Second Report of Chronic Granulomatous Disease in Jordan: Clinical and Genetic Description of 31 Patients From 21 Different Families, Including Families From Lybia and Iraq.

Chronic granulomatous Disease (CGD) is a rare innate immunodeficiency disorder caused by mutations in one of the six genes (CYBA, CYBB, NCF1, NCF2, NCF4, and CYBC1/EROS) encoding the superoxide-producing nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase complex in phagocytes. In the Western population, the most prevalent form of CGD (about two-thirds of all cases) is the X-linked form (X-CGD) caused by mutations in CYBB. The autosomal recessive forms (AR-CGD), due to mutations in the other genes, collectively account for the remaining one-third of CGD cases. We investigated the clinical and molecular features of 22 Jordanian, 7 Libyan, and 2 Iraqi CGD patients from 21 different families. In addition, 11 sibling patients from these families were suspected to have been died from CGD as suggested by their familial and clinical history. All patients except 9 were children of consanguineous parents. Most of the patients suffered from AR-CGD, with mutations in CYBA, NCF1, and NCF2, encoding p22 phox , p47 phox , and p67 phox proteins, respectively. AR-CGD was the most frequent form, in Jordan probably because consanguineous marriages are common in this country. Only one patient from non-consanguineous parents suffered from an X910 CGD subtype (0 indicates no protein expression). AR670 CGD and AR220 CGD appeared to be the most frequently found sub-types but also the most severe clinical forms compared to AR470 CGD. As a geographical clustering of 11 patients from eight Jordanian families exhibited the c.1171_1175delAAGCT mutation in NCF2, segregation analysis with nine polymorphic markers overlapping NCF2 indicates that a common ancestor has arisen ~1,075 years ago.

NOX4 is the main NADPH oxidase involved in the early stages of hematopoietic differentiation from human induced pluripotent stem cells.

Reactive oxygen species (ROS) produced in hematopoietic stem cells (HSCs) are involved in the balance between quiescence, self-renewal, proliferation and differentiation processes. However the role of NOX enzymes on the early stages of hematopoietic differentiation is poorly investigated. For that, we used induced pluripotent stem cells (iPSCs) derived from X-linked Chronic Granulomatous Disease (X0CGD) patients with deficiency in NOX2, and AR220CGD patients with deficiency in p22phox subunit which decreases NOX1, NOX2, NOX3 and NOX4 activities. CD34+ hematopoietic progenitors were obtained after 7, 10 and 13 days of iPS/OP9 co-culture differentiation system. Neither NOX expression nor activity was found in Wild-type (WT), X0CGD and AR220CGD iPSCs. Although NOX2 and NOX4 mRNA were found in WT, X0CGD and AR220CGD iPSC-derived CD34+ cells at day 10 and 13 of differentiation, NOX4 protein was the only NOX enzyme expressed in these cells. A NADPH oxidase activity was measured in WT and X0CGD iPSC-derived CD34+ cells but not in AR220CGD iPSC-derived CD34+ cells because of the absence of p22phox, which is essential for the NOX4 activity. The absence of NOX4 activity and the poor NOX-independent ROS production in AR220CGD iPSC-derived CD34+ cells favored the CD34+ cells production but lowered their hematopoietic potential compared to WT and X0CGD iPSC-derived CD34+ cells. In addition we found a large production of primitive AR220CGD iPSC-derived progenitors at day 7 compared to the WT and X0CGD cell types. In conclusion NOX4 is the major NOX enzyme involved in the early stages of hematopoietic differentiation from iPSCs and its activity can modulate the production, the hematopoietic potential and the phenotype of iPSC-derived CD34+.

Ex Vivo Models of Chronic Granulomatous Disease.

Induced pluripotent stem cells (iPSCs) are pluripotent stem cells that can be established from dedifferentiation of all somatic cell types by epigenetic phenomena. iPSCs can be differentiated into any mature cells like neurons, hepatocytes, or pancreatic cells that have not been easily available to date. Thus, iPSCs are widely used for disease modeling, drug discovery, and cell therapy development. Here, we describe a protocol to obtain human mature and functional neutrophils and macrophages as ex vivo models of X-linked chronic granulomatous disease (X-CGD). This method can be applied to model the other genetic forms of CGD. We also describe methods for testing the characteristics and functions of neutrophils and macrophages by morphology, phagocytosis assay, release of granule markers or cytokines, cell surface markers, and NADPH oxidase activity.