ANO5非移码插入突变型GDD家系临床分析及其诱导多能干细胞的构建

Clinical analysis of ANO5 non-frameshift insertion mutation GDD family and generation of the induced pluripotent stem cells

目的:建立和鉴定临床发现的一个由ANO5基因新的致病性突变引起的颌骨骨干发育不良(gnathodiaphyseal dysplasia,GDD)家系中患病者和非患病者来源的诱导多能干细胞(induced pluripotent stem cells,i PSCs)。方法 :纳入以颌骨畸形为主要表现的1个GDD家系5例患者,评估患者临床表现、骨影像学、骨密度、骨量等相关生化指标。收集家系中1例患病者与非患病者外周血样本,将表达Oct4、Sox2和Klf4转录因子的仙台病毒转染患病者和非患病者外周血单核细胞,将其重编程为i PSCs,并通过碱性磷酸酶染色、细胞免疫荧光、qPCR、核型分析等检测其干细胞多能性。结果:GDD主要表现为双侧上下颌骨弥漫性多发肿胀,并伴有全身骨密度下降。通过检测,证明患病者及非患病者外周血来源的i PSCs具有多能性。结论:GDD患者外周血单核细胞在体外可被成功诱导为无外源性基因整合的具有多能分化特性的i PSCs,为GDD的发病机制研究提供了高效直接的细胞模型。

PURPOSE:To generate and identify gnathodiaphyseal dysplasia(GDD)patient and healthy donor derived induced pluripotent stem cells from a Chinese GDD family caused by a mutation in ANO5 gene.METHODS:The clinical manifestations,skeletal radiographic features,bone mineral density(BMD),and bone turn over biomarkers were investigated of 5 patients from a Chinese GDD family with facial deformities.Peripheral blood mononuclear cells(PBMCs)from one patient and one non-patient were collected and transfected with sendai virus carrying Oct4,Sox2 and Klf4 to be reprogrammed into iPSCs.The pluripotency was tested by alkaline phosphatase staining,immune-fluorescence,qPCR,and karyotyping analysis.RESULTS:GDD was mainly manifested as diffuse expansive swelling of maxilla and mandible with reduced bone mineral density throughout the body.iPSCs derived from PBMCs of the patient and the healthy donor maintained pluripotency.CONCLUSIONS:PBMCs of GDD patient are successfully reprogrammed into integration-free iPSCs with the pluripotency.These iPSCs provide a valuable cell model for mechanism exploration.