Abstract
Chronic granulomatous disease is an inherited disorder in which phagocytes lack a functional NADPH oxidase and cannot produce superoxide anions. The most common form is caused by mutations in CYBB encoding gp91phox. We investigated 24 CGD patients and their families. Twenty-one mutations in CYBB were classified as X910, X91+ or X91− variants according to cytochrome b 558 expression. Point mutations in encoding regions represented 50 % of the mutations found in CYBB, splice site mutations 27 %, deletions and insertions 23 %. Eight mutations in CYBB were novel leading to X910CGD cases. Two of these were point mutations: c493G>T and a double mutation c625C>G in exon 6 and c1510C>T in exon 12 leading to a premature stop codon at Gly165 in gp91phox and missense mutations His209Arg/Thr503Ile respectively. Two novel splice mutations in 5′intronic regions of introns 1 and 6 were found. A novel deletion/insertion c1024_1026delCTG/insT results in a frameshift introducing a stop codon at position 346 in gp91phox. The last novel mutation was the insertion of a T at c1373 leading to a frameshift and a premature stop codon at position 484 in gp91phox. For the first time the precise size of two large mutations in CYBB was determined by array-comparative genomic hybridization and carriers’ status were evaluated by multiplex ligation-dependent probe amplification assay. No clear correlation between clinical severity and CYBB mutations could be established. Of three mutations in CYBA, NCF1 and NCF2 leading to rare autosomal recessive CGD, one nonsense mutation c29G>A in exon 1 of NCF2 was new.
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References
Malech HL, Hickstein DD. Genetics, biology and clinical management of myeloid cell primary immune deficiencies: chronic granulomatous disease and leukocyte adhesion deficiency. Curr Opin Hematol. 2007;14:29–36.
Takeya R, Sumimoto H. Molecular mechanism for activation of superoxide-producing NADPH oxidases. Mol Cells. 2003;31:271–7.
Stasia MJ, Li XJ. Genetics and immunopathology of chronic granulomatous disease. Semin Immunopathol. 2008;30:209–35.
Matute JD, Arias AA, Wright NA, Wrobel I, Waterhouse CC, Li XJ, Marchal CC, Stull ND, Lewis DB, Steele M, Kellner JD, Yu W, Meroueh SO, Nauseef WM, Dinauer MC. A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40phox and selective defects in neutrophil NADPH oxidase activity. Blood. 2009;114:3309–15.
Roos D, Kuhns DB, Maddalena A, Bustamante J, Kannengiesser C, de Boer M, van Leeuwen K, Köker MY, Wolach B, Roesler J, Malech HL, Holland SM, Gallin JI, Stasia MJ. Hematologically important mutations: the autosomal recessive forms of chronic granulomatous disease (second update). Blood Cells Mol Dis. 2010;44:291–9.
Roos D, Kuhns DB, Maddalena A, Roesler J, Lopez JA, Ariga T, Avcin T, de Boer M, Bustamante J, Condino-Neto A, Di Matteo G, He J, Hill HR, Holland SM, Kannengiesser C, Köker MY, Kondratenko I, van Leeuwen K, Malech HL, Marodi L, Nunoi H, Stasia MJ, Ventura AM, Witwer CT, Wolach B, Gallin JI. Hematologically important mutations: X-linked chronic granulomatous disease (third update). Blood Cells Mol Dis. 2010;1545:246–65.
Van den Berg JM, van Koppen E, Ahlin A, Belohradsky BH, Bernatowska E, Corbeel L, Español T, Fischer A, Kurenko-Deptuch M, Mouy R, Petropoulou T, Roesler J, Seger R, Stasia MJ, Valerius NH, Weening RS, Wolach B, Roos D, Kuijpers TW. Chronic granulomatous disease: the European experience. PLoS One. 2009;4:e5234.
Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, Marciano BE, Uzel G, DeRavin SS, Long Priel DA, Soule BP, Zarember KA, Malech HL, Holland SM, Gallin JI. Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med. 2010;363:2600–10.
Stasia MJ, Bordigoni P, Floret D, Brion JP, Bost-Bru C, Michel G, Gatel P, Durant-Vital D, Voelckel MA, Li XJ, Guillot M, Maquet E, Martel C, Morel F. Characterization of six novel mutations in the CYBB gene leading to different sub-types of X-linked chronic granulomatous disease. Hum Genet. 2005;116:72–82.
Böyum A. Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Lab Investig. 1968;21:77–89.
Cohen-Tanugi L, Morel F, Pilloud-Dagher MC, Seigneurin JM, François P, Bost M, Vignais PV. Activation of O −2 generating oxidase in heterologous cell-free system derived from Epstein-Barr-virus-transformed human B lymphocytes and bovine neutrophils. Eur J Biochem. 1991;202:649–55.
Stasia MJ, Brion JP, Martel C, Morel F. Severe clinical forms of cytochrome b-negative chronic granulomatous disease (X91-) in three children with a point mutation in the promoter region of the CYBB gene. J Infect Dis. 2003;188:1597–604.
Carrichon L, Picciocchi A, Debeurme F, Defendi F, Beaumel S, Jesaitis AJ, Dagher MC, Stasia MJ. Characterization of superoxide overproduction by the D-Loop(Nox4)-Nox2 cytochrome b(558) in phagocytes-differential sensitivity to calcium and phosphorylation events. Biochim Biophys Acta. 2011;1808:78–90.
Zhou M, Diwu Z, Panchuk-Voloshina N, Haugland RP. A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal Biochem. 1977;253:162–8.
Picciocchi A, Debeurme F, Beaumel S, Dagher MC, Grunwald D, Jesaitis AJ, Stasia MJ. Role of the putative second transmembrane region 45LLGSALALARAPAACLNFNCMLILL69 of Nox2 in its structural stability and electron transfer in the phagocytic NADPH oxidase. J Biol Chem. 2011;286:28357–69.
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227:680–5.
Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some application. Proc Natl Acad Sci USA. 1979;76:4350–435.
Verhoeven AJ, Bolscher GJM, Meerhof L, van Zwieten R, Keijer J, Weening RS, Roos. Characterization of two monoclonal antibodies against cytochrome b 558 of human neutrophils. Blood. 1989;73:1686–94.
Vergnaud S, Paclet MH, El Benna J, Pocidalo MA, Morel F. Complementation of NADPH oxidase in p67-phox/p40-phox interaction. Eur J Biochem. 2000;267:1059–67.
Bakri F, Martel C, Khuri-Bulos N, Mahafzah A, El-Khateeb MS, Al-Wahadneh AD, Hayajneh WA, Hamamy HA, Maquet E, Molin M, Stasia MJ. First report of clinical, functional, and molecular investigation of chronic granulomatous disease in nine Jordanian families. J Clin Immunol. 2009;29:215–30.
Dekker J, de Boer M, Roos D. Gene-scan method for the recognition of carriers and patients with p47phox-deficient autosomal recessive chronic granulomatous disease. Exp Hematol. 2001;29:1319–25.
Menten B, Maas N, Thienpont B, Buysse K, Vandesompele J, Melotte C, de Ravel T, Van Vooren S, Balikova I, Backx L, Janssens S, De Paepe A, De Moor B, Moreau Y, Marynen P, Fryns JP, Mortier G, Devriendt K, Speleman F, Vermeesch JR. Emerging patterns of cryptic chromosomal imbalance in patients with idiopathic mental retardation and multiple congenital anomalies: a new series of 140 patients and review of published reports. J Med Genet. 2006;43:625–33.
Coutton C, Monnier N, Rendu J, Lunardi J. Development of a multiplex ligation-dependent probe amplification (MLPA) assay for quantification of the OCRL1 gene. Clin Biochem. 2010;43:609–14.
Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54.
Araujo A, Pagnier A, Frange P, Wroblewski I, Stasia MJ, Morand P, Plantaz D. Lymphohistiocytic activation syndrome and Burkholderia cepacia complex infection in a child revealing chronic granulomatous disease and chromosomal integration of the HHV-6 genome. Arch Pediatr. 2011;18:416–9.
El Kares R, Barbouche MR, Elloumi-Zghal H, Bejaoui M, Chemli J, Mellouli F, Tebib N, Abdelmoula MS, Boukthir S, Fitouri Z, M’Rad S, Bouslama K, Touiri H, Abdelhak S, Dellagi MK. Genetic and mutational heterogeneity of autosomal recessive chronic granulomatous disease in Tunisia. J Hum Genet. 2006;51:887–95.
Noack D, Rae J, Cross AR, Ellis BA, Newburger PE, Curnutte JT, Heyworth PG. Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes. Blood. 2001;97:305–11.
Brunson T, Wang Q, Chambers I, Song Q. A copy number variation in human NCF1 and its pseudogenes. BMC Genet. 2010;11–3.
Nisimoto Y, Freeman JL, Motalebi SA, Hirshberg M, Lambeth JD. Rac binding to p67(phox). Structural basis for interactions of the Rac1 effector region and insert region with components of the respiratory burst oxidase. J Biol Chem. 1997;272:18834–41.
Debeurme F, Picciocchi A, Dagher MC, Grunwald D, Beaumel S, Fieschi F, Stasia MJ. Regulation of NADPH oxidase activity in phagocytes: relationship between FAD/NADPH binding and oxidase complex assembly. J Biol Chem. 2010;285:33197–208.
Cooper DN, Krawczak M. The mutational spectrum of single base-pair substitutions causing human genetic disease:patterns and predictions. Hum Genet. 1990;85:55–74.
Wolach B, Scharf Y, Gavrieli R, de Boer M, Roos D. Unusual late presentation of X-linked chronic granulomatous disease in an adult female with a somatic mosaic for a novel mutation in CYBB. Blood. 2005;105:61–6.
Lewis EM, Singla M, Sergrant S, Koty PP, McPhail LC. X-linked chronic granulomatous diseasesecondary to skewed X chromosome inactivation in a female with a novel CYBB mutation and late presentation. Clin Immunol. 2008;129:372–80.
Henderson LM. Role of histidines identified by mutagenesis in the NADPH oxidase-associated H+ channel. J Biol Chem. 1998;273:33216–23.
Li XJ, Fieschi F, Paclet MH, Grunwald D, Campion Y, Gaudin P, Morel F, Stasia MJ. Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly. J Leukoc Biol. 2007;81:238–49.
Stasia MJ, Lardy B, Maturana A, Rousseau P, Martel C, Bordigoni P, Demaurex N, Morel F. Molecular and functional characterization of a new X-linked chronic granulomatous disease variant (X91+) case with a double missense mutation in the gp91-phox-cytosolique C-terminal tail. Biochem Biophys Acta. 2002;1586:316–30.
Krawczak M, Cooper DN. Gene deletions causing human genetic disease: mechanisms of mutagenesis and the role of the local DNA sequence environment. Hum Genet. 1991;86:425–41.
Cooper DN, Krawczak M. Mechanisms of insertional mutagenesis in human genes causing genetic disease. Hum Genet. 1991;87:409–15.
Krawczak M, Reiss J, Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet. 1992;90:41–54.
Simon KC, Noack D, Rae J, Curnutte J, Sarraf S, Kolev V, Blancato JK. Long polymerase chain reaction-based fluorescence in situ hybridization analysis of female carriers of X-linked chronic granulomatous disease deletions. J Mol Diagn. 2005;7:183–6.
Di Matteo G, Giordani L, Finocchi A, Ventura A, Chiriaco M, Blancato J, Sinibaldi C, Plebani A, Soresina A, Pignata C, Dellepiane RM, Trizzino A, Cossu F, Rondelli R, Rossi P, De Mattia D, Martire B, IPINET (Italian Network for Primary Immunodeficiencies). Molecular characterization of a large cohort of patients with chronic granulomatous disease and identification of novel CYBB mutations: an Italian multicenter study. Mol Immunol. 2009;46:1935–41.
Redman CM, Marsh WL. The Kell blood group system and the McLeod phenotype. Semin Hematol. 1993;30:209–18.
Martinez CA, Shah S, Shearer WT, Rosenblatt HM, Paul ME, Chinen J, Leung KS, Kennedy-Nasser A, Brenner MK, Heslop HE, Liu H, Wu MF, Hanson IC, Krance RA. Excellent survival after sibling or unrelated donor stem cell transplantation for chronic granulomatous disease. J Allergy Clin Immunol. 2012;129:176–83.
Grez M, Reichenbach J, Schwäble J, Seger R, Dinauer MC, Thrasher AJ. Gene therapy of chronic granulomatous disease: the engraftment dilemma. Mol Ther. 2011;19:28–35.
Haldane JBS. The ratio of spontaneous mutation of a human gene. J Genet. 1935;31:317–26.
Den Dunnen JT, Antonarakis SE. Mutation nomenclature extensions and suggestions to describe complex mutations: a discussion. Hum Mut. 2000;15:7–12.
Acknowledgments
MJS is grateful for support from the University Joseph Fourier, Faculty of Medicine; the Ministry of Education and Research, MENRT; the Regional Clinical Research Department, DRCI, Grenoble University Hospital, the CGD Research Trust grant award reference J4G/09/09 and from the National Institutes of Health Grant N01–AI-30070 from the Immunodeficiency Network and the Primary Immunodeficiency Disease Consortium.
The authors are grateful to Dr D. Roos and Dr A. J. Verhoeven for the generous gift of mAb449 and mAb48 antibodies against the two subunits of the cytochrome b 558. We also thank Dr Alison Foote for editing the manuscript.
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Cécile Martel and Michelle Mollin contributed equally to the work
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Martel, C., Mollin, M., Beaumel, S. et al. Clinical, Functional and Genetic Analysis of Twenty-Four Patients with Chronic Granulomatous Disease – Identification of Eight Novel Mutations in CYBB and NCF2 Genes. J Clin Immunol 32, 942–958 (2012). https://doi.org/10.1007/s10875-012-9698-8
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DOI: https://doi.org/10.1007/s10875-012-9698-8