Dirk Roos1, Jaap D van Buul1, Anton Tj Tool1, Juan D Matute2, Christophe M Marchal2, Bu'Hussain Hayee3, M Yavuz Köker4, Martin de Boer1, Karin van Leeuwen1, Anthony W Segal3, Edgar Pick5, Mary C Dinauer2. 1. Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. 2. Departments of Pediatrics (Hematology/Oncology), Microbiology/Immunology, and Medical and Molecular Genetics, Riley Hospital for Children, Indiana University School of Medicine, Indianapolis, IN, USA. 3. Department of Medicine, University College London, London, United Kingdom. 4. Department of Immunology and Immunology Laboratory, Faculty of Medicine, University of Erciyes, Kayseri, Turkey. 5. Julius Friedrich Cohnheim Laboratory of Phagocyte Research, Sackler School of Medicine, Tel Aviv University, Israel.
Abstract
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 CYBB, 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 in NCF2, 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' NCF2 gene was expressed as Ala202Val-p67phox in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67phox 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 p67phox was clearly hypomorphic: substantial expression of p67phox 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-p67phox translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67phox in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67phox. CONCLUSION: The mutation found in NCF2 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 p67phox, resulting in reduced activation of gp91phox.
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 CYBB, 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 in NCF2, 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' NCF2 gene was expressed as Ala202Val-p67phox in K562 cells to measure its effect on NADPH oxidase activity. Translocation of the mutated p67phox 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 p67phox was clearly hypomorphic: substantial expression of p67phox 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-p67phox translocation to the plasma membrane during cell activation was also stimulus dependent. Ala202Val-p67phox in K562 cells mediated only about 3% of normal oxidase activity compared to cells transfected with the wild-type p67phox. CONCLUSION: The mutation found in NCF2 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 p67phox, resulting in reduced activation of gp91phox.
Authors: Marianne O Price; Linda C McPhail; J David Lambeth; Chang-Hoon Han; Ulla G Knaus; Mary C Dinauer Journal: Blood Date: 2002-04-15 Impact factor: 22.113
Authors: Annemarie van de Geer; Alejandro Nieto-Patlán; Douglas B Kuhns; Anton Tj Tool; Andrés A Arias; Matthieu Bouaziz; Martin de Boer; José Luis Franco; Roel P Gazendam; John L van Hamme; Michel van Houdt; Karin van Leeuwen; Paul Jh Verkuijlen; Timo K van den Berg; Juan F Alzate; Carlos A Arango-Franco; Vritika Batura; Andrea R Bernasconi; Barbara Boardman; Claire Booth; Siobhan O Burns; Felipe Cabarcas; Nadine Cerf Bensussan; Fabienne Charbit-Henrion; Anniek Corveleyn; Caroline Deswarte; María Esnaola Azcoiti; Dirk Foell; John I Gallin; Carlos Garcés; Margarida Guedes; Claas H Hinze; Steven M Holland; Stephen M Hughes; Patricio Ibañez; Harry L Malech; Isabelle Meyts; Marcela Moncada-Velez; Kunihiko Moriya; Esmeralda Neves; Matias Oleastro; Laura Perez; Vimel Rattina; Carmen Oleaga-Quintas; Neil Warner; Aleixo M Muise; Jeanet Serafín López; Eunice Trindade; Julia Vasconcelos; Séverine Vermeire; Helmut Wittkowski; Austen Worth; Laurent Abel; Mary C Dinauer; Peter D Arkwright; Dirk Roos; Jean-Laurent Casanova; Taco W Kuijpers; Jacinta Bustamante Journal: J Clin Invest Date: 2018-08-06 Impact factor: 14.808