Analytical validation of a high-resolution melting assay for UGT1A1 TATA-box polymorphisms

Area of Science:

• Molecular Diagnostics
• Genetics
• Pharmacogenomics

Background:

• UGT1A1 promoter TATA-box polymorphisms are linked to Gilbert syndrome.
• These polymorphisms significantly influence irinotecan-related toxicity.
• There is a need for efficient and reliable genotyping methods in diagnostics.

Purpose of the Study:

• To analytically validate a High-Resolution Melting (HRM) assay for UGT1A1 TATA-box allele discrimination.
• To assess the assay's performance under routine diagnostic conditions using clinical samples.

Main Methods:

• Analytical validation using plasmid controls, reference genomic DNA, and Sanger sequencing confirmed samples.
• Testing of various UGT1A1 TATA-box genotypes (TA5, TA6, TA7, TA8) and heterozygotes.
• Assessment of assay reproducibility through intra-test and inter-test analyses.

Main Results:

• The HRM assay successfully discriminated tested UGT1A1 TATA-box genotypes based on melting temperature and curve morphology.
• Complete concordance was observed between HRM genotype calls and expected or reference genotypes.
• High reproducibility was demonstrated, with low coefficient of variation (CV) in melting temperature measurements.

Conclusions:

• The developed UGT1A1 TATA-box HRM assay provides reproducible genotype discrimination.
• This assay is a practical and potentially valuable tool for routine UGT1A1 promoter genotyping in molecular diagnostics.