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
文摘目的:阐明细丝蛋白B基因(filamin B,FLNB)第1542位异亮氨酸(Ile/I)→苏氨酸(Thr/T)突变(FLNB p.Ile1542Thr,FLNB^(I1542T))在休门氏后凸畸形(Scheuermann′s kyphosis,SK)中的机制。方法:对一个汉族SK家系进行标准化临床评估,包括脊柱X线、CT三维重建及MRI检查。通过全外显子测序与Sanger测序筛选并验证致病性变异,依据美国医学遗传学与基因组学学会(American College of Medical Genetics and Genomics,ACMG)指南进行致病性评估。采用CRISPR-Cas9技术构建携带FLNB^(I1542T)突变的小鼠模型,通过外观观察、显微CT成像及组织学染色(HE、Masson、Safranin O/Fast Green)对模型表型进行评估,以确认其是否重现SK特异的软骨终板缺陷与局部后凸畸形。利用单细胞RNA测序(scRNA-seq)结合生物信息学分析,解析软骨终板病理发育过程中关键的致病软骨细胞亚群及其靶分子调控网络。通过单细胞流式分选致病性软骨细胞亚群,并进一步利用免疫组化(immunohistochemistry,IHC)、免疫共沉淀(co-immunoprecipitation,Co-IP)验证FLNB^(I1542T)对c-Jun蛋白表达水平的影响;利用定量聚合酶链式反应(quantitative polymerase chain reaction,qPCR)、双荧光素酶报告基因等实验分析c-Jun对细胞外基质(extracellular matrix,ECM)稳态关键分子Serpinh1的转录激活调控,并利用免疫组化、Western blot分析Jun对ECM稳态的调控作用。结果:通过对先证者全外显子测序,鉴定出一个杂合FLNB c.4625T>C(p.Ile1542Thr)突变,与患者表型共分离,且计算预测提示其破坏蛋白稳定性。利用CRISPR-Cas9技术构建的FLNB^(I1542T)基因敲入小鼠,在生长发育过程中表现出与人SK患者相似的进行性胸腰段后凸、椎体体积与长度减小、骨化延迟及终板凹陷等影像学与组织病理学特征。单细胞转录组测序分析揭示,突变主要靶向并损害了终板软骨中的软骨形成细胞亚群的功能,导致该群细胞中与“软骨发育”和“细胞外基质组织”相关的基因表达程序显著下调。分子机制研究表明,FLNB^(I1542T)突变削弱了转录因子c-Jun的蛋白水平及其转录活性。c-Jun可直接结合并激活分子伴侣Serpinh1的启动子,而FLNB^(I1542T)突变导致的Jun功能抑制致使SERPINH1表达显著下降。SERPINH1的下游关键客户蛋白,包括COL2A1、COL9A3和COL11A2等维持ECM稳态的胶原蛋白,其表达也随之降低,从而破坏了软骨终板的ECM完整性。功能挽救实验证实,过表达Jun可逆转由FLNB^(I1542T)突变引起的SERPINH1及其下游胶原蛋白的表达下调。结论:FLNB^(I1542T)通过损害Jun的转录活性,抑制SERPINH1表达,从而破坏ECM胶原稳态,驱动SK的软骨终板缺陷与后凸畸形。
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。
