Integrated enzymatic and molecular diagnosis of G6PD deficiency: a one-year experience identifying five novel variants.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, caused by pathogenic variants in the G6PD gene, is the most common X-linked enzymopathy in the world. The clinical manifestations range from drug-induced hemolytic anemia to neonatal hyperbilirubinemia and chronic hemolysis. The G6PD status needs to be accurately assessed in several clinical settings, including drug prescription, assisted reproductive technologies, and the diagnostic workflow of hemolytic anemia. In this study, the contribution of molecular analysis in the diagnostic workflow of patients with suspected G6PD deficiency was retrospectively evaluated over one year. A total of 340 samples were analyzed for the enzymatic activity of G6PD, including 28 organ donors, 62 pediatric patients, 157 hospitalized patients, and 93 referred from the Genetics, Internal Medicine, Gastroenterology, and Hematology units. Molecular analysis was performed for 52 cases (17 males and 35 females): 37 with reduced enzymatic activity, five with inconclusive results due to recent blood transfusions, and 10 with specific clinical indications. Through molecular testing, eight previously reported and five novel disease-causing variants (c.769 C > T, p.(Arg257Gly); c.488G > T, p.(Gly163Val); c.1124 A > G, p.(Asn375Gly); c.1152G > C p.(Gln384His); c.582 C > G, p.(Asp194Glu)) were identified in 36 patients, along with three common polymorphic variants in 11 other patients. No variants were identified in five patients, consistent with borderline or difficult-to-interpret activity results. Overall, lower G6PD activity was found in 66 of 340 samples (19.4%), while novel variants accounted for 13.5% of cases (5/37). These findings indicate that molecular analysis should be integrated into routine diagnostic workflows for G6PD deficiency and highlight its contribution to expanding the mutational spectrum of the disease.