Expanding the spectrum of hypomorphic NCF2 variants causing Chronic Granulomatous Disease

Background: p67phox is a cytosolic subunit of the NADPH oxidase (NOX) complex encoded by the NCF2 gene. The NOX enzyme is responsible for superoxide production in a variety of cell types, being a key defense mechanism against pathogens in leukocytes. Activation of the complex depends on p67phox interaction with the active form of the GTPase RAC1. Mutations in NCF2 impair reactive oxygen species production, leading to an autosomal recessive form of rare primary immunodeficiency, Chronic Granulomatous Disease (CGD; OMIM 233700). Most CGD cases are caused by loss-of-function mutations, while a subset of individuals carrying hypomorphic variants has also been described. Aim: Here, we describe a case of an individual diagnosed with CGD, carrying a previously unreported homozygous missense p67phox variant (c.197G>C, p. Arg66Pro). Methods: Clinical suspicion arisen in childhood due to granulomas on lymph node biopsy and recurrent infections. All tests assessing oxidative burst function (NBT, cytochrome b, and DHR) yielded pathological results. To identify the variant and assess its pathogenicity, exome sequencing was performed, followed by in silico predictions and in vitro functional assays in cell lines overexpressing either wildtype or mutant p67phox together with RAC1. Results and conclusions: Arg66 is conserved among p67phox orthologues and its paralog protein, with the in silico analyses predicting a detrimental effect on protein stability. Notably, the residue is located within the RAC1-binding domain, suggesting that the amino acid substitution may impair protein-protein interaction. Consistently, functional assays demonstrated that the p.Arg66Pro variant reduces p67phox stability, leading to substantial protein degradation mediated both by proteasomal and lysosomal machineries. In addition, the residual mutant protein exhibits a decreased binding capability to active RAC1. Overall, these findings widen the spectrum of pathogenic NCF2 variants and support a hypomorphic mechanism underlying the pathogenicity of p.Arg66Pro.