Functional Characterization of a Novel IRF6 Frameshift Mutation From a Van Der Woude Syndrome Family
Functional Characterization of a Novel IRF6 Frameshift Mutation From a Van Der Woude Syndrome Family
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范德沃德综合征家族的新型 IRF6 移码突变的功能表征
DOI：
10.3389/fgene.2020.00562
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发表时间：
2020-06
影响因子：
3.7
通讯作者：
Feng Chen
中科院分区：
文献类型：
--
作者：
Mengqi Zhang;Jieni Zhang;Huaxiang Zhao;Vitaly Ievlev;Wenjie Zhong;Wenbin Huang;Robert A. Cornell;Jiuxiang Lin;Feng Chen
背景干扰素调节因子6（IRF 6）的功能缺失突变导致约70%的货车-德-沃德综合征（VWS）病例，VWS是一种常染色体显性遗传发育障碍，以下唇凹陷和/或窦以及唇裂、腭裂或两者兼有为特征。方法收集一个中国汉族VWS家系，进行基因测序和突变筛查。最初，物种保守性分析和同源蛋白质建模用于预测突变的潜在致病性。为了测试IRF 6的VWS家族衍生的突变变体是否保留功能，我们在irf 6母体无效突变斑马鱼胚胎中进行了拯救测定。为了评估蛋白质稳定性，我们在体外过表达IRF 6的参考和家族变体。结果我们关注了一个VWS家族，包括一个儿子患有双侧唇窝，悬雍垂裂隙和他的父亲患有双侧唇腭裂。测序和筛选后，IRF 6的移码突变被鉴定为潜在的致病变体（NM. 006147. 3，c.1088- 1091 delTCTA; p.Ile363ArgfsTer33）。该位置的残基在物种之间是高度保守的，同源性建模表明该变体改变了蛋白质结构。在irf 6母体缺失突变的斑马鱼胚胎中，周壁分化异常，胚胎在原肠胚形成期间破裂并死亡。注射编码人IRF 6的参考变体而不是移码变体的mRNA通过原肠胚形成拯救了这些胚胎。在HEK 293 FT细胞中过表达后，IRF 6移码突变体相对不稳定，并且与参考变体相比优先靶向蛋白酶体。结论在该VWS家系中，IRF 6基因可能存在一种新的移码突变。它是一个功能缺失突变，不能挽救irf 6缺失的斑马鱼异常围产期表型，并通过蛋白酶体依赖性降解导致蛋白质不稳定。
Background Loss-of-function mutations in interferon regulatory factor-6 (IRF6) are responsible for about 70% of cases of Van Der Woude Syndrome (VWS), an autosomal dominant developmental disorder characterized by pits and/or sinuses of the lower lip and cleft lip, cleft palate, or both. Methods We collected a Chinese Han VWS pedigree, performed sequencing and screening for the causal gene mutant. Initially, species conservation analysis and homology protein modeling were used to predict the potential pathogenicity of mutations. To test whether a VWS family-derived mutant variant of IRF6 retained function, we carried out rescue assays in irf6 maternal-null mutant zebrafish embryos. To assess protein stability, we overexpressed reference and family-variants of IRF6 in vitro. Results We focused on a VWS family that includes a son with bilateral lip pits, uvula fissa and his father with bilateral cleft lip and palate. After sequencing and screening, a frameshift mutation of IRF6 was identified as the potential causal variant (NM.006147.3, c.1088-1091delTCTA; p.Ile363ArgfsTer33). The residues in this position are strongly conserved among species and homology modeling suggests the variant alters the protein structure. In irf6 maternal-null mutant zebrafish embryos the periderm differentiates abnormally and the embryos rupture and die during gastrulation. Injection of mRNA encoding the reference variant of human IRF6, but not of the frame-shift variant, rescued such embryos through gastrulation. Upon overexpression in HEK293FT cells, the IRF6 frame-shift mutant was relatively unstable and was preferentially targeted to the proteasome in comparison to the reference variant. Conclusion In this VWS pedigree, a novel frameshift of IRF6 was identified as the likely causative gene variant. It is a lost function mutation which could not rescue abnormal periderm phenotype in irf6 maternal-null zebrafish and which causes the protein be unstable through proteasome-dependent degradation.
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作者：
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