Orofacial clefts (OFCs) are the most common congenital craniofacial disorders, of which the etiology is closely related to rare coding variants. Filamin B (FLNB) is an actin-binding protein implicated in bone formatio...Orofacial clefts (OFCs) are the most common congenital craniofacial disorders, of which the etiology is closely related to rare coding variants. Filamin B (FLNB) is an actin-binding protein implicated in bone formation. FLNB mutations have been identified in several types of syndromic OFCs and previous studies suggest a role of FLNB in the onset of non-syndromic OFCs (NSOFCs). Here, we report two rare heterozygous variants (p.P441T and p.G565R) in FLNB in two unrelated hereditary families with NSOFCs. Bioinformatics analysis suggests that both variants may disrupt the function of FLNB. In mammalian cells, p.P441T and p.G565R variants are less potent to induce cell stretches than wild type FLNB, suggesting that they are loss-of-function mutations. Immunohistochemistry analysis demonstrates that FLNB is abundantly expressed during palatal development. Importantly, Flnb^(−/−) embryos display cleft palates and previously defined skeletal defects. Taken together, our findings reveal that FLNB is required for development of palates in mice and FLNB is a bona fide causal gene for NSOFCs in humans.展开更多
目的通过酵母双杂交实验筛选与环指蛋白216(ring finger protein 216,RNF216)相互作用的蛋白,进一步阐明RNF216在GnRH缺陷疾病中的作用。方法构建pGBKT7-RNF216重组表达载体,将其转化到Y2HGold酵母中,与人cDNA文库进行杂交,筛选与RNF21...目的通过酵母双杂交实验筛选与环指蛋白216(ring finger protein 216,RNF216)相互作用的蛋白,进一步阐明RNF216在GnRH缺陷疾病中的作用。方法构建pGBKT7-RNF216重组表达载体,将其转化到Y2HGold酵母中,与人cDNA文库进行杂交,筛选与RNF216相互作用的蛋白,然后在Y2HGold酵母中进行验证。结果成功构建了pGBKT7-RNF216重组表达载体,并在Y2HGold酵母中成功表达;通过酵母双杂交实验,筛选到了一个与RNF216相互作用的蛋白——丝状蛋白B(filamin B,FLNB),并在Y2HGold酵母中验证了它们之间的相互作用。结论成功筛选到一个与RNF216相互作用的FLNB蛋白,RNF216可能通过调节FLNB或FLNB/FLNA异源二聚体影响GnRH神经元的增殖和迁移。展开更多
基金supported by the National Natural Science Foundation of China(No.81870747,82170916,81900984,and 82001030)the Fundamental Research Funds for the Central Universities(PKU2022XGK001)+2 种基金Natural Science Foundation of Beijing Municipality(7182184)Xi'an“Science and Technology+”Action Plan-Medical Research Project(20YXYJ0010[1])the Fundamental Research Funds for the Central Universities(xzy012020110).
文摘Orofacial clefts (OFCs) are the most common congenital craniofacial disorders, of which the etiology is closely related to rare coding variants. Filamin B (FLNB) is an actin-binding protein implicated in bone formation. FLNB mutations have been identified in several types of syndromic OFCs and previous studies suggest a role of FLNB in the onset of non-syndromic OFCs (NSOFCs). Here, we report two rare heterozygous variants (p.P441T and p.G565R) in FLNB in two unrelated hereditary families with NSOFCs. Bioinformatics analysis suggests that both variants may disrupt the function of FLNB. In mammalian cells, p.P441T and p.G565R variants are less potent to induce cell stretches than wild type FLNB, suggesting that they are loss-of-function mutations. Immunohistochemistry analysis demonstrates that FLNB is abundantly expressed during palatal development. Importantly, Flnb^(−/−) embryos display cleft palates and previously defined skeletal defects. Taken together, our findings reveal that FLNB is required for development of palates in mice and FLNB is a bona fide causal gene for NSOFCs in humans.
文摘目的通过酵母双杂交实验筛选与环指蛋白216(ring finger protein 216,RNF216)相互作用的蛋白,进一步阐明RNF216在GnRH缺陷疾病中的作用。方法构建pGBKT7-RNF216重组表达载体,将其转化到Y2HGold酵母中,与人cDNA文库进行杂交,筛选与RNF216相互作用的蛋白,然后在Y2HGold酵母中进行验证。结果成功构建了pGBKT7-RNF216重组表达载体,并在Y2HGold酵母中成功表达;通过酵母双杂交实验,筛选到了一个与RNF216相互作用的蛋白——丝状蛋白B(filamin B,FLNB),并在Y2HGold酵母中验证了它们之间的相互作用。结论成功筛选到一个与RNF216相互作用的FLNB蛋白,RNF216可能通过调节FLNB或FLNB/FLNA异源二聚体影响GnRH神经元的增殖和迁移。
