case_images: PMC11368735_01
case_images: Clinical findings. (A) Photosensitive malar rash sparing the nasolabial folds. (B) Chest imaging showing right middle and lower lobe consolidation and parapneumonic effusion and right upper lobe atelectasis at the time of presentation with acute hypoxemic respiratory failure requiring management with bilevel positive airway pressure.
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case_images: F1
case_images: Additional relevant medical history post-pSLE diagnosis includes multiple bacterial urinary tract infections including one associated with presumed sepsis, acyclovir-responsive blepharitis, persistent right eyelid hordeolum, facial folliculitis, a consolidative pneumonia with parapneumonic effusion requiring drainage (Figure 1B), and fungal urinary tract infections secondary to Candida spp.
case_images: NCF4. (A) Familial segregation of NCF4 variants. (B) RT-PCR of NCF4 mRNA flanking exon 9 in the patient and a control. (C) Gene structure of NCF4 and protein domains of p40phox with patients' variants indicated. bp, base pair; HD, healthy donor.
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case_images: F2
case_images: However, the patient was found to have biallelic variants in NCF4, each inherited from a different parent (Figure 2A). The presence of an alternate transcript with shorter length was observed (Figure 2B); Sanger sequencing of the fragment confirmed an alternate splicing event resulting in in-frame skipping of all of exon 9. Exon 9 partially encodes the SH3 domain (Figure 2C) essential for p40phox binding to other NADPH subunits, such as p67phox.
case_images: Functional studies. (A) DHR assay in neutrophils (top) and monocytes (bottom) upon PMA stimulation in the patient and a control. (B) Percentage (left) and Index MFI (right) of cells able to oxidize the DHR. (C) H2O2 release from patient and control neutrophils following different stimuli. DHR, dihydrorhodamine 1,2,3; HD, healthy donor; MFI, mean florescence intensity; NS, non-stimulated; PMA, phorbol 12-myristate 13-acetate; PMN, polymorphonuclear cell; RLU, relative light units.
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case_images: F3
case_images: The DHR oxidation assay showed defective oxidation upon PMA stimulation in patient neutrophils, although all neutrophils and monocytes were capable of oxidizing DHR (Figure 3A). However, patient cells showed half the index of oxidation compared to control (Figure 3B). Further evaluation revealed a profound defect in H2O2 release upon zymosan and S. aureus stimulation, consistent with the cellular phenotype of p40phox deficiency (Figure 3C).
case_images: Protein expression of NADPH oxidase subunits. Protein expression of the NADPH subunits in neutrophil lysates from controls, the patient's mother (heterozygous for the NCF4 c.824+1G>A variant) and the patient. Actin protein was used as loading control.
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case_images: F4
case_images: Moreover, protein electrophoresis of neutrophil lysate from the patient confirmed a reduction of approximately 50% in p40phox expression compared to controls, while the other NADPH subunits (gp91phox, p67phox, p47phox, and p22phox) exhibited expression more similar to healthy individuals (Figure 4).