Rapid detection of UGT1A1 gene polymorphisms by newly developed Invader assay.

To the Editor: Recent progress in human genome analysis has been providing tools for a new approach to disease treatment based on individual differences identified by use of genetic information. The feasibility of genotyping for DNA polymorphisms before treatment depends on the availability of rapid, accurate, and efficient geno-typing methods. We previously reported that genetic polymorphisms of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene were significantly related to severe toxicity of irinotecan (1). We now report that we have succeeded in detecting a 2-bp insertion of repeated sequence in the UGT1A1 gene by use of Invader assay technology. Sixty patients who had received irinotecan-containing chemotherapy from July 1994 to June 1999 were enrolled in this study. All gave informed consent in writing for their peripheral blood to be used for the research. We used the QIAamp Blood Kit (QIAGEN GmbH) to prepare genomic DNA from whole blood (100 –200 ␮L) and genotyped three sites of DNA polymorphisms in UGT1A1 (UGT1A1*28, UGT1A1*6, and UGT1A1*27) by the previously described method (1). UGT1A1*28 was distinguished from the most common allele (UGT1A1*1) by direct sequencing (nucleotides Ϫ147 to ϩ106) of the 253-to 255-bp fragments produced by PCR. UGT1A1*6 and UGT1A1*27 were distinguished from UGT1A1*1 by direct sequencing combined with PCR-restriction fragment length polymorphism (RFLP) analysis. The Invader assay detects single-nucleotide polymorphisms (SNPs) by use of Cleavase enzyme and a fluorescence resonance energy transfer cassette (2, 3). Two sets of probes were designed for detecting the SNPs associated with UGT1A1*6 and *27 (see Table 1 in the Data Supplement that accompanies the online version of this letter at The UGT1A1*6 target site was ϩ211G/A, and the *27 target site was ϩ686C/A. In addition, a set of probes was designed to differentiate between the UGT1A1*28 polymorphism and its reference allele (UGT1A1*1; Table 1 in the online Data Supplement) based on the number of TA repeats; UGT1A1*28 has seven TA repeats, and UGT1A1*1 has six. An important portion of the probe was designed with the help of Third Wave Technologies , Inc. With these detection systems, the reference and variant alleles are indicated by the Redmond Red (Epoch Biosciences) and 6-car-boxyfluorescein (FAM) fluorescent signals, respectively, which were released from the fluorescence resonance energy transfer cassette. The 60 samples included 4 homozy-gous [(TA) 7 /(TA) 7 ] and 11 heterozy-gous [(TA) 6 /(TA) 7 ] for UGT1A1*28; the remaining 45 samples were ho-mozygous for the reference allele [(TA) 6 /(TA) …