# Functional Characterization of HGD Gene Variants by Minigene Splicing Assay

## TL;DR

This study uses a minigene splicing assay to evaluate the impact of HGD gene variants on splicing, improving the diagnosis and understanding of alkaptonuria.

## Contribution

The study introduces a systematic functional analysis of HGD variants using a minigene assay, leading to reclassification of variant pathogenicity.

## Key findings

- 13 HGD variants were reclassified based on splicing assay results, with 9 upgraded and 4 downgraded in pathogenicity.
- 5 variants retained their original pathogenicity classification after functional analysis.
- Ten missense/nonsense variants showed no significant splicing disruption and were not reclassified.

## Abstract

The HGD gene encodes homogentisate 1,2-dioxygenase. A deficiency of this enzyme causes alkaptonuria (AKU; OMIM 203500), a monogenic autosomal recessive metabolic disorder. The global incidence of alkaptonuria is estimated at 1 in 250,000 to 1,000,000 live births. A large number of pathogenic nucleotide variants disrupt pre-mRNA splicing, leading to hereditary diseases. Many potentially splice-disruptive variants, including those in coding regions, remain uncharacterized. This lack of data makes clinical interpretation more difficult and can complicate diagnosis. We systematically analyzed 27 HGD variants predicted to affect splicing. Candidate variants from public databases (ClinVar, HGDdatabase) and our patient cohort were prioritized using in silico splicing predictions and evaluated with a minigene splicing assay in HEK293T cells. Based on the obtained functional analysis data, the variants were reclassified according to ACMG/AMP guidelines. In total, 13 variants changed their classification (9 were upgraded and 4 were downgraded), while 5 variants retained their pathogenicity class after analysis. Ten missense/nonsense variants were not reclassified, as no significant splicing disruption was detected. These findings improve the pathogenicity assessment of HGD variants, support more accurate diagnosis, and lay the foundation for future therapeutic strategies targeting splicing defects in AKU.