UGT1A1 Fragment Analysis: Genotyping the (TA)_n Variable Repeat Polymorphism for Clinical Applications

Area of Science:

• Pharmacogenomics
• Molecular Diagnostics
• Clinical Chemistry

Background:

• The UGT1A1 gene is crucial for drug metabolism, with specific variants (rs3064744) impacting irinotecan and atazanavir dosing.
• Existing genotyping methods may lack the precision to accurately determine UGT1A1 TA repeat lengths, affecting pharmacogenomic applications.

Purpose of the Study:

• To develop and validate a precise Polymerase Chain Reaction (PCR) and fragment analysis assay for UGT1A1 genotyping.
• To improve the accuracy of UGT1A1 variant determination compared to existing assays like PharmacoScan.

Main Methods:

• A CLIA-validated PCR assay using SuperFi Master Mix and GC Enhancer with a FAM-labeled forward primer.
• Fragment analysis via capillary electrophoresis with HiDi Formamide and LIZ 500 size standard.
• Data analysis using GeneMapper software, identifying peaks by area under the curve (AUC) against known controls.

Main Results:

• The assay achieved 100% concordance across all CLIA validation parameters: accuracy, precision, reportable range, reference range, analytical specificity, and sensitivity.
• Clinical implementation revealed discrepancies in 74 out of 940 samples (7.8%) when compared to PharmacoScan results for UGT1A1 rs3064744.

Conclusions:

• The developed assay serves as a valuable supplemental tool in pharmacogenomics, significantly improving the accuracy of UGT1A1 genotyping.
• The assay identified a 7.8% correction rate in patient samples, including a 2.77% miscalling rate of the UGT1A1*37 variant by previous methods.