Functional characterization of compound heterozygous HLCS variants reveals exon skipping and impaired biotin responsiveness in holocarboxylase synthetase deficiency.

Holocarboxylase synthetase deficiency (HLCSD) is a rare inherited metabolic disorder caused by variants in the HLCS gene. Functional characterization can aid the interpretation of rare HLCS variants. Clinical, biochemical, genetic, transcriptional, functional, and structural analyses were performed, including urine organic acid analysis, targeted next-generation sequencing, Sanger validation, RT-PCR, cDNA sequencing, fibroblast propionyl-CoA carboxylase (PCC) activity before and after biotin treatment, and AlphaFold3-based structural modeling. A 29-month-old child with late-onset HLCSD presented with severe metabolic acidosis. Urine organic acid analysis revealed elevated levels of lactic acid, 3-hydroxyisovaleric acid, 3-methylcrotonylglycine, propionylglycine, and methylcitric acid. PCC activity increased from 34 to 142 pmol/min/mg protein following biotin supplementation, confirming preserved biotin responsiveness. The patient showed marked clinical and metabolic improvement after treatment. Genetic analysis identified compound heterozygous HLCS variants: a novel splice-acceptor site variant, c.1796-2 A > G, paternal, and a previously reported missense variant, c.2126C > T, p.(Pro709Leu), maternal. RT-PCR and cDNA sequencing demonstrated exon 11 skipping caused by c.1796-2 A > G, predicting a truncated protein. Comparative AlphaFold3-based structural modeling of wild-type and p.(Pro709Leu) HLCS suggested variant-associated local conformational changes affecting the HLCS-substrate interaction interface and active-site architecture, providing additional structural evidence supporting the pathogenicity of p.(Pro709Leu). These findings support the pathogenicity of the novel splice-site variant, provide new structural evidence for the previously reported p.(Pro709Leu) variant, and expand the molecular and functional spectrum of HLCSD.