A MUTATION LOCATED AT THE 5-BETA SPLICE JUNCTION SEQUENCE OF INTRON-3IN THE P67(PHOX) GENE CAUSES THE LACK OF P67(PHOX) MESSENGER-RNA IN APATIENT WITH CHRONIC GRANULOMATOUS-DISEASE

Chronic granulomatous disease (CGD) is due to a functional defect of t he O-2(-)-generating NADPH oxidase of neutrophils. Mutations resulting in CGD have been shown to occur in only four genes, thus identifying the main components of the oxidase complex, namely the two subunits of a membrane-bound cytochrome b and two cytosolic factors of activation of 67 kD (p67(phox)) and 47 kD (p47(phox)). The present study deals w ith the biochemical and genetic analysis of the defect in a patient su ffering from a p67(phox)-deficient form of CGD. The p67(phox) deficien cy was ascertained by immunochemistry and the ability of recombinant p 67(phox) to restore NADPH oxidase activity using a cell-free system of oxidase activation. The cellular extracts from the proband contained no p67(phox) protein and no p67(phox) mRNA when assayed by Western and Northern blot analysis. However, reverse transcription of mRNA and su bsequent cDNA amplification by polymerase chain reaction using specifi c p67(phox) primers showed that trace amounts of a p67(phox) mRNA dele ted for exon 3 were synthesized in the patient immortalized B lymphocy tes. Sequence analysis of the genomic DNA showed a T-to-C transition a t position +2 of intron 3. This point mutation in the consensus 5' spl ice site of the intron 3 was probably responsible for lack of accumula tion of mRNA and also for the skipping of exon 3 detected in the few m RNA molecules that escaped cellular degradation. (C) 1995 by The Ameri can Society of Hematology.