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Technical Briefs
manual, 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1989:7.28 –7.52. 7. Glazer A, Rye HS. Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection. Nature 1992;359:859 – 61. 8. Ogura M, Keller C, Koo K, Mitsuhashi M. Use of the fluorescent dye YOYO-1 to quantify oligonucleotides immobilized on plastic plates. Biotechniques 1994;16:1032– 4.
Genotyping in Urine: An Interesting Tool for Epidemiological Studies, Vincent Haufroid,* Andre´ Clippe, Bernard Knoops, Alfred Bernard, and Dominique Lison (Industrial Toxicology and Occupational Medicine Unit, Catholic University of Louvain, Clos Chapelle-aux-Champs, 30.54, 1200 Brussels, Belgium; * author for correspondence: fax 32-2-764-3228, e-mail
[email protected]) Many enzymes involved in the biotransformation of xenobiotics have been shown to be genetically polymorphic, as reviewed recently (1). The rapidly growing list of polymorphic genes coding for xenobiotic-metabolizing enzymes includes various cytochrome P450s, flavin monooxygenases, epoxide hydrolase, NAD(P)H:quinone oxidoreductase, UDP-glucuronosyltransferases, N-acetyltransferases, glutathione-S-transferases, and paraoxonase. Among these are enzymes of great importance in the field of environmental and occupational toxicology; therefore, an effort should be made to develop valid genotyping techniques easily applicable on a large scale, e.g., in the framework of epidemiological studies in industrial settings. Although in clinical practice genotyping is usually carried out on DNA extracted from white blood cells, the necessity of blood sampling probably contributes to limit the use of genotyping techniques in large cohort studies. The principal objective of this study is, therefore, to validate a reliable and robust protocol aimed to obtain, from urinary samples, a biological material on which molecular biology techniques such as restriction fragment length polymorphism (RFLP) analysis could be applied to determine the genotypic status of individuals. Indeed, in occupational and environmental medicine urine is a biological medium that is more easily sampled than blood and is very often obtained for biomonitoring purposes. The protocols used for DNA isolation from whole blood and urine samples were as follows: Genomic DNA was isolated from whole blood with the QIAamp blood kit (QIAGEN, cat. no. 29104) according to the manufacturer’s instructions. To extract genomic DNA from urine, the procedure of the QIAamp Viral RNA kit (QIAGEN, cat. no. 29504) was followed: 4 mL of urine was centrifuged for 5 min at 20 000g. The supernatant was decanted, and the pellet was suspended in 140 mL of buffer AVL supplemented with carrier RNA (QIAamp Viral RNA kit). Nucleic acids were then extracted according to the manufacturer’s instructions and eluted in a final volume of 50 mL of water (urine extract). A practical application of this urinary extraction protocol is illustrated by the determination of three RFLPs already described for an important drug-metabolizing
enzyme in industrial toxicology, i.e., cytochrome P4502E1 (CYP2E1). RFLP analysis of the human CYP2E1 gene displays many polymorphisms (2), and we have selected three of them, detectable with the restriction endonucleases RsaI, PstI, DraI, and TaqI, for the validation process: polymorphism c13c2 (3), polymorphism D3 C (4), and polymorphism A13 A2 (5). Four Caucasian subjects were selected on the basis of their CYP2E1 genotype status, determined previously following classic blood extraction. Subject 1 is homozygous c1/c1 and D/D, subject 2 is heterozygous c1/c2 and D/C, subject 3 is homozygous A1/A1, and subject 4 is heterozygous A1/A2. Each subject was asked to provide a blood sample (blood a) and two different urinary samples. The first urine sample corresponded to the first morning void (urine b), which is usually more concentrated in exfoliated urinary tract cells; the second urine sample was an end-of-workday void or spot sample (urine c), such as the samples usually obtained in the framework of biomonitoring programs applied in occupational medicine. The extraction protocols described above were applied to the respective specimens. The protocol of this study was approved by the local ethics committee, and all subjects gave their informed consent. Blood and urinary samples were amplified by PCR under the following conditions: PCR was carried out on a Perkin-Elmer 2400 thermocycler. The reaction mixture contained 13 PCR buffer (75 mmol/L Tris-HCl, pH 9, 20 mmol/L ammonium sulfate, 0.1 mL/L Tween 20), 2.5 mmol/L MgCl2, 200 mmol/L each dNTP, 0.5 mmol/L each primer, and 2.5 U of Goldstar Red DNA polymerase (Advanced Biotechnologies). The starting material was 10 mL of urine extract or 1 mL of blood extract, and the final reaction volume was 50 mL. The mixture was submitted to the following temperature profile: initial denaturation for 5 min at 95 °C, 40 cycles with denaturation for 30 s at 94 °C, annealing at 60 °C for 30 s, and extension at 72 °C for 1 min 30 s; the final extension was at 72 °C for 5 min. Restriction enzyme digestion. Twenty microliters of the PCR products was digested with 5 U of enzyme (New England Biolabs) in a final volume of 50 mL of 13 buffer, according to the manufacturer’s instructions. The reaction was at 37 °C for 1 h, except for TaqI, which was at 65 °C for 1 h. The whole digestion mixture was electrophoresed in a 2% agarose gel for 1 h at 5V/cm in 13 Tris-borate-EDTA buffer. Primers and restrictions for polymorphism c13c2. DNA was amplified with primers c1pol1 (59-CCACCTTCTATGAAGGTAGTCC) and c1pol2 (59-GCCAGTCGAGTCTACATTGTCAG). The product was 489 bp long and, depending on the allele considered, was cut into two fragments of 368 bp and 121 bp by the PstI enzyme (c2) or into two fragments of 354 bp and 135 bp by the RsaI enzyme (c1). Primers and restrictions for polymorphism D3 C. DNA was amplified with primers dra1pol1 (59-TCGTCAGTTCCTGAAAGCAG) and dra1pol2 (59-AGTAGCTGTGACTATAGGCG). The product was 488 bp long and, depending on the allele considered, was either cut (D) or not cut
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The specific patterns of bands on the electrophoresis gel after the PCR products were digested with the respective restriction enzymes are shown in Fig. 1. The pattern obtained after classic blood extraction (lane a) is compared with those obtained after extraction of the first morning void (lane b) and of a spot urine sample obtained at the end of a workday (lane c). Results clearly show that a blood sampling for genotype determination can easily be replaced by classic PCR analysis performed on a urinary sample. However, it is important to mention that, generally, the band intensity obtained from urine b was of better quality than that obtained from urine c. In some cases however, and without any apparent reason, the material obtained from the first morning void did not yield better results than that from a spot sample (Fig. 1B, subject 2). Therefore, although genotyping is feasible from a spot urine sample, it is more appropriate to sample the first urine of the morning. The protocol described above has been applied to .40 urine samples to test the recovery of DNA. All samples gave satisfying results when tested by PCR, and the amplified DNA allowed determination of the genotype for the different RFLPs. Some urine samples (n 5 12) have also been examined after 1 month of storage at 4 °C to test the robustness of the extraction protocol. The results were excellent for urine b, whereas the band intensity was less (not always detectable) with urine c. The good stability at 4 °C is an another reason to prefer a first urine of the morning. In conclusion, the application of molecular biology techniques used routinely after extraction of the genomic DNA from blood cells could be transferred to DNA extracted from a first morning void. This approach is particularly suited for genotyping large cohorts of individuals, such as in epidemiological studies conducted in environmental and occupational settings.
Fig. 1. Agarose gel electrophoresis of PCR-amplified and restriction endonuclease-digested fragments (ethidium bromide staining). (A) Polymorphism c13c2: genomic DNAs amplified by PCR were digested with RsaI and PstI. Subject 1 is homozygous c1/c1 (allele containing the RsaI site but not the PstI site), and subject 2 is heterozygous c1/c2. (B) Polymorphism D3 C: genomic DNAs amplified by PCR and digested with DraI. Subject 1 is homozygous D/D (allele containing the DraI site), and subject 2 is heterozygous D/C. (C) Polymorphism A13 A2: genomic DNAs amplified by PCR and digested with TaqI. Subject 3 is homozygous A1/A1 (allele containing the TaqI site), and subject 4 is heterozygous A1/A2. A–C: lane a, classic blood extraction; lane b, extraction of the first morning urine; lane c, extraction of a spot urine sample.
(C) into two fragments of 305 bp and 183 bp by the DraI enzyme. Primers and restrictions for polymorphism A13 A2. DNA was amplified with primers taq1pol1 (59-TCCAGGAGTGCTCACATTGG) and taq1pol2 (59-GTTGTCAATAGAAACAGGGCA). The product was 622 bp long and, depending on the allele considered, was either cut (A1) or not cut (A2) into two fragments of 396 bp and 226 bp by the TaqI enzyme.
This study was supported by the Belgian Government (OSTC contract MD DD 006) and by the National Fund for Scientific Research, Belgium.
References 1. Puga A, Nebert DW, McKinnon RA, Menon AG. Genetic polymorphisms in human drug-metabolizing enzymes: potential uses of reverse genetics to identify genes of toxicological relevance. Crit Rev Toxicol 1997;27:199 – 222. 2. Rannug A, Alexandrie AK, Persson I, Ingelman-Sundberg M. Genetic polymorphism of cytochromes P450 1A1, 2D6 and 2E1: regulation and toxicological significance. J Occup Environ Med 1995;37:25–36. 3. Watanabe J, Hayashi SI, Nakachi K, Imai K, Suda Y, Sekine T, Kawajiri K. PstI and RsaI RFLPs in complete linkage disequilibrium at the CYP2E gene. Nucleic Acids Res 1990;18:7194. 4. Uematsu F, Kikuchi H, Ohmachi T, Sagami I, Motomiya M, Kamataki T, et al. Two common RFLPs of the human CYP2E gene. Nucleic Acids Res 1991; 19:2803. 5. McBride OW, Umeno M, Gelboin HV, Gonzalez FJ. A TaqI polymorphism in the human P450IIE1 gene on chromosome 10 (CYP2E). Nucleic Acids Res 1987;15:10071.
