BACKGROUND: Aplastic anemia (AA) and myelodysplastic syndrome (MDS) are marrow failure states that may be associated with chromosomal instability. An absence of the glutathione S-transferase (GST) enzyme may genetically predispose individuals to AA or MDS. PROCEDURE AND RESULTS: To test this hypothesis, we determined the GSTM1 and GSTT1 genotypes in a total of 196 patients using multiplex PCR. The GSTT1 null genotype was found to be overrepresented in Caucasian, Asian, and Hispanic patients with either AA or MDS. We confirmed a difference in the expected frequency of the GSTM1 null genotype in Caucasian MDS patients. The double null GSTM1/GSTT1 genotype was also overrepresented in Caucasian AA and MDS patients. In our population, 26% of AA patients and 40% of MDS patients had a chromosomal abnormality identified by karyotype or FISH analyses for chromosomes 7 and 8. Patients with AA and the GSTT1 null genotype had an increased frequency of chromosomal abnormalities (P = 0.003). CONCLUSION: There seems to be an increased risk for AA and MDS in individuals lacking GSTT1 or both GSTM1/GSTT1

Kearns, William G.

Liu, Johnson M.

Roeg, Thomas S.

Stacey, Michael

Sutton, Joanne F.

Young, Neal S.

English

Old Dominion University

Old Dominion UniversityODU Digital CommonsBioelectrics Publications Frank Reidy Research Center for Bioelectrics2004Increased Risk for Aplastic Anemia andMyelodysplastic Syndrome in Individuals LackingGlutathione S-Transferase GenesJoanne F. SuttonMichael StaceyOld Dominion University, mstacey@odu.eduWilliam G. KearnsThomas S. RoegNeal S. YoungSee next page for additional authorsFollow this and additional works at: https://digitalcommons.odu.edu/bioelectrics_pubsPart of the Hematology Commons, Oncology Commons, and the Pediatrics CommonsThis Article is brought to you for free and open access by the Frank Reidy Research Center for Bioelectrics at ODU Digital Commons. It has beenaccepted for inclusion in Bioelectrics Publications by an authorized administrator of ODU Digital Commons. For more information, please contactdigitalcommons@odu.edu.Repository CitationSutton, Joanne F.; Stacey, Michael; Kearns, William G.; Roeg, Thomas S.; Young, Neal S.; and Liu, Johnson M., "Increased Risk forAplastic Anemia and Myelodysplastic Syndrome in Individuals Lacking Glutathione S-Transferase Genes" (2004). BioelectricsPublications. 212.https://digitalcommons.odu.edu/bioelectrics_pubs/212Original Publication CitationSutton, J. F., Stacey, M., Kearns, W. G., Rieg, T. S., Young, N. S., & Liu, J. M. (2004). Increased risk for aplastic anemia andmyelodysplastic syndrome in individuals lacking glutathione s-transferase genes. Pediatric Blood Cancer, 42(2), 122-126. doi:10.1002/pbc.10479AuthorsJoanne F. Sutton, Michael Stacey, William G. Kearns, Thomas S. Roeg, Neal S. Young, and Johnson M. LiuThis article is available at ODU Digital Commons: https://digitalcommons.odu.edu/bioelectrics_pubs/212Pediatr Blood Cancer 2004;42:122–126Increased Risk for Aplastic Anemia and MyelodysplasticSyndrome in Individuals Lacking GlutathioneS-Transferase GenesJoanne F. Sutton, MD,1,2,3 Michael Stacey, PhD,4 William G. Kearns, PhD,1 Thomas S. Rieg, PhD,3Neal S. Young, MD,1 and Johnson M. Liu, MD1*BACKGROUNDAcquired aplastic anemia (AA) and myelodysplasticsyndrome (MDS) are distinct but related bone marrowfailure syndromes, diagnosed by marrow morphology.Approximately 20% of MDS cases present with a hypo-cellular marrow and, in turn, AA may evolve to MDS ordevelop cytogenetic abnormalities [1]. For each disorder,the etiology is unknown, however, there may be gene-environment interactions that predispose to disease [1,2].For example, an increased incidence of AA has beenobserved in Asia [3]. Glutathione S-transferase (GST) is akey biometabolic enzyme for a number of chemical toxins,including the organophosphates, alkylating agents, epox-ides, and polycyclic aromatic hydrocarbons [4]. Low GSTactivity may genetically predispose some individuals tomarrow failure, perhaps as a result of increased suscept-ibility to endogenous or environmental toxins.Glutathione S-transferase M1 (GSTM1) and T1(GSTT1) genes are polymorphic in humans. It has beenwell established that the frequencies of GSTT1 null andGSTM1 null genotypes in healthy human subjects varygreatly among different ethnic populations and can reachfrequencies as high as 0.58 (GSTT1 null) and 0.67 (GSTM1null) in Asians [5–10]. Racial or ethnic variation in bonemarrow failure risk may reflect innate biological suscept-ibility as well as differences in environmental exposures orsocioeconomic and demographic factors [1,11].Recently, increased frequencies of GSTM1 and GSTT1gene deletions were reported in 57 Korean patients withAA [12]. Similarly, a higher risk for MDS was noted inindividuals with GSTT1 gene deletions, although subse-quent studies of patients with different ethnicities yieldedconflicting results [13–19]. Here, we have determined thefrequency of homozygous GSTM1 and GSTT1 genedeletions in an ethnically heterogeneous cohort of AAand MDS patients referred to the National Institutes ofHealth. We also expanded our study to determine if thereBackground. Aplastic anemia (AA) andmyelodysplastic syndrome (MDS) are marrowfailure states that may be associated with chro-mosomal instability. An absence of the glu-tathione S-transferase (GST) enzyme maygenetically predispose individuals to AA orMDS. Procedure and Results. To test thishypothesis, we determined the GSTM1 andGSTT1 genotypes in a total of 196 patients usingmultiplex PCR. The GSTT1 null genotype wasfound to be overrepresented in Caucasian,Asian, and Hispanic patients with either AA orMDS. We confirmed a difference in the expec-ted frequency of the GSTM1 null genotype inCaucasianMDSpatients.ThedoublenullGSTM1/GSTT1 genotype was also overrepresented inCaucasian AA and MDS patients. In our popula-tion, 26%ofAApatients and40%ofMDSpatientshad a chromosomal abnormality identified bykaryotypeorFISHanalysesforchromosomes7and8. Patients with AA and theGSTT1 null genotypehad an increased frequency of chromosomalabnormalities (P¼ 0.003). Conclusion. Thereseems to be an increased risk for AA and MDS inindividualslackingGSTT1orbothGSTM1/GSTT1.Pediatr BloodCancer 2004;42:122–126.Published 2003Wiley-Liss, Inc.{Key words: aplastic anemia; chromosomal abnormality; genotype; glutathioneS-transferase; myelodysplastic syndrome——————1Hematology Branch, NHLBI, Bethesda, Maryland2Department of Pediatrics, Uniformed Services University of theHealth Sciences, Bethesda, Maryland3Naval Medical Center Portsmouth, Portsmouth, Virginia4Center for Pediatric Research, Eastern Virginia Medical School,Norfolk, VirginiaJoanne F. Sutton and Michael Stacey contributed equally to the work.Grant sponsor: Division of Intranural Research, NHLBI, NIH.*Correspondence to: Johnson M. Liu, Division of Hematology/Oncology, Mount Sinai School of Medicine, One Gustave L. LevyPlace, Box 1079, New York, NY 10029.E-mail: Johnson.Liu@mssm.eduReceived 25 June 2003; Accepted 13 November 2003Published 2003 Wiley-Liss, Inc.yThis article is a US government work and, as such, is in the public domain of the United States of America.DOI 10.1002/pbc.10479Published online 15 December 2003 in Wiley InterScience (www.interscience.wiley.com)was a difference in the frequency of chromosomal abnor-malities in patients with GSTM1 or GSTT1 null genotypescompared to those with GSTM1 or GSTT1 present.STUDY DESIGN AND METHODSThe study population included 196 patients, 108diagnosed with either moderate or severe AA and 88 withMDS, referred to the National Heart Lung and BloodInstitute, Hematology Branch between 1997 and 2000(age range, 3–85 years; median age, 41 years; 112 malesand 84 females). Standard diagnostic criteria wereutilized, and no patients with congenital bone marrowfailure (Fanconi anemia) were included. Race was self-reported by each patient (142 Caucasian, 28 Hispanic,16 Black, and 10 Asian). To assure patient anonymity, noother patient identifiers were used. Cytogenetic data byconventional karyotype of the bone marrow were availablefor 185 (94%), and a bone marrow pellet was available for113 (57%). Control values were based on selecting themedian value of previously published gene frequencies ofhealthy human subjects sorted by race [5–10].DNA was isolated from bone marrow samples usingQiagen tissue prep kit according to the manufacturer’sguidelines (Qiagen, Valencia, CA). Analysis for theGSTM1 and GSTT1 genes was performed by multiplexPCR, as previously described [5]. In brief, 30 ng of isolatedDNA was analyzed in a single assay for GSTM1, GSTT1,andCYP1A1. All primers were custom synthesized at LifeTechnologies (Invitrogen, Corp, Rockville, MD). Primerswere: GSTM1 (50-GAACTCCCTGAAAAGCTAAA-GC-30) and (50-GTTGGGCTCAAATATACGGTGG-30);GSTT1 (50-TTCCTTACTGGTCCTCACATCTC-30) and(50-TCACCGGATCATGGCCAGCA-30); CYP1A1 (50-GAACTGCCACTTCAGCTGTCT-30) and (50-CAGCT-GCATTTGGAAGTGCTC-30). A 50 ml reaction mixturecontaining 30 pmol of each primer, 200 mmol dNTPs, 5 mlof 10 PCR buffer, 2 U Taq DNA polymerase (QiagenPCR Core Kit) was amplified in a PTC-200 PeltierThermal Cycler. PCR conditions were as follows: initialmelting 948C 5 min, followed by 35 cycles of melting948C 2 min, annealing 598C 1 min, extension 728C1 min, and a final extension at 728C 10 min. The PCRproducts were analyzed on an ethidium bromide stained2% agarose gel. The presence or absence of GSTT1 andGSTM1 genes was determined by examination forcharacteristic bands at 480 bp (GSTT1) and 215 bp(GSTM1). A band at 312 bp corresponding to the internalcontrol (CYP1A1) was used to confirm successful PCRamplification.Fluorescent in situ hybridization (FISH) was performedon 113 patients with available bone marrow pellets and14 normal bone marrow controls, using alpha satelliteDNA probes for chromosomes 7 and 8 (VYSIS, Inc.,Downer’s Grove, IL). A probe mixture was applied to thetarget area and codenatured at 758C for 3 min followed byhybridization at 428C. Approximately 300 interphasenuclei per slide were scored for monosomy 7 and trisomy 8as per criteria established by Vysis. Aneuploidy wasestablished for monosomy 7 as >2.91% and for trisomy 8as >2.8% of cells, based on results obtained in the normalsamples.Descriptive analyses included gene frequencies andcontingency tables. Associations between variables weretested with chi-square and Fisher exact test whereappropriate. Standard odds ratio and power were com-puted. Significance was obtained for P< 0.05.RESULTS AND DISCUSSIONFrequency of GSTM1 and GSTT1 Null Genotypes inBone Marrow Failure Patients Stratified by RaceControl values were selected (see ref. [8], expectedfrequencies, Table I) for each genotype based on themedian value of previously published gene frequencies ofhealthy human subjects sorted by race. A recent studyreported metabolic gene polymorphism frequencies forover 15,000 control subjects. Significant differences wereobserved between races, as expected, but much lessheterogeneity was observed between White populationsfrom different countries and no differences were seen byage, sex, or type of controls (hospital patients vs. popu-lation controls).The GSTT1 null genotype was overrepresented inCaucasian, Hispanic, and Asian patients with either AA orMDS (Table I). In contrast, the GSTM1 null genotype wassignificantly overrepresented only in Caucasian MDSpatients. No significant association was found for the nullgenotypes in Black patients. The double null genotype wassignificantly overrepresented only in Caucasians witheither AA or MDS.Data on the GSTM1 and GSTT1 null genotypes inmarrow failure patients had previously been reported inrelatively homogeneous populations, many of which didnot include Black or Hispanic patients. For example, anincreased frequency of the GSTT1 null genotype wasreported in White American and Japanese MDS patients[13,19]. Subsequent studies were unable to confirm thisresult in British, French, and Japanese populations [14–17]. Overrepresentation of the GSTM1 null genotype wasalso reported in Greek MDS patients [18]. Acquired AAhas not been as extensively examined for GST genedeletions. A study of 57 Korean AA patients reported asignificant increase in frequency of the GSTM1 or GSTT1null genotypes [12], however, a Brazilian study of 37 AApatients found no difference [20]. Ethnic variation infrequency of GST polymorphisms and different environ-mental exposures may account for the lack of concor-dance in the literature. Our referral population wasethnically and geographically diverse, and we haveAA, MDS, and G-ST Genes 123TABLEI.FrequencyofGSTM1andGSTT1NullGenotypeStratifiedbyRaceandDiagnosisTotalGSTM1nullGSTT1nullGSTM1/GSTT1nullNo.OddsratioExpfreqFreqP-valuePowerNo.OddsratioExpfreqFreqP-valuePowerNo.OddsratioExpfreqFreqP-valuePowerCaucasianTotal142911.780.520.64<0.0030.095470.490.150.33<0.0010.789340.320.070.24<0.0010.998AA69401.380.520.580.28210.440.150.30<0.0010.265150.280.070.22<0.0010.724MDS73512.320.520.700.0020.163260.550.150.36<0.0010.723190.350.070.26<0.0010.995Black Total1640.330.320.250.5860.600.240.380.2120.140.040.13AA1320.180.320.150.2150.630.240.390.2210.080.040.076MDS3a22.000.320.660.1910.500.240.330.05110.500.040.330.0080.957HispanicTotal28130.870.510.460.6380.400.10.29<0.0010.4734a0.17NA0.14AA1880.800.510.440.5750.380.10.280.010.2743a0.20NA0.17MDS1051.000.510.500.9530.430.10.300.030.2461aNA0.1Asian Total1061.500.550.600.7584.000.480.800.0430.19540.670.250.40.27AA851.670.550.630.6763.000.480.750.1330.600.250.380.41MDS2a11.000.550.50NA20.481.0NA10.250.5AA,aplasticanemia;MDS,myelodysplasticsyndrome;Expfreq,expectedfrequency;NA,notavailable.aNumberofpatientstoolowtodeterminestatisticalsignificance.124 Sutton et al.confirmed a difference in expected frequency of theGSTT1 null genotype and AA or MDS in White andHispanic patients. However, unlike the Korean study, wefound no significant difference in frequency of theGSTM1null genotype and AA.A striking difference in frequency of the double nullGSTM1/GSTT1 genotype in Caucasian AA and MDSpatients was also observed. The double null genotype is animportant variable to investigate because nullizygosity forGSTM1 and GSTT1 has been linked independently tovarious cancers [4], and deletion of both genes may havedifferent significance from that of either gene alone.High Frequency of Chromosomal Abnormalities Seenin AA Patients With GSTT1 Null GenotypeOf the 187 patients with cytogenetics available, 26 of102 (26%) patients with AA and 34 of 85 (40%) patientswith MDS had a chromosomal abnormality identified byeither conventional karyotype or FISH (Table II). Of thosepatients with bone marrow pellets available, FISHanalyses identified monosomy 7 or trisomy 8 in 26 (44%)AA patients, of whom 18 had no chromosomal abnorm-alities identified by conventional karyotype. FISH ana-lyses identified 27 (50%) MDS patients with eithermonosomy 7 or trisomy 8, of whom 8 had a normal con-ventional karyotype. The karyotype of each patient wasnot known at the time that FISH was performed, in order toprevent bias in scoring.FISH analysis is known to be more sensitive thanconventional cytogenetics. In this setting, FISH was limit-ed to determining aneuploidy of chromosomes 7 and 8,which are two frequent chromosomal abnormalitiesidentified in MDS and AA [1,2]. Among the MDS pa-tients, there was no significant correlation between theGSTM1 null and GSTT1 null genotypes and chromosomalabnormalities. However, a significant difference infrequency of the GSTT1 null genotype was observed inAA patients with chromosomal abnormalities, consistentwith findings from a Korean study [12]. The etiology ofchromosomal aberrations in MDS cannot be correlated toa deletion of either theGSTT1 orGSTM1 gene. However, itis possible that the GSTT1 null genotype decreasesan individual’s ability to biometabolize toxins, whichmay lead to an increased risk for chromosomal aberrationsin AA.ACKNOWLEDGMENTWe thank Kathleen L. McManaman (United StatesDepartment of Veterans Affairs, Washington, DC) andDean Follmann (National Institutes of Health, Bethesda,MD) for their assistance in the statistical analyses. TheChief, Navy Bureau of Medicine and Surgery, Washing-ton, DC, Clinical Investigation Program sponsored LCDRJoanne F. Sutton, MC, USNR. The views expressed in thisstudy are those of the authors and do not reflect the officialpolicy or position of the Department of the Navy,Department of Defense, or the United States Government.REFERENCES1. Young NS. Acquired aplastic anemia. Ann Intern Med 2002;136:534–546.2. Barrett J, Saunthararajah Y, Molldrem J. Myelodysplastic syn-drome and aplastic anemia: Distinct entities or diseases linked by acommon pathophysiology? Semin Hematol 2000;37:15–29.3. Young NS. Acquired aplastic anemia. JAMA 1999;282:271–278.4. Hayes JD, Pulford DJ. The glutathione S-transferase supergenefamily: Regulation of GST and the contribution of the isoenzymesto cancer chemoprotection and drug resistance. Crit Rev BiochemMol Biol 1995;30:445–600.5. Abdel-Rahman SZ, El Zein RA, Anwar WA, et al. A multiplexPCR procedure for polymorphic analysis of GSTM1 andGSTT1 genes in population studies. Cancer Lett 1996;107:229–233.6. Au WW, Oh HY, Grady J, et al. Usefulness of genetic susceptibilityand biomarkers for evaluation of environmental health risk.Environ Mol Mutagen 2001;37:215–225.7. Chen CL, Liu Q, Relling MV. Simultaneous characterization ofglutathione S-transferase M1 and T1 polymorphisms by polymer-ase chain reaction in American whites and blacks. Pharmacoge-netics 1996;6:187–191.8. Garte S, Gaspari L, Alexandrie AK, et al. Metabolic genepolymorphism frequencies in control populations. Cancer Epide-miol Biomarkers Prev 2001;10:1239–1248.9. Nelson HH, Wiencke JK, Christiani DC, et al. Ethnic differences inthe prevalence of the homozygous deleted genotype of glutathioneS-transferase theta. Carcinogenesis 1995;16:1243–1245.10. Rebbeck TR. Molecular epidemiology of the human glutathione S-transferase genotypes GSTM1 and GSTT1 in cancer susceptibility.Cancer Epidemiol Biomarkers Prev 1997;6:733–743.11. Perera FP. Environment and cancer: Who are susceptible? Science1997;278:1068–1073.12. Lee KA, Kim SH, Woo HY, et al. Increased frequencies ofglutathione S-transferase (GSTM1 and GSTT1) gene deletions inKorean patients with acquired aplastic anemia. Blood 2001;98:3483–3485.TABLE II. Patient Karyotype Stratified by Genotypeand DiagnosisGSTM1null GSTT1 nullGSTM1/T1 nullGSTM1/T1 presentAANL karyotypea 38 22 14 32Abn karyotypeb 13 13 7 7P-value (power) 0.160 0.003 (0.243) 0.066MDSNL karyotypea 36 17 11 12Abn karyotypeb 23 15 11 7P-value 0.733 0.239 0.200AA, aplastic anemia; MDS, myelodysplastic syndrome; NL, normalkaryotype; Abn, abnormal karyotype.aKaryotype from conventional and FISH analysis on bone marrow cells.bIncludes aneuploidy and structural defects.AA, MDS, and G-ST Genes 12513. Chen H, Sandler DP, Taylor JA, et al. Increased risk formyelodysplastic syndromes in individuals with glutathionetransferase theta 1 (GSTT1) gene defect. Lancet 1996;347:295–297.14. Basu T, Gale RE, Langabeer S, et al. Glutathione S-transferasetheta 1 (GSTT1) gene defect in myelodysplasia and acute myeloidleukaemia. Lancet 1997;349:1450.15. Atoyebi W, Kusec R, Fidler C, et al. Glutathione S-transferase genedeletions in myelodysplasia. Lancet 1997;349:1450–1451.16. Preudhomme C, Nisse C, Hebbar M, et al. Glutathione S transferasetheta 1 gene defects in myelodysplastic syndromes and theircorrelation with karyotype and exposure to potential carcinogens.Leukemia 1997;11:1580–1582.17. Okada M, Okamoto T, Wada H, et al. Glutathione S-transferasetheta 1 gene (GSTT1) defect in Japanese patients with myelodys-plastic syndromes. Int J Hematol 1997;66:393–394.18. Tsabouri SE, Georgiou I, Alamanos I, et al. Increased prevalence ofGSTM(1) null genotype in patients with myelodysplastic syn-drome: A case-control study. Acta Haematol 2000;104:169–173.19. Sasai Y, Horiike S, Misawa S, et al. Genotype of glutathione S-transferase and other genetic configurations in myelodysplasia.Leuk Res 1999;23:975–981.20. Arruda VR, Lima CS, Grignoli CR, et al. Increased risk for acutemyeloid leukaemia in individuals with glutathione S-transferasemu 1 (GSTM1) and theta 1 (GSTT1) gene defects. Eur J Haematol2001;66:383–388.126 Sutton et al.

Increased Risk for Aplastic Anemia and Myelodysplastic Syndrome in Individuals Lacking Glutathione S-Transferase Genes

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Increased Risk for Aplastic Anemia and Myelodysplastic Syndrome in Individuals Lacking Glutathione S-Transferase Genes

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