A genetic and molecular approach to understanding allelic and phenotypic heterogeneity in Acrofacial dysostosis, Cincinnati-type

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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.