Study on the mechanisms by which cleft palate shows its phenotypic polymorphism in mice

腭裂是人类最常见的先天性颅面畸形。人类腭裂的原因尚不清楚，但已被假定为多因素。然而，最近使用转基因小鼠的研究倾向于得出结论，腭裂是由某种基因突变引起的。另外，我们对腭裂的表型和突变率为什么会发生变化以及为什么用特定基因缺陷小鼠进行突变分析并不一定能预测其表型结果知之甚少，在这项研究中，我们通过回交8代将Tgfb 3缺陷等位基因转移到各种小鼠品系中，如C57 BL/6 J、129/Sv、FVB/N、SJL/J和ICR，并实际分析了Tgfb 3缺失胎儿（每个品系n>50）中腭裂表型的方差。因此，我们发现所有Tgfb 3基因敲除的胎儿在C57 BL/6 J中仅显示完全腭裂，而在ICR中仅显示不完全腭裂。在129/Sv、FVB/N和SJL/J小鼠品系中，腭裂表现为完全性和不完全性两种类型。此外，C57 BL/6 J完全腭裂通过与ICR回交转化为不完全腭裂，表明腭裂表型在完全腭裂和不完全腭裂之间是可逆的，并依赖于遗传背景。此外，C57 BL/6 J品系Tgfb 3基因敲除小鼠中的严重（完全）腭裂在DNA甲基转移酶抑制剂5-Aza-2 '-脱氧胞苷的培养中得到部分挽救，并导致与ICR品系中观察到的不完全腭裂相当的不完全腭裂。我们的研究结果有力地表明，缺乏Tgfb 3诱导腭裂的表型修饰因子是印记基因，其特定的甲基化模式在近交系中是可遗传的。因此，腭裂的表型可以通过基因突变和表观遗传修饰物的协同作用来确定。

Cleft palate is the most frequent congenital craniofacial birth defect in humans. The cause of human cleft palate is still unclear but has been assumed to be multi-factorial. However, recent studies using transgenic mice tend to conclude that cleft palate results from mutation of a certain gene. In addition, very little is known about why cleft palate is variable in its phenotype and penetrance and why mutation analysis using specific gene-deficient mice does not necessarily predict its phenotypic consequences.In this study, we transferred the Tgfb3-deficient allele into various mouse strains, such as C57BL/6J, 129/Sv, FVB/N, SJL/J, and ICR by backcrossing over 8 generations, and actually analyzed the variance of the cleft palate phenotypes in the Tgfb3-null fetuses (n>50 of each strain). We thereby found that all the Tgfb3-null fetuses show only compete cleft palate in C57BL/6J, but they show only incomplete cleft type in ICR. In 129/Sv, FVB/N, and SJL/J mouse strains, the cleft palate showed both of complete and incomplete cleft types. In addition, the complete cleft palate in C57BL/6J was changed into incomplete cleft palate by re-backcrossing with ICR, indicating that the cleft palate phenotype is reversible between complete and incomplete cleft types and is dependent on the genetic background. Furthermore, the severe (complete) cleft palate in the Tgfb3-null mice of C57BL/6J strain was partially rescued in culture with 5-Aza-2'-deoxycytidine, a DNA methyltransferase inhibitor, and resulted in the incomplete cleft palate equivalent to that observed in ICR strain. Our results strongly suggest that the phenotypic modifier of the lack of Tgfb3-induced cleft palate is the imprinting gene(s) and its specific methylation pattern is inheritable in inbred strains. Thus, the phenotype of cleft palate can be determined by synergy of genetic mutation and epigenetic modifier(s).