You are on page 1of 9

Article pubs.acs.

org/crt

Identification of the GST-T1 and GST-M1 Null Genotypes Using High Resolution Melting Analysis
Zuzana Drobna,́ *,† Luz Maria Del Razo,‡ Gonzalo Garcia-Vargas,§ Blanca San ́ chez-Ramírez,|| || || ⊥ Carmen Gonzaĺ ez-Horta, Lourdes Ballinas-Casarrubias, Dana Loomis, and Miroslav Styb ́ lo†

Department of Nutrition, Gillings School of Global Public Health, University of North Carolina at Chapel Hill, North Carolina, United States ‡ Department of Toxicology, CINVESTAV-IPN, Mexico § Faculty of Medicine, UJED, Gomez Palacio, Durango, Mexico || Faculty of Chemical Sciences, Autonomous University of Chihuahua, Chihuahua, Mexico ⊥ School of Community Health Sciences/MS-274, University of Nevada, Reno, Nevada, United States ABSTRACT: Glutathione S-transferases, including GST-T1 and GST-M1, are known to be involved in the phase II detoxification pathways for xenobiotics as well as in the metabolism of endogenous compounds. Polymorphisms in these genes have been linked to an increased susceptibility to carcinogenesis and associated with risk factors that predispose to certain inflammatory diseases. In addition, GST-T1 and GSTM1 null genotypes have been shown to be responsible for interindividual variations in the metabolism of arsenic, a known human carcinogen. To assess the specific GST genotypes in the Mexican population chronically exposed to arsenic, we have developed a multiplex High Resolution Melting PCR (HRMPCR) analysis using a LightCycler480 instrument. This method is based on analysis of the PCR product melting curve that discriminates PCR products according to their lengths and base sequences. Three pairs of primers that specifically recognize GST-T1, GST-M1, and β-globin, an internal control, to produce amplicons of different length were designed and combined with LightCycler480 High Resolution Melting Master Mix containing ResoLight, a completely saturating DNA dye. Data collected from melting curve analysis were evaluated using LightCycler480 software to determine specific melting temperatures of individual melting curves representing target genes. Using this newly developed multiplex HRM-PCR analysis, we evaluated GST-T1 and GST-M1 genotypes in 504 DNA samples isolated from the blood of individuals residing in Zimapan, Lagunera, and Chihuahua regions in Mexico. We found that the Zimapan and Lagunera populations have similar GST-T1 and GST-M1 genotype frequencies which differ from those of the Chihuahua population. In addition, 14 individuals have been identified as carriers of the double null genotype, i.e., null genotypes in both GST-T1 and GST-M1 genes. Although this procedure does not distinguish between biallelic (+/+) and monoallelic (+/−) genotypes, it can be used in an automated workflow as a simple, sensitive, and time and money saving procedure for rapid identification of the GST-T1 and GST-M1 positive or null genotypes.

INTRODUCTION Glutathione S-transferases (GSTs) represent a group of enzymes that are involved in the phase II detoxification reactions. Three families of GSTs have been described: the cytosolic GSTs, the mitochondrial GSTs, and the microsomal GSTs (membrane-associated proteins in eicosanoids and glutathione metabolism). These enzymes primarily catalyze the conjugation of reduced glutathione (GSH) to the electrophilic center of endo- or exogenous compounds (e.g., dopamine, prostaglandins, products of lipid peroxidation, chemotherapeuticals, and environmental carcinogens). The conjugation with GSH increases solubility and facilitates excretion of these compounds.1 Apart from GSH conjugation, several other biological functions have been described for different GSTs, such as involvement in the biosynthesis of
© 2011 American Chemical Society

hormones, tyrosine degradation, peroxide breakdown, or dehydroascorbate reduction.2 GSTs have also been shown to play a role in the metabolism of inorganic arsenic (iAs), a common drinking water contaminant and known human carcinogen. Four classes of cytosolic GSTs (GST-P1, GSTO1, GST-M1, and GST-T1) are suggested to take part in iAs metabolism. GST-O1 and GST-O2 have been shown to reduce monomethylarsonate.3,4 An increased expression of GST-P1 has been observed in several arsenic-resistant cell lines and linked to the formation of arsenic−glutathione conjugates that are excreted from the cells by the multidrug resistant protein transporters.5−9 Genetic polymorphism in GST-T1 and GSTReceived: October 20, 2011 Published: December 2, 2011
216
dx.doi.org/10.1021/tx200457u | Chem. Res. Toxicol. 2012, 25, 216−224

a fluorescence dye known to specifically bind double-stranded DNA (dsDNA).10−13 Epidemiological studies demonstrate that the absence or aberrant activities of GST-T1 and GST-M1 enzymes due to genetic polymorphism render a potential risk for the development of multiple diseases such as cancer. and GST-M1 reverse primer. All procedures were approved by the Institutional Review Boards of Cinvestav-IPN. Toxicol. MATERIALS AND METHODS DNA Samples. Recently. where association between chronic exposures to iAs in drinking water and prevalence of diabetes is being examined. To validate the HRM assay and to assess its feasibility in population studies. a trimodal genotype (+/+. The final amplicon extension has been performed at 72 dx. Mexico. +/−. 5′-CAA CTT CAT CCA CGT TCA CC3′. Conventional Singleplex and Multiplex PCR for GST-M1 and GST-T1 Genotypes. followed by 40 cycles of denaturation at 94 °C for 1 min. DNA was precipitated by ethanol and purified on silica-based membrane spin column. The PCR reaction was performed using Eppendorf Gradient Thermal Cycler. 5′-TCA CCG GAT CAT GGC CAG CA3′. 200 μM of each dNTPs. 25. Briefly. other techniques using realtime PCR for GST-T1 and GST-M1 null genotype evaluation have been implemented.25.27 A quantitative real-time PCR method has been developed to evaluate the copy number polymorphisms. and −/−) for GST-T1 or GST-M1.26 However. and elongation at 72 °C for 1 min. Recently.23. and epigenetic differences in dsDNA samples with a high accuracy. Pittsboro. cultured primary human hepatocytes were digested by Proteinase K.17 were used to determine GST-M1 and GST-T1 genotypes in primary human hepatocytes from seven donors and to validate results of the multiplex HRM analysis (95 samples randomly selected from a population study representing 18. two different procedures are used to identify GST-T1 and GST-M1 null genotypes.5 mM MgCl2. GST-T1 and GST-M1.30 This technique is based on genotype discrimination by melting curve analysis that uses SYBR Green I. and 263 in Chihuahua). This type of deletion leads to a complete loss of enzymatic activity and is associated with an increased susceptibility of individuals to genotoxic and carcinogenic agents plausibly due to an impaired detoxification capability. high-throughput.27−30 Gene specific hybridization probes have been used to identify the carriers of at least one functional GST-T1 or GST-M1 allele and to distinguish them from the individuals who are homozygous for the GST-T1 or GST-M1 null genotype.4).17. and University of North Carolina at Chapel Hill. 1 μM of each primer. all recruited study subjects were provided with details of the research project and gave written informed consent.31 Depending on PCR conditions. 216−224 . we used genomic DNA (gDNA) isolated from primary human hepatocytes obtained from CellzDirect (Invitrogen Corporation. like the method that uses gene specific hybridization probes. a low concentration of SYBR Green I may limit the detection of multiple amplicons by melting peaks due to a low fluorescent signal30 or dye migration from small amplicons to large ones (“dye jumping.39 Demographic description of the donors of human hepatocytes is provided in Table 1. and reliable analysis of GST-null genotypes. To develop the HRM assay for GST-M1 and GSTT1 null genotypes.29 However.20 or diabetes.. Res.22 GST-T1 and GST-M1 share a common polymorphism represented by a complete absence of the gene.28. Mexico City. In general. We have developed a highthroughput multiplex genotyping analysis for GST-T1 and GSTM1 that is based on high resolution melting (HRM) analysis using a completely DNA saturating dye. polymorphisms.” the process in which dye from a melted small DNA duplex may be reincorporated into regions of dsDNA which have not yet melted) affecting accuracy of the analysis.36 The HRM analysis is a powerful technique used for the detection of mutations.34. PCR conditions were as follows: initial denaturation at 94 °C for 5 min. 5′-GAA GAG CCA AGG ACA GGT AC-3′. The culture conditions for hepatocytes were described elsewhere.21. As of today. annealing at 57 °C for 1 min. The PCR mixture (50 μL total volume) consisted of 25 ng of gDNA. USA. Quality and yield of DNA was verified on 1% agarose gel and by UV reading at 260 and 280 nm using Eppendorf BioPhotometer. NC). 5′-CTT GGG CTC AAA TAT ACG GTG G-3′. the null genotype. The final concentration and quality of DNA was determined by reading the absorbance at 260 and 280 nm using Eppendorf Biophotometer. subjects can be falsely identified as the null genotype carriers.14−19 cardiovascular diseases. especially when short fragments are to be amplified.27 this technique cannot analyze both genes. 5′-GAA CTC CCT GAA AAG CTA AAG C-3′. The most common method is a conventional multiplex PCR. Before blood collection. 20 mM Tris·HCl (pH 8. North Carolina. SYBR Green I must be used at subsaturating concentrations as it can inhibit PCR reaction. A conventional singleplex (one target gene) and multiplex (three target genes) PCR as described by Liu et al.Chemical Research in Toxicology M1 has been associated with distinct urinary profiles of arsenic metabolites in populations chronically exposed to iAs in drinking water. ResoLight.org/10.e. and β-globin reverse primer.24 Studies examining gene−environment interactions or gene− disease associations require a simple. the poor detection limit for the ethidium bromide stained PCR amplicons (5−10 ng/band) may yield false-negative results. 2012. Demographic Description of Primary Human Hepatocyte Donors and Their GST-M1 and GST-T1 Genotypes human hepatocytes Hu228 Hu229 Hu230 Hu0503 Hu0507 Hu0531 Hu0534 a gender male female female male male female male ethnicity Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian Caucasian age 73 39 74 64 51 70 56 GST-M1 genotype negative negative negative positivea positivea negative positivea GST-T1 genotype positivea positivea positivea positivea negative positivea negative Biallelic (+/+) or monoallelic (+/−) positive. this technique that requires electrophoretic analysis of PCR amplicons is time-consuming and cannot provide the high throughput needed in large population studies. 1. 5′-TTC CTT ACT GGT CCT CAC ATC TC-3′.40 DNA was extracted and purified using DNA Blood Mini Kit (QIAGEN). i.doi.5 U of Taq polymerase (Invitrogen). in one reaction vessel. Another drawback is that ethidium bromide as a potent mutagen requires special handling and disposal.8% from total of 504 samples analyzed by HRM).1021/tx200457u | Chem. a multiplex assay for the simultaneous analysis of the GST-T1 and GST-M1 genotype has been described. and purified using QIAamp DNA Mini Kit from QIAGEN following the manufacturer’s protocol.38. 105 in Lagunera.37 This newly developed technique was applied to the assessment of specific 217 Article ■ GST’s genotypes in our ongoing cross-sectional studies in Mexico. DNA has been isolated Table 1. The following sets of primers were used to amplify target genes (GST-M1 and GST-T1) and the internal control (β-globin): GST-M1 forward primer. and 50 mM KCl.32−35 Availability of completely DNA saturating dyes might overcome some limitations. we used gDNA isolated from the whole blood collected from 504 study subjects living in arsenicosis areas in Mexico (136 in Zimapan. 0. GSTT1 forward primer. β-globin forward primer. and GST-T1 reverse primer. In addition.

218 ■ RESULTS Conventional Singleplex PCR Analysis of GST-T1 and GST-M1 Genotypes in Primary Human Hepatocytes. For the amplification of GST-T1. Hu0503. and 268 bp for β-globin. The internal control. The PCR reaction (20 μL total volume) consisted of 25 ng of gDNA. jsp?id=UP030000). The amplification of gDNA for individual target genes showed that only three donors were positive for GST-M1. The multiplex PCR analysis was performed side by side with the individual (singleplex) PCR to ensure sensitivity and reproducibility of the results and to confirm that no interference between primers would occur. forward (5′-CCAGGATGTGAGGCTGTTCT-3′) and reverse (5′GAAACCAACTAAACCTGCAACC-3′) primers producing a 69 bp long PCR fragment were used (Table 2).8 87. Hu229.1021/tx200457u | Chem. 480 bp for GST-T1.2 for GST-T1 . HRM Analysis of GST-M1 and GST-T1 Genotypes. singleplex (one target gene) and multiplex (three target genes) HRM analyses were performed using LightCycler480 (Roche). negative (Figure 1B) indicating null genotype. 0. 2. 5.1 μM of each primer (forward and reverse) for GST-M1. Sequencing verified authenticity of the PCR products (amplicons) for all three target genes. while four donors were carriers of null genotype (Figure 1A).5 °C increments per cycle) and a high resolution melting curve analysis protocol were used to amplify the target genes. Conventional Multiplex PCR Analysis of GST-T1. Figure 2 shows a representative images of multiplex PCR for donors who are dx. and HPLC purified primers. forward primer. 3. Hu0531. Hu228. Five were positive for GST-T1. After testing different sets of GST-T1 primers and several MgCl2 concentrations. Genomic DNA (gDNA) isolated from primary human hepatocytes obtained from seven individuals (4 males and 3 females) were screened for the presence or absence of GST-T1 and GST-M1 genes by conventional singleplex PCR analysis. °C for 7 min. 3. collected data were analyzed using the LightCycler480 “Tm calling” algorithm to determine the presence of melting peaks and their maxima. and two were Figure 1.3 for GST-M1 . base pair. and β-globin in Primary Human Hepatocytes. R. The primers for amplification of GST-M1 and β-globin were those described above. Both.2 μM of each primer (forward and reverse) for GST-T1 and β-globin. Demographic data (gender. The length of amplicons was 215 bp for GST-M1. and 0. the best conditions were applied to evaluate results from conventional multiplex PCR. http://www. and Hu0534) were used as positive controls for HRM analysis of GST-T1 and GST-M1 genotypes in gDNA samples collected from Mexican study subjects. DNA samples isolated from human hepatocytes with unique GST’s genotype (Hu0503. new sets of GST-T1 primers needed to be designed as the size of amplicon produced by primers used for conventional PCR was too long (480 bp) and in general unsuitable for HRM analysis. Tm.3 F. 4.Chemical Research in Toxicology Table 2. bp. After PCR amplification and melting curve analysis.8 79. 8. This modification allowed production of a shorter GST-T1 PCR fragment with lower melting temperature and with a melting curve shape distinct from the other two target genes. Hu230. 216−224 . negative control (no DNA template). The amplicon sequences were identical with the corresponding sequences in the NCBI database. GST-M1 and GST-T1 genotypes in primary human hepatocytes. To ensure the authenticity of the PCR products. Res. Primers Used for Conventional and HRM PCR Analysesa PCR analysis conventional PCR targeted gene GST-M1 GST-T1 β-globin HRM analysis GST-M1 GST-T1 β-globin a Article primer sequence F: 5′-GAACTCCCTGAAAAGCTAAAGC-3′ R: 5′-CTTGGGCTCAAATATACGGTGG-3′ F: 5′-TTCCTTACTGGTCCTCACATCTC-3′ R: 5′-TCACCGGATCATGGCCAGCA-3′ F: 5′-CAACTTCATCCACGTTCACC-3′ R: 5′-GAAGAGCCAAGGACAGGTAC-3′ F: 5′-GAACTCCCTGAAAAGCTAAAGC-3′ R: 5′-GTTGGGCTCAAATATAGGGTGG-3′ F: 5′-CCAGGATGTGAGGCTGTTCT-3′ R: 5′-GAAACCAACTAAACCTGCAACC-3′ F: 5′-CAACTTCATCCACGTTCACC-3′ R: 5′-GAAGAGCCAAGGACAGGTAC-3′ size of amplicon (bp) 215 480 268 215 69 268 amplicon Tm (°C) 82. GSTM1. ethnicity. a High Resolution Melting Master Mix (Roche) containing ResoLight (a completely saturating DNA dye). reverse primer. Results of the conventional singleplex PCR analysis of GST-M1 (A) and GST-T1 (B) for seven donors of primary human hepatocytes: 1. Hu230. was amplified for all donors (data not shown). Gene Scanning 96-II LightCycler480 program with a touchdown PCR (covering a range of annealing temperature from 65 to 53 °C with 0. The sequences of PCR fragments were verified using the GST ’ s sequences accessed from NCBI database (NM_000561. However. 7. β-globin. The primers used for the PCR amplification and length of the amplicons are described in Table 2. 6. Using the Universal ProbeLibrary Assay Design Center (Roche. individual PCR reaction for each gene was performed under the PCR conditions described above and resulting amplicons were sequenced in the UNC-Chapel Hill Genome Analysis Facility using Big Dye Terminator chemistry.4 for β-globin). 2012. 25.5 mM MgCl2. and age) for all donors of hepatocytes are summarized in Table 1. and 1× of LightCycler480 High Resolution Melting Master Mix. specific melting temperatures.org/10.doi. Hu0507. Toxicol. we designed four new sets of primers for amplification of GST-T1 which produced amplicons with lengths between 60 − 250 bp. PCR amplicons were separated by electrophoresis in 2% agarose gel and visualized by ethidium bromide staining. Hu0534.com/sis/rtpcr/upl/index. melting temperature. NM_000853. NM_000518.roche-applied-science.

GST-M1. and carriers of the GST-M1 gene and null for the GST-T1 gene (Figure 3F). The differences were due to the lack of amplification in conventional multiplex PCR resulting in a missing signal for one or both target genes visualized by ethidium bromide in agarose gel. Almost 5% of the Zimapan and Lagunera residents. 25. representing 25% of reanalyzed samples.1 °C for GST-T1. In addition. ethidium bromide. this method is not environment friendly and makes genotype screening in large-population studies difficult. GST-T1. and β-globin in Primary Human Hepatocytes. With the exception of one sample results of the singleplex analysis were in agreement with results obtained by the multiplex HRM analysis.3 ± 0.34. DISCUSSION Most epidemiological studies have assessed specific GST-T1 and GST-M1 genotypes using the conventional multiplex PCR technique that requires electrophoretic separation of PCR amplicons in agarose gel. HRM Analysis of GST-T1 and GST-M1 Genotypes in the Mexican Population. βglobin. carriers of the GST-T1 gene and null for the GST-M1 gene (Figure 3E). respectively) than in residents from Zimapan and Lagunera. Toxicol. The HRM analysis was used to genotype gDNA collected from 504 individuals living in the Zimapan. This one sample was positive for GST-T1 and negative for GST-M1 by the singleplex analysis. and Hu0534 (C) primary human hepatocytes are shown as examples. However. three were carriers of the GST-M1 gene. these techniques require expensive hybridization probes or complex calculations of the copy-number variation and cannot determine GST-T1 and GST-M1 genotypes simultaneously in one reaction mixture. were carriers of null genotypes for both GST-T1 and GST-M1. GST-M1 and GST-T1 genotypes in Hu0503 (A). Inc.E.8% from total) using conventional multiplex PCR analysis. suggesting that poor gDNA quality or the presence of an unknown inhibitor might interfere with PCR amplification.37 This technique was developed in collaboration by University of Utah and Idaho Technology. carriers of GST-T1 gene and null in GST-M1 gene (Figure 2B). or carriers of GST-M1 gene and null in GST-T1 gene (Figure 2C).25. We also reanalyzed 95 samples (18. GST-M1. 5. The method uses high datadensity acquisition during the melting analysis of PCR fragments with a potential to detect small sequence differences. The HRM analysis requires a fluorescent dye to detect low Tm dx. Among the seven donors of human hepatocytes. β-globin.1021/tx200457u | Chem. mutations. Results were then compared with the results of the multiplex HRM analysis. 216−224 ■ . Thus. but only 1% of the Chihuahua residents. 2.B.26 This approach is timeconsuming and increases handling errors as multiple steps from the beginning to the end of the process are required. including the GST-T1 and GST-M1 null genotypes. Figure 3D. It also produces a large amount of hazardous waste represented by agarose gel and buffers containing a carcinogen.1 °C for β-globin.17.1 °C for GST-M1. β-globin + GST-M1.org/10. fourteen samples (out of 504 analyzed samples) that were identified by multiplex HRM analysis to be null for both GST-T1 and GST-M1 genotypes gave the same results after reanalysis by singleplex HRM.C). each target gene was evaluated separately under the same HRM-PCR cycling conditions described in Materials and Methods) to verify the results of multiplex HRM analysis. Singleplex and Multiplex HRM Analysis of GST-T1. UT) and introduced in 2003. or Chihuahua regions in Mexico.41 It is a close-tube system for genotyping and mutation analysis that does not require labeled oligonucleotides. Conventional singleplex and multiplex PCR.Chemical Research in Toxicology Article Figure 2. 82. (Salt Lake City. Res. The conventional PCR was performed using the following sets of primers with sequences described in Material and Methods and summarized in Table 2: 1. carriers of both GST-T1 and GST-M1 genotypes (Figure 2A).doi. and β-globin were present) from three samples of gDNA by either the multiplex or singleplex HRM analyses.27−29 The high resolution melting method is a powerful technique used to analyze genetic variations (SNPs. and 87. Representative melting peak profiles obtained by the HRM analysis are shown in Figure 4.e. Lagunera. 2012. No PCR amplicons were obtained (no melting curves for GST-T1. A specific melting temperature (Tm) was determined for each gene PCR fragment as the maximum of the negative first derivative of the change in fluorescence vs temperature (−dF/dT. The singleplex HRM confirmed results obtained by the conventional PCR method.8 ± 0. The calculated melting temperatures for PCR fragments were 79.. and the internal control. On the basis of our results. β-globin + GST-M1 + GST-T1. Hu230 (B). All seven samples of primary human hepatocytes were tested for the presence of the three target genes in a singleplex HRMPCR reaction using the gene-specific sets of primers (Figure 3A. There were disagreements between these two methods for 24 samples.F shows representative melting peak profiles for the donors who were carriers of both GST-T1 and GST-M1 genotypes (Figure 3D). β-globin + GST-T1. 7. Fifteen percent of randomly selected samples (75 in total) were analyzed also by singleplex HRM analysis (i. The rapid development of real-time PCR has introduced multiple techniques and approaches that can be used to examine genetic polymorphisms.8 ± 0. 3. the rate of change of fluorescence). 4. GST-M1. five were carriers of the GST-T1 gene. Zimapan and Lagunera populations have similar GST-T1 and GST-M1 genotype frequencies (Table 3) which differ from the frequencies found in the Chihuahua population. and DNA methylation) in PCR amplicons employing the melting curve data analysis. was amplified for all donors. 6. but no melting curves for GST-T1 or GST-M1 were present in the multiplex analysis. The individual null genotypes for GST-T1 and GST-M1 are less frequent in residents from Chihuahua (62% 219 and 34%. negative control (no DNA template).

hard to detect melting peaks.9 (N = 45) 91.7 (N = 175) 65. N.6 (N = 58) 57.8 (N = 73) 72. However. Melting peak profiles and melting temperatures (Tm) as determined by “Tm calling” analysis of the melting curve for seven donors of primary human hepatocytes. The traditional fluorescent dye SYBR Green I has been also used for real-time PCR and melting analysis.2 (N = 190) 1. “saturation dyes” such as LC Green Plus. +/+. (C) melting peaks for β-globin with Tm = 87.1 (N = 7) Lagunera % (N = 105) 13. Toxicol.doi. (F). it may not work well for the HRM analysis in situations when short PCR fragments are amplified as it results in weak fluorescence emission.1 (N = 459) 34.4 (N = 78) 5. (Left) Singleplex HRM-PCR: (A) melting peaks for GST-T1 with Tm = 79. The advantage of these dyes is that maximum fluorescence is achieved at the saturation point. Table 3.1021/tx200457u | Chem.9 (N = 13) 95.3 (N = 329) 3 (N = 15) −/−. products and heteroduplexes in DNA during melting. Here.2 (N = 18) 86. Eva Green.org/10.8 (N = 118) 42. we employed the principles of the HRM analysis to develop a procedure for simultaneous GST-T1 and GST-M1 genotyping in a single reaction vessel. 25. melting peak profiles for Hu0534 donor. (Right) Multiplex HRM-PCR: (D).9 (N = 44) 58. (E).8 (N = 5) Chihuahua % (N = 263) 4. or ResoLight have been developed to be used with the HRM technique. melting peak profiles for Hu0503 donor. (B) melting peaks for GST-M1 with Tm = 82. melting peak profiles for Hu230 donor. yielding small.30−35 Thus. null genotype.1 (N = 3) total % (N = 504) 8. Res.3 (N = 14) 86.36 These dyes do not inhibit PCR 220 amplification even at concentrations that completely saturate DNA. monoallelic positive genotype. 2012.7 (N = 91) 41. SYTO9.1 (N = 250) 27. providing high sensitivity to HRM analysis. 216−224 .Chemical Research in Toxicology Article Figure 3.8 °C (N = 5). Singleplex and multiplex high resolution melting analysis. +/−. We used a LightCycler480 system and a high resolution melting application available from Roche.1 (N = 61) 4.8 °C (N = 3).33.3 °C (N = 7). biallelic positive genotype. GST-T1 and GST-M1 Genotype Frequencies in Mexican Populationsa genotype GST-T1 (−/−) GST-T1 (+/+. +/−) GST-T1 (−/−) GST-M1 (−/−) a Zimapan % (N = 136) 13. number of subjects. +/−) GST-M1 (−/−) GST-M1 (+/+. This system uses ResoLight dye with dx.

homogeneous staining properties in the target sequence that helps to generate a strong melting fluorescence signal. 2012. (B) results for 6 randomly selected samples representing GST-M1 positive and GST-T1 null genotype plus internal control (βglobin). (A) Results for 10 randomly selected samples representing GST-M1 and GST-T1 positive genotypes and internal control (β-globin). The drawback of this method is that it does not determine the copy-number of the target gene.org/10. Using the multiplex HRM analysis in our Mexican cohort.9% of the study subjects are carriers of the GST-T1 null genotype. (C) results for 10 randomly selected samples representing GST-T1 positive and GST-M1 null genotype and internal control (β-globin).doi. and provides high specificity and sensitivity. eliminating the need for a post-PCR evaluation of genotype by determining specific melting temperature using the 221 “Tm calling” algorithm. we found that only 8. This is one of the lowest frequencies dx. it allows a high-throughput analysis at relatively low cost. In addition.Chemical Research in Toxicology Article Figure 4. Res.. Representative melting peak profiles for the multiplex HRM-PCR analysis of GST-T1 and GST-M1 genotypes in DNA samples collected from the Mexican study subjects. Toxicol. The overall advantages of this real-time PCR close-tube system is that it avoids end-product contamination. i. 216−224 . which is a part of the LightCycler480 instrument software.1021/tx200457u | Chem.e. does not distinguish between a homozygote (+/+) and a heterozygote (+/−). 25. and (D) results for 7 randomly selected samples representing GST-T1 and GST-M1 null genotype and internal control (β-globin). improves amplification efficiency.

Salavaggione. S. S. EPA/STAR grant no. Kala. Drug Metab. (2) Hayes. 275. Webber. (7) Zhou. Pharmacol.48..edu. the frequency of the GST-M1 null genotype (34. M. A. Drug Metab.. Hernandez. (2000) The MRP2/cMOAT transporter and arsenic-glutathione complex formation are required for biliary excretion of arsenic. (9) Leslie. Z.. polymerase chain reaction. R.. 32700−32708. 183. high resolution melting. PCR. 51−88. (4) Chowdhury. I.. S. Chihuahua) displays European ancestry. CB# 7461. Wieben. P. J. 1237−1246. When a subset of samples (95 samples in total) was reanalyzed by multiplex conventional PCR and compared with results obtained by the multiplex HRM-PCR analysis. and Weinshilboum. Achanzar. Pharamcol.g... and the National Institute of Health grant R01 ES015326-01A2 to M. (2006) Gluthahione-Stransferase-omega [MMA(V) reductase] knockout mice: enzyme and arsenic species concentrations in tissues after arsenate administration. Toxicol. genomic DNA. H. Pharmacol. J. Phone: (919) 843-2719. people of mixed European and Native American ancestry. Avram. dx.. we found that individuals living in the Chihuahua region have significantly lower occurrence of null genotype in both genes than subjects from Zimapan and Lagunera regions. Thus. and Waalkes. These results suggest that admixture among Mexicans should be considered in studies where gene−environment interaction or gene− disease association is examined for GST-T1 and GST-M1 polymorphs. 99−107.. Toxicol. Flanagan. L. Zakharyan. 33404−33408..42. P. (8) Kala.S. C.. U. Funding This work has been supported in part by American Institute for Cancer Research/World Cancer Research Fund (AICR/ WCRF) to Z. Qu. S. D. gDNA. E. high-performance liquid chromatography.S. Moon. J. D.. (2010) Genetic polymorphisms in glutathione S-transferase (GST) superfamily and arsenic metabolism in residents of the Red River Delta.. Biol. W. GST-M1. M. T. Article Chapel Hill. W. glutathione-S-transferase theta 1. Atwood. 60. A similar explanation is possible for the misinterpretation of GST-M1 genotype when the multiplex conventional PCR method is used. ■ ■ ■ ACKNOWLEDGMENTS We acknowledge Peter Bent and Dr. and Aposhian. W. W. E. V. ABBREVIATIONS HRM. S. Chen. UNC Gillings School of Global Public Health at Chapel Hill. and Waalkes.. J. M. dsDNA. (2004) Arsenic transport by the human multidrug resistance protein I (MRPI/ ABCCI): Evidence that a tri-glutathione conjugate is required. 222 (1) Oakley... Eckloff. P. M. (3) Mukherjee. Iwata. 138−151. Keefer.. E.. B. 45. M. Mol.. J. A. L. Viet. Johnson.. L. Similar discrepancies between the conventional and real-time PCR (using SYBR Green I) methods were reported by Marin et al. and Lieberman. 5-times more subjects from Zimapan and Lagunera regions were carriers of the GST-T1/GST-M1-double null genotype than in Chihuahua. B... R. Fax: (919) 843-0776.. i. Dispos. H.D. Tm... Toxicol. Rev. Vietnam.. Even though the same amounts of gDNA were used for the amplification of all samples. J. (10) Agusa. E. the final amount of PCR product for GST-T1 determined after ethidium-bromide staining in agarose gel vary among the samples (Figure 1A). M. 25.Chemical Research in Toxicology reported for any ethnic group. Pharmacol. M. D.doi.. while the Center and Southeast (e. D.. P. Appl. A. GST-T1. 216−224 . (2005) Glutathione transferases.1021/tx200457u | Chem. Styblo. 446−457. melting temperature REFERENCES ■ AUTHOR INFORMATION Corresponding Author *Department of Nutrition. P..g. NC 27599-7461...g. an excellent agreement between the results obtained by singleplex and multiplex HRM-PCR analysis for DNA samples collected from a Mexican population (only 1 sample out of 75 was in disagreement) suggests that multiplex HRM-PCR analysis is a suitable method for GST-T1 and GST-M1 genotype evaluation and might become a method of choice.. 34.. R. J. 279. D. (6) Brambila. M..51−53 Considering the fact of the admixed population.49 The majority of Mexicans are represented by “Mestizos”..50. Blood 105. Haimeur. Toxicol. The sensitivity of the multiplex HRM-PCR analysis is superior to that of the multiplex conventional PCR. C. M. Neely. by the U. Annu. a certain level of genetic diversity in different regions of Mexico can be expected. I.51 Unfortunately. Prater. T.. We also thank to Olivia Dong. A. Toxicol. D. information about the ethnic origin is scarce in Mexico. L. the primer set used for GST-T1 amplification by conventional multiplex PCR yields a larger PCR fragment (480 bp) than does the set of primers used for multiplex HRM-PCR (69 bp). 242. O. Miller. Rev.. 832735. G. T.. K.44−47 Interestingly. E. Pelleymounter. University of North Carolina at Chapel Hill. Some studies showed that the GST-T1/GST-M1-double null genotype may be associated with even higher risk for the development of certain type of diseases (e. Y. Jing. M. In particular. Fujihara. W. M. H. NC. (2011) Glutathione transferases: a structural perspective.. and Waxman. Chem.. reduced efficiency for the amplification of the larger fragment might be responsible for falsely identified GST-T1 null genotypes by conventional multiplex PCR. K. E-mail: drobnazu@med. glutathione-S-transferase mu 1. M. 2012. E. and Tanabe. 1198−1203. J. 352−362.. Biol..unc. Minh. (2001) Overexpression of glutathione S-transferase II and multidrug resistance transport proteins is associated with acquired tolerance to inorganic arsenic. U. HPLC.. Rice. Appl. B. Zimapan and Lagunera) Amerindian ancestry. H.7%) is similar to those previously reported for other ethnicities (30−70%). the limited sensitivity of ethidium bromide to visualize certain amounts of final PCR product might influence the genotype identification. In addition.30 In our study.e. 16. Kunito. 2003 Michael Hooker Research Center. Klaassen. (5) Liu. W. I. we observed 25% differences in genotype. J. and Jowsey.. H. 43. T. Schaid. Rupal Cutting from Roche Applied Science in USA for their assistance and support. (2005) Glutathione-S-transferase Π inhibits As2O3-induced apoptosis in lymphoma cells: involvement of hydrogen peroxide catabolism... Chen. Res. Kopplin. D. (2006) Gluthahione S-transferase omega1 and omega 2 pharmacogenomics. for helping with gel electrophoresis. Trang. double-stranded DNA. (2002) Chronic arsenic-exposed human prostate epithelial cells exhibit stable arsenic tolerance: mechanistic implications of altered cellular glutathione and glutathione S-transferase. However. The variations may reflect different copy-number of the target gene. a graduate student at the Department of Nutrition. Appl.org/10.43 In contrast. cancer and liver injury) than a single GSTs null genotype. 302−309. J. Chem. Pharmacol. Recent studies using autosomal Y-STRs (short-tandem repeats linked to the Ychromosome) to identify human origin showed that the Northwest Mexican territory (e.. while only ∼7% belong to more conserved Mexican tribes. Takeshita.. M. Saavedra.. D. and Waalkes.. R. V.

B. (14) Moyer. S. Chem. 471−482. Christiani.. R. and Styblo. Health. M. 299−312. Gaspar. J. Y. X. W. Drobna. V.de/LC480Technical-Note-01-HRM. J.. Carranza.. D. and Bruning. Bruhn. and Andreassi. and Dobrovic. cigarette smoking and gastric cancer risk. (27) Ko. H. and Weinshilboum. 247−283.. A. L. Y. Warholm. (2007) Arsenic methylation. Environ.. and Rueff... M.. S.. S. Vandersteen. Pu. Health. Sachinidis. 312−320. K.. R. Barton. N.. 134−138. Z. Smith. L. Chen. Biagini. Pharmacogenetics 10. Brockmoller. (2011) Genetic polymorphisms in glutathione Stransferase (GST) superfamily and risk of arsenic-induced urothelial carcinoma in residents of Southwestern Taiwan. (2000) Rapid analysis of GSTM1. T1 and P1 genetic polymorphisms. Li. Salavaggione. Hoffmeister. C. Schlagenhaufer. S. J. M. 341. Carcinogenesis 16. and Saint.. Clin. Atik. M. J. Phillips.pdf (37) Wojdacz. 853−860. 23. Cheng. J. U. Hildebrandt. C. and GSTP1 gene variants: simultaneously detecting GSTM1 and GSTT1 gene copy number and the allelic status of the GSTP1 Ile105Val genetic variant. Chen.. Su.. T. (12) McCarthy. Clin. P. Wiencke. 341−345. R. G. S.. D. C. S. A. G.. Camdeviren. J. 201. and Aydin. (28) Timofeeva. J. 84. Y. and Camacho-Carranza. Brenner.. I. C.. A. K. Henderson. and Nagakawa. dx. 79.1021/tx200457u | Chem. Faber. (29) Rose-Zerilli. (21) Wang. L. and saint.... K. Environ..... N... J. Hopenhayn. J. K. Rahman.. 3−16.. Mohler.. 159−170. M.. Biomed. J. Timofeeva.. D.. M. Toxicol. C.. A. and Smith. (2007) Genotype frequencies of polymorphic GSTM1.. Scheartz. M... Yin.. 12.. (2009) A multiplex real-time PCR method for detection of GSTM1 and GSTT1 copy numbers. LeCluyse.. 32... P. L. H. Wang. (2000) Mammalian class theta GST and differential susceptibility to carcinogens: A review. N.. M.. R. Res. E. 31. Yildirim. J. (33) Monis.. 500−509.. Diagn. C. Z.. T. K. I. Xing. F. Gonzalez. G. (42) Nelson. S.. Garcia. and Styblo. 463. Concha. T.. Clin. Salinas. J. (25) Buchard. C.. Schaid. Risch. 68298 Mannheim. Res. Gupta. Shipp. Zhang. Environ. E. and NQO1 genes and diabetes mellitus risk in Chinese population. T.. R.. O. and Christiani. C.. B. W. Environ. and Pryor.. D. J. Part A 73. A. and Li. Biochem. W. P. Liang.. L. H. Ates. Hernandez Castellanos.. Sci. Mahiuddin... P. Xu.. J. M. 115.. 85. and Aggarwal. L. e41.. T. Mejia-Loza.. Wu. Rey.. L. Appl.. Health 10. T. GSTT1. M.. D. D.. Environ. E.. 55.. K. Kent. Dalhoff. H. and Vahter. Calvi. Nermell. Pharmacogenetics 10. A. J. M. Nucleic Acids Res. (2010) Functional alterations in the glutathione S-transferase family associated with enhanced occurrence of esophageal carcinoma in China. (34) Reed. Pharmacogenomics 10.. Dev. O. Broberg. H. Botto. and cytochrome P450 CYP1A1 in Mexicans. (2007) Methylation-sensitive high resolution melting (MS-HRM): a new approach for sensitive and high-throughput assessment of methylation. Bickeboller. G. (2007) Glutathione S-transferase T1 and M1: gene sequence variation and functional genomics. Araujo. L.. J. H. Part A 70. F. and Chen.. GSTT1. M. e136. O. and Brinkmann. Lo. Hardwick. C. (35) Winder. (2003) Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR.. A.. Wiencke. D. Mutagen... Chen.. Lee. 29−34. Methods Ecol. Commun. V. Zheng. and Lucier... Gundry. and skin lesions. R. 42.. GSTT1. Robertson. Walton. Paulson. Eckloff... Q. 18. R. B.. J.. R.. GSTT1.. Y. M.. Toxicol.doi. Yadav.. D.. Shaheen. Paul. (2007) Multiplex PCR detection of GSTM1. D. B. M. Rosenberger.. C. (2010) Simultaneous genotyping of GSTT1 and GSTM1 null polymorphisms by melting curve analysis in presence of SYBR Green I. 557−565. E. S... G.. A. (1995) Ethnic differences in the prevalence of the homozygous deleted genotype of glutathione S-transferase theta. K... A. (26) Sprenger. X. Q. Thomas.. G. (30) Marin. S.. (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Albores.. B. (2009) Glutathione S-transferase T1. Elipek. L. 1−12. H.. 267−272. F.. 24−34.. Cell Biochem. M. Anal. W. P. 310−313. T.. G. M. (32) Giglio. Currier. (2004) Interindividual variation in the metabolism of arsenic in cultured primary human hepatocytes. G. T. J.. S. and Zhang. 2012. Y. X. K.. Ryan. (2007) Genetic polymorphisms in MTHFR 677 and 1298. L. Diagn. Monis. Sanchez. and Rasmussen. (16) Tamer. Loomis. A. and GSTP1 genotypes and the genotoxicity of hydroquinone in human lymphocytes. Ates. M. and Risch... Mol. J. M. L. Toxicol. T. Taylor. C. J.. (2000) Characterization of the glutathione S-transferase GSTT1 deletion: discrimination of all genotypes by polymerase chain reaction indicates a trimodular genotype-phenotype correlation. Garcia-Vargas. Chiou. Yang. Toxicol. K. and Kelsey. S. Biochem. J. N. H.. H. Y. 13. Herrmann. M... Germany (2008).. 258−264.. L.. J... G. Harth. 1680−1685. F. Biol.. del Fiandra. T.. 2. 49. W. P. R.. Hum... Z. (1993) Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J. and Wittwer..... Thomas. N. Wang. M. J. Biochem. Y. (18) Funke... 33−41. Kerb. B. G. and Morling. A. Hebbring. 340. Liao. R. Sanchez-Pena. D. and Goldson. J.. C. 43... (2005) Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reacytion and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Quamruzzaman. F. Pharmacol. Li. 9. 216−224 . (2007) Genetic polymorphisms influencing arsenic metabolism: evidence from Argentina. 7207−7216. N. T. Wieben. 599−605. Oniki. Gautam. H.. 271−274.. K. (39) Drobna.. Mol. Koch.and M1null genotypes and coronary artery disease risk in patients with type 2 diabetes mellitus. C. Mutat. GSTM1. Arch. K. Roche Applied Science. Hsueh. Moore. A. Biotechniques 130−131. Pharmacol. Natl. 115. A. Spitz. D. (2004) GSTM1.. K. J.. Health Perspect. A.. Goto. T. Y. Environ. Reed. (22) Hori. (38) Drobna. Cancer Inst.. 612−617. S. Vink.. Waters. Environ. Toxicol.... 25. Health Perspect. C. Published by Roche Diagnostics GmbH. Mol. GSTM1 and T1. (2010) Genetic polymorphisms in GST genes and survival of colorectal cancer patients treated with chemotherapy. G. (36) LightCycler 480 Real-Time PCR System.. I. Cancer Res. Serrano. Roots. R. K. O. Biggs.. N. P. J. H.. C. K. M. Moon. W. A. (2010) Metabolism of arsenic in human liver: the role of membrane transporters. Evol. A.. J. A.org/10. L. A. 300−304. (2011) Effect of genetic polymorphism of GSTM1 and GSTT1 genotypes on cytogenetic biomarkers among coaltar workers. (2005) Glutathione Stransferase M1. and Sala. A. Y. Jager.... L. H.. E. A. A. (17) Liu.. Duarte Silva. 597−608. Toxicol. Munoz. (2007) Combined glutathione S-transferase T1 and M1 positive genotypes afford protection against type 2 diabetes in Japanese. C. (20) Manfredi. (2007) Highresolution DNA melting analysis for simple and efficient molecular diagnostics. (24) Silva Mdo. Ochoa-Corona. H. T.1. B... S. 128−135. Y. Kalman... S. C. G. M. Vetter.. S. A. Chem. H... Their. Marubayashi. S.. Ueda. (2011) Evaluation of DNA melting analysis as a tool for species identification. Z. Moss.Chemical Research in Toxicology (11) Schlawicke. GSTM1. Nucleic Acids Res.. (13) Steinmaus. W.. Zheng-FA. Ercan. Hsueh.. Pharmacogenomics 8. E.gene-quantification. M. (23) Kumar. C. E. http://www. (2009) Copy-number variation genotyping of GSTT1 and GSTM1 gene deletions by real-time PCR. (44) Bell. J. (2011) Exposure to arsenic in drinking water is associated with increased prevalence of diabetes: A cross-sectional study in the Zimapan and Lagunera regions in Mexico. Wichmann. Capella... Pharmacogenomics 11. High Resolution Melting: Optimization Strategies. Technical Note No. U. C. and Styblo. (15) Landi. R. M. Biophys.. Agudo... Stegmaier. Valenzuela. C. 1159−1164. C. L. (31) Wittwer. Seiler. Funct.. N. 1307−1314. M. M... J. H.. 1243−1245. and metabolism of arsenic. 51. (2006) Genetic polymorphisms of GSTT1. Sauter. B. and Holloway. M. Bolt. Y. Giri. T. C. D. S. M. P. E. Pharmacogenomics 8. s. 166−177.. 73−83... C. (19) Hsu. Giglio. N.. Bates. J. (2003) High-resolution genotyping by amplicon melting analysis using LCGreen. Walton. 35. (40) Del Razo. S. S. and Chang-Claude. I. J. Res. N. G... A. M. S... (41) Wittwer. M.. M. O. 223 Article GSTT1 and GSTP1 polymorphisms using real-time polymerase chain reaction. Mihara. Clin. J. Richards. K. A. (43) Montero. GSTP1 polymorphisms..

. Toxicol.. A. Anaya-Palafox. Alexandrie.. M..doi. S. J. L... C.. V... Biol. J.. G. Oda.. Am. X. 52−54.. D. L. M.. Xu. D. Gaspari. Costa. H.... J. Nazar-Stewart.... (46) Zhang. Hildesheim. Dell’Omo. (1995) Cytochrome P4501A1 gene polymorphism and homozygous deletion of the glutathione S-transferase M1 gene in urothelial cancer patients. J. F. M. J. L.. Boffetta. S. A.. R. Anaya-Palafox. E. L.. L. and Taioli. Coutelle. Benhamou. X. Rannug. J. (2007) Allele and haplotype distribution for 16 Y-STRs. M. E. Dolzan. 8.. Campos-Gutierrez. H. FloresFlores... L. W. Anthropol. G. J.. Inatomi. C. 284−294. M. (47) Garte. K. Hao. G.. I.. A. 216−224 . R. G.. 25. Med. (2009) Pre-Hispanic Mesoamerican demography approximates te present-day ancestry of Mestizos throughout the territory of Mexico. Haugen... J. A.. and Sugita. Lechner. Risch. J. Romkes. Hum. Ingelman-Sundberg. Ryberg. F. Zhao. I. Shields. F.. Agundez.Chemical Research in Toxicology (45) Katoh... (52) Gutierrez-Alarcon. Cerda-Flores. M.. 847−857. A. Epidemiol.. Romero-Gomez. Sandoval. C. A. Martinez.org/10. M. J. Stücker. Z. smoking.. P.. L. Gonzalez-Martin. and Spanish Group for the Study of Drug-Induced Liver Disease (2008) Glutathione S-transferase M1 and T1 null genotypes increase susceptibility to idiosyncratic drug-induced liver injury. L.. Le Marchand. Kihara. J... S. Maugard. M. G.. J. 1239−1248. Biomarkers Prev. O’Brien.. C..... H. (2001) Metabolic gene polymorphism frequencies in control populations. Y.. Kremers. D. A.. P.. Kalina... Leg. Morita. Jiang.. GarciaMartin. Hsieh. (50) Torres-Rodriguez. and Rangel-Villalobos. Nagaoka. K. R. B. T. Sugimura. A..... Y.. Am.. M. 10. Kiyohara. Sim. 588−596. Kang. R. Brockmoller. Lazarus. and Rangel-Villalobos. J. Peters. Carcinogenesis 16. Paez-Riberos. A.... J.. Salagovic. Noda.. Bathum. Strange.. (49) Lucena. Article 224 dx. M. T. R. Schoket. van Lieshout.. (2006) Forensic potential of the STR DXYS156 in Mexican populations: inference of X-linked allele null. Ambrosone. Martinez-Corteas. G. R. I. Ceballos-Quintal. and Rangel-Villalobos.. D. Song. E. J. H. Yu.. Med. A. Y-filter kit) in the state of Chihuahua at North Center of Mexico. (2008) GSTM1 and GSTT1 gene deletions and the risk for nasopharyngeal carcinoma in Han Chinese. 401− 409. M. Zeng. C.. (AmpF1STR. Ulzurrun.. Martinez-Cortes. Planas. A. Rubi-Castellanos. Parl. Seidegard. 139. J. H.. A.. E.. L. Munoz-Valle. L. (48) Guo.. Borraz. Cuellar-Nevarez. Cascorbi. (51) Salazar-Flores... (2010) Population structure and paternal admixture landscape on present-day MexicanMestizos revealed by Y-STR haplotypes. P. G. M. I. H. Daly. N. and Ma. S. S.1021/tx200457u | Chem. and Winkler. Rebbeck. Canseco-Avila. M.. Barojas-Perez. Autrup. Morales-Vallejo. X. Phys. D. Y. (2011) Glutathione S-transferase M1 (GSTM1) and glutathione S-transferase T1 (GSTT1) null polymorphisms.. Dondiego-Aldape.. K. Fernandez.. C.. Hepatology 48. E. C. Pastorelli. Fryer. J.. I. Hein. G. 9... M. M.... K.. A. Muñoz-Valle.. F. Breskvar. Am. 1760−1763. W. R... 154−157. Cancer Epidemiol. (53) Rubi-Castellanos.. P.. R. M.. Cancer Epidemiol.. Hirvonen. M. 22. PintoEscalante... V.. Biomarkers Prev. Anzola. Moguel-Torres. A. 17. Castiella. Clapper. C. B. E. Persson. J. Baranova. F. J.. M.. Bouchardy. M.. H... London. W.. and Rangel-Villalobos. 655−657. A. M. Autrup. Leon-Jimenez.. Sinnet.. 173. Andrade.. Res. S. Dresler. 2012. G. R.. M. To-Figueras. C. Leg. K. P. A. I. J.. Muñoz-Valle. C. Nuño-Arana. H. and their interaction in oral cancer: A HuGE review and meta-analysis.... A.. J. C.. Vineis. Y... H. R. C.. Nelson. E. Shi.. L. Roelandt. Manni..