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Technical Briefs |
1 Project Team for Pharmacogenetics,
2
Division of Biochemistry and Immunochemistry,
3
Division of Environmental Chemistry,
4
Division of Medicinal Safety Science, and
8
Division of Pharmacology, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
5 Division of Cardiology,
6 Department of Cardiovascular Dynamics Research Institute, and
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Department of Pharmacy, National Cardiovascular Center, 5-7-1, Fujishirodai, Suita, Osaka 565-8565, Japan
aaddress correspondence to this author at: Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; fax 81-3-3707-6950, e-mail yoshiro{at}nihs.go.jp
Glucuronidation, catalyzed by UDP-glucuronosyltransferases (UGTs), is important in the detoxification and enhanced elimination of a large number of endogenous and exogenous substrates. The human UGT1A gene complex contains at least nine variations of exon 1, common exons 2–5, and a single exon 1 splices to exons 2–5 (1).
Of the UGT1A isoforms, UGT1A1 is primarily responsible for the glucuronidation of bilirubin in the human liver and can also conjugate phenols, anthraquinones, flavonoids, and a variety of therapeutic drugs and their metabolites (e.g., SN-38, an active irinotecan metabolite) (2)(3). Several functional polymorphisms in UGT1A1 are associated with reduced bilirubin glucuronidation activity and cause hyperbilirubinemia (Gilbert and Crigler–Najjar syndromes).
UGT1A1 TATA box variants [A(TA)6TAA>A(TA)5/7/8TAA] are associated with enhanced [(TA)5] or reduced [(TA)7/8] UGT1A1 transcription (4). Among them, the (TA)6 and (TA)7 repeats have been reported in Asians. The variant (TA)7 is associated with reduced glucuronidation of SN-38 and bilirubin, as well as the pathogenesis of Gilbert syndrome (5). In addition, a T-to-G substitution at nucleotide -3279 (A of the translational start codon in GenBank accession no. AF297093.1 is nucleotide number 1) in the UGT1A1 phenobarbital-responsive enhancer module reduces transcriptional activity (6).
The most common nonsynonymous single-nucleotide polymorphism (SNP) (211G>A) that causes an amino acid alteration (glycine to arginine at codon 71) is found in Asian populations at frequencies of 13–23% (7)(8). The 686C>A (P229Q) variation in the Taiwanese has a frequency of 2.8% (8). Also associated with Gilbert syndrome are 211G>A (G71R) and 686C>A (P229Q). Rare in Japanese and Taiwanese patients is 1456T>G (Y486D), which is associated with the more severe type II Crigler–Najjar syndrome (8)(9). Our previous study demonstrated that the in vitro intrinsic clearance (Vmax/Km) of SN-38 was decreased to 47% for G71R, 52% for P229Q, and 5% for Y486D compared with that of the wild type (10). Recently, a novel SNP, 247T>C (F83L), was shown to be associated with Gilbert syndrome patients in Thailand. This variation was also found in the Japanese at a frequency of 1.4% (11).
The detection of genotypic differences in the coding and promoter regions of this enzyme thus is important for understanding the metabolism of various compounds and for clinical diagnosis. Several methods have been reported for genotyping the TA repeat polymorphism (4)(5), but no single method has been available for simultaneous genotyping of all known polymorphisms. DNA sequencing is the most accurate and informative technique, but is time-consuming. Other high-throughput genotyping methods, such as PCR-restriction fragment length polymorphism analysis, Taq-Man, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, are not completely suitable for typing repeat polymorphisms.
Pyrosequencing is a real-time, nonelectrophoretic method for DNA sequencing that is based on enzymatic reactions catalyzed by ATP sulfurylase and luciferase. The inorganic pyrophosphates that are released after deoxynucleotide incorporation are monitored. Unreacted nucleotides are degraded by apyrase, allowing iterative nucleotide addition. It has been shown that SNP typing can be efficiently performed by pyrosequencing (12).
In this study, we developed a method for genotyping the common UGT1A1 polymorphisms [-3279T>G, TA repeat, 211G>A (G71R), 247T>C (F83L), 686C>A (P229Q), and 1456T>G (Y486D)]. DNA was extracted from the blood leukocytes of 48 Japanese patients who had been administered beta blockers. The ethics committees of the National Cardiovascular Center and the National Institute of Health Sciences approved this study. Written informed consent was obtained from all participants.
The genotypes of the 48 patients were determined by pyrosequencing and compared with direct sequencing results. For pyrosequencing, fragments were directly amplified from genomic DNA (10–15 ng) by Ex-Taq (1 U; Takara Shuzo) with specific amplification primer pairs (either primer was biotinylated; Table 1
). The PCR conditions were 94 °C for 5 min, followed by 50 cycles of 94 °C for 30 s, 58 °C for 45 s, and 72 °C for 30 s.
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Biotinylated single-stranded DNA fragments were generated by the following procedure at room temperature. PCR products were mixed with Streptavidin-Sepharose High Performance (Amersham Biosciences AB) in Binding Buffer (2x; 10 mmol/L Tris-HCl, 1 mmol/L EDTA, 2 mol/L NaCl, 1 mL/L Tween 20, pH 7.6) for 10 min. The beads were transferred to a MultiScreen-HV Plate (Millipore Corporation), and the Binding Buffer was removed by application of reduced pressure. DNA attached to the beads was denatured for 1 min in 50 µL of Denaturation Buffer (0.2 mol/L NaOH) and washed twice with 150 µL of Wash Buffer (10 mmol/L Tris acetate, pH 7.6). The beads were suspended in 50 µL of Annealing Buffer (20 mmol/L Tris acetate, 2 mmol/L magnesium acetate, pH 7.6) and transferred to the 96-well PSQ plate (Pyrosequencing AB); 10 pmol of the sequencing primer (polyacrylamide gel electrophoresis-purified grade; Table 1
) for SNP analysis was then added to the single-stranded fragments. The mixture was incubated at 95 °C for 2 min, and then cooled to room temperature for annealing. An automated pyrosequencing instrument, the PSQTM 96MA (Pyrosequencing AB), and the PSQ 96 SNP reagent set (Pyrosequencing AB) were used for genotyping. The total procedure for 96 samples took 
4 h, including 25 min to run the samples. In addition, direct sequencing was performed as described previously, using the primers listed in Table 1
(13). The time required for the entire sequencing procedure for 96 samples was 12 h, including 3 h of run time on the ABI Prism 3700 DNA Analyzer (Applied Biosystems).
Two polymorphisms in the promoter region (-3279T>G and TA repeat) and four additional SNPs that lead to reduced activity [211G>A (G71R), 247T>C (F83L), 686C>A (P229Q), and 1456T>G (Y486D)] were analyzed. All samples were successfully genotyped at all polymorphic sites. The genotyping results were identical to those obtained by direct sequencing. Fig. 1
shows representative sequencing patterns (pyrograms) for the UGT1A1 TA repeat (TA)6>(TA)7 (Fig. 1A
) and 211G>A (G71R; Fig. 1B
). DNA samples derived from established cell lines or in vitro-mutated cDNA plasmids were also used as templates for the genotypes that were not detected, such as the homozygous (TA)7 repeat, heterozygous and homozygous 247T>C (F83L), homozygous 686C>A (P229Q), and heterozygous and homozygous 1456T>G (Y486D). Correct genotyping results were successfully obtained from these control samples (data for the TA repeat are shown). This genotyping method was also applicable to the other TA repeat variants, (TA)5 and (TA)8, which have been found in African Americans, with the same primers and only a slight modification to the sequencing program (data not shown).
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The allelic frequencies of these polymorphisms were 0.281 for -3279T>G (4 homozygous G/G and 19 heterozygous T/G patients), 0.135 for (TA)6>(TA)7 (13 heterozygous 6/7 repeat patients) and 211G>A (G71R; 2 homozygous A/A and 9 heterozygous G/A patients), and 0.010 for 686C>A (P229Q; 1 heterozygous C/A patient), respectively. These frequencies were similar to those reported previously (4)(6)(7)(8). Two low-frequency SNPs, 247T>C (F83L) and 1456T>G (Y486D) (9)(11), were not detected in this study. We also did not find any Japanese patients with the (TA)5 or (TA)8 repeat.
In summary, we developed a pyrosequencing-based genotyping method for six functionally significant polymorphisms that are especially important in the Japanese. The pyrosequencing data were identical to those obtained from direct sequencing. Pyrosequencing thus can expedite studies on the association between genetic polymorphisms and pharmacokinetic or clinical data.
Acknowledgments
This study was supported in part by the Program for Promotion of Fundamental Studies in Health Sciences (MPJ-3 and MPJ-6) of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan and the Program for Promotion of Studies in Health Sciences of the Ministry of Health, Labor and Welfare.
References
The following articles in journals at HighWire Press have cited this article:
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E. Rouits, M. Boisdron-Celle, A. Dumont, O. Guerin, A. Morel, and E. Gamelin Relevance of Different UGT1A1 Polymorphisms in Irinotecan-Induced Toxicity: A Molecular and Clinical Study of 75 Patients Clin. Cancer Res., August 1, 2004; 10(15): 5151 - 5159. [Abstract] [Full Text] [PDF]
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K. E. Kruckeberg and S. N. Thibodeau Pyrosequencing Technology as a Method for the Diagnosis of Multiple Endocrine Neoplasia Type 2 Clin. Chem., March 1, 2004; 50(3): 522 - 529. [Abstract] [Full Text] [PDF]
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A (G71R; B).