ABSTRACT Congenital heart defects (CHD) are the most common class of birth defects, with a prevalence of approximately 1% of live births and a wide range of morbidity and mortality. Disorders of ribosome biogenesis (“ribosomopathies”) are congenital malformation syndromes variably associated with both CHD and craniofacial anomalies. The mechanism by which disruption of a ubiquitous process (ribosome biogenesis) leads to tissue- specific phenotypes such as CHD is unknown. Acrofacial dysostosis, Cincinnati type (AFD-CIN) is a recently identified autosomal dominant ribosomopathy caused by mutations in POLR1A. Among our cohort of patients with AFD-CIN we observed an increased incidence of CHD. The objective of this application is to study how POLR1A-mediated perturbation of ribosome biogenesis disrupts cardiac development, and to determine the mechanisms underlying tissue-specific effects of different POLR1A alleles. The central hypothesis is that distinct POLR1A alleles cause lineage-specific alteration of translational regulation of protein expression. We will test this hypothesis with the following three specific aims: [1] Analyze the requirement for Polr1a in neural crest cells and the second heart field, [2] Analyze phenotype and function of an allelic series of Polr1a, and [3] Quantify the lineage-specific effects of distinct Polr1a alleles on ribosome biogenesis and mRNA translation. Methods will include [1] detailed phenotyping of conditional knock-out alleles of Polr1a, [2] generating in vitro and in vivo (mouse) models of human mutations, and [3] assessing tissue-specific effects of Polr1a disruption with RNA-seq and Ribo-Seq. Through completion of these three aims, I will gain experience and skills as an independent investigator leading clinical, translational, and basic research. Successful completion of the goals of this grant will [1] define a role for ribosome biogenesis in cardiac development, [2] confirm pathogenicity of POLR1A genetic variants in mice, [3] provide insight into pathogenic domains of POLR1A, and [4] elucidate novel pathways that mediate tissue-specific phenotypes associated with loss of Polr1a. Collectively this could enable intervention to reduce severity or even prevent malformations associated with defects in ribosome biogenesis.

NARRATIVE Ribosomopathies such as Acrofacial dysostosis, Cincinnati-type are genetic diseases associated with a variety of congenital malformations. The mechanism by which disruption of a ubiquitous process like ribosome biogenesis leads to tissue-specific phenotypes is largely unknown. The proposed project seeks to understand how different missense variants in POLR1A lead to different cardiovascular phenotypes, therefore providing insight into how disruption of ribosome biogenesis leads to congenital heart disease.