Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.

Amsterdam UMC University of Amsterdam Heart Center Department of Clinical and Experimental Cardiology Amsterdam Cardiovascular Sciences

Children's Heart Centre 2nd Faculty of Medicine Charles University Prague Motol University Hospital Prague Czech Republic

Cyprus School of Molecular Medicine Nicosia Cyprus

Department of Biochemistry and Cell Biology Stony Brook University Stony Brook New York USA

Department of Cell and Developmental Biology Vanderbilt University School of Medicine Nashville Tennessee USA

Department of Clinical Genetics and

Department of Clinical Genetics The Cyprus Institute of Neurology and Genetics Nicosia Cyprus

Department of Genetics Hadassah Hebrew University Medical Center Jerusalem Israel

Department of Human Genetics Leiden University Medical Center Leiden Netherlands

Department of Human Genetics Leiden University Medical Centre Leiden Netherlands

Department of Medical Biology Amsterdam UMC Amsterdam Netherlands

Department of Obstetrics and

Department of Obstetrics Gynecology and Women's Health

Department of Pathology Isala Women and Children's Hospital Zwolle Netherlands

Department of Pediatric Cardiology Emma Children's Hospital Amsterdam UMC University of Amsterdam Amsterdam Netherlands

Department of Pediatric Cardiology Hadassah Hebrew University Medical Center Jerusalem Israel

Department of Pediatric Cardiology Leiden University Medical Centre Leiden Netherlands

Department of Pediatrics Division of Pediatric Cardiology and

Department of Pediatrics Ochsner Clinic Tulane University University of Queensland New Orleans Louisiana USA

Department of Pediatrics University of Minnesota Minneapolis Minnesota USA

Department of Pharmacological Sciences and Graduate Program in Molecular and Cellular Pharmacology Stony Brook University Stony Brook New York USA

Department of Pharmacology Vanderbilt University School of Medicine Nashville Tennessee USA

Division of Cardiology Ann and Robert H Lurie Children's Hospital of Chicago Chicago Illinois USA

Division of Genetics Birth Defects and Metabolic Disorders Ann and Robert H Lurie Children's Hospital of Chicago Chicago Illinois USA

Feinberg School of Medicine Northwestern University Chicago Illinois USA

Genetic and Rare Disease Research Division Bambino Gesù Children's Hospital IRCCS Rome Italy

Makarios Medical Centre Nicosia Cyprus

Medical Genetics Department UPMC Children's Hospital of Pittsburgh Pittsburgh Pennsylvania USA

Research Unit for Rare Diseases Department of Pediatrics and Adolescent Medicine 1st Faculty of Medicine Charles University and General University Hospital Prague Czech Republic

Service de Génétique Médicale CHU Estaing Clermont Ferrand France

Ultrasound and Fetal Medicine Diagnostic Centre Nicosia Cyprus

UMR 1231 INSERM GAD Université Bourgogne Franche Comté Dijon France

Unité Fonctionnelle d'Innovation en Diagnostique Génomique des Maladies Rares FHU TRANSLAD Centre Hospitalier Universitaire Estaing Dijon Bourgogne Dijon France

University Medical Center Groningen Department of Genetics University of Groningen Groningen Netherlands

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