(p47)-deficient Chronic Granulomatous Disease: Comprehensive Genetic and Flow Cytometric Analysis

Mutations in (p47) cause autosomal recessive chronic granulomatous disease (CGD) with abnormal dihydrorhodamine (DHR) assay and absent p47 protein. Genetic identification of mutations is complicated by adjacent highly conserved (>98%) pseudogenes ( and ). has GTGT at the start of exon 2, whereas the pseudogenes each delete 1 GT (ΔGT). In p47 CGD, the most common mutation is ΔGT in (c.75_76delGT; p.Tyr26fsX26). Sequence homology between and its pseudogenes precludes reliable use of standard Sanger sequencing for mutations and for confirming carrier status. We first established by flow cytometry that neutrophils from p47 CGD patients had negligible p47 expression, whereas those from p47 CGD carriers had ∼60% of normal p47 expression, independent of the specific mutation in We developed a droplet digital polymerase chain reaction (ddPCR) with 2 distinct probes, recognizing either the wild-type GTGT sequence or the ΔGT sequence. A second ddPCR established copy number by comparison with the single-copy telomerase reverse transcriptase gene, We showed that 84% of p47 CGD patients were homozygous for ΔGT The ddPCR assay also enabled determination of carrier status of relatives. Furthermore, only 79.2% of normal volunteers had 2 copies of GTGT per 6 total () copies, designated 2/6; 14.7% had 3/6, and 1.6% had 4/6 GTGT copies. In summary, flow cytometry for p47 expression quickly identifies patients and carriers of p47 CGD, and genomic ddPCR identifies patients and carriers of ΔGT , the most common mutation in p47 CGD.