The RNA polymerase II complex (pol II) is responsible for transcription of all ∼21,000 human protein-encoding genes. Here, we describe sixteen individuals harboring de novo heterozygous variants in POLR2A, encoding RPB1, the largest subunit of pol II. An iterative approach combining structural evaluation and mass spectrometry analyses, the use of S. cerevisiae as a model system, and the assessment of cell viability in HeLa cells allowed us to classify eleven variants as probably disease-causing and four variants as possibly disease-causing. The significance of one variant remains unresolved. By quantification of phenotypic severity, we could distinguish mild and severe phenotypic consequences of the disease-causing variants. Missense variants expected to exert only mild structural effects led to a malfunctioning pol II enzyme, thereby inducing a dominant-negative effect on gene transcription. Intriguingly, individuals carrying these variants presented with a severe phenotype dominated by profound infantile-onset hypotonia and developmental delay. Conversely, individuals carrying variants expected to result in complete loss of function, thus reduced levels of functional pol II from the normal allele, exhibited the mildest phenotypes. We conclude that subtle variants that are central in functionally important domains of POLR2A cause a neurodevelopmental syndrome characterized by profound infantile-onset hypotonia and developmental delay through a dominant-negative effect on pol-II-mediated transcription of DNA.

Center for Applied Genomics the Children's Hospital of Philadelphia Philadelphia PA 19104 USA

Center for Applied Genomics the Children's Hospital of Philadelphia Philadelphia PA 19104 USA; Division of Human Genetics the Children's Hospital of Philadelphia Philadelphia PA 19104 USA; Perelman School of Medicine University of Pennsylvania Philadelphia PA 19104 USA

Department of Biology and Medical Genetics Charles University 2nd Faculty of Medicine and University Hospital Motol 150 06 Prague Czech Republic

Department of Biomedical Genetics Wilhelmina Children's Hospital University Medical Center Utrecht Utrecht University 3584 EA Utrecht the Netherlands

Department of Clinical and Medical Genetics Our Lady's Hospital for Sick Children D12 N512 Dublin Ireland

Department of Clinical Genetics and Pediatrics Walter Reed National Military Medical Center Bethesda Maryland MD 20814 USA

Department of Clinical Genetics Erasmus Medical University Center Rotterdam 3000 CA Rotterdam the Netherlands

Department of Clinical Genetics Odense University Hospital 5000 Odense Denmark

Department of Clinical Genetics University Medical Center Groningen 9713 GZ Groningen the Netherlands

Department of Genetics and Reproduction Centre Hospitalier Universitaire de Grenoble 38700 Grenoble France

Department of Genetics Centre Hospitalier Universitaire de Dijon 21000 Dijon France

Department of Genetics Rouen University Hospital Centre de Référence Anomalies du Développement Normandy Centre for Genomic and Personalized Medicine 76000 Rouen France

Department of Genomic Medicine Oxford Centre for Genomic Medicine Oxford University Hospitals National Health Service Foundation Trust OX3 7LE Oxford UK

Department of Human Genetics Donders Institute for Brain Cognition and Behaviour Radboud University Medical Center Nijmegen 6525 HR Nijmegen the Netherlands

Department of Medical Genetics BC Children's Hospital Research Institute University of British Columbia BC V6H 3N1 Vancouver Canada

Department of Pathology Rouen University Hospital Centre de Référence Anomalies du Développement 76000 Rouen France

Department of Pediatrics Amphia Hospital Breda 4818 CK Breda the Netherlands

Department of Pediatrics Duke University School of Medicine Durham North Carolina NC 27710 USA

Department of Pediatrics Duke University School of Medicine Durham North Carolina NC 27710 USA; AstraZeneca Centre for Genomics Research Precision Medicine and Genomics IMED Biotech Unit AstraZeneca CB4 0WG Cambridge United Kingdom; Department of Medicine the University of Melbourne VIC 3010 Melbourne Australia

Department of Pediatrics Section of Genetics and Metabolism University of Arkansas for Medical Sciences Little Rock Arkansas AR 72223 USA

Department of Pediatrics Wilhelmina Children's Hospital University Medical Center Utrecht Utrecht University 3584 EA Utrecht the Netherlands

Department of Pediatrics Wilhelmina Children's Hospital University Medical Center Utrecht Utrecht University 3584 EA Utrecht the Netherlands; Department of Biomedical Genetics Wilhelmina Children's Hospital University Medical Center Utrecht Utrecht University 3584 EA Utrecht the Netherlands; German Cancer Consortium 79106 Heidelberg Germany

Department of Translational Medicine Federico 2 University 80126 Naples Italy; Telethon Institute of Genetics and Medicine Pozzuoli 80126 Naples Italy

Departments of Pediatrics and Human Genetics University of Michigan Medical School Ann Arbor Michigan MI 48109 USA

Division of Human Genetics the Children's Hospital of Philadelphia Philadelphia PA 19104 USA; Mitochondrial Medicine Frontier Program Division of Human Genetics the Children's Hospital of Philadelphia PA 19104 Philadelphia USA

Expertise Center for Structural Biology University Medical Center Utrecht Utrecht University 3584 CT Utrecht the Netherlands; Molecular Cancer Research Center for Molecular Medicine University Medical Center Utrecht Utrecht University Oncode Institute 3584 CT Utrecht the Netherlands

H C Andersen Children Hospital Odense University Hospital 5000 Odense Denmark

Mitochondrial Medicine Frontier Program Division of Human Genetics the Children's Hospital of Philadelphia PA 19104 Philadelphia USA

Molecular Cancer Research Center for Molecular Medicine University Medical Center Utrecht Utrecht University Oncode Institute 3584 CT Utrecht the Netherlands

National Institute for Health Research Oxford Biomedical Research Centre Wellcome Centre for Human Genetics University of Oxford OX3 7BN Oxford UK

Regenerative Medicine Center and Center for Molecular Medicine University Medical Center Utrecht Utrecht University 3584 CT Utrecht the Netherlands; Department of Urology University Medical Center Freiburg University of Freiburg 79110 Freiburg Germany

Regenerative Medicine Center and Center for Molecular Medicine University Medical Center Utrecht Utrecht University 3584 CT Utrecht the Netherlands; German Cancer Consortium 79106 Heidelberg Germany

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