CRISPR-gene-engineered CYBB Knock-out PLB-985 cells, a useful model to study functional impact of X-linked Chronic Granulomatous Disease mutations: Application to the G412E X91 +-CGD mutation.

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 gp91 phox. 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 cell model obtained by homologous recombination in the original PLB-985 human myeloid cell line, in order to study the functional impact of such mutations [1]. Although the PLB-985 cell line was originally described as a distinct cell line isolated from a patient with acute nonlymphocytic leukemia [2], 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.