Clinical Medical Research Center of The Second Clinical Medical College, Jinan University, Shenzhen People's Hospital, No.1017, Dongmen North Road, Luohu District, Shenzhen, 518020, China.
Guangxi Key Laboratory of Metabolic Disease Research, Central Laboratory of Guilin No. 181 Hospital, No. 1, Xinqiaoyuan Road, Guilin, 541002, China.
Stem Cell Res Ther. 2019 Aug 14;10(1):251. doi: 10.1186/s13287-019-1369-8.
Autosomal dominant osteopetrosis type II (ADO2) is a rare human genetic disease that has been broadly studied as an important osteopetrosis model; however, there are no disease-specific induced pluripotent stem cells (ADO2-iPSCs) that may be valuable for understanding the pathogenesis and may be a potential source of cells for autologous cell-based therapies.
To generate the first human ADO2-iPSCs from a Chinese family with ADO2 and to identify their characteristics, blood samples were collected from the proband and his parents and were used for genotyping by whole-exome sequencing (WES); the urine-derived cells of the proband were reprogrammed with episomal plasmids that contained transcription factors, such as KLF4, OCT4, c-MYC, and SOX2. The proteome-wide protein quantification and lysine 2-hydroxyisobutyrylation detection of the ADO2-iPSCs and normal control iPSCs (NC-iPSCs) were performed by high-resolution LC-MS/MS and bioinformatics analysis.
WES with filtering strategies identified a mutation in CLCN7 (R286W) in the proband and his father, which was absent in the proband's mother and the healthy controls; this was confirmed by Sanger sequencing. The ADO2-iPSCs were successfully generated, which carried a normal male karyotype (46, XY) and the mutation of CLCN7 (R286W); the ADO2-iPSCs positively expressed alkaline phosphatase and other surface markers; and no vector and transgene were detected. The ADO2-iPSCs could differentiate into all three germ cell layers, both in vitro and in vivo. The proteomic profiling revealed similar expression of pluripotency markers in the two cell lines and identified 7405 proteins and 3664 2-hydroxyisobutyrylated peptides in 1036 proteins in the ADO2-iPSCs.
Our data indicated that the mutation CLCN7 (R286W) may be a cause of the osteopetrosis family. The generated vector-free and transgene-free ADO2-iPSCs with known proteomic characteristics may be valuable for personalized and cell-based regenerative medicine in the future.
常染色体显性遗传骨硬化症 2 型（ADO2）是一种罕见的人类遗传性疾病，已被广泛研究为重要的骨硬化症模型；然而，目前尚无针对该疾病的特异性诱导多能干细胞（ADO2-iPSCs），这些细胞可能对于理解发病机制具有重要价值，并且可能成为自体细胞疗法的潜在细胞来源。
为了从一个患有 ADO2 的中国家庭中生成首例人类 ADO2-iPSCs，并鉴定其特征，我们从先证者及其父母采集血样，通过全外显子组测序（WES）进行基因分型；先证者的尿源性细胞通过含有转录因子（如 KLF4、OCT4、c-MYC 和 SOX2）的附加型质粒进行重编程。通过高分辨率 LC-MS/MS 和生物信息学分析，对 ADO2-iPSCs 和正常对照 iPSCs（NC-iPSCs）进行全蛋白质组定量和赖氨酸 2-羟基异丁酰化检测。
通过过滤策略的 WES 鉴定出先证者及其父亲 CLCN7 基因（R286W）的突变，而先证者的母亲和健康对照者中不存在该突变；该突变通过 Sanger 测序得到了证实。成功生成了 ADO2-iPSCs，它们携带正常的男性核型（46，XY）和 CLCN7（R286W）突变；ADO2-iPSCs 阳性表达碱性磷酸酶和其他表面标志物；并且未检测到载体和转基因。ADO2-iPSCs 可在体外和体内分化为所有三个胚层。蛋白质组学分析显示，两种细胞系中多能性标志物的表达相似，并在 1036 种蛋白质中鉴定出 7405 种蛋白质和 3664 种 2-羟基异丁酰化肽。
我们的数据表明，CLCN7（R286W）突变可能是该骨硬化症家族的致病原因。生成的无载体和无转基因的 ADO2-iPSCs 具有已知的蛋白质组学特征，可能对未来的个性化和基于细胞的再生医学具有重要价值。
