Two Novel PKLR Variants in Pyruvate Kinase Deficiency: Insights From Clinical, Molecular and Functional Analysis

Pyruvate kinase deficiency (PKD) is a rare cause of hereditary non-spherocytic hemolytic anemia. This study reports the clinical and molecular characterization of three pediatric PKD cases, focusing on two novel PKLR variants.

Clinical and laboratory data were reviewed. PKLR gene was analyzed by next-generation sequencing and validated by Sanger sequencing. Pathogenicity was predicted using in silico tools and ACMG criteria. Structural consequences were modeled with AlphaFold3 and Chimera-X. Functional impact was assessed by Western blot and immunofluorescence in transfected 293T cells.

All patients presented with neonatal hemolytic anemia. Genetic analysis revealed compound heterozygosity for PKLR mutations. Two novel variants were identified: c.1708G>A (p.Val570Met) and c.1430C>T (p.Thr477Ile). In silico analysis predicted both as damaging. Structural modeling suggested p.Val570Met disrupts inter-subunit hydrogen bonds, while p.Thr477Ile lies within the fructose-1,6-bisphosphate (FBP) binding loop. In vitro, both mutants showed reduced protein expression compared to wild-type but normal cytoplasmic localization.

We identified two novel PKLR variants expanding the mutational spectrum of PKD. Integrated analysis suggests p.Val570Met may impair tetramer stability, whereas p.Thr477Ile likely affects allosteric regulation. These findings underscore the value of combining clinical phenotyping with functional studies for accurate variant interpretation in PKD.