Validation of Rapid Polymerase Chain Reaction-based Detection of All Length Polymorphisms in the UGT 1A1 Gene Promoter

Abstract

UDP glucuronosyltransferase (UGT) 1A1 gene promoter polymorphism can affect the expression level of the UGT 1A1 enzyme. The polymorphism consists of an insertion of a TA nucleotide sequence into a (TA)₆TAA sequence in the gene promoter resulting in (TA)₇TAA (UGT1A1*28). This results in a reduced UGT 1A1 expression with 70% less glucuronidation capacity for bilirubin and other UGT1A1 substrates. Other polymorphisms include (TA)₈TAA (UGT1A1*37) and (TA)₅TAA (UGT1A1*36). The longer the TA repeats the lower the enzyme expression level. The anticancer agent irinotecan is metabolized to the active SN-38, which is further glucuronidated and detoxified by UGT 1A1. Decreased glucuronidation leads to SN-38 accumulation with severe neutropenia and diarrhea. We have developed a rapid polymerase chain reaction (PCR)-based detection of all length polymorphisms in the UGT 1A1 gene promoter. It uses PCR and DNA fragment analysis using an ABI Genetic Analyzer. Thirty-two blood samples were analyzed for UGT 1A1 promoter polymorphism. We found 2 (TA)₅TAA/(TA)₅TAA, 4 (TA)₅TAA/(TA)₆TAA, 2 (TA)₅TAA/(TA)₇TAA, 9 (TA)₆TAA/(TA)₆TAA, 11 (TA)₆TAA/(TA)₇TAA, 2 (TA)₇TAA/(TA)₇TAA, and 2 (TA)₇TAA/(TA)₈TAA in our sample group. To confirm the results, 6 samples with different repeats were also analyzed by DNA sequencing method. This is a rapid and reliable method for analysis of the promoter length polymorphisms of UGT 1A1 gene.