Severe congenital neutropenia, type 4 results from a mutation in the G6PC3 gene. This gene encodes the enzyme glucose-6-phosphatase (G6Pase) which is ubiquitously expressed in endoplasmic reticulum and is responsible for the final step of gluconeogenesis and glycogenolysis. G6pc3-/- mouse models have demonstrated decreased neutrophil (PMN) function including impaired chemotaxis and superoxide anion production and increased apoptosis. Metabolically, g6pc3-/-mice have impaired intracellular glucose transport and reduced levels of ATP and lactic acid. We present the most comprehensive functional and metabolic analysis to date on a patient with a novel compound heterozygous mutation in the G6PC3 gene, c.325G>A and c.758G>A, validating mouse model findings in a human.
PMNs were isolated from heparinized whole blood by dextran sedimentation and hypotonic lysis of red cells. Chemotactic assays were performed under agarose techniques with zymosan activated serum (ZAS), fMLF, and buffer control. Expression of CD18 and F-actin assembly were analyzed after incubation with buffer, PMA, and fMLF using standard flow cytometric techniques. Bactericidal activity was measured using variable ratios of S Aureus:PMNs in the presence of 10% normal human serum. Superoxide anion production was measured with SOD-inhibitable chemiluminescence with Diogenes reagent. Chemiluminescence was employed to measure caspase expression. Metabolomics and tracing experiments were performed incubating cells with U-13C-glucose prior to UHPLC/MS analysis.
Cell motility was significantly impaired in human G6Pase deficient (patient) PMNs. Non-directed migration was reduced by over 50% compared to controls. Directed migration of patient PMNs was only 35% and 23% of control when stimulated with ZAS and 0.1 µM fMLF respectively. Surface expression of CD18 in patient cells did not increase in response to either stimulus but more than doubled in normal PMNs. F-actin was present at significantly higher levels in unstimulated patient PMNs and did not increase in the presence of 200 ng/mL PMA or 1 µM fMLF.
Bactericidal activity was normal at a bacteria:PMN ratio of 1:1 but abnormal at ratios > 5:1 with over a 20% increase in viable bacteria at 120 minutes for patient PMNs. Superoxide anion production was markedly impaired with over 70% reduction compared to control when stimulated with either 200 ng/mL PMA or 1 µM fMLF. Caspase expression was not significantly different between patient and control PMNs, but the patient was on filgrastim which has antiapoptotic effects.
Tracing experiments with 13C6-glucose showed decreased flux through glycolysis (significant reductions in labeled isotopologues for G6P and lactate) and marked inhibition of the NADPH-generating pentose phosphate pathway (PPP) in patient PMNs stimulated with 1µM fMLF. Glutaminolysis appears to be increased with increased glutamine consumption in patient PMNs, decreased glucogenic amino acids, and increased TCA cycle intermediates.
Metabolic reprogramming is a recently appreciated hallmark of immune cell activation. Decreases in G6P affect multiple metabolic pathways including glycolysis, PPP and TCA cycle. Deficiency of NADPH-dependent superoxide anion production likely results from impairment in the NADPH-generating PPP. Interestingly the bactericidal defect is not as severe as would be predicted given the degree of impairment in superoxide anion production. The most striking functional defect is with PMN motility. Defects in the glycolytic/PPP likely contribute to this phenotype. ATP-dependent actin assembly is critical for cell migration. Our data demonstrate that in human patient PMNs, nearly all actin is in the filamentous form and does not increase further in response to chemoattractants. This could result in impaired hydrolysis of the F-actin filament and reassembly at the leading edge. We also see reduction in surface expression of CD11b/CD18 which is vital for PMN adhesion and migration.
In conclusion, metabolic defects resulting from G6PC3 deficiency contribute to global PMN dysfunction and absolute neutropenia. Patients may be at increased risk for infections in spite of correction of neutropenia with filgrastim. These novel findings in human patient cells elucidate further the pathogenesis of this disease which has largely been dependent on data from mouse models.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.