A multiplex real-time PCR method for detection of GSTM1 and GSTT1 copy numbers

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Clinical Biochemistry 42 (2009) 500 – 509

A multiplex real-time PCR method for detection of GSTM1 and GSTT1 copy numbers Maria Timofeeva a , Birgit Jäger a , Albert Rosenberger b , Wiebke Sauter c , Heinz-Erich Wichmann c,d KORA Study Group c , Heike Bickeböller b , Angela Risch a,⁎ a

German Cancer Research Center (DKFZ), Division of Epigenomics and Cancer Risk Factors (C010), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b Department of Genetic Epidemiology, Georg-August University of Göttingen, Medical School, Germany c Institute of Epidemiology, Helmholtz Centre Munich, Germany d LMU Munich, Germany Received 27 August 2008; received in revised form 20 November 2008; accepted 9 December 2008 Available online 30 December 2008

Abstract Objectives: Deletion polymorphisms of Glutathione-S-transferase (GST) M1 and T1 are considered risk factors for various diseases. However, most previous studies only distinguished “null” and “non-null” genotypes. Our aim was to develop a reliable, high-throughput GSTM1/T1 genotyping method able to determine allele copy numbers. Design and methods: We developed a multiplex real time PCR method to distinguish between heterozygous (1/0) and homozygous (1/1) GSTM1 and GSTT1 genotypes. The principle of relative quantification was applied and an expectation–maximisation (EM) algorithm was developed to assign one of 3 possible genotypes: 1/1, 1/0 or 0/0 for each of the two genes. Results: 1320 Caucasians were genotyped using the newly developed method. The observed genotype distributions did not deviate from the expected and were in Hardy–Weinberg equilibrium. GSTM1 duplication was detected in one sample. Conclusion: This new semiquantitative genotyping method is a sensitive and promising tool for large-scale molecular epidemiological and clinical studies. © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. Keywords: Glutathione-S-transferase M1; Glutathione-S-transferase T1; Real time PCR quantification; Copy number polymorphism

Introduction The glutathione-S-transferase θ 1 (GSTT1) and glutathioneS-transferase μ 1 (GSTM1) belong to the superfamily of multifunctional enzymes, which catalyze conjugation of glutathione to electrophilic compounds, including products of oxidative stress, carcinogens and some chemotherapeutic drugs [1,2]. Deletion polymorphisms of GSTM1 and GSTT1 have previously been shown to be present with different frequencies in various populations [3]. Thus, approximately 50% and 20% of Caucasians lack the enzyme activity due to the GSTM1 and GSTT1 deletions, respectively [4–6].

⁎ Corresponding author. Fax: +49 6221 42 3359. E-mail address: [email protected] (A. Risch).

Due to the importance of glutathione conjugation for Phase II of carcinogen detoxification and drug metabolism and due to the high prevalence of GSTT1 and GSTM1 deletions in the human population, deletion polymorphisms of these genes have been considered as potential risk and prognostic factors for multifactorial diseases such as different types of cancer [7–11], asthma and chronic obstructive pulmonary disease [12,13] or various cardio-vascular diseases [12,14,15]. The majority of the molecular epidemiological and clinical studies has been based on the comparison of “null genotype” or “non-conjugator” with “non-null genotype” or “conjugator”. However, previously a trimodal phenotype distribution has been shown for GSTM and GSTT1 enzyme activity in the human population [4,16,17], suggesting the existence of a gene– dosage effect. Indeed, correlation between the enzyme activity and the number of functional alleles was detected for both genes

0009-9120/$ - see front matter © 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.clinbiochem.2008.12.011

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Fig. 1. Structure of the GSTM1 region and location of primers. The GSTM1 gene, which is approximately 6 kb, is located at 1p13.3 and consists of 8 exons (numbered boxes) and 7 introns. PCR primers (→ and ←), used in various methods to characterize GSTM1 deletion polymorphisms: R — primers for detection of the “null allele", described and used by Roodi et al. (2004) [22]. R primers yield a single product of 14-kb in the presence of at least one “null allele". K — primers for detection of the “null allele", described and used by Kerb et al. (1999) [21]. K primers yield a single product of 13-kb in the presence of at least one “null allele". LC — primers, used in the current method on the Light Cycler 480. In the probe area of GSTM1 (black box) a SNP rs737497 is located. G — primers, used by Girault et al. (2005) for GSTM1 copy number quantification [23]. BA — primers, used by Brasch-Andersen et al. (2004), for GSTM1 copy number quantification [13]. M1 — primers to distinguish between “null" and “non-null" GSTM1 genotypes in a multiplex reaction [33,34]. GSTM2 — glutathione S-transferase mu 2; GSTM5 — glutathione S-transferase mu 5.

[16,18]. It is expected that the effect of these two deletions on the various clinical outcomes might also best be described in a dosage specific manner.

Several methods for distinguishing between GSTM1 and GSTT1 homo- and heterozygous gene carriers have been described [16,19–22] (Fig. 1 and Fig. 2). These methods are

Fig. 2. Structure of the GSTT1 region and location of primers. The GSTT1 gene, which is approximately 8 kb, is located at 22q11.23 and consists of 5 exons (numbered boxes) and 4 introns. HA5 and HA3 are two ∼ 18 kb highly homologous regions upstream and downstream of GSTT1. It is supposed that deletion of GSTT1 is a result of homologous recombination between HA5 and HA3 regions [16]. PCR primers (→ and ←), used in various methods to characterize GSTT1 deletion polymorphisms: S0 and S1 — primers for detection “null" and “non-null alleles", respectively, described by Sprenger et al. (2000). In case of deletion the S0 primers yield a product of 1450 bp and no product in the presence of at least one “non-null allele". The S1 primers yield a product of 466 bp in the presence of at least one “nonnull allele". LC — primers, used by Girault et al. (2005) and in the current method on the LightCycler 480 [23]. BA — primers, used by Brasch-Andersen et al. (2004), for GSTT1 copy number quantification [13]. T1 — primers to distinguish between “null" and “non-null" GSTT1 genotypes in a multiplex reaction [33,34]. GSTT2 — glutathione S-transferase theta 2; GSTTP1 — glutathione S-transferase theta pseudogene 1, GSTTP2 — glutathione S-transferase theta pseudogene 2.

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based on PCR followed by gel-electrophoresis for product separation and detection. For genotyping GSTM1, only long range PCR based methods are available, where sizes of amplification products range from ∼ 5000 bp to 14000 bp [19,21,22] (Fig. 1). These methods are often technically difficult and time consuming, making them unsuitable for large scale epidemiological and clinical studies. Brasch-Andersen (2004) [13] had developed two separate assays for relative quantification of gene copy number of GSTM1 and GSTT1, which were used to genotype approximately 1000 individuals in 246 families. More recently, two new semiquantitative real time PCR methods for trimodal GSTT1 and GSTM1 genotyping were described by Girault et al. (2005) [23], who genotyped 29 individuals. We modified these methods by multiplexing them into one reaction, which significantly decreases the time of genotyping, and we introduced a more reliable and more precise PCR efficiency (E) correction approach for interpretation of the results. We developed and applied an expectation–maximisation (EM) algorithm to assign one of three possible genotypes: 1/1, 1/0 or 0/0 for each of two genes. The newly developed technique was applied to genotyping 1320 Caucasians from a population based cohort study. Material and methods Samples DNA was extracted using a commercially available kit (Gentra, Minneapolis MN) according to the manufacturer's protocol from EDTA anticoagulated blood of 1320 Caucasian individuals from the population-based KORA study (Cooperative Health Research in the Augsburg Region) [24,25]. Additionally, for method development, DNA of blood samples from Caucasian volunteers from Heidelberg was isolated using the QIAamp DNA blood midi kit according to the manufacturer's instructions (Quiagen GmbH, Hilden, Germany). As a larger amount of DNA was available for these samples than for the KORA samples, they were later used as positive controls and/or a calibrator. Informed consent was obtained from all study participants and the study was approved by the ethics committee of the Bayerische Landesärztekammer, the corresponding local ethics committees of the participating clinics and the ethics committee of the University of Heidelberg (Ref. Nr. 182/96 and 201/98). Principle of relative quantification with efficiency correction for distinguishing GSTT1 and GSTM1 hetero- and homozygous genotype carriers The principle of the gene quantification based on a real-time PCR is described elsewhere [23,26–29]. To calculate the initial amount of the genomic DNA (X0) a threshold cycle parameter (Ct) is calculated during the real time PCR. Ct is considered to be the most reliable measurement proportional to the initial concentration of the genomic DNA template in the reaction mix. To normalize the input amount of genomic DNA in every reaction, a reference gene R (ALB, location 4q11–q13), which is

characterized by a constant copy number [30], is amplified together with both target genes of interest T (GSTM1 and GSTT1) in the same reaction vessel. In order to obtain a normalized measure for the amount of the amplified DNA at cycle Ct (Xn), the amplification of target gene (T) is related to the reference gene (R): XTn XT 0
ð1 + ET ÞCTt ð1 + ET ÞCTt = = X
NR XRn XR0
ð1 + ER ÞCRt ð1 + ER ÞCRt

ð1Þ

The final step is the so called calibration, where XNR, of a sample of interest (S) is related to the XNR of a sample with known copy number of GSTM1 and GSTT1, the so-called calibrator-sample (C). The calibrated normalized ratio (NR) is given by: NR =

XNRðS Þ = ð1 + ER ÞCRtðSÞ CRtðCÞ
ð1 + ET ÞCTtðCÞ CTtðSÞ XNRðC Þ

ð2Þ

In the current study a sample homozygous for GSTM1 and GSTT1 wild type alleles was applied as a calibrator. Therefore, samples with a calibrated normalized ratio NR of 1 are considered as homozygous for GSTM1 1/1 or GSTT1 1/1, respectively. Individuals with NR values of about 0.5 are tentatively considered as heterozygotes (1/0) and those with value about 0 as tentative homozygotes (0/0). All calculations of Ct values and normalized ratios NR were carried out using the LightCycler 480 (Roche, Mannheim, Germany) software for relative quantification. Semiquantitative GSTM1/T1 genotyping Genotyping of GSTM1 and GSTT1 was performed on the LightCycler 480 in a multiplex reaction with the reference gene albumin. Primers and probes were designed with the help of TIB MOLBIOL Syntheselabor GmbH (Berlin, Germany) (Table 1). For this new GST genotyping method a hydrolysis probe assay was chosen as a real-time PCR detection format with the following reporter–quencher pairs: FAM-Dabcyl for GSTT1, Cy5-BBQ for GSTM1, and LCRed 610 - BBQ for ALB (TIB MOLBIOL) (Table 1). The principle of hydrolysis probe chemistry for real-time detection of PCR products is explained elsewhere [27,31,32]. In the probe region of GSTM1 there is a polymorphism A/G rs737497, with a frequency of 0.65 for the A allele in the Caucasian population (Fig. 1). Thus, for the GSTM1 gene two probes for A and G alleles respectively were designed to minimize the effect of mismatching due to the polymorphism (Table 1). For GSTM1 genotyping a “wobble” probe was applied, which is, in fact, a 1:1 mixture of the GSTM1 probes for the A and G alleles. The 10 μL PCR mix for GSTM1 and GSTT1 semiquantitative genotyping contained 1xLightCycler 480 Probe Master (Roche, Mannheim, Germany), 10–100 ng DNA template, 0.5 μmol/L each of the primers and 0.15 μmol/L of each probe, including the GSTM1 “wobble” probe. The amplification of DNA was carried out in the LightCycler 480 (Roche, Mannheim, Germany) under the following conditions: initial denaturation of DNA for 5 min at 95 °C ,

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Table 1 Primers and probes used for PCR Gene

Sequence

GSTT1 for LC480

5′ - CAA GTC CCA GAG CAC CTC ACC TC - 3′ 5′ - GTG TGC ATC ATT CTC ATT GTG GCT T -3′ Probe: 6FAM-CAT CCC CAC CCT GTC TTC CAC AGC C-DB a 5′ - CAT CCC CTT CCC ATA AGC AAG - 3′ 5′ - TGC ATT CGT TCA TGT GAC AGT ATT CT - 3′ Probe_A: Cy5 - CAG AGA GGA GAC CGG GCA CTC AC-BBQ b Probe_G: Cy5 - CAG GGA GGA GAC CGG GCA CTC AC-BBQ b 5′ - CTG GAA GTC GAT GAA ACA TAC GTT - 3′ 5′ - CTC TCC TTC TCA GAA AGT GTG CAT A - 3′ Probe:- LC Red610-TGC TGA AAC ATT CAC CTT CCA TGC AGA-BBQ c 5′ - AAG AAG TAC ACG ATG GGG G - 3′ 5′ - GGG AAG GGT AAT GAT GGG AG 3′ 5′ - GCC CTG GCT AGT TGC TGA AG - 3′ 5′ - GCA TCT GAT TTG GGG ACC ACA -3′ 5′ - CGC CAT CTT GTG CTA CAT TGC CCG- 3′ 5′ -TTC TGG ATT GTA GCA GAT CA- 3′ 5′ -CAA CTT CAT CCA CGT TCA CC- 3′ 5′ - GAA GAG CCA AGG ACA GGT AC - 3′

GSTM1 for LC480

Albumin for LC 480

GSTM cDNA GSTT1 GSTM1 Β-globin

Length of the amplification products 89 bp

100 bp

92 bp

814 bp 111 bp 231 bp 268 bp

Hydrolysis probe labelled with the reporter dye FAM (absorption — 495 nm, emission — 520 nm) and quencher dye DABCYL (quenching range 380–650 nm). Hydrolysis probe labelled with the reporter dye Cy5 (absorption — 643 nm, emission — 667 nm) and quencher dye BBQ (quenching range 550–650 nm). Due to the SNP rs737497 in the probe region, which influences interpretation of genotyping results, two probes for A and G alleles were designed. For genotyping a 1:1 mix of these probes was applied. c Hydrolysis probe labelled with the reporter dye LCRed 610 (absorption — 590 nm, emission — 610 nm) and quencher dye BBQ (quenching range 550–650 nm). a

b

followed by 35–38 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 20 s and elongation at 72 °C for 2 s. After the cycles a final cooling step was performed at 40 °C for 30 s. The fluorescent signal was measured for every sample in every cycle during the elongation step. PCR reactions were performed on 96 well plates on LightCycler 480. On each plate the calibrator was amplified in triplicate. All samples were genotyped in triplicate. To adjust for differences in PCR efficiencies between target and reference genes, standard curves for all genes of interest and for albumin were generated using serial dilutions of a single sample (undiluted — approx. 200 ng/μL, 1:10, 1:100, 1:1000). The normalized ratio was calculated using the LightCycler 480 software for relative quantification. Statistical analysis of results The average normalized ratio (NR) and the standard deviation (SD) for every sample was calculated. Mean NRs, and SD for all GSTM1 and GSTT1 genotypes were calculated. As normal deviation of NR values is possible, there is a need to assign each genotype employing a statistically sound approach. Therefore, after calculation of the mean NR from the triplicate analysis an EM algorithm was applied to decompose the mixed distribution into the three latent normal distributions. This approach is based on the assumption that the mixed distribution of NRs can be decomposed into three normal distributions Ni (mi, si²), i = 0 to 2, each corresponding to one of the three genotypes, where mi is the expected value and si is variance. The expected values mi for the genotypes 0/0, 0/1 and 1/1 have to be close to 0, 0.5 and 1, respectively. The variances si2 of the normal distributions do not necessarily have to be equal. Given mi and si the density dNRj ;i for an

observed value NRj can be calculated. This value is weighted ni  1 , which reflects the by an a-priori probability pj;i = n1 genotype frequencies ni in the sample of size n apart from observation j. The probability for NRj to belong to genotype i (pNRj ;i ), is finally estimated by the posterior probability d

NR ;i d pNRj ;i = P j

pj;i

dNRj ;l d pj;l

. The critical values of genotype allocation

l = 0;1;2

are such values of NR, where these posterior probabilities for two genotypes are equal: NR01 : pNR01 ;0 = pNR01 ;1 and NR12 : pNR12 ;1 = pNR12 ;2 . Hence NR0–1 and NR1–2 are called borders of allocation. We also applied a bootstrap-method with 2000 replications to estimate the “area of uncertain genotype allocation” by the 95%-confidence intervals for the allocation borders NR0–1 and NR1–2. Observations with the probability of genotype allocation (pNRj ;i ) less than 0.99 were considered as samples with uncertain genotype and genotyped again in triplicate or with the help of additional validation methods. By this criterion more samples of uncertain genotype were chosen for precautionary replication, than by choosing those within “the areas of uncertain genotype allocation”. All calculations were done using the Statistical Analysis Software (SAS) 9.1. The Hardy–Weinberg equilibrium was tested using χ2 test of goodness of fit with 1 degree of freedom, checking for GSTM1 and GSTT1 genotype distribution. Validation methods Genotyping methods In order to genotype the calibrator and to validate the new method, previously published methods to distinguish between hetero- and homozygous carriers of GSTM1 and GSTT1 were applied [16,22].

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Standard genotyping to identify GSTT1 and GSTM1 deletions was carried out using a modified multiplex PCRbased method [33,34]. The 15 μL PCR mix contained 1xPCR buffer (containing 15 mmol/L Mg2+), 200 μmol/L dNTPs, 1.0 U HotStar Taq polymerase, ∼ 10 ng DNA template, 0.6 μmol/L each of the GSTT1 and GSTM1 primers and 0.4 μmol/L each of the β-globin primers (Table 1). The cycling conditions were as follows: initial denaturation 95 °C for 15 min, followed by 35 cycles of denaturation at 94 °C, annealing at 58 °C for 30 s and elongation at 72 °C for 60 s. This and similar multiplex methods are commonly used in epidemiological studies and is able to distinguish between two deleted alleles (“null genotype”) and carriers of at least one normal allele (“non-null genotype”) of both GSTT1 and GSTM1. Additionally, a long-range PCR based method was applied for detection of the GSTM1 “null allele" as described elsewhere [22]. A 14-kb PCR product was observed if a “null allele" was present. This method was applied in combination with the standard multiplex methods for GSTT1 and GSTM1 deletion genotyping to distinguish between heterozygous and homozygous GSTM1 carriers. Additionally a genotyping method was carried out to distinguish between individuals homozygous and heterozygous for GSTT1 as described by Sprenger et al. (2000) [16]. Summary diagrams of the location of primers used in various GSTM1 and GSTT1 genotyping methods are presented in Fig. 1 for GSTM1 and Fig. 2 for GSTT1. Southern blotting To check GSTM1 genotype and possible duplication of the calibrator, Southern blotting as described by McLellan et al. (1997) was applied [18]. To prepare a GSTM1 cDNA probe of 814 bp for hybridisation, total cellular RNA was isolated from the whole blood using PAXgene Blood RNA Kit according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany for PreAnalytix GmbH, Hombrechtikon, Switzerland). The one-step reverse transcription–PCR was performed using the QIAGEN OneStep RT–PCR Kit (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instruction. Primers, designed with the help of TIB MOLBIOL Syntheselabor GmbH (Berlin, Germany) are presented in Table 1. To prepare the probe for Southern blotting the PCR product of 814 bp was purified by isolation from an agarose gel and labelled with digoxigenin (DIG) (Roche, Mannheim, Germany) by a random primer labelling according to the manufacturer's instructions. Approximately 5 μg of DNA was digested with 2 U EcoRI to distinquish between GSTM1 1/1, 1/0 and 0/0 genotypes (Fermentas GmbH, St.Leon-Rot, Germany) or 2 U EcoRV to detect GSTM1 duplication (New England Biolabs, Hertfordshire, UK), at 37 °C for 3 h. After separation of 2–5 μg of DNA on a 0.9% agarose pulse-electrophoresis gel for a period of 14 h the DNA was transferred on to a nitrocellulose membrane (Qiagen, GmbH, Hilden, Germany) and hybridizied at 65 °C with the DIG labelled GSTM1 probe. For visualisation, the membrane was washed for 30 min with the 75 mU/ml AntiDigoxigenin-AP antibody (Boehringer Mannheim, Germany)

and subjected to chromogenic alkaline phosphatase substrate (18.75 mg/ml nitro blue tetrazolium chloride/9.4 mg/ml 5bromo-4-chloro-3-indolyl-phosphate). Results Evaluation of real-time PCR efficiency The efficiency of the PCR characterises the kinetics during the reaction. The PCR efficiency depends on many factors, which can vary from reaction to reaction (e.g. concentration of reagents in the reaction mix, quality of DNA). To compensate for differences in PCR efficiency of target and reference gene amplification, the efficiencies of GSTT1, GSTM1 and ALB amplification were determined from the corresponding standard curves. The reaction efficiencies for GSTT1, GSTM1 and ALB were 1.891, 1.889 and 1.801, respectively. We assumed that there was no or little between sample variations in the PCR efficiency. No significant influence of the GSTM1 rs737497 polymorphism on the efficiency of the PCR reaction was observed (data are not presented). The genotype of the calibrator is GSTM1 1/1, GSTT1 1/1 To obtain the normalized ratio for result interpretation, the concentration ratio of an unknown sample has to be normalized to the concentration ratio of the calibrator, which is a sample with a known genotype. Several samples were genotyped using previously described methods to distinguish between heteroand homozygous GSTM1 and GSTT1 gene carriers [16,22]. A sample with the genotype GSTT1 1/1 and GSTM1 1/1 was chosen as a calibrator. It was previously demonstrated that duplication of the GSTM1 gene could be observed in the human population [18]. To eliminate the possibility of GSTM1 duplication (genotype GSTM1 11/1) in the calibrator, Southern blotting was applied as described by McLellan et al., 1997 [18]. This confirmed the GSTM1 genotype of the calibrator as 1/1. Method validation To validate the new established method, 15 samples were additionally genotyped with the method described by Sprenger et al. (2000) [16] for GSTT1 genotypes and with the long-range PCR described by Roodi et al. (2004) [22] or by Southern blotting followed Eco RI restriction, described by McLellan et al. (1997) [18] for GSTM1 genotype. No discrepancies were observed between the new LC480 method and previously published methods. Trimodal distribution of GSTM1 and GSTT1 genotypes To genotype 1320 samples from the population-based KORA study, the new semiquantitative method was applied. All samples were genotyped three times and the average NR was calculated for every sample. Results were checked, failed samples, samples with the NRs between 0 and 0.2 and samples

M. Timofeeva et al. / Clinical Biochemistry 42 (2009) 500–509 Table 2 Summary of genotyping results Genotype Number of Observed Expected Mean observations (n) frequencies (%) frequencies (%) NR

SD

GSTT1 0/0 1/0 1/1 Failed

225 646 434 15

17.2 49.5 33.3

16 48 36

0.00 0.59 1.06

0.01 0.06 0.07

GSTM1 0/0 1/0 1/1 Failed

675 540 93 12

51.6 41.3 7.1

50 41 9

0.00 0.58 1.03

0.01 0.06 0.03

with SD more then 10% for heterozygous genotype were repeated again in triplicate. Overall 101 samples were genotyped again on the LightCycler 480. To obtain final results from the average NRs, the EM algorithm was applied to decompose the mixed distribution into the three latent normal distributions with NR = 0, NR = 0.5 and NR = 1.0. The genotype of samples was determined with the probability of pNRj ;i z0:99, genotypes with a probability of belonging to one of three genotypes less than 0.99 were considered as uncertain. Uncertain GSTT1 and GSTM1

505

genotypes were observed for 27 and 13 samples, respectively. Additionally 16 samples were recognized as failed for GSTT1 genotype and 14 for GSTM1 genotype. Such samples were genotyped one more time. In cases of repeat unclear genotype results long range PCR was applied to distinguish between GSTM1 homo- and heterozygous carriers [22], and the method previously published by Sprenger et al. (2000) [16] was applied to obtain GSTT1 genotype of the samples. Overall these methods failed to obtain results for 15 samples for GSTT1 genotype and for 12 samples for GSTM1 genotype. For 11 of these samples genotyping failed for both methods, probably, due to the low DNA quality. The summary of genotyping results is presented in Table 2. The mean NRs for GSTT1 1/1 and 1/0 genotypes were 1.06 and 0.59 respectively with the corresponding SD of 0.06 and 0.07. The mean NR for GSTM1 1/1 genotype was 1.03 with the SD of 0.03 and 0.58 with the SD of 0.06 for genotype GSTM1 1/0. The “null genotype" was characterized by mean NR of 0. To visualize results we plot the NR of the graphic (Figs. 3C and D). A clear trimodal distribution can be observed for both genes of interests. Overall, in the current population the distribution of GSTM1 genotypes was 51.6%, 41.3% and 7.1% for 0/0, 0/1 and 1/1 genotypes, respectively. The distribution of GSTT1 genotypes was 17.2%, 49.5% and 33.3% for 0/0, 0/1 and 1/1 genotypes,

Fig. 3. Distribution of normalized ratios (NRs) for all samples. NRs, calculated for every sample, were plotted on the graph, where ▵ — genotype 0/0, ● — genotype 1/0 and ○ — genotype 1/1. Dotted line marks areas of uncertain genotype allocation. (A) No clear trimodal distribution was observed after GSTM1 genotyping of 81 samples, applying the probe for the A allele only. (B) the same 81 samples, genotyped with a “wobble“ probe for GSTM1. (C) trimodal distribution of GSTM1. (D) trimodal distribution of GSTT1.

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respectively. The frequency of the GSTM1 deleted allele is 0.72 and of the GSTT1 deleted allele is 0.42. Quality control For quality control an additional 10% of samples were repeated and no discrepancies were observed. To confirm reproducibility of real-time PCR, the within sample variation was measured by calculation of mean SD for the samples after 3 repeats. The mean within sample variation for GSTT1 1/1 genotypes was 0.09 ± 0.04 and for GSTT1 1/0 was 0.05 ± 0.02. The same parameter for GSTM1 1/1 genotypes was 0.09 ± 0.05 and for GSTM1 1/0 was 0.06 ± 0.03. The observed genotype frequencies are comparable with the expected frequencies (Table 2). The genotype distributions of both polymorphisms are in accordance with the HWE (p = 0.286 for GSTM1, p = 0.561 for GSTT1). Evaluation of gene duplication, using the new developed method Southern blotting was applied to determine GSTM1 genotype and to check for possible GSTM1 duplication of the calibrator and of 10 samples, which we intended to use as positive controls. After the EcoRV restriction for one of the samples an additional band of 56 kb was detected (Fig. 4), which indicates an additional copy of GSTM1. The results of semiquantitative genotyping on LC 480 indicated a NR value of 1.48 for this sample. The highest GSTM1 NR detected among the 1320 samples analyzed in the current study was 1.25. Unfortunately, the available amount of DNA of the sample with the NR = 1.25 was not enough to perform the Southern blotting and to confirm the genotype. Long range PCR can not give sufficient information to determine copy numbers. Interestingly,

Fig. 4. EcoRV analysis of the μ class GST genes from genomic DNA samples. Hybridisation was done with the human GSTM1 cDNA probe; 29 kb fragment — GSTM4 and GSTM2 genes; 38 kb fragment indicates non deleted copy of GSTM1; 56 bp — duplication of GSTM1 region. Marker — lambda DNA/HindIII, sample 1, sample 3 — genotype 1/0, sample 2 — genotype 0/0, sample 4 — genotype 1/11.

the highest NR value for GSTT1 was 1.87 and was observed only in one sample. All other values were less then 1.38. To summarize, an increased NR of approx. 1.5 can be used as an indicator of possible copy number duplication. Discussion The majority of studies searching for the effect of GSTM1 and GSTT1 deletion polymorphisms are based on the comparison of “null genotype” with “non-null genotype”, due to historical methodological constraints. However, the need to determine GSTT1 and GSTM1 copy numbers has become increasingly obvious. There is evidence from large meta- and pooled analyses, including only data from “null”/“non-null” genotyping, that GSTM1 and GSTT1 genotypes affect the risk of lung cancer [35–37], with a small but significant effect. There is also some evidence that GSTM1 copy number may affect treatment outcome [38] — such possible clinical relevance must be investigated in more detail. Distinction of three possible genotypes (1/1, 0/1 and 0/0) will enable researchers to consider not only the dominant model of inheritance (“non-null genotype” vs. “null genotype”), but recessive (0/0 and 1/0 vs. 1/1) and codominant models of inheritance and therefore increases chances to detect weak and modest effect of the deletions. Several methods to distinguish between 1/1 and 1/0 genotypes have been described and successfully applied in epidemiological studies [13,39]. Thus, Roodi et al. (2004) carried out a long range PCR to detect GSTM1 copy numbers in a breast cancer case-control study and observed an increased risk of breast cancer only for carriers of two normal GSTM1 alleles (genotype 1/1), which would not have been possible applying standard method for GSTM1 “null allele" detection. Applying this method as well as other previously published PCR based methods to distinguish between homo- and heterozygous carriers [16,19–22] is, however, challenging in large scale epidemiological studies, as they are often technically difficult, time consuming and require post PCR gel-electrophoresis. Recently, two semiquantitative GSTT1 and GSTM1 genotyping assays based on real-time PCR were described in literature [13,23]. Brasch-Andersen et al. (2004) compared the threshold cycle parameter (Ct) (or crossing point) of the candidate gene and the reference gene and calculated the difference ΔCt, which they used to determine copy number of allele [13]. Girault et al. (2005) presented genotyping results as normalized unit-less parameter-normalized ratio, which is easier to interpret [23]. We modified the method published by Girault et al. (2005) [23]. Like this paper, we applied a hydrolysis probe format for product detection. We used the same GSTT1 primers sequences for genotyping. However, in silico analysis revealed a high binding stem loop in the region of the GSTM1 probe described by Girault et al. (2005), which might influence on binding to the DNA sequence. Therefore, a new GSTM1 forward primer and probe were developed and applied for genotyping (Fig. 1). The newly developed probe contained a SNP rs737497, which had been in the primer region for the GSTM1 genotyping method described by Girault et al. (2005). Interestingly, first experiments

M. Timofeeva et al. / Clinical Biochemistry 42 (2009) 500–509

on method development showed that this SNP in our GSTM1 probe sequence significantly influenced signal detection, probably due to the mismatching and inefficient binding of probes to templates, and therefore results could not be properly evaluated. To minimize the effect of the mismatching due to the SNP, a “wobble” probe, i.e., a mix of two probes for both polymorphic variants, was used for genotyping (Figs. 3A and B). One of the advantages of the modified method is multiplexing. Amplification of reference and target gene in one reaction tube corrects for variation in initial DNA amount, pipetting errors and differences in DNA quality. Additionally, it makes the reaction not sensitive to the initial DNA concentration, which can vary between 10 and 100 ng per tube. Like Girault et al. (2005) we also applied normalization with the calibrator to get unit-less normalized ratios. Applying the same calibrator in all runs compensates for constant differences between detection of target and reference genes (e.g. probe annealing, FRET efficiency) and provides a constant calibration point between all PCR runs. In contrast to the previously published methods, we applied efficiency correction for result calculation (Eq. (2)). Our experiment showed that GSTT1, GSTM1 and ALB have different efficiency of amplification: 1.891, 1.889 and 1.801, respectively. Our approach is thus more precise than the widely used ΔΔCT method, which assumes that target and reference genes have similar efficiency of 2 [27,28]. Genotyping results, expressed as NR, showed clear trimodal distribution of both GSTT1 and GSTM1 genotypes. Cut-offs for NRs distribution for every genotype were established and one of three possible genotypes for each of two genes was assigned, applying an EM algorithm. This statistical approach significantly strengthens the method described here and making it more reliable. For the majority of samples one of the 3 possible genotypes was assigned with a probability of genotype allocation pNRj ;i z0:99. The observed genotyping call rate after implementation of the developed EM algorithm was comparable with those previously published by Brasch-Andersen et al. (2004). The observed NR values (Table 2) were slightly higher than the expected 0.5 for heterozygous gene carriers and 1.0 for homozygous gene carriers. This can be explained by different PCR efficiency of the calibrator sample and other study samples. The calibrator DNA was isolated using different kit than all other study samples. It is very probable that the method of isolation influences the DNA quality and thereby the efficiency of the PCR reaction. The frequencies of the observed genotypes are comparable with the expected frequencies (Table 2). Similar results were obtained by other investigators. Thus, Buchard et al. (2007) genotyped 200 healthy Danes and observed 40% of GSTT1 1/1 genotype, 46% GSTT1 1/0 and 14% 0/0 [19]. The distribution of the GSTM1 1/1, GSTM1 1/0 and GSTM1 0/0 in the same population was 7%, 40.5% and 52.5%, respectively. Girault et al. (2005) genotyped 29 healthy Caucasians and observed the following distribution of GSTM1 genotypes: 3.4% of 1/1, 41.4% of 1/0 and 55.2% of 0/0. The distribution of GSTT1 1/1, 0/1 and 0/0 genotypes in the same study was 31.0%, 41.4% and 27.6%, respectively [23].

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The method described here was successfully applied for genotyping samples from the case-control study searching for the effect of metabolizing enzymes on the risk of early onset lung cancer, [40] as well as in other ongoing epidemiological studies. Duplication of GSTM1 was first described by McLellan et al. (1997), who applied Southern blotting to check the GSTM1 genotype of two Saudi Arabians with ultrarapid GSTM1 activities [18]. GSTM1 duplication was also detected by Southern blotting in one of our samples, which we intended to use as a positive control. The result of Southern blotting was confirmed by the newly developed method. For the sample with the GSTM1 duplication, NR (GSTM1) = 1.48, was detected. An NR of approximately 1.5 would be expected in case of 3 copies of the GSTM1 allele. All other DNA samples had an NR less then 1.25. Therefore we assumed that the observed sample was the only one with the 3 copies of GSTM1 allele in our study group. The method described here is not able to distinguish between 11/0 genotype and 1/1 genotype. Previously, it was suggested that taking into account the high frequency of the GSTM1 “null allele", the genotype 11/0 might be possible [18]. To the authors' knowledge there is no evidence of GSTT1 duplication described in the literature. However, taking into account the structure of the GSTT1 gene, it is easily to suggest a hypothetical unequal crossover event between two highly homologous sequences HA5 and HA3 downstream and upstream of the GSTT1 gene, which would lead to duplication of the region (Fig. 2). Interestingly, among our genotyped samples there was a subject with a very high NR of 1.87 compared to all other NR values which were not higher than 1.38. This finding may indicate a possible GSTT1 duplication. Additionally, the methodological approach described here, can be applied for quantification of any other copy number polymorphisms (CNPs). Association of CNPs with various multifactorial diseases has been shown [41–43]. However, high throughput genome screening methods applied for detection of CNPs differ significantly in resolution and ability to detect various structural variations [44,45]. Therefore, it is essential to confirm results of genome wide association studies and to develop suitable methods for clinical studies using alternative approaches, e.g. a real-time quantitative PCR-based method. To summarize, the modified semiquantitative real-time PCR method for evaluation of GSTT1 and GSTM1 copy numbers is a very reproducible and reliable multiplex assay, which in combination with the described statistical approach is a promising tool for future large scale epidemiological and clinical studies. Acknowledgments We acknowledge the KORA research platform (KORA, Cooperative Research in the Region of Augsburg), which was initiated and financed by the Helmholtz Centre Munich, which is funded by the German Federal Ministry of Education and Research and by the State of Bavaria.

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