Two CGD Families with a Hypomorphic Mutation in the Activation Domain of p67phox.
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 inCYBB, the X-linked gene that encodes gp91phox, the catalytic subunit of the leukocyte NADPH oxidase. We report here three autosomal recessive CGD patients from two families with a homozygous mutation inNCF2, the gene that encodes p67phox, 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'NCF2gene was expressed as Ala202Val-p67phoxin K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67phoxfrom 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 p67phoxwas clearly hypomorphic: substantial expression of p67phoxprotein 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-p67phoxtranslocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67phoxin K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67phox.


CONCLUSIONS
The mutation found inNCF2is 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 p67phox, resulting in reduced activation of gp91phox.
Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Dirk
D
Roos
Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Jaap D
JD
van Buul
Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Anton Tj
AT
Tool
Departments of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN, USA.
Juan D
JD
Matute
Departments of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN, USA.
Christophe M
CM
Marchal
Department of Medicine, University College London, London, United Kingdom.
Bu'Hussain
B
Hayee
Department of Immunology and Immunology Laboratory, Faculty of Medicine, University of Erciyes, Kayseri, Turkey.
M Yavuz
MY
Köker
Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Martin
M
de Boer
Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands.
Karin
K
van Leeuwen
Department of Medicine, University College London, London, United Kingdom.
Anthony W
AW
Segal
Julius Friedrich Cohnheim Laboratory of Phagocyte Research, Sackler School of Medicine, Tel Aviv University, Israel.
Edgar
E
Pick
Departments of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN, USA.
Mary C
MC
Dinauer
Chronic granulomatous disease; NADPH oxidase; NCF2; hypomorphic mutation; p67phox; p67phox activation domain; p67phox translocation