Identification of a donor splice site mutation leading to loss of p22-phox exon 5 in autosomal chronic granulomatous disease
✍ Scribed by Colin D. Porter; Mohamed H. Parkar; Christine Kinnon
- Publisher
- John Wiley and Sons
- Year
- 1996
- Tongue
- English
- Weight
- 118 KB
- Volume
- 7
- Category
- Article
- ISSN
- 1059-7794
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✦ Synopsis
Defects in the phagocyte NADPH oxidase give rise to chronic granulomatous disease (CGD) (Roos, 1994). Two components, gp91-phox and p22-phox, comprise the membrane-associated cytochrome bSss. Defects in the former account for the most common, X-linked form of CGD, for which a large number of mutations have been characterised (de Boer et al., 1992a:l. In contrast, autosomal CGD due to mutations in p22-phox is rare. We report that the mutation in 1 case of p22-phox deficiency is a splice site mutation resulting in the skipping of exon 5.
The patient whose mutation is described is female and presented at the age of 7 years, having a history of recurrent infections. Her parents are first cousins but there is no family history of such infections. A diagnosis of CGD was made following failure of her neutrophils to respond in assays for NBT reduction, 0,-production and cell killing. Complementation using :a patient-derived EBV-transformed B cell line confirmed that the disease was due to p22-phox deficiency (Porter et al., 1994).
Southern blot analysis indicated no overt p22-phox gene deletion or rearrangement. RNA was prepared from the patient B cell line and the p22-phox cDNA was amplified by PCR using the specific primers 5'-AGTCTCGAgCAOTGTCCCAGCCGGGTTC (position 1 of the sequence published by Parkos et al. ( 1988) is in lower case) and 5'-ACGAGATCtGCACCTGGTGGGAGGGCAG (the base complementary to position 649 of the published sequence is in lower case), designed to introduce terminal XhoI and BglII restriction sites, respectively. Thirty-five cycles of PCR (1 min each at 94, 60, and 72°C) were performed: the inclusion of 5% formamide was required in order to obtain a specific product, owing to the high GC-content of the p22-phox cDNA. Its :size was reduced by approximately 80 bp relative to a control (665 bp), consistent with a reduction in transcript size detected by Northern blotting. The PCR product was cloned into the plasmid pSP72 and the nucleotide sequence of pooled clones was determined with flanking; SP6 and T7 primers, revealing the absence of exon 5 (82 bp). The consequent frame shift generates 66 C-terminal amino-acids unrelated in sequence to the normal 72 amino-acid translation product of exon 6. The sequence was otherwise identical to that published (Parkos et al., 1988), including the known polymorphic sites at nucleotide positions 242, 508, and 640 (de Boer et al., 199213;Dinauer et al., 1990).
A 216 bp fragment encompassing p22-phox exon 5 was successfully amplified from genornic DNA, confirming that its absence from the cDNA was due to altered mRNA splicing. Thirty cycles of PCR (30 sec at 9TC, 1 min at 5TC, 30 sec at 72°C) were performed, using the specific primers 5'-TCCGTTCCCCTTTCTGAGT(iC from intron 4 and 5'-CATGCGTGTCCGAGGGTGTC from intron 5. Sequence analysis of the PCR product by "cycle-sequencing" detected a G to C point mutation in the fllanking genomic DNA at position + 1 of intron 5, disrupting the G T donor splice site. The presence of a single sequence confirmed the homozygosity of the gene defect. This is the tenth mutation characterised for p22-phox. As for gpgl-phox, they are heterogeneous in nature (reviewed in Roos, 1994). One
other mutation involves altered splicing, leading to loss of exon 4 due to a G to A point mutation at the + 1 position of intron 4 donor splice site (de Boer et al., 199210). Aberrant splicing accounts for 10 of the 47 known gp91-phox defects (de Boer et al., 1992a; Roos, 1994) and may similarly prove to be a common cause of p22-phox-deficiency.