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

Area of Science:

• Genetics
• Molecular Biology
• Hematology

Background:

• Pyruvate kinase deficiency (PKD) is a rare genetic disorder causing hereditary non-spherocytic hemolytic anemia.
• This study focuses on the clinical and molecular aspects of three pediatric PKD cases.

Purpose of the Study:

• To characterize novel variants in the PKLR gene associated with pyruvate kinase deficiency.
• To investigate the functional and structural impact of these new variants.

Main Methods:

• Clinical and laboratory data review.
• Next-generation and Sanger sequencing for PKLR gene analysis.
• In silico pathogenicity prediction, structural modeling (AlphaFold3, Chimera-X), and in vitro functional assays (Western blot, immunofluorescence).

Main Results:

• Three pediatric patients with neonatal hemolytic anemia were identified with compound heterozygous PKLR mutations.
• Two novel variants, c.1708G>A (p.Val570Met) and c.1430C>T (p.Thr477Ile), were discovered and predicted as damaging.
• In vitro studies showed both mutants had reduced protein expression but normal localization, with p.Val570Met potentially affecting tetramer stability and p.Thr477Ile impacting allosteric regulation.

Conclusions:

• Two novel PKLR variants were identified, broadening the known mutation spectrum for pyruvate kinase deficiency.
• The findings suggest distinct mechanisms for the identified variants: p.Val570Met may impair protein stability, while p.Thr477Ile likely affects enzyme function.
• Integrating clinical data with functional studies is crucial for accurate variant interpretation in PKD.