NCF1 (p47phox)-deficient chronic granulomatous disease: comprehensive genetic and flow cytometric analysis

Mutations in NCF1 (p47(phox)) cause autosomal recessive chronic granulomatous disease (CGD) with abnormal dihydrorhodamine (DHR) assay and absent p47(phox) protein. Genetic identification of NCF1 mutations is complicated by adjacent highly conserved (>98%) pseudogenes (NCF1B and NCF1C). NCF1 has GTGT at the start of exon 2, whereas the pseudogenes each delete 1 GT (Delta GT). In p47(phox) CGD, the most common mutation is DGT in NCF1 (c. 75_76delGT; p.Tyr26fsX26). Sequence homology between NCF1 and its pseudogenes precludes reliable use of standard Sanger sequencing for NCF1 mutations and for confirming carrier status. We first established by flow cytometry that neutrophils from p47(phox) CGD patients had negligible p47(phox) expression, whereas those from p47(phox) CGD carriers had; 60% of normal p47(phox) expression, independent of the specific mutation in NCF1. We developed a droplet digital polymerase chain reaction (ddPCR) with 2 distinct probes, recognizing either the wild-type GTGT sequence or the Delta GT sequence. A second ddPCR established copy number by comparison with the single-copy telomerase reverse transcriptase gene, TERT. We showed that 84% of p47(phox) CGD patients were homozygous for Delta GT NCF1. 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 (NCF1/NCF1B/NCF1C) copies, designated 2/6; 14.7% had 3/6, and 1.6% had 4/6 GTGT copies. In summary, flow cytometry for p47(phox) expression quickly identifies patients and carriers of p47(phox) CGD, and genomic ddPCR identifies patients and carriers of Delta GT NCF1, the most common mutation in p47(phox) CGD.