Generation of Powerful Biological Tools for Understanding the Pathophysiology of Chronic Granulomatous Disease.

Overview

Chronic granulomatous disease (CGD) is a rare genetic disease of innate immune due to the malfunction of phagocytic cells unable to destroy pathogens during infection. The four genes implicated are CYBB, CYBA, NCFA and NCF2 respectively encoding Nox2, p22phox, p47phox and p67phox. Nox2 analogs have recently been discovered in cells other than phagocytes. So the question arises on physiopathological impact of the absence of theses proteins not only in phagocytes but also in other cells types such as fibroblasts or neurons. The principal objective is thus to study the impact of protein deficits Nox2 and p22phox, in the pathophysiology of neurons from inducible pluripotent bone marrow cells (iPSC). For this purpose, a collection was built of fibroblasts and keratinocytes from patients with different forms of CGD to get iPSC similar to embryonic marrow cells and differentiable into several cell types (neurons, phagocytes).

Non randomised pilot descriptive multicentric study. Since recently it has been shown that Nox2p22phox protein is expressed not only in phagocytic cells but also in non-phagocytic cells such as fibroblasts, epithelial cells ,vascular cells, neurons. If pathological consequences of the deficiency Nox2 and p22phox, essential to the production of bactericidal toxic derivatives at the level of phagocytic cells, is well documented, impact of their absence in other types of non-phagocytic cells is not known. A better understanding of the impact of the absence of these proteins in these tissues could improve the management of CGD patients by providing a more specific monitoring of their condition. Similarly the formation of different cell models of all genetic forms of CGD that do not exist at present will be of great use to study the physiopathology of this disease and as tools for future studies. The study requires the inclusion of minor subjects as CGD is usually diagnosed in early childhood ( \<2 years), it is rare (frequency 1/200 000) and the life expectancy is reduced. To elaborate the cells collection, hair and skin biopsy are necessary. They will be performed under local anesthesia for adults, and during a planned general anesthesia for minors. Fibroblasts and keratinocytes in culture will be obtained by conventional control methods and the absence of expression of p22phox or Nox2 will be checked. Measurement of the kinetics of neuronal development and apoptosis iPSC will be performed in a differentiation system 2 dimensions on stromal cells MS5. For that, markers of neuronal differentiation of each step will be measured. Measurement of Reactive Oxygen Species (ROS) in phagocytes and p22phox deficient Nox2 from differentiation from iPSC (chemiluminescence, flow cytometry) will be performed. Measurement of the effectiveness of phagocytosis (phagocytic function) in phagocytes deficient p22phox and Nox2 from differentiation from iPSC (flow cytometry) will be performed. The absence of protein and p22phox Nox2 in phagocytes and p22phox deficient Nox2 from differentiation from the iPSC (western blot, flow cytometry) will be verified. Kinetic of transformation of fibroblasts derived from CGD patients with deficiency or p22phox Nox2 in myofibroblasts will be measured. To answer the principal objective of this study the recruitment of 10 patients will be necessary.