Chronic granulomatous disease (CGD) is an inherited immunodeficiency caused by defects in any of four genes encoding components of the leukocyte nicotinamide dinucleotide phosphate, reduced (NADPH) oxidase. One of these is the autosomal neutrophil cytosolic factor I (NCF1) gene encoding the p47(phox) protein. Most (> 97%) CGD patients without p47(phox) (A47 degrees CGD) are homozygotes for one particular mutation in NCF1, a GT deletion in exon 2. This is due to recombination events between NCF1 and its two pseudogenes (psi NCF1) that contain this GT deletion. We have previously set up a gene,scan method to establish the ratio of NCF1 genes and pseudogenes. With this method we now found, in three CGD families patients with the normal number of two intact NCF1 genes (and four psi NCF1 genes) and in six CGD families, patients with one intact NCF1 gene (and five psi NCF1 genes). All patients lacked p47(phox) protein expression. These results indicate that other mutations were present in their NCF1 gene than the GT deletion. To identify these mutations, we designed PCR primers to specifically amplify the cDNA or parts of the genomic DNA from NCF1 but not from the psi NCF1 genes. We found point mutations in NCF1 in eight families. In another family, we found a 2,860-bp deletion starting in intron 2 and ending in intron 5. In six families the patients were compound heterozygotes for the GT deletion and one of these other mutations; in two families the patients had a homozygous missense mutation; and in one family the patient was a compound heterozygote for a splice defect and a nonsense mutation. Family members with either the GT deletion or one of these other mutations were identified as carriers. This knowledge was used in one of the families for prenatal diagnosis.