Identification of novel disease-causing variants and their influence on clinical presentation in dihydrofolate reductase deficiency.

Dihydrofolate reductase (DHFR) deficiency is a rare autosomal recessive disorder of folate metabolism, which is crucial for DNA/RNA synthesis and normal growth. Only seven molecularly confirmed cases, all presenting with megaloblastic anemia and neurological symptoms, have been reported. We report two cases from a non-consanguineous Chinese family. The proband presented with neonatal seizures; folic acid improved neurological symptoms but did not prevent fatal respiratory failure. His sister had severe megaloblastic anemia. Whole-exome sequencing identified two compound heterozygous variants in DHFR (p.Gly18Val and p.Pro26Leu) in both patients. Structural modeling suggested these variants to compromise protein stability/function. Variant protein expression plasmids produced significantly less DHFR protein than wild-type plasmid as indicated by western blot and ELISA analyses. Methotrexate fluorescence binding assays indicated reduced binding capacity, suggesting potential structural alterations in the variant proteins. These results demonstrate that the variants lead to decreased DHFR protein abundance and perhaps altered molecular interactions, supporting their pathogenicity. Our findings underscore the critical importance of early genetic testing for patients with megaloblastic anemia or neurological symptoms to diagnose DHFR deficiency. Early detection and treatment are essential to improve prognosis and prevent severe outcomes.