CGDilemma: Fitting Together the Puzzle Pieces for Definitive Diagnosis

Abstract

IntroductionChronic granulomatous disease (CGD) is a rare inherited primary immunodeficiency. ΔGT mutations in NCF1, which encodes phagocyte nicotinamide adenine phosphate (NADPH) p47-phox protein, accounts for 23% of CGD. Oftentimes testing focuses on ΔGT defects. However, 20% of p47 CGD patients have mutations at other sites in NCF1 which can be missed. Further, NCF1 has 2 pseudogenes near chromosomal locus 7q11.23 which may undergo reciprocal crossover between functional genes exceeding expected frequency, complicating diagnosis, especially in atypical presentations. Case PresentationA 4-year-old male from Saudi Arabia presented with a necrotic lesion on his left cheek, unresponsive to antibiotics. Two years prior he had a Rhizopus infection on his right leg necessitating debridement. Biopsy revealed recurrent Rhizopus infection. He received debridement, linezolid, posaconazole and amphotericin. He was discharged on posaconazole and trimethoprim/sulfamethoxazole, after a protracted hospital stay. His wound improved, however margins remained positive.Immune workup revealed an oxidative burst assay with a broad abnormal peak of intermediate granulocyte dihydrorhodamine fluorescence suggesting autosomal recessive CGD. Genetic panels identified no variants in CGD genes. Western blot showed absent p47phox protein, confirming NCF1-deficient CGD. Genetic testing did not show the common ΔGT deletion at the start of exon 2 in 6 copies of NCF1/NCF1 pseudogenes. Sanger sequencing revealed a single nucleotide change distinct to the patient. This coded for NCF1 c.579 G > A, p. Trp193X, a previously reported mutation in NCF1. ConclusionDinucleotide deletion at the beginning of exon 2 of NCF1 pseudogenes leads to a frameshift and stop. When a pseudogene replaces the authentic NCF1, the overall ratio of functional genes to pseudogenes is altered. This potentially masks CGD carriers who possess pseudogene to functional gene ratios that renders them indistinguishable from healthy individuals with most testing. Non-ΔGT mutations occur in ~20% of p47phox patients. Because of the extensive sequence homology of NCF1 with the pseudogenes, Sanger sequencing may be insufficient to determine the underlying defect in NCF1. Therefore, gene panels and even whole genome sequencing may miss p47phox deficiency. This multimodality approach allowed us to identify p47phox-deficient autosomal recessive CGD.