PDF(Pubmed)
Abstract：
Chronic granulomatous disease (CGD) is a rare primary immune disorder caused by mutations in one of the five subunits of the NADPH oxidase complex expressed in phagocytes. Two-thirds of CGD cases are caused by mutations in CYBB that encodes NOX2 or gp91phox. Some rare X91+-CGD point mutations lead to a loss of function but with a normal expression of the mutated NOX2 protein. It is therefore necessary to ensure that this mutation is indeed responsible for the loss of activity in order to make a safe diagnosis for genetic counselling. We previously used the X-CGD PLB-985 cell model of M.C. Dinauer obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations. Although the PLB-985 cell line was originally described by K.A. Tucker et al. in1987 as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia, it is actually identified as a subclone of the HL-60 cells. In order to use a cellular model that meets the quality standard for the functional study of X91+-CGD mutations in CGD diagnosis, we developed our own model using the CRISPR-Cas9 technology in a certified PLB-985 cell line from DSMZ-German Collection of Microorganisms and Cell Cultures. Thanks to this new X-CGD model, we demonstrated that the G412E mutation in NOX2 found in a X91+-CGD patient prohibits access of the electron donor NADPH to its binding site explaining the absence of superoxide production in his neutrophils.
摘要：
慢性肉芽肿病（CGD）是一种罕见的原发性免疫疾病，由吞噬细胞中表达的NADPH氧化酶复合物的五个亚基之一的突变引起。三分之二的CGD病例是由编码NOX2或gp91phox的CYBB突变引起的。一些罕见的X91+-CGD点突变导致功能丧失,但具有突变的NOX2蛋白的正常表达。因此，有必要确保这种突变确实是活性丧失的原因，以便为遗传咨询做出安全的诊断。我们先前使用了在原始PLB-985人髓系细胞系中通过同源重组获得的X-CGD细胞模型，为了研究这种突变的功能影响[1]。尽管PLB-985细胞系最初被描述为分离自急性非淋巴细胞白血病患者的独特细胞系[2]，它实际上被鉴定为HL-60细胞的亚克隆。为了使用符合质量标准的细胞模型进行CGD诊断中X91+-CGD突变的功能研究，我们使用CRISPR-Cas9技术在DSMZ-德国微生物和细胞培养保藏所认证的PLB-985细胞系中开发了我们自己的模型.多亏了这个新的X-CGD模型，我们证明,在1例X91+-CGD患者中发现的NOX2中的G412E突变阻止了电子供体NADPH进入其结合位点,这解释了其中性粒细胞中没有产生超氧化物.
