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

摘要 先天性心脏病（CHD）是最常见的一类出生缺陷，患病率约为 1%的活产婴儿和各种各样的发病率和死亡率。核糖体生物发生障碍 核糖体病（“核糖体病”）是先天性畸形综合征，其与CHD和颅面神经疾病都相关。 异常一个普遍存在的过程（核糖体生物合成）的破坏导致组织- 诸如CHD的特定表型是未知的。肢面骨发育不全，辛辛那提型（AFD-CIN）是近年来 鉴定了由POLR 1A突变引起的常染色体显性核糖体病。在我们的患者队列中， 与AFD-CIN，我们观察到冠心病的发病率增加。本申请的目的是研究如何 POLR 1A介导的核糖体生物合成干扰心脏发育， 不同POLR 1A等位基因的组织特异性作用机制。核心假设是， POLR 1A等位基因引起蛋白质表达的翻译调节的谱系特异性改变。我们将测试 这个假设有以下三个具体目标：[1]分析Polr 1a在神经嵴细胞中的需求 和第二心脏领域，[2]分析Polr 1a的等位基因系列的表型和功能，以及[3]定量 不同Polr 1a等位基因对核糖体生物合成和mRNA翻译的谱系特异性影响。方法将 包括[1] Polr 1a条件性敲除等位基因的详细表型分析，[2]在体外和体内产生 （小鼠）人类突变模型，以及[3]用RNA-seq评估Polr 1a破坏的组织特异性效应 和Ribo-Seq。通过这三个目标的完成，我将获得作为独立工作者的经验和技能。 领导临床、转化和基础研究的研究者。成功完成本补助金的目标 将[1]确定核糖体生物合成在心脏发育中的作用，[2]证实POLR 1A的致病性 小鼠遗传变异，[3]提供了对POLR 1A致病结构域的了解，[4]阐明了新的 途径介导与Polr 1a缺失相关的组织特异性表型。总的来说，这可以使 干预以降低严重程度或甚至预防与核糖体生物合成缺陷相关的畸形。

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.