Division of Evolution and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UK.
Laboratory of Embryology and Genetics of Human Malformation, Institut National de la Santé et de la Recherche Médicale (INSERM) UMR 1163, Institut Imagine, Paris, France.
Hum Mutat. 2020 Aug;41(8):1372-1382. doi: 10.1002/humu.24027. Epub 2020 May 3.
Pathogenic variants in the core spliceosome U5 small nuclear ribonucleoprotein gene EFTUD2/SNU114 cause the craniofacial disorder mandibulofacial dysostosis Guion-Almeida type (MFDGA). MFDGA-associated variants in EFTUD2 comprise large deletions encompassing EFTUD2, intragenic deletions and single nucleotide truncating or missense variants. These variants are predicted to result in haploinsufficiency by loss-of-function of the variant allele. While the contribution of deletions within EFTUD2 to allele loss-of-function are self-evident, the mechanisms by which missense variants are disease-causing have not been characterized functionally. Combining bioinformatics software prediction, yeast functional growth assays, and a minigene (MG) splicing assay, we have characterized how MFDGA missense variants result in EFTUD2 loss-of-function. Only four of 19 assessed missense variants cause EFTUD2 loss-of-function through altered protein function when modeled in yeast. Of the remaining 15 missense variants, five altered the normal splicing pattern of EFTUD2 pre-messenger RNA predominantly through exon skipping or cryptic splice site activation, leading to the introduction of a premature termination codon. Comparison of bioinformatic predictors for each missense variant revealed a disparity amongst different software packages and, in many cases, an inability to correctly predict changes in splicing subsequently determined by MG interrogation. This study highlights the need for laboratory-based validation of bioinformatic predictions for EFTUD2 missense variants.
核心剪接体 U5 小核核糖核蛋白基因 EFTUD2/SNU114 中的致病性变异导致颅面畸形-颌面部发育不良 Guion-Almeida 型（MFDGA）。EFTUD2 中的 MFDGA 相关变异包括包含 EFTUD2 的大片段缺失、基因内缺失以及单核苷酸截断或错义变异。这些变异预计通过变异等位基因的功能丧失导致杂合不足。虽然 EFTUD2 内的缺失对等位基因功能丧失的贡献是显而易见的，但错义变异导致疾病的机制尚未在功能上得到表征。我们结合生物信息学软件预测、酵母功能生长测定和小基因（MG）剪接测定，研究了 MFDGA 错义变异如何导致 EFTUD2 功能丧失。在酵母中建模时，只有 19 个评估的错义变异中的 4 个通过改变蛋白质功能导致 EFTUD2 功能丧失。在其余 15 个错义变异中，有 5 个主要通过外显子跳跃或隐蔽剪接位点激活改变了 EFTUD2 前信使 RNA 的正常剪接模式，导致提前终止密码子的引入。对每个错义变异的生物信息预测器进行比较后发现，不同软件包之间存在差异，并且在许多情况下，无法正确预测随后通过 MG 询问确定的剪接变化。这项研究强调了需要对 EFTUD2 错义变异的生物信息预测进行基于实验室的验证。
