Functional Characterization of HGD Gene Variants by Minigene Splicing Assay.

Area of Science:

• Genetics
• Molecular Biology
• Biochemistry

Background:

• Alkaptonuria (AKU) is a rare autosomal recessive metabolic disorder caused by HGD gene mutations.
• Pathogenic variants affecting pre-mRNA splicing are a significant cause of genetic diseases, yet many remain uncharacterized.
• Uncharacterized HGD variants complicate AKU diagnosis and interpretation.

Purpose of the Study:

• To systematically analyze HGD variants predicted to affect splicing.
• To functionally evaluate these variants using minigene splicing assays.
• To reclassify HGD variants based on ACMG/AMP guidelines to improve diagnostic accuracy.

Main Methods:

• In silico splicing predictions were used to prioritize candidate HGD variants.
• A minigene splicing assay in HEK293T cells was employed for functional evaluation.
• Variants were reclassified according to ACMG/AMP guidelines based on functional data.

Main Results:

• Functional analysis was performed on 27 HGD variants predicted to affect splicing.
• Thirteen variants had their classification changed (9 upgraded, 4 downgraded).
• Ten missense/nonsense variants showed no significant splicing disruption and retained their classification.

Conclusions:

• Functional analysis of HGD variants significantly improves pathogenicity assessment.
• Accurate variant classification aids in more precise diagnosis of AKU.
• These findings provide a foundation for developing targeted therapies for splicing defects in AKU.