Heterozygous variants in POLR1A cause diverse human phenotypes

Heterozygous pathogenic variants in POLR1A were identified as the cause of Acrofacial Dysostosis, Cincinnati-type in 2015. Craniofacial anomalies reminiscent of Treacher Collins syndrome were the predominant phenotype observed in the first 3 affected individuals. We have subsequently identified 17 additional individuals with 12 unique (11 novel) heterozygous variants in POLR1A and observed numerous additional phenotypes including developmental delay, infantile spasms, and structural cardiac defects. To understand the pathogenesis of this pleiotropy, we created an allelic series of POLR1A using a combination of in vivo (mouse) and in vitro models. We describe distinct spatiotemporal requirements for Polr1a during mouse embryogenesis and identify a requirement for Polr1a for survival of pre migratory and migratory neural crest cells, forebrain precursors, and the second heart field. We used CRISPR/Cas9 to recapitulate two human alleles in mouse, demonstrating pathogenicity of one and likely benign nature of the other. Our work greatly expands the phenotype of human POLR1A-related disorders, provides new evidence of reduced penetrance and variable expression of POLR1A heterozygous variants, and demonstrates a multi-faceted approach to characterize and define pathogenicity of variants. Overall design: Comparing differentially expressed genes in two Polr1a mutants; a null allele and a p.C1559F missense allele.

2015年，研究人员确认POLR1A基因的杂合致病性变异是辛辛那提型面肢发育不全（Acrofacial Dysostosis, Cincinnati-type）的致病原因。
最初的3名受累个体主要表现出类似于特雷彻·柯林斯综合征（Treacher Collins syndrome）的颅面畸形。
后续我们又鉴定出17名额外患者，其携带12种独特（其中11种为新发）的POLR1A杂合变异，并观察到诸多其他表型，包括发育迟缓、婴儿痉挛症以及结构性心脏缺损。
为阐明这种多效性的发病机制，我们结合体内（小鼠）与体外模型构建了POLR1A的等位基因系列。
我们明确了小鼠胚胎发生过程中Polr1a存在不同的时空需求，并证实Polr1a对迁移前、迁移期神经嵴细胞、前脑前体细胞以及第二心区的存活至关重要。
我们利用CRISPR/Cas9技术在小鼠体内重现了两种人类等位基因，证明其中一种具有致病性，而另一种大概率为良性变异。
本研究极大拓展了人类POLR1A相关疾病的表型谱，为POLR1A杂合变异的外显率降低及表现度差异提供了新证据，并展示了一套多维度的方法来表征和明确变异的致病性。
总体实验设计：对两种Polr1a突变体（即无效等位基因与p.C1559F错义等位基因）中的差异表达基因进行比较分析。