先天性多数牙齿缺失家系PAX9基因两个新突变功能的初步研究

Functional analysis of novel mutations in PAX9 associated with familial oligodontia

目的对成对盒基因(pairedboxgene,PAX)PAX9两个新突变109(InsG)及139(C→T)分子生物学功能及与先天性多数牙齿缺失的发病机理进行初步探讨。方法分别以先天性多数牙齿缺失家系A[109(InsG)]、家系B[139(C→T)]中患者及健康成员基因组DNA为模板,PCR扩增PAX9基因DNA结合域序列(paireddomain,PD),构建原核表达质粒GST,进一步在大肠杆菌中诱导表达,纯化表达产物,分离制备PAX9PD蛋白。凝胶迁移试验检测PAX9PD蛋白与靶DNA(CD192和Pax6CON)结合活性。结果野生型PAX9PD蛋白与靶DNA结合,109(InsG)及139(C→T)突变的PAX9PD蛋白皆失去与靶DNA结合能力。结论109(InsG)及139(C→T)突变造成PAX9基因DNA结合域功能丧失,可能是导致多数牙齿先天性缺失的原因。

Objective To gain new insights into the molecular pathogenesis of the 109（InsG） and 139（C→T） mutations and their roles in familial oligodontia. Methods The region of PAX9 paired domain （PAX9PD） was amplified and the expression plasmids were constructed in pGEXλ-1T by PCR-based cloning. PAX9PD proteins were prepared on the basis of GST instruction. The binding of wild type and two novely mutant PAX9 paired domain to double-stranded DNA targets were analyzed by gel mobility shift assay. Results Wild type PAX9PD protein bind to the high affinity paired domain recognition sequences, CD19-2（A-ins） and Pax6CON, the 109（InsG） and 139（C→T） mutant PAX9PD protein were unable to bind to these cognate DNA-binding sites. Conclusion The functional defects in DNA binding of mutant 109（ InsG）PAX9 and 139（C→T）PAX9, as well as loss-of-fimction of PAX9 most likely result in its haploinsufficiency during the patterning of dentition and the subsequent loss of posterior teeth.