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

腭裂是人类头面部最常见的先天性出生缺陷。人类腭裂的原因尚不清楚，但已被认为是多因素的。然而，最近使用转基因小鼠的研究倾向于得出结论，腭裂是特定基因突变的结果。此外，为什么腭裂的表型和外显率是不同的，以及为什么使用特定基因缺失的小鼠的突变分析不一定能预测其表型结果，我们还知之甚少。在本研究中，我们通过回交将TGFB3缺乏的等位基因转移到不同的小鼠品系，如C57BL/6J，129/Sv，FVB/N，SJL/J和ICR，并实际分析了TGFB3缺失的胎儿(n&gt；每个品系50个)的腭裂表型的差异。由此我们发现TGFB3缺失型胎儿在C57BL/6J中仅表现为竞争型腭裂，而在ICR中仅表现为不完全唇裂型。在129/Sv、FVB/N和SJL/J小鼠品系中，腭裂均表现为完全性和不完全性唇裂。此外，C57BL/6J的完全性腭裂通过与ICR的再回交而转变为不完全性腭裂，这表明腭裂表型在完全性和不完全性腭裂类型之间是可逆的，并取决于遗传背景。此外，用DNA甲基转移酶抑制剂5-aza-2‘-脱氧胞苷部分挽救了C57BL/6J品系TGFB3基因缺失小鼠的严重(完全)腭裂，并导致了相当于ICR品系的不完全性腭裂。我们的结果有力地表明，TGFB3诱导的腭裂缺失的表型修饰基因是印记基因(S)，其特有的甲基化模式在近交系中是可遗传的。因此，可以通过遗传突变和表观遗传修饰物的协同作用来确定腭裂的表型(S)。

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).