ArticleMolecular biology reports2026
Analytical validation of a high-resolution melting assay for UGT1A1 TATA-box polymorphisms.
Article in Molecular biology reports, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
backgroundPolymorphisms in the TATA-box of the UGT1A1 promoter are responsible for Gilbert syndrome and play a key role in irinotecan-related toxicity. Reliable, rapid, and cost-effective genotyping methods are therefore required in routine molecular diagnostics. METHODS AND
resultsWe performed an analytical validation of a High-Resolution Melting (HRM) assay designed to discriminate UGT1A1 TATA-box alleles. A total of 106 neonatal clinical samples were analyzed to assess genotype distribution under routine diagnostic conditions. Analytical validation was performed using plasmid controls, reference genomic DNA samples, and sequencing-confirmed in-house samples. Plasmid-derived TA5, TA6, TA7, and TA8 genotypes and their heterozygous combinations were tested in quintuplicate. Reference genomic DNA samples carrying rare or non-observed genotypes were tested in triplicate, and 30 in-house samples representative of TA6/TA6, TA6/TA7, and TA7/TA7 genotypes were tested in triplicate and confirmed by Sanger sequencing. The HRM assay discriminated the tested genotypes based on melting temperature (Tm) and curve morphology. HRM genotype calls showed complete concordance with expected or reference genotypes. Intra-test analysis of the TA6/TA7 standard showed a mean Tm of 43.36 °C, SD of 0.20 °C, and CV of 0.46%, while inter-test analysis showed identical Tm values across replicates.
conclusionsThe UGT1A1 TATA-box HRM assay showed reproducible genotype discrimination in the tested validation panel and may represent a practical approach for routine UGT1A1 promoter genotyping.
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