Dore

A decade ago, Gheita et al. reported a high frequency of glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49) deficiency in patients with rheumatoid arthritis (R.A.) and concurrent metabolic syndrome.[1] More recently, in a retrospective cohort study using a large, nationwide database of individuals with known G6PD status, Israel et al. confirmed a significant association between the occurrence of R.A. and G6PD enzyme deficiency.[2] In Sardinia, Italy, hereditary G6PD deficiency reaches a frequency as high as 8% and 12% in the male and female population, respectively,[3] which is among the highest in the world. It has already been proven that 95% of cases are due to the G6PD Med mutation (Ser¹⁸⁸Phe),[3] which entails a class II severe deficiency, according to the World Health Organization, because it is associated with a more than 90% reduction of catalytic activity.[4] Moreover, the prevalence of R.A. has been estimated at 552 × 10⁵ in the general Sardinian population,[5] i.e., considerably higher than that recorded in the Italian peninsula (330 × 10⁵).[6]
Based on this claim and the particularly favourable setting, the association between R.A. and G6PD deficiency was tested in the population of northern Sardinia.

This was a retrospective case-control study recruiting outpatients referred for upper endoscopy to the Gastroenterology section of a teaching hospital (University of Sassari, Italy). The section is the main referral centre for rheumatological patients with dyspeptic complaints. Data from January 2014 and January 2022 were retrieved from an electronic database. Collected data included sex, age, smoking habits, anthropometric parameters, and the presence of a defined diagnosis of R.A.
Rheumatoid arthritis. R.A. was diagnosed by the rheumatologist according to national and international guidelines/expert consensuses progressively developed and used in clinical practice.[7] Rheumatoid factor (R.F.) levels and anti-citrullinated protein antibody (ACPA) titres were also available in a subset (n=291) of patients with R.A. Serum R.F. titers were measured in the hospital reference lab using a commercial ELISA kit (Abcam®, Cambridge, MA, U.S.A.),[8] and ACPA titres (Euro Diagnostica Immunoscan CCPlus®, Arnhem, The Netherlands) according to the manufacturer’s instructions.
Glucose-6-phosphate dehydrogenase deficiency. In all study participants, G6PD status had been assessed using a previously described laboratory test based on the measurement of the G6PD/6PGD ratio in red blood cells of peripheral venous blood samples.[9] Enzyme deficiency was classified as severe (<10% residual G6PD activity) or intermediate (between 10% and 80% residual activity). Molecular testing was not available.
Statistical analysis. The body mass index (B.M.I.), calculated by using the formula weight (kg) / height (m)², allowed to stratify participants into normal, overweighted (B.M.I. between 25 and 29.9 kg/m²), and obese (B.M.I.≥ 30 kg/m²). According to smoking habits, patients and controls were divided into never smokers or former smokers/current smokers. Both severe and intermediate G6PD deficiency were merged into the same category to strengthen the analysis. Differences between means were evaluated by the Student’s t-test for continuous variables and by the Pearson χ² test for categorical variables. The association between G6PD deficiency and R.A. was determined using univariable and multivariable logistic regression models by calculating unadjusted and adjusted odds ratios (O.R.s) and their 95% confidence intervals (CI). The analysis was conducted separately in males and females. All statistical analyses were performed using SPSS statistical software (version 22.0, Chicago, IL, U.S.A.). P-values lower than 0.05 were considered statistically significant.
Ethical Considerations. Ethical review and approval were waived for this study due to observational retrospective design by the Italian law (GU No. 76 31/Mar/2008). The procedures followed in the study were in accordance with the ethical standards of the Declaration of Helsinki.

A total of 5,279 study participants (mean age 53.0 ± 17.1 years; 66.5% female) were included in the analysis. In 594 participants, a diagnosis of R.A. (11.3%) was posed by the rheumatologist. Table 1 shows the features of the study participants separately in males and females. There were 661 patients (12%) with G6PD deficiency, reflecting the regional frequency,[3] with an F: M ratio of 1.86, and 4618 patients without. No significant differences were observed in B.M.I. and smoking habits. The prevalence of R.A. was 14.4% in males and 16.6% in females, respectively, among carriers of G6PD deficiency, whereas it was 8.5% and 11.7% among males and females with normal enzyme activity (p < 0.0001).

Table 1. Features of the 5279 study participants stratified by G6PD status and sex.

The results of univariable and multivariable logistic regression analysis, using the presence or absence of R.A. as the outcome, according to the exposure to G6PD deficiency, are reported in Table 2. The odds for an R.A. diagnosis in subjects with G6PD deficiency were statistically significant (OR 2.21, 95%CI 1.54‒3.18) after adjusting for the covariates included in the study (Table 2) and higher in males [OR 1.82, 95%CI 1.13–2.93] compared with females [OR 1.49, 95%CI 1.15–1.94].

Table 2. Unadjusted and adjusted odds ratios (ORs) and 95% confidence intervals (CI) for rheumatoid arthritis in 5,279 study participants.

The subset of 291 patients with R.A. and availability of immunological markers was stratified according to R.F. and ACPA positivity (Table 3). Interestingly, the frequency of G6PD deficiency was significantly increased in R.A. patients positive for both markers compared with controls (17.6% vs 12.1%, p = 0.029). In comparison, the frequency of G6PD deficiency was non-significantly increased (14.8% vs 12.1%, p = 0.537) in patients negative for RA-specific markers. However, the small number of patients with G6PD deficiency in this subset did not allow us to draw definitive conclusions.

Table 3. Selected variables in 291 rheumatoid arthritis patients stratified according to immunological markers including rheumatoid factor (RA) and anti-citrullinated protein antibodies (ACPA).

The present study involved a large cohort of patients. Our findings suggest that similar to Israel's results,[2] in the Northern Sardinian population, G6PD deficiency significantly increased the odds of R.A. This association was found in both sexes, especially in male patients. It is reasonable considering that among females, a higher frequency of heterozygosity was reported, resulting in greater residual enzyme activity. Interestingly, in a subanalysis, the increased frequency of G6PD deficiency was detected in R.A. patients with at least one positive immunologic marker.
Antioxidant mechanisms protecting the body from the harmful action of reactive oxygen species (R.O.S.) are hinged on several enzymes found in most cells, one of the most important being G6PD. More specifically, in the first reaction of the pentose phosphate pathway, G6PD supplies reducing equivalents (NADPH) necessary to maintain high intracellular levels of reduced glutathione (G.S.H.), a thiol-containing tripeptide active against R.O.S., especially the superoxide anion.[10] Subjects with G6PD deficiency are generally asymptomatic, but they may experience more or less serious haemolytic crises following the intake of specific drugs, especially NSAIDs, or the consumption of certain foods, such as fava beans.[4] Beyond haemolytic crises and neonatal jaundice, more recent literature has reported how G6PD deficiency, affecting any cell of the organism, can be implicated in other disorders, including the cardiovascular system,[11] as well as various autoimmune diseases[12] and, more specifically, R.A. Pathogenic pathways underlying R.A. onset and progression are largely unknown, with different mechanisms, including genetic predisposition, environmental risk factors, microbial exposure, and increased oxidative stress, being proposed until now.[13,14] The R.A. is characterized by impaired antioxidant defense, although the precise mechanisms involved are relatively poorly understood. Experimental[15] and human studies[16] seem to corroborate the notion that a defect in antioxidant mechanisms, whatever the cause, concurs with inflammation in determining joint tissue injury as well as systemic damage.
Several studies and meta-analyses have shown that impaired antioxidant defense is one of the pathogenic hallmarks of R.A. disease.[17] Suggested hypotheses to explain how failure to counteract oxidative stress can contribute to maintaining the autoimmune mechanism in R.A. are structured along two lines of reasoning: (i) a decrease in the intracellular glutathione, whose sulfhydryl (‒SH) moiety is responsible for R.O.S. neutralization, and (ii) the establishment of a chronic inflammatory dysregulation, globally affecting cell signalling including that of the immune system. Evidence from different studies highlighted the large amount of R.O.S. produced by monocytes from R.A. patients. In particular, oxygen and nitrogen-reactive species can directly degrade some components of the synovial tissues―more specifically, hyaluronic acid and other proteoglycans―contributing to joint damage. Furthermore, activated T-cells themselves are highly sensitive to R.O.S. damage, and this might contribute to the establishment of an altered immune response in R.A. via a self-sustained pathogenic loop. In such circumstances, it is reasonable to assume that any alteration of the antioxidant system due to inherited defects can exacerbate the intracellular redox status, thereby interfering with the mechanisms involved in immune tolerance and increasing the chance of developing R.A. Consequently, tentative speculation to explain our findings could be that G6PD deficiency, via intracellular NADPH depletion, may hamper the conversion of GSSG to G.S.H. necessary for R.O.S. disposal. Some intracellular components, critical for immune tolerance, may be permanently modified and trigger the sustained immune activation conducive to R.A.
G6PD deficiency might also enhance R.A. risk via additional mechanisms, such as dysregulation of the inflammatory response. Several in vitro studies have demonstrated that G6PD-deficient cells release several regulatory cytokines in excess, such as the transforming growth factor beta (TGF-β), which plays a major role in inflammation and oxidative stress, as confirmed by the lowering R.O.S. effect of TGF-β inhibitors.[18] Clinical evidence suggests that TGF-β regulates the function of fibroblasts and might have a pathogenic role in R.A..[19] Bira et al. reported an increased TGF-β in the synovial tissue and fluids of R.A. patients.[20] Through its action on pivotal mechanisms of innate (natural killer cells) and adaptive (Tregs cells) immunity, TGF-β upregulation may contribute to the autoimmune response mounted in R.A..[20]
The present study has some limitations due to its retrospective design and the lack of molecular typing of G6PD deficiency. However, it is reasonable to assume that the majority of patients carried the Mediterranean variant.[3] The frequency of G6PD deficiency in patients without R.A. was comparable to that reported for the Sardinian population in the same area,[12] making a bias unlike. Although the database used included patients with clinical complaints requiring endoscopy, we are confident there were no reasons to think that the comorbidity distribution was dissimilar between subjects with and without G6PD deficiency.

Association of Rheumatoid Arthritis with Glucose-6-Phosphate Dehydrogenase Deficiency: Results from a Case-Control Study