Heterozygous pathogenic variants in POLR1A, which encodes the largest subunit of RNA Polymerase I, were previously identified as the cause of acrofacial dysostosis, Cincinnati-type. The predominant phenotypes observed in the cohort of 3 individuals were craniofacial anomalies reminiscent of Treacher Collins syndrome. We subsequently identified 17 additional individuals with 12 unique heterozygous variants in POLR1A and observed numerous additional phenotypes including neurodevelopmental abnormalities and structural cardiac defects, in combination with highly prevalent craniofacial anomalies and variable limb defects. To understand the pathogenesis of this pleiotropy, we modeled an allelic series of POLR1A variants in vitro and in vivo. In vitro assessments demonstrate variable effects of individual pathogenic variants on ribosomal RNA synthesis and nucleolar morphology, which supports the possibility of variant-specific phenotypic effects in affected individuals. To further explore variant-specific effects in vivo, we used CRISPR-Cas9 gene editing to recapitulate two human variants in mice. Additionally, spatiotemporal requirements for Polr1a in developmental lineages contributing to congenital anomalies in affected individuals were examined via conditional mutagenesis in neural crest cells (face and heart), the second heart field (cardiac outflow tract and right ventricle), and forebrain precursors in mice. Consistent with its ubiquitous role in the essential function of ribosome biogenesis, we observed that loss of Polr1a in any of these lineages causes cell-autonomous apoptosis resulting in embryonic malformations. Altogether, our work greatly expands the phenotype of human POLR1A-related disorders and demonstrates variant-specific effects that provide insights into the underlying pathogenesis of ribosomopathies.

Atrium Health's Levine Children's Hospital Charlotte NC USA

CCA AHU de génétique clinique et de neurogénétique Service de Génétique et de Neurologie CHU de Caen Caen France

Department of Clinical Genetics Erasmus MC Rotterdam the Netherlands

Department of Neurology Charles University 1st Faculty of Medicine and General University Hospital Prague Prague Czech Republic

Department of Neurology Comprehensive Epilepsy Center New York University Grossman School of Medicine New York NY USA

Department of Neurology P J Safarik University Kosice Slovak Republic; Department of Neurology University Hospital of L Pasteur Kosice Slovak Republic

Department of Pathology and Laboratory Medicine Children's Mercy Kansas City 2401 Gillham Road Kansas City MO USA; Genomic Medicine Center Children's Mercy Research Institute 2401 Gillham Road Kansas City MO USA; School of Medicine University of Missouri Kansas City 2411 Holmes Street Kansas City MO USA

Department of Pediatrics Penn State Health Children's Hospital Hershey PA USA

Department of Pediatrics University of Washington Seattle WA USA

Department of Women's Health University of Texas Austin Dell Medical Center Austin TX USA

Division of Clinical Genetics Department of Pediatrics Children's Mercy Kansas City 2401 Gillham Road Kansas City MO USA

Division of Human Genetics Cincinnati Children's Hospital Medical Center Cincinnati OH USA

Division of Human Genetics Cincinnati Children's Hospital Medical Center Cincinnati OH USA; Department of Pediatrics University of Cincinnati College of Medicine Cincinnati OH USA

Division of Medical Genetics Department of Pediatrics University of California San Francisco San Francisco CA USA

GeneDx LLC Gaithersburg MD USA

Genetic Department APHP Sorbonne Université Pitié Salpêtrière Hospital 47 83 Boulevard de l'Hôpital 75013 Paris France

Genome Dynamics Laboratory National Institute of Genetics Mishima Shizuoka Japan; Department of Genetics School of Life Science Sokendai Mishima Shizuoka Japan

Institute of Neurogenomics Helmholtz Zentrum München Munich Germany; Institute of Human Genetics School of Medicine Technical University of Munich Munich Germany

Institute of Neurogenomics Helmholtz Zentrum München Munich Germany; Institute of Human Genetics School of Medicine Technical University of Munich Munich Germany; Lehrstuhl für Neurogenetik Technische Universität München Munich Germany; Munich Cluster for Systems Neurology SyNergy Munich Germany

Paediatric Neuroscience Research Group Royal Hospital for Children Glasgow G667AB UK

Sibley Heart Center Atlanta GA USA

Steve and Cindy Rasmussen Institute for Genomic Medicine Nationwide Children's Hospital Columbus OH USA; Department of Pediatrics The Ohio State University School of Medicine Columbus OH USA

Steve and Cindy Rasmussen Institute for Genomic Medicine Nationwide Children's Hospital Columbus OH USA; Division of Developmental Biology Cincinnati Children's Hospital Medical Center Cincinnati OH USA

Stowers Institute for Medical Research Kansas City MO USA

Stowers Institute for Medical Research Kansas City MO USA; Department of Anatomy and Cell Biology University of Kansas Medical Center Kansas City KS USA

The Cancer Institute of JFCR Tokyo Japan

University of São Paulo São Paulo Brazil

Victorian Clinical Genetics Services Murdoch Children's Research Institute Flemington Road Melbourne VIC Australia

Victorian Clinical Genetics Services Murdoch Children's Research Institute Flemington Road Melbourne VIC Australia; University of Melbourne Melbourne VIC Australia

Victorian Clinical Genetics Services Murdoch Children's Research Institute Flemington Road Melbourne VIC Australia; University of Melbourne Melbourne VIC Australia; Australian Genomics Melbourne VIC Australia

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