Emerging data from COVID19 show that the case fatality rate (CFR) is higher in males, indicating possible X-linked mediated differences in the crosstalk between SARS-CoV-2 and immune effectors2,5,6. We illustrate the potential role of G6PD deficiency in patients hospitalized for COVID19 pneumonia affecting the severity of the disease and clinical management. The gene encoding G6PD is located near the telomeric region of the distal arm of the X chromosome (band Xq28), a well-documented hot spot of a group of genes that includes fragile X, color vision, hemophilia A, and congenital dyskeratosis8. There are more than 200 point mutations identified in the G6PD gene resulting in many biochemical variants and deficiency of the gene product G6PD, which is the rate-limiting enzyme in the pentose-phosphate pathway8,9. The variants are grouped into four classes: a) Class I variants- comprise the most severe form of G6PD deficiency and lead to chronic non-spherocytic haemolytic anaemia and typically occur with enzyme activity <10% of normal; b) Class II variants typically have <10% residual enzyme activity but no haemolytic anaemia; c) Class III and IV variants (10–60% and 60–150% activity, respectively) have milder phenotypes, and haemolysis occurs only after extreme oxidative stress11. Very severe G6PD deficiencies are sporadic and rare, whereas less severe deficiencies are polymorphic and more common in tropical areas, postulated to evolve as protection against malaria7,8. Males are more commonly affected when hemizygous and can be either phenotypically normal or deficient11. Homozygous females are as deficient as hemizygous males, whereas heterozygous females are mosaics with intermediate levels of deficiency as a result of random X-chromosome inactivation (lyonization)12. Thus far, there are no reports of an association between G6PD deficiency and COVID19, likely because G6PD deficiency was either rare or of mild variety or not reported in the respective populations2-4. Nevertheless, given the high prevalence of this mutation in African Americans (1 in 10) and Italians,5,7 it is important to elucidate the biological differences in the outcomes of COVID19 infection. In our study, a total of 17 patients were enrolled where the G6PD levels were known. Six were identified with G6PD deficiency; we used the 11 patients with normal levels as the comparator group. In the deficiency group, two males (both African Americans) had levels less than 4.5, and the other four patients, two males and three females (likely hemizygous), had levels just below the lower limit of the established reference (9.9 to 16.6 U/g Hb)13.
G6PD converts glucose-6-phosphate into 6-phosphogluconolactone and catalyzes the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, NADPH is the critical cofactor for the enzyme glutathione reductase, which reduces glutathione disulfide (GSSG) into reduced glutathione (GSH). GSH eliminates ROS by scavenging hydroxyl radicals, singlet oxygen, and electrophiles. The deficiency is most pronounced on erythrocytes, which depend solely on the cytosolic pentose phosphate pathway and generation of GSH for oxidative protection. Generally, the outcomes for G6PD-deficient patients are favorable if they can avoid oxidative triggers commonly encountered due to drugs (including some antimalarial drugs, sulfonamides, and rasburicase)7,9. Fatigue is the most common symptom, followed by dyspnea, dizziness, headache, pallor, chest pain, and jaundice if hemolysis is severe. A quantitative analysis of G6PD activity13 can provide a definitive diagnosis of G6PD deficiency so that individuals can be advised to avoid drugs, foods, or other oxidizing agents that may precipitate a hemolytic crisis. The severity of hemolysis is variable and depends on the G6PD allelic mutation and factors such as the dose of the inciting drug. The usual management is supportive care and blood transfusion if hemolysis is severe.
The reasons for testing G6PD in our patients was proactive baseline measurement in preparation for hydroxychloroquine use. As expected, in our study, there were significant differences in the G6PD levels that also correlated with the differences in haemoglobin and haematocrit (Table 1). More importantly, there is an increase in the severity of the pulmonary process, as demonstrated by the higher requirements of oxygen and longer time on mechanical ventilation. The differences seen as a measure of the lowest PaO2/FiO2 are also shown by the similarity of the Rothman index between admission and the time of mechanical ventilation with subsequent deterioration in the deficiency group (Table 2 and Figure 1 A-G). The limitation of this study is the retrospective review, the sample size and the lack of power to detect the differences due to intervention. Nevertheless, given the high prevalence of this mutation in African Americans (1 in 10) and Italians,3,5 it is important to elucidate the biological differences in the outcomes of COVID19 infection.
Two questions that are of utmost clinical importance that need answers are: 1) Does the deficiency increase the susceptibility and/or the severity to COVID19? 2) How do we judiciously use the pharmacologic array of medications that can potentially worsen the deficiency? One of the severe manifestations of COVID19 is ARDS characterized by the acute onset of hypoxemia, reduced lung compliance, diffuse lung inflammation and bilateral opacities on chest imaging attributable to noncardiogenic pulmonary edema14. As details of the pathobiology are still emerging, our understanding of similar viral-mediated lung injury from the previous human coronavirus (hCoV) epidemics of severe acute respiratory syndrome (SARS) and middle eastern respiratory syndrome (MERS) points to the central oxidative stress on the pulmonary vasculature by ROS production causing further alveolar damage limiting gas exchange and setting up right to left shunting of venous blood15-17. The pathophysiologic process of pulmonary vascular endothelial diathesis is caused by increased oxidant stress and reduced bioavailable nitric acid15-17. This has also been reported in influenza infection, which provokes a pro-oxidant condition by increasing ROS production in the host cell to facilitate viral proliferation18,19. ROS further inactivate nitric oxide, resulting in nitric oxide insufficiency20. The primary determinant of protection against oxidative stress is the ability to maintain GSH stores through the synthesis of NADPH. Since NADPH concentrations are primarily maintained by G6PD encoded by its gene located on Xq28, it follows that its deficiency could result in an inability to eliminate ROS, as seen in our patients.
There is now accumulating evidence that G6PD deficiency affects cells other than erythrocytes 10,21-24. The replication and spread of respiratory viruses normally involves activation of the antiviral innate immune responses and culminates in the production of type I interferons (IFNs) and proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), which in turn upregulate antiviral proteins24. In an in vitro study, when G6PD-deficient human fibroblasts and human lung epithelial carcinoma A549 cells were treated with G6PD-RNA interference (RNAi), they showed reduced viability and a 3-fold increase in viral replication21. In another study, the authors reported a marked reduction in antiviral genes, such as TNF-α, in G6PD-deficient A549 cells after infection with HCoV-229E compared to the parental cell lines23. Their observations also include decreased nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) activation in virus-infected host cells, pointing to a reduced ability to activate timely antiviral responses. Furthermore, a few studies indicate that oxidative stress increases the susceptibility of non-erythroid G6PD-deficient cells to viral infection, which could be ameliorated by antioxidant agents, such as lipoic acid21.
In another study, G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells showed impaired inflammasome activation. In particular, G6PD knockdown reduced the expression of mature interleukin (IL)-1β but not the expression of caspase-1 or the components of the inflammasome (NLRP3, ASC, and pro-Caspase-1) pathway10. Additionally, there was a differential expression of cytokines between the G6PD-deficient cells and the normal cells. Furthermore, as reported in severe influenza pneumonia17,19, this pro-oxidant condition can induce secretion of inflammatory cytokines, including interleukin (IL)-1β, IL-6, IFN, and TNF-α, from the microenvironment. This uncontrolled pro-inflammatory response, referred to as the cytokine storm, is abetted by the action of monocytes/macrophages and neutrophils on infected lung epithelial cells22. Taken together, these results indicate that G6PD deficiency can allow viral proliferation even as it impairs the cellular immune response through abnormal redox homeostasis. It is imperative that further studies be performed to have an enhanced understanding of the interplay between the viral and host factors in G6PD deficiency that may lead to disparity in outcomes. This will have significant clinical implications in the management of patients with COVID19 infection.