussion Paper ItemData - Gene Expression Omnibus ItemDataset Itemscripts for the single cell analysis and BPNet analysis for the Cardiogenesis dataset ItemCardiogenesis dataset
Sundaram, Laksshman0000-0002-8953-3940
Banerjee, Abhimanyu0000-0003-3047-6803
Nair, Surag0000-0002-6216-2457
Shcherbina, Anna0000-0002-5080-5494
Gu, Mingxia0000-0001-7405-8473
Vadgama, Nirmal0000-0001-6226-3867
Balsubramani, Akshay0000-0003-1545-9837
Wu, Joseph C.0000-0002-6068-8041
Engreitz, Jesse0000-0002-5754-1719
Farh, Kyle0000-0001-6947-8537
Karakikes, Ioannis0000-0002-4348-600X
Quertermous, Thomas0000-0002-7645-9067
Greenleaf, William J.0000-0003-1409-3095
Kundaje, Anshul0000-0003-3084-2287
Additional Information:The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license. This work was supported by grants from the NIH 1DP2GM123485 (AK), U01HG012069 (AK), R01 HL139478 (TQ), R01 HL145708 (TQ), R01 HL134817 (TQ), R01 HL151535 (TQ), R01 HL156846 (TQ), 1UM1 HG011972 (TQ), RM1-HG007735, (WJG) UM1-HG009442 (WJG), UM1-HG009436 (WJG), R01-HG00990901 (WJG), and U19-AI057266 (WJG.), R01 GM136737 (K.C.W.), R61 AR076815 (K.C.W.), a Human Cell Atlas grant from the Chan Zuckerberg Foundation (TQ), K08 HL119251 (K.D.W.), K99 HL135258 (M.G.); S10 OD018220 (Stanford Functional Genomics), NHGRI Genomic Innovator Award (R35HG011324 to J.M.E.); Gordon and Betty Moore and the BASE Research Initiative at the Lucile Packard Children’s Hospital at Stanford University (J.M.E.); and the Stanford Maternal & Child Health Research Institute and Additional Ventures (to J.M.E.), NSF Graduate Research Fellowship Program (M.A.) and The Bio-X Bowes Fellowship (L.S.). K.C.W. is a New York Stem Cell Foundation–Robertson Investigator, and the Stephen Bechtel Endowed Faculty Scholar in Pediatric Translational Medicine, Stanford Maternal and Child Health Research Institute. This work was also supported by funding from the Rita Allen Foundation (W.J.G.), the Human Frontiers Science (RGY006S) (W.J.G.). W.J.G. is a Chan Zuckerberg Biohub investigator and acknowledges grants 2017-174468 and 2018-182817 from the Chan Zuckerberg Initiative and funding from Emerson Collective. Author Contributions. M.A., L.S., I.K., K.C.W. and A.K. conceived the project. L.S., M.A., T.Q, W.J.G and A.K. generated figures and wrote the manuscript with input from authors. M.A. designed and performed all experimental data generation for the manuscript with inputs from L.S., M.C., K.D.W, M.G., I.K., K.C.W., T.Q., A.K. and W.J.G.. L.S designed and performed all analyses for the manuscript with inputs from M.A, A.Ban., S.K., S.N., A.S., A.V., N.V., A.Bal, J.E., K.F., T.Q, W.J.G & A.K.. Data availability: Aligned fragment files from single cell chromatin assays are deposited in the Gene Expression Omnibus database with the SuperSeries reference number GSE181346. The cell by gene accessibility scores matrices along with cluster 5’ insertion bigWig tracks for the human heart samples are deposited to UCSC cell browser portal under reference url : to enable visualization of cell markers and genes for the broader community. Code used for single cell analysis, training BPNet models and reproducing results for all figures can be found at: Interactive HiGlass browser sessions with cell-type resolved tracks for measured base-resolution scATAC-seq coverage profiles and predicted base-resolution scATAC-seq coverage profiles from BPNet models as well as model-derived nucleotide-resolution contribution scores in peak regions can be found at: (Please press the Alt key or Option key + mouse scroll to scroll down or up through the tracks). Competing Interest Statement. W.J.G. is named as an inventor on patents describing ATAC-seq methods. 10x Genomics has licensed intellectual property on which WJG is listed as an inventor. WJG holds options in 10x Genomics, and is a consultant for Ultima Genomics and Guardant Health. WJG is a scientific co-founder of Protillion Biosciences. A.S. is an employee of Insitro and is a consultant at Myokardia. A.K. is a consulting Fellow with Illumina, a member of the SAB of OpenTargets (GSK), PatchBio, SerImmune and a scientific co-founder of RavelBio. K.F. is an employee of Illumina. J.C.W. is a co-founder of Khloris Biosciences but has no competing interests, as the work presented here is completely independent. The other authors declare no competing interests.
Funding AgencyGrant Number
NIHR01 HL139478
NIHR01 HL145708
NIHR01 HL134817
NIHR01 HL151535
NIHR01 HL156846
NIH1UM1 HG011972
NIHR01 GM136737
NIHR61 AR076815
Chan Zuckerberg FoundationUNSPECIFIED
NIHK08 HL119251
NIHK99 HL135258
NIHS10 OD018220
Gordon and Betty MooreUNSPECIFIED
Lucile Packard Children's HospitalUNSPECIFIED
Stanford Maternal & Child Health Research InstituteUNSPECIFIED
NSF Graduate Research FellowshipUNSPECIFIED
Bio-X Bowes FellowshipUNSPECIFIED
New York Stem Cell FoundationUNSPECIFIED
Rita Allen FoundationUNSPECIFIED
Human Frontier Science ProgramRGY006S
Chan Zuckerberg Biohub2017-174468
Chan Zuckerberg Biohub2018-182817
Emerson CollectiveUNSPECIFIED
Record Number:CaltechAUTHORS:20220706-965769000
Persistent URL:
Official Citation:Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease Mohamed Ameen, Laksshman Sundaram, Abhimanyu Banerjee, Mengcheng Shen, Soumya Kundu, Surag Nair, Anna Shcherbina, Mingxia Gu, Kitchener D. Wilson, Avyay Varadarajan, Nirmal Vadgama, Akshay Balsubramani, Joseph C. Wu, Jesse Engreitz, Kyle Farh, Ioannis Karakikes, Kevin C Wang, Thomas Quertermous, William Greenleaf, Anshul Kundaje bioRxiv 2022.06.29.498132; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:115359
Deposited By: George Porter
Deposited On:08 Jul 2022 22:44
Last Modified:25 Jul 2022 23:13

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