A splice-supporting intronic mutation in the last bp position of a cryptic exon within intron 6 of the CYBB gene induces its incorporation into the mRNA causing chronic granulomatous disease (CGD) Andreas Rump a , Angela Rösen-Wolff b , Manfred Gahr b , Jürgen Seidenberg c , Christian Roos d , Lutz Walter d , Viola Günther a , Joachim Roesler b, ⁎ a Institute of Human and Clinical Genetics, University Clinic Carl Gustav Carus, Dresden, Germany b Department of Pediatrics, University Clinic Carl Gustav Carus, Dresden, Fetscherstr. 74, 01307 Dresden, Germany c Department of Pediatrics, Clinic of Oldenburg, Germany d Department of Primate Genetics, German Primate Center, Göttingen, Germany Received 28 June 2005; received in revised form 17 October 2005; accepted 16 November 2005 Available online 3 March 2006 Abstract Chronic granulomatous disease (CGD) is caused by a defect in both the host's defenses and its regulation of inflammation normally provided by phagocytes and other leukocytes. As in the case described here, it is not uncommon that CGD patients are diagnosed late, only after organ- damaging manifestations have already progressed. In this patient, we found that CGD arose due to a splice-supporting mutation in the last position of a cryptic exon towards the middle of intron 6 of the CYBB (gp91-phox) gene. The mutation led to the insertion of 56 bp into most of the CYBB mRNA of leukocytes causing a frame shift and a premature stop codon. The normal cryptic exon was also found to be mildly active in some tissues other than leukocytes in healthy donors, to be conserved in many primates, and to a lesser degree in other mammals. Some sequence similarity suggests that the cryptic exon may have originated from a mammalian interspersed repetitive (MIR) element. Taken together, we clarify an unusual disease-causing mutation, indicate its evolutionary background and emphasize the importance of a timely diagnosis of CGD. © 2005 Elsevier B.V. All rights reserved. Keywords: NADPH-oxidase; gp91-phox; Pulmonary fibrosis; X-inactivation; Reactive oxygen; Exon evolution; Splice-supporting mutation 1. Introduction In chronic granulomatous disease (CGD, OMIM #306400, #233690, #233700, #233710), phagocytes are unable to generate sufficient amounts of microbicidal reactive oxygen metabolites (ROM), rendering patients highly susceptible to life- threatening bacterial and fungal infections (Curnutte, 1993; Winkelstein et al., 2000; Heyworth et al., 2003; Segal, 2005). Furthermore, such patients tend to develop pulmonary fibrosis following granulomatous inflammation triggered by infections, but this can also develop spontaneously (Moskaluk et al., 1994; Segal et al., 2000; Winkelstein et al., 2000; Kobayashi et al., 2004). In CGD the lack or significant decrease in the production of ROM is due to a defective phagocyte NADPH-oxidase. The failure of this multienzyme complex can result from mutations in any of the four genes CYBB (OMIM ⁎ 300481, gp91-phox, phagocyte oxidase), CYBA ( ⁎ 608508, p22-phox), NCF1 ( ⁎ 608512, p47-phox) or NCF2 ( ⁎ 608515, p67-phox), all en- coding important components of the enzyme. CYBB is located on the X-chromosome whereas all other components are on autosomes (Curnutte, 1993; Segal et al., 2000; Winkelstein et al., 2000; Segal, 2005). Carrier mothers of the most frequent form of CGD, gp91- phox deficiency accounting for approx. two-thirds of all cases, normally have two subsets of neutrophils in their peripheral Gene 371 (2006) 174 – 181 www.elsevier.com/locate/gene Abbreviations: BSA, bovine serum albumin; CAT scan, computerized axial tomography or CT scan; CGD, chronic granulomatous disease; CYBB, cytochrome b-245, beta polypeptide; ddH 2 O, double-distilled water; MIR, mammalian interspersed repeat; NADPH, nicotinamide adenine dinucleotide phosphate; PBS, phosphate-buffered saline; ROM, reactive oxygen metabolites. ⁎ Corresponding author. Tel.: +49 351 458 2267; fax: +49 351 458 6333. E-mail address: roeslerj@rcs.urz.tu-dresden.de (J. Roesler). 0378-1119/$ - see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2005.11.036