Environmental Genomics and Systems Biology Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
CHU Lille, University of Lille, EA7364, F-59000, Lille, France.
Cell. 2020 Mar 19;180(6):1262-1271.e15. doi: 10.1016/j.cell.2020.02.031. Epub 2020 Mar 12.
Establishing causal links between non-coding variants and human phenotypes is an increasing challenge. Here, we introduce a high-throughput mouse reporter assay for assessing the pathogenic potential of human enhancer variants in vivo and examine nearly a thousand variants in an enhancer repeatedly linked to polydactyly. We show that 71% of all rare non-coding variants previously proposed as causal lead to reporter gene expression in a pattern consistent with their pathogenic role. Variants observed to alter enhancer activity were further confirmed to cause polydactyly in knockin mice. We also used combinatorial and single-nucleotide mutagenesis to evaluate the in vivo impact of mutations affecting all positions of the enhancer and identified additional functional substitutions, including potentially pathogenic variants hitherto not observed in humans. Our results uncover the functional consequences of hundreds of mutations in a phenotype-associated enhancer and establish a widely applicable strategy for systematic in vivo evaluation of human enhancer variants.
在非编码变异体和人类表型之间建立因果关系是一个日益严峻的挑战。在这里，我们引入了一种高通量的小鼠报告基因检测方法，用于评估人类增强子变异体在体内的致病潜能，并在一个与多指畸形反复相关的增强子中检测了近千个变异体。我们表明，以前被认为是致病原因的所有罕见非编码变异体中有 71%导致报告基因表达模式与其致病作用一致。观察到改变增强子活性的变异体进一步被证实会导致 knockin 小鼠出现多指畸形。我们还使用组合和单核苷酸诱变来评估影响增强子所有位置的突变的体内影响，并确定了其他功能取代，包括迄今为止在人类中未观察到的潜在致病变异体。我们的研究结果揭示了与表型相关的增强子中数百个突变的功能后果，并建立了一种广泛适用于系统评估人类增强子变异体的体内评估策略。
