- Email: [email protected]
GSTT1 and GSTM1 gene copy number analysis in paraffin-embedded tissue using quantitative real-time PCR
Analytical Biochemistry 378 (2008) 221–223
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
GSTT1 and GSTM1 gene copy number analysis in paraffin-embedded tissue using quantitative real-time PCR Naiara G. Bediaga a, Miguel A. Alfonso-Sánchez a, Mertxe de Renobales b, Ana M. Rocandio c, Marta Arroyo c, Marian M. de Pancorbo a,* a
Servicio de Genómica, Banco de ADN, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria, Spain Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria, Spain c Departamento de Nutrición y Bromatología, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria, Spain b
a r t i c l e
i n f o
Article history: Received 4 March 2008 Available online 9 April 2008
a b s t r a c t GSTT1 and GSTM1 genes possess an inherited deletion associated with a lack of enzyme activity. The heterozygous condition of this deletion is difficult to determine in low-quality DNA with existing PCR protocols. We designed and validated a multiplex real-time PCR assay by adapting the DDCt relative quantification method for the analysis of GSTT1 and GSTM1 markers to accurately differentiate the three genotypes (*1/1, *1/0, and *0/0) in degraded DNA from formalin-fixed paraffin-embedded tissue. Gene copy number values obtained provide for unambiguous homozygous and heterozygous differentiation. The efficacy shown by the PCR assay endorses its usefulness for complete genotyping of glutathione Stransferases in archival tissues. Ó 2008 Elsevier Inc. All rights reserved.
Formalin-fixed paraffin-embedded (FFPE)1 tissue samples represent the largest source of archival biological material for epidemiological studies based on molecular genetic analysis. Therefore, there is a growing need to implement PCR methods allowing an accurate genotyping of DNA samples extracted from fixed tissues. Genes belonging to the glutathione S-transferase (GST) superfamily are commonly explored as susceptibility and tumor markers in molecular epidemiological surveys. GSTT1 and GSTM1 genes are known to be polymorphic, with an inherited deletion (GSTT1*0 and GSTM1*0 null alleles, respectively) associated with a lack of enzyme activity that is present in approximately 20% (GSTT1) and 50% (GSTM1) of Caucasians [1]. Prevalence of the heterozygous condition (deletion on a single chromosome) for these loci has never been reported in FFPE tissue, probably because the nonquantitative endpoint PCR assays used to genotype these deletions [2,3], which require amplicon sizes greater than 13 kb, cannot be applied efficiently on degraded DNA. Earlier GSTT1 analyses based on semiquantitative PCR assays unveiled a gene dose effect on GSTT1 activity [4], suggesting that accurate differentiation between homozygous (high enzymatic activity) and heterozygous (medium enzymatic activity) could be important to correlate clinical phenotypes with GST genotypes. Real-time PCR is a recommendable option for gene copy number analysis in DNA from FFPE tissues [5]. Real-time PCR assays
* Corresponding author. E-mail address: [email protected] (M.M. de Pancorbo). 1 Abbreviations used: FFPE, formalin-fixed paraffin-embedded; GST, glutathione S-transferase; Ct, cycle threshold; CI, confidence interval; SD, standard deviation. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.04.010
have been recently devised for complete genotyping of GSTT1 and GSTM1 loci [6–9], but only Covault and coworkers [6] and Brasch-Andersen and coworkers [7] have employed the DDCt method of relative quantification, which is easier to implement than the absolute method because standard curves are unneeded [10]. Nevertheless, validity of this method demands PCR amplification efficiencies for both target and reference genes close to 100%, which is complicated when working with low-quality starting DNA. Indeed, neither of the cited PCR assays has been employed so far for GSTM1 and GSTT1 gene dose determination in FFPE tissue despite their obvious theoretical advantages, probably due to low reproducibility in archival tissues. Therefore, we designed and tested a new real-time PCR assay based on the DDCt method to overcome potential complications arising from the analysis of FFPE tissue with the existing protocols. Quantitative real-time PCR was performed using a TaqMan (50 nuclease) assay system with signal from a GSTT1- or GSTM1-specific probe that is normalized to the signal for a reference gene (b-globin). A total of 240 DNA samples from archival (FFPE) and fresh blood tissues were screened. FFPE samples (n = 63) belonged to surgical specimens of primary liver cancers from patients diagnosed with hepatocellular carcinoma between 1996 and 1999. The control group (blood samples) consisted of 177 unrelated donors without manifest neoplasms. All samples belong to the DNA Bank at the University of the Basque Country (Spain). The study protocol was approved by the relevant ethics committees of the institutions involved. Primer and probe sequences for the genotyping of GSTT1 (NM_000853), GSTM1 (NM_000561), and b-globin (U_01317)
222
Notes & Tips / Anal. Biochem. 378 (2008) 221–223
genes were designed to reach maximum efficiency of the amplification process and, thereby, of the PCR assay. Because GSTT1 and GSTM1 markers are members of a multigene family, primer pairs and probe were selected to be unique when compared with the sequences of closely related genes. To that end, Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA) was used. To assay GSTT1 gene copy number relative to reference gene bglobin, triplicate PCRs (15 ll) using 15 ng of genomic DNA, 0.9 lM of each GSTT1 primer (forward, 50 -AAGTCCCAGAGCACCTCACCTCC30 ; reverse, 50 -CAGTGTGCATCATTCTCATTGTGGC-30 ), 0.4 lM of bglobin primers (forward, 50 -GAGGGTTTGAAGTCCAACTCCTAA-30 ; reverse, 50 -CAGGGTGAGGTCTAAGTGATGACA-30 ), and fluorescent product-specific oligonucleotide probes (GSTT1, 6FAM-CACCATC CCCACCCTGTCTTCCA-TAMRA, 0.2 lM; b-globin, VIC-CAGTGCCAG AAGAGCCAAGGACAGGT-TAMRA, 0.1 lM) were prepared in 1 Master Mix (Applied Biosystems). Thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. PCR was performed in a 96well optical plate (ABgene) using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). To test GSTM1 gene dose relative to b-globin, we used 1.2 lM of GSTM1 primers (forward, 50 -CACCTGCATTCGTTCATGTGAC-30 ; reverse, 50 -CACCTGCATTCGT TCATGTGAC-30 ), 0.4 lM of b-globin primers and fluorescent product-specific probes (GSTM1, 6FAM-TTCAGTCCTGCCATGAGCAGGCA CA-TAMRA, 0.15 lM; b-globin, VIC-CAGTGCCAGAAGAGCCAAGGA CAGGT-TAMRA, 0.15 lM) using thermal cycling conditions identical to those for GSTT1 primers. Data analysis was conducted by adapting the DDCt method of relative quantification [10] to estimate copy numbers of GSTT1 and GSTM1 genes. Ct (cycle threshold) is defined as the point at which the fluorescence level rises above a baseline. Two known control samples (carrying both alleles) were analyzed on each reaction tray as calibrators. In addition, a number of no-template controls were also included in the analysis. The samples employed as calibrators were previously analyzed by standard and long-range PCR to confirm possession of two copies of the genes examined, whereas b-globin was the reference gene in all experiments. The result for each target gene, expressed as n-fold copy number of the target gene relative to b-globin (nGST), was obtained through the equation 2–DDCt, where DDCt = (Ct GSTsample – Ct GLOBINsample) – (Ct GSTcalibrator – Ct GLOBINcalibrator). As argued above, for the DDCt method to be valid, two essential conditions must be satisfied: (i) all PCR amplifications must be close to 100% and (ii) the relative efficiency must be optimal (i.e., the amplification efficiency of the target and reference genes must be approximately equal). Real-time efficiencies (E) were calculated by means of standard calibration curves (Fig. 1), using threefold serial dilutions of blood and FFPE tissue DNA with known GSTT1*1/1 and GSTM1*1/1 genotypes, through the equation E = 10(–1/slope) – 1. The observed real-time PCR efficiency (E) was always between 92 and 100% for all target genes in all tissues considered. To check whether the amplification efficiencies of the target and reference genes were similar, we verified that the slope of DCt versus input DNA was less than 0.1 over DNA dilution series. To estimate the range of nGST values for a reliable determination of the gene copy number, 40 fresh tissue samples with known genotype, analyzed by methods mentioned above [2,3], were used to determine confidence intervals (CIs) of 95% (P < 0.05) for each genotype. Samples that did not fit with their respective nGST ranges were reamplified per triplicate. For GSTT1, three groups of samples were clearly distinguishable: (i) nGSTT1 values greater than 0.84 were characteristics of samples that showed no deletion (*1/1), (ii) nGSTT1 values between 0.47 and 0.62 appeared in cases with one-copy deletion genotype (*0/1), and (iii) nGSTT1 values less than 0.01 were typical of null genotypes (*0/0). Very similar ranges were estimated for GSTM1: (i) nGSTM1 values greater than 0.84 for no al-
Fig. 1. TaqMan GSTT1 and b-globin standard curves obtained by real-time PCR applied on DNA from FFPE tissue. (A) Amplification plots for five serial dilutions of a GSTT1*1/1 DNA sample. DNA concentration decreases from left to right from 50 to 0.63 ng. (B) Standard curves plotting the log dilution (x) against Ct. The dashed line represents the standard curve obtained for b-globin. Regression parameters of the standard curves are displayed: b, slope; a, y intercept; r2, coefficient of determination.
lele deletion genotypes, (ii) nGSTM1 values between 0.43 and 0.62 for heterozygous genotypes, and (iii) nGSTM1 values less than 0.02 for null genotypes. The efficiency of the method to accurately discriminate among the three genotypes is endorsed by the fact that nGST 95% CI showed no overlapping. To test the real-time PCR assay developed using the nGST ranges estimated, we compared the genotype distribution obtained for FFPE tissue DNA samples (n = 63) with the obtained one for fresh (nondegraded) DNA (n = 177) as well as with genotypic frequencies reported in further Caucasian populations. Results of GSTT1 and GSTM1 copy number determination in both types of specimens (blood and FFPE tissues) are presented in Fig. 2. Regarding GSTT1 marker, the mean nGSTT1*1/1 value for DNA samples extracted from blood was 1.02 (95% CI = 0.84–1.20) with a standard deviation (SD) of 0.09. The mean nGSTT1*1/0 value was 0.55 (95% CI = 0.47–0.63) with an SD of 0.04. All samples with the null genotype showed an nGSTT1*0/0 value of 0 (Ct 40). Frequencies for the homozygous-present, heterozygous, and null genotypes were 0.299, 0.492, and 0.209, respectively. In DNA from FFPE tissue, the mean value of nGSTT1*1/1 was 1.01 with an SD of 0.10 (95% CI = 0.81–1.21). For GSTT1*1/0 genotypes, the mean nGST value was 0.48 with an SD of 0.08 (95% CI = 0.32–0.64), whereas samples with the GSTT1*0/0 genotype had an nGSTT1*0/0 value of 0. Genotypic frequencies were 0.388 (homozygous-present), 0.469 (heterozygous), and 0.143 (null allele). Results of a likelihood ratio test (G test) showed no statistically significant differences between these two samples (blood vs. FFPE tissue) as for the GSTT1 genotypic frequencies (G = 1.84, df = 2, P = 0.399).
Notes & Tips / Anal. Biochem. 378 (2008) 221–223
A
223
B
Fig. 2. Results of GSTT1 and GSTM1 genotyping assay in blood and FFPE tissue samples. Mean nGST values, standard deviations (in parentheses), and observed frequencies for the three different genotypes (homozygous-present, heterozygous, and null) are displayed for both GSTT1 (A) and GSTM1 (B) genes. Theoretical nGST values of 0.0, 0.5, and 1.0 were considered to have zero, one, and two copies of the target genes, respectively.
Concerning the GSTM1 gene, the mean nGSTM1*1/1 value estimated for blood DNA samples was 1.15 with an SD of 0.07 (95% CI = 1.01–1.29), whereas for GSTM1*1/0, the mean nGST value was 0.51 with an SD of 0.04 (95% CI = 0.43–0.59). Once again, all samples with the GSTM1*0/0 genotype exhibited an nGSTT1*0/0 value of 0. Genotypic frequencies were 0.107 (homozygous-present), 0.434 (heterozygous), and 0.46 (null allele). In the case of DNA extracted from FFPE tissues, the mean nGST value for homozygouspresent genotype was 1.07 with an SD of 0.11 (95% CI = 0.85– 1.29). For the heterozygous genotype, the mean nGSTM1*1/0 value was 0.49 with an SD of 0.05 (95% CI = 0.39–0.59), whereas samples with the null genotype showed nGSTM1*0/0 values of 0. Genotypic frequencies in this collection were 0.122 (GSTM1*1/1), 0.367 (GSTM1*1/0), and 0.531 (GSTM1*0/0). No significant difference was found between these two samples (blood vs. FFPE tissue) regarding GSTM1 genotype distribution (G = 0.43, df = 2, P = 0.806). In addition, no statistically significant differences were detected when we compared the genotyping results obtained for FFPE DNA samples with GST population data reported for other Caucasian collections by Covault and coworkers [6] (GSTT1, G = 2.20, df = 2, P = 0.33; GSTM1, G = 3.03, df = 2, P = 0.22) and by Girault and coworkers [8] (GSTT1, G = 2.11, df = 2, P = 0.34; GSTM1, G = 1.69, df = 2, P = 0.43). To further validate our results, endpoint PCR products from 10 subjects for each of the three GSTT1 and GSTM1 genotypes were analyzed using ethidium bromide-stained gels. Quantitative results achieved by real-time PCR fully coincided with those obtained through the methods taken as reference (standard and long-range PCR). In summary, the most notable findings of the current study are as follows. First, amplification efficiency and relative efficiency were always close to 100% for both the target and reference genes in both types of specimens (blood and FFPE tissue). Second, nGSTM1 and nGSTT1 values were clear-cut with small SDs, permitting unambiguous differentiation between homozygous and heterozygous genotypes. Third, no departure from Hardy–Weinberg equilibrium expectations were found for GSTT1 and GSTM1 loci. Fourth, no statistical differences were detected between the genotype distributions of the two collections examined. Fifth, genotyping results emerging from the analysis of FFPE DNA samples did not differ from GST population data reported for other Caucasian samples [6,8]. These results confirm applicability of the PCR assay developed here based on the comparative Ct method, which constitutes
a rapid, sensitive, and reliable procedure for complete genotyping of GSTT1 and GSTM1 genes in FFPE tissue. Acknowledgments This study was funded by research grants Saiotek SPE03UN20 and IT-424-07 from the Basque Government and GIU 05/51 from Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/ EHU). N.G.B. has a doctoral fellowship from the Basque Government. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2008.04.010. References [1] J. Brockmoller, I. Cascorbi, R. Kerb, I. Roots, Combined analysis of inherited polymorphisms in arylamine N -acetyltransferase 2, glutathione S-transferases M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk, Cancer Res. 56 (1996) 3915–3925. [2] R. Sprenger, R. Schlagenhaufer, R. Kerb, C. Bruhn, J. Brockmoller, I. Roots, U. Brinkmann, Characterization of the glutathione S-transferase GSTT1 deletion: Discrimination of all genotypes by polymerase chain reaction indicates a trimodular genotype–phenotype correlation, Pharmacogenetics 10 (2000) 557–565. [3] N. Roodi, W.D. Dupont, J.H. Moore, F.F. Parl, Association of homozygous wildtype glutathione S-transferase M1 genotype with increased breast cancer risk, Cancer Res. 64 (2004) 1233–1236. [4] F.A. Wiebel, A. Dommermuth, R. Thier, The hereditary transmission of the glutathione transferase hGSTT1-1 conjugator phenotype in a large family, Pharmacogenetics 9 (1999) 251–256. [5] U. Lehmann, H. Kreipe, Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies, Methods 25 (2001) 409–418. [6] J. Covault, C. Abreu, H. Kranzler, C. Oncken, Quantitative real-time PCR for gene dosage determinations in microdeletion genotypes, BioTechniques 35 (2003) 594–598. [7] C. Brasch-Andersen, L. Christiansen, Q. Tan, A. Haagerup, J. Vestbo, T.A. Kruse, Possible gene dosage effect of glutathione-S-transferases on atopic asthma: Using real-time PCR for quantification of GSTM1 and GSTT1 gene copy numbers, Hum. Mutat. 24 (2004) 208–214. [8] I. Girault, R. Lidereau, I. Bieche, Trimodal GSTT1 and GSTM1 genotyping assay by real-time PCR, Intl. J. Biol. Markers 20 (2005) 81–86. [9] L.E. Moore, W.Y. Huang, N. Chatterjee, M. Gunter, S. Chanock, M. Yeager, B. Welch, P. Pinsky, J. Weissfeld, R.B. Hayes, GSTM1, GSTT1, and GSTP1 polymorphisms and risk of advanced colorectal adenoma, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 1823–1827. [10] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2–DDCt method, Methods 25 (2001) 402–408.
Contents lists available at ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
GSTT1 and GSTM1 gene copy number analysis in paraffin-embedded tissue using quantitative real-time PCR Naiara G. Bediaga a, Miguel A. Alfonso-Sánchez a, Mertxe de Renobales b, Ana M. Rocandio c, Marta Arroyo c, Marian M. de Pancorbo a,* a
Servicio de Genómica, Banco de ADN, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria, Spain Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria, Spain c Departamento de Nutrición y Bromatología, Facultad de Farmacia, Universidad del País Vasco, 01006 Vitoria, Spain b
a r t i c l e
i n f o
Article history: Received 4 March 2008 Available online 9 April 2008
a b s t r a c t GSTT1 and GSTM1 genes possess an inherited deletion associated with a lack of enzyme activity. The heterozygous condition of this deletion is difficult to determine in low-quality DNA with existing PCR protocols. We designed and validated a multiplex real-time PCR assay by adapting the DDCt relative quantification method for the analysis of GSTT1 and GSTM1 markers to accurately differentiate the three genotypes (*1/1, *1/0, and *0/0) in degraded DNA from formalin-fixed paraffin-embedded tissue. Gene copy number values obtained provide for unambiguous homozygous and heterozygous differentiation. The efficacy shown by the PCR assay endorses its usefulness for complete genotyping of glutathione Stransferases in archival tissues. Ó 2008 Elsevier Inc. All rights reserved.
Formalin-fixed paraffin-embedded (FFPE)1 tissue samples represent the largest source of archival biological material for epidemiological studies based on molecular genetic analysis. Therefore, there is a growing need to implement PCR methods allowing an accurate genotyping of DNA samples extracted from fixed tissues. Genes belonging to the glutathione S-transferase (GST) superfamily are commonly explored as susceptibility and tumor markers in molecular epidemiological surveys. GSTT1 and GSTM1 genes are known to be polymorphic, with an inherited deletion (GSTT1*0 and GSTM1*0 null alleles, respectively) associated with a lack of enzyme activity that is present in approximately 20% (GSTT1) and 50% (GSTM1) of Caucasians [1]. Prevalence of the heterozygous condition (deletion on a single chromosome) for these loci has never been reported in FFPE tissue, probably because the nonquantitative endpoint PCR assays used to genotype these deletions [2,3], which require amplicon sizes greater than 13 kb, cannot be applied efficiently on degraded DNA. Earlier GSTT1 analyses based on semiquantitative PCR assays unveiled a gene dose effect on GSTT1 activity [4], suggesting that accurate differentiation between homozygous (high enzymatic activity) and heterozygous (medium enzymatic activity) could be important to correlate clinical phenotypes with GST genotypes. Real-time PCR is a recommendable option for gene copy number analysis in DNA from FFPE tissues [5]. Real-time PCR assays
* Corresponding author. E-mail address: [email protected] (M.M. de Pancorbo). 1 Abbreviations used: FFPE, formalin-fixed paraffin-embedded; GST, glutathione S-transferase; Ct, cycle threshold; CI, confidence interval; SD, standard deviation. 0003-2697/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2008.04.010
have been recently devised for complete genotyping of GSTT1 and GSTM1 loci [6–9], but only Covault and coworkers [6] and Brasch-Andersen and coworkers [7] have employed the DDCt method of relative quantification, which is easier to implement than the absolute method because standard curves are unneeded [10]. Nevertheless, validity of this method demands PCR amplification efficiencies for both target and reference genes close to 100%, which is complicated when working with low-quality starting DNA. Indeed, neither of the cited PCR assays has been employed so far for GSTM1 and GSTT1 gene dose determination in FFPE tissue despite their obvious theoretical advantages, probably due to low reproducibility in archival tissues. Therefore, we designed and tested a new real-time PCR assay based on the DDCt method to overcome potential complications arising from the analysis of FFPE tissue with the existing protocols. Quantitative real-time PCR was performed using a TaqMan (50 nuclease) assay system with signal from a GSTT1- or GSTM1-specific probe that is normalized to the signal for a reference gene (b-globin). A total of 240 DNA samples from archival (FFPE) and fresh blood tissues were screened. FFPE samples (n = 63) belonged to surgical specimens of primary liver cancers from patients diagnosed with hepatocellular carcinoma between 1996 and 1999. The control group (blood samples) consisted of 177 unrelated donors without manifest neoplasms. All samples belong to the DNA Bank at the University of the Basque Country (Spain). The study protocol was approved by the relevant ethics committees of the institutions involved. Primer and probe sequences for the genotyping of GSTT1 (NM_000853), GSTM1 (NM_000561), and b-globin (U_01317)
222
Notes & Tips / Anal. Biochem. 378 (2008) 221–223
genes were designed to reach maximum efficiency of the amplification process and, thereby, of the PCR assay. Because GSTT1 and GSTM1 markers are members of a multigene family, primer pairs and probe were selected to be unique when compared with the sequences of closely related genes. To that end, Primer Express 2.0 software (Applied Biosystems, Foster City, CA, USA) was used. To assay GSTT1 gene copy number relative to reference gene bglobin, triplicate PCRs (15 ll) using 15 ng of genomic DNA, 0.9 lM of each GSTT1 primer (forward, 50 -AAGTCCCAGAGCACCTCACCTCC30 ; reverse, 50 -CAGTGTGCATCATTCTCATTGTGGC-30 ), 0.4 lM of bglobin primers (forward, 50 -GAGGGTTTGAAGTCCAACTCCTAA-30 ; reverse, 50 -CAGGGTGAGGTCTAAGTGATGACA-30 ), and fluorescent product-specific oligonucleotide probes (GSTT1, 6FAM-CACCATC CCCACCCTGTCTTCCA-TAMRA, 0.2 lM; b-globin, VIC-CAGTGCCAG AAGAGCCAAGGACAGGT-TAMRA, 0.1 lM) were prepared in 1 Master Mix (Applied Biosystems). Thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min. PCR was performed in a 96well optical plate (ABgene) using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems). To test GSTM1 gene dose relative to b-globin, we used 1.2 lM of GSTM1 primers (forward, 50 -CACCTGCATTCGTTCATGTGAC-30 ; reverse, 50 -CACCTGCATTCGT TCATGTGAC-30 ), 0.4 lM of b-globin primers and fluorescent product-specific probes (GSTM1, 6FAM-TTCAGTCCTGCCATGAGCAGGCA CA-TAMRA, 0.15 lM; b-globin, VIC-CAGTGCCAGAAGAGCCAAGGA CAGGT-TAMRA, 0.15 lM) using thermal cycling conditions identical to those for GSTT1 primers. Data analysis was conducted by adapting the DDCt method of relative quantification [10] to estimate copy numbers of GSTT1 and GSTM1 genes. Ct (cycle threshold) is defined as the point at which the fluorescence level rises above a baseline. Two known control samples (carrying both alleles) were analyzed on each reaction tray as calibrators. In addition, a number of no-template controls were also included in the analysis. The samples employed as calibrators were previously analyzed by standard and long-range PCR to confirm possession of two copies of the genes examined, whereas b-globin was the reference gene in all experiments. The result for each target gene, expressed as n-fold copy number of the target gene relative to b-globin (nGST), was obtained through the equation 2–DDCt, where DDCt = (Ct GSTsample – Ct GLOBINsample) – (Ct GSTcalibrator – Ct GLOBINcalibrator). As argued above, for the DDCt method to be valid, two essential conditions must be satisfied: (i) all PCR amplifications must be close to 100% and (ii) the relative efficiency must be optimal (i.e., the amplification efficiency of the target and reference genes must be approximately equal). Real-time efficiencies (E) were calculated by means of standard calibration curves (Fig. 1), using threefold serial dilutions of blood and FFPE tissue DNA with known GSTT1*1/1 and GSTM1*1/1 genotypes, through the equation E = 10(–1/slope) – 1. The observed real-time PCR efficiency (E) was always between 92 and 100% for all target genes in all tissues considered. To check whether the amplification efficiencies of the target and reference genes were similar, we verified that the slope of DCt versus input DNA was less than 0.1 over DNA dilution series. To estimate the range of nGST values for a reliable determination of the gene copy number, 40 fresh tissue samples with known genotype, analyzed by methods mentioned above [2,3], were used to determine confidence intervals (CIs) of 95% (P < 0.05) for each genotype. Samples that did not fit with their respective nGST ranges were reamplified per triplicate. For GSTT1, three groups of samples were clearly distinguishable: (i) nGSTT1 values greater than 0.84 were characteristics of samples that showed no deletion (*1/1), (ii) nGSTT1 values between 0.47 and 0.62 appeared in cases with one-copy deletion genotype (*0/1), and (iii) nGSTT1 values less than 0.01 were typical of null genotypes (*0/0). Very similar ranges were estimated for GSTM1: (i) nGSTM1 values greater than 0.84 for no al-
Fig. 1. TaqMan GSTT1 and b-globin standard curves obtained by real-time PCR applied on DNA from FFPE tissue. (A) Amplification plots for five serial dilutions of a GSTT1*1/1 DNA sample. DNA concentration decreases from left to right from 50 to 0.63 ng. (B) Standard curves plotting the log dilution (x) against Ct. The dashed line represents the standard curve obtained for b-globin. Regression parameters of the standard curves are displayed: b, slope; a, y intercept; r2, coefficient of determination.
lele deletion genotypes, (ii) nGSTM1 values between 0.43 and 0.62 for heterozygous genotypes, and (iii) nGSTM1 values less than 0.02 for null genotypes. The efficiency of the method to accurately discriminate among the three genotypes is endorsed by the fact that nGST 95% CI showed no overlapping. To test the real-time PCR assay developed using the nGST ranges estimated, we compared the genotype distribution obtained for FFPE tissue DNA samples (n = 63) with the obtained one for fresh (nondegraded) DNA (n = 177) as well as with genotypic frequencies reported in further Caucasian populations. Results of GSTT1 and GSTM1 copy number determination in both types of specimens (blood and FFPE tissues) are presented in Fig. 2. Regarding GSTT1 marker, the mean nGSTT1*1/1 value for DNA samples extracted from blood was 1.02 (95% CI = 0.84–1.20) with a standard deviation (SD) of 0.09. The mean nGSTT1*1/0 value was 0.55 (95% CI = 0.47–0.63) with an SD of 0.04. All samples with the null genotype showed an nGSTT1*0/0 value of 0 (Ct 40). Frequencies for the homozygous-present, heterozygous, and null genotypes were 0.299, 0.492, and 0.209, respectively. In DNA from FFPE tissue, the mean value of nGSTT1*1/1 was 1.01 with an SD of 0.10 (95% CI = 0.81–1.21). For GSTT1*1/0 genotypes, the mean nGST value was 0.48 with an SD of 0.08 (95% CI = 0.32–0.64), whereas samples with the GSTT1*0/0 genotype had an nGSTT1*0/0 value of 0. Genotypic frequencies were 0.388 (homozygous-present), 0.469 (heterozygous), and 0.143 (null allele). Results of a likelihood ratio test (G test) showed no statistically significant differences between these two samples (blood vs. FFPE tissue) as for the GSTT1 genotypic frequencies (G = 1.84, df = 2, P = 0.399).
Notes & Tips / Anal. Biochem. 378 (2008) 221–223
A
223
B
Fig. 2. Results of GSTT1 and GSTM1 genotyping assay in blood and FFPE tissue samples. Mean nGST values, standard deviations (in parentheses), and observed frequencies for the three different genotypes (homozygous-present, heterozygous, and null) are displayed for both GSTT1 (A) and GSTM1 (B) genes. Theoretical nGST values of 0.0, 0.5, and 1.0 were considered to have zero, one, and two copies of the target genes, respectively.
Concerning the GSTM1 gene, the mean nGSTM1*1/1 value estimated for blood DNA samples was 1.15 with an SD of 0.07 (95% CI = 1.01–1.29), whereas for GSTM1*1/0, the mean nGST value was 0.51 with an SD of 0.04 (95% CI = 0.43–0.59). Once again, all samples with the GSTM1*0/0 genotype exhibited an nGSTT1*0/0 value of 0. Genotypic frequencies were 0.107 (homozygous-present), 0.434 (heterozygous), and 0.46 (null allele). In the case of DNA extracted from FFPE tissues, the mean nGST value for homozygouspresent genotype was 1.07 with an SD of 0.11 (95% CI = 0.85– 1.29). For the heterozygous genotype, the mean nGSTM1*1/0 value was 0.49 with an SD of 0.05 (95% CI = 0.39–0.59), whereas samples with the null genotype showed nGSTM1*0/0 values of 0. Genotypic frequencies in this collection were 0.122 (GSTM1*1/1), 0.367 (GSTM1*1/0), and 0.531 (GSTM1*0/0). No significant difference was found between these two samples (blood vs. FFPE tissue) regarding GSTM1 genotype distribution (G = 0.43, df = 2, P = 0.806). In addition, no statistically significant differences were detected when we compared the genotyping results obtained for FFPE DNA samples with GST population data reported for other Caucasian collections by Covault and coworkers [6] (GSTT1, G = 2.20, df = 2, P = 0.33; GSTM1, G = 3.03, df = 2, P = 0.22) and by Girault and coworkers [8] (GSTT1, G = 2.11, df = 2, P = 0.34; GSTM1, G = 1.69, df = 2, P = 0.43). To further validate our results, endpoint PCR products from 10 subjects for each of the three GSTT1 and GSTM1 genotypes were analyzed using ethidium bromide-stained gels. Quantitative results achieved by real-time PCR fully coincided with those obtained through the methods taken as reference (standard and long-range PCR). In summary, the most notable findings of the current study are as follows. First, amplification efficiency and relative efficiency were always close to 100% for both the target and reference genes in both types of specimens (blood and FFPE tissue). Second, nGSTM1 and nGSTT1 values were clear-cut with small SDs, permitting unambiguous differentiation between homozygous and heterozygous genotypes. Third, no departure from Hardy–Weinberg equilibrium expectations were found for GSTT1 and GSTM1 loci. Fourth, no statistical differences were detected between the genotype distributions of the two collections examined. Fifth, genotyping results emerging from the analysis of FFPE DNA samples did not differ from GST population data reported for other Caucasian samples [6,8]. These results confirm applicability of the PCR assay developed here based on the comparative Ct method, which constitutes
a rapid, sensitive, and reliable procedure for complete genotyping of GSTT1 and GSTM1 genes in FFPE tissue. Acknowledgments This study was funded by research grants Saiotek SPE03UN20 and IT-424-07 from the Basque Government and GIU 05/51 from Universidad del País Vasco/Euskal Herriko Unibertsitatea (UPV/ EHU). N.G.B. has a doctoral fellowship from the Basque Government. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2008.04.010. References [1] J. Brockmoller, I. Cascorbi, R. Kerb, I. Roots, Combined analysis of inherited polymorphisms in arylamine N -acetyltransferase 2, glutathione S-transferases M1 and T1, microsomal epoxide hydrolase, and cytochrome P450 enzymes as modulators of bladder cancer risk, Cancer Res. 56 (1996) 3915–3925. [2] R. Sprenger, R. Schlagenhaufer, R. Kerb, C. Bruhn, J. Brockmoller, I. Roots, U. Brinkmann, Characterization of the glutathione S-transferase GSTT1 deletion: Discrimination of all genotypes by polymerase chain reaction indicates a trimodular genotype–phenotype correlation, Pharmacogenetics 10 (2000) 557–565. [3] N. Roodi, W.D. Dupont, J.H. Moore, F.F. Parl, Association of homozygous wildtype glutathione S-transferase M1 genotype with increased breast cancer risk, Cancer Res. 64 (2004) 1233–1236. [4] F.A. Wiebel, A. Dommermuth, R. Thier, The hereditary transmission of the glutathione transferase hGSTT1-1 conjugator phenotype in a large family, Pharmacogenetics 9 (1999) 251–256. [5] U. Lehmann, H. Kreipe, Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies, Methods 25 (2001) 409–418. [6] J. Covault, C. Abreu, H. Kranzler, C. Oncken, Quantitative real-time PCR for gene dosage determinations in microdeletion genotypes, BioTechniques 35 (2003) 594–598. [7] C. Brasch-Andersen, L. Christiansen, Q. Tan, A. Haagerup, J. Vestbo, T.A. Kruse, Possible gene dosage effect of glutathione-S-transferases on atopic asthma: Using real-time PCR for quantification of GSTM1 and GSTT1 gene copy numbers, Hum. Mutat. 24 (2004) 208–214. [8] I. Girault, R. Lidereau, I. Bieche, Trimodal GSTT1 and GSTM1 genotyping assay by real-time PCR, Intl. J. Biol. Markers 20 (2005) 81–86. [9] L.E. Moore, W.Y. Huang, N. Chatterjee, M. Gunter, S. Chanock, M. Yeager, B. Welch, P. Pinsky, J. Weissfeld, R.B. Hayes, GSTM1, GSTT1, and GSTP1 polymorphisms and risk of advanced colorectal adenoma, Cancer Epidemiol. Biomarkers Prev. 14 (2005) 1823–1827. [10] K.J. Livak, T.D. Schmittgen, Analysis of relative gene expression data using realtime quantitative PCR and the 2–DDCt method, Methods 25 (2001) 402–408.