Neonatal genetic screening of Glucose-6-phosphate dehydrogenase deficiency through next-generation sequencing

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited enzymopathy worldwide. Current neonatal screening, based on enzymatic assays, often fails to identify heterozygous females due to X-chromosome inactivation. This study aimed to characterize by genotype, enzymatic activity and haematological parameters infants carrying pathogenic G6PD variants and to explore genotype-phenotype correlations. Within the NeoGen project, 4067 newborns underwent whole-exome sequencing (WES). Infants with pathogenic or likely pathogenic variants of the G6PD gene were tested for quantitative enzyme activity and haematological indices. Pathogenic or likely pathogenic G6PD variants were found in 123 infants (3.0%); 107 completed full laboratory assessment. Enzymatic deficiency was observed in hemizygous males, while over 60% of heterozygous females showed normal enzyme levels and would have been missed by enzymatic screening. Males exhibited lower G6PD activity, higher reticulocyte counts and lower haemoglobin than females. Enzymatic and haematological variability was greater among females, underscoring the complexity of genotype-phenotype relationships. Enzymatic screening alone misses a significant proportion of affected females. Incorporating molecular testing into neonatal screening improves diagnostic accuracy, supports preventive strategies and promotes equity in early-life healthcare. These findings support the feasibility of combining molecular and enzymatic screening within large-scale genomic screening programmes.