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Letters to the Editor |
1 Department of Medicine, Division of Respiratory Diseases, Nagoya University, Graduate School of Medicine, Nagoya, Japan. 2 Genomic Business Planning Department, Daiichi Pure Chemicals Co., Tokyo, Japan. 3 Department of Clinical Oncology, Saitama Medical School, Saitama, Japan
aAddress correspondence to this author at: Department of Medicine, Division of Respiratory Diseases, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. Fax 81-52-744-2176; e-mail yhasega{at}med.nagoya-u.ac.jp.
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 genotyping 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 µL) 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 –147 to +106) 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 http://www.clinchem.org/content/vol50/issue8/). The UGT1A1*6 target site was +211G/A, and the *27 target site was +686C/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-carboxyfluorescein (FAM) fluorescent signals, respectively, which were released from the fluorescence resonance energy transfer cassette.
The 60 samples included 4 homozygous [(TA)7/(TA)7] and 11 heterozygous [(TA)6/(TA)7] for UGT1A1*28; the remaining 45 samples were homozygous for the reference allele [(TA)6/(TA)6]. The distribution of genotypes showed 1 homozygous and 17 heterozygous for UGT1A1*6 and 42 homozygous for the reference allele. Two samples heterozygous for UGT1A1*27 and 58 homozygous for the reference allele were assayed. These data were determined by direct DNA sequencing of UGT1A1*28 and by direct DNA sequencing combined with PCR-RFLP analysis for UGT1A1*6 and UGT1A1*27, and the results obtained from the Invader assay were compared with them (see Table 2 in the online Data Supplement).
Three of the 60 samples could not be assayed for UGT1A1*28 by the Invader assay. These samples showed low fluorescence intensity because of their small genomic DNA content (DNA concentration, 15 ng/10 µL, 16 ng/10 µL, and 21 ng/10 µL, respectively). The minimum input of DNA for which UGT1A1*28 was measured by the Invader assay was 21 ng/10 µL. The genotyping of 57 samples measured by the Invader assay agreed completely with the results obtained by direct DNA sequencing.
Six of the 60 samples could not be measured for UGT1A1*6 by the Invader assay. These samples showed low fluorescence intensity because of the small content of genomic DNA (content, per 10 µL, of 15, 16, 21, 21, 38, and 46 ng, respectively). The minimum input of DNA for which UGT1A1*6 was measured by the Invader assay was 29 ng/10 µL. The results for the remaining samples obtained by the Invader assay agreed well with those obtained by the PCR-RFLP assay.
Forty-seven of the 60 samples were genotyped correctly for UGT1A1*27 by the Invader assay, and the results were consistent with those obtained by the PCR-RFLP assay. Three samples could not be measured by the Invader assay because of their low fluorescence intensity (DNA per 10 µL, 15, 16, and 21 ng, respectively). In addition, 10 samples could not be measured because of an indistinct fluorescence signal ratio (range, 2.861–4.995; median, 4.718; Table 1 in the online Data Supplement). These samples also contained small amounts of genomic DNA (range, 21–59 ng/10 µL; median, 44 ng/10 µL). Thirty-nine samples containing genomic DNA >60 ng/µL were correctly genotyped by the Invader assay. The minimum input of DNA for which UGT1A1*27 was measured by the Invader assay was 39 ng/10 µL.
In conclusion, we demonstrated that genotyping of UGT1A1*28, *6, and *27 by the newly developed Invader assay agreed almost completely with the genotyping results obtained with established methods and that the Invader assay provides rapid detection of polymorphisms. We recommend use of 100 ng/10 µL for accurate screening. We previously suggested that determination of the UGT1A1 polymorphisms before irinotecan treatment could be clinically useful and important for predicting and preventing related toxicities (1). Our newly developed method for detecting UGT1A1 polymorphisms is feasible and has the potential to be widely used for rapid and accurate screening before irinotecan treatment.
References
The following articles in journals at HighWire Press have cited this article:
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  J. M. Hoskins, R. M. Goldberg, P. Qu, J. G. Ibrahim, and H. L. McLeod UGT1A1*28 Genotype and Irinotecan-Induced Neutropenia: Dose Matters J Natl Cancer Inst, September 5, 2007; 99(17): 1290 - 1295. [Abstract] [Full Text] [PDF]
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