Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67.

STUDY BACKGROUND

Chronic granulomatous Disease (CGD) is a rare immunodeficiency caused by a defect in the leukocyte NADPH oxidase. This enzyme generates superoxide, which is needed for the killing of bacteria and fungi by phagocytic leukocytes. Most CGD patients have mutations in , the X-linked gene that encodes gp91, the catalytic subunit of the leukocyte NADPH oxidase. We report here three autosomal recessive CGD patients from two families with a homozygous mutation in , the gene that encodes p67, the activator subunit of the NADPH oxidase.

METHODS

Leukocyte NADPH oxidase activity, expression of oxidase components and gene sequences were measured with standard methods. The mutation found in the patients' gene was expressed as Ala202Val-p67 in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67 from the cytosol of the patients' neutrophils to the plasma membrane was measured by confocal microscopy and by Western blotting after membrane purification.

RESULTS

The exceptional feature of the A67 CGD patients reported here is that the p.Ala202Val mutation in the activation domain of p67 was clearly hypomorphic: substantial expression of p67 protein was noted and the NADPH oxidase activity in the neutrophils of the patients was 20-70% of normal, dependent on the stimulus used to activate the cells. The extent of Ala202Val-p67 translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67 in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67.

CONCLUSION

The mutation found in is the cause of the decreased NADPH oxidase activity and the (mild) clinical problems of the patients. We propose that the p.Ala202Val mutation has changed the conformation of the activation domain of p67, resulting in reduced activation of gp91.