A variant allele of Growth Factor Independence 1 (GFI1) is associated with acute myeloid leukemia

A variant allele of Growth Factor Independence 1 (GFI1) is associated with acute myeloid leukemia
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  MYELOID NEOPLASIA Avariantalleleof  GrowthFactorIndependence1 ( GFI1 )isassociatedwithacutemyeloidleukemia Cyrus Khandanpour, 1-3 Christian Thiede, 4 Peter J. M. Valk, 5 Ehssan Sharif-Askari, 1 Holger Nu¨ckel, 3 Dietmar Lohmann, 6 Bernhard Horsthemke, 6 Winfried Siffert, 7 Andreas Neubauer, 8 Karl-Heinz Grzeschik, 9 Clara D. Bloomfield, 10 Guido Marcucci, 10 Kati Maharry, 10,11 Marilyn L. Slovak, 12 BertA. van der Reijden, 13 Joop H. Jansen, 13 Hans K. Schackert, 14 KhashayarAfshar, 1 Susanne Schnittger, 15 Justine K. Peeters, 5 Frank Kroschinsky, 4 Gerhard Ehninger, 4 Bob Lowenberg, 5 Ulrich Du¨hrsen, 3 and Tarik Mo¨ro¨y 1,2 1 Institut de Recherches Cliniques de Montre´al (IRCM) and De´partement de Microbiologie et Immunologie, Universite´ de Montre´al, Montre´al, QC;  2 Institut fu¨rZellbiologie (Tumorforschung), Universita¨tsklinikum Essen and Zentrum fu¨r Medizinische Biologie, Universita¨t Duisburg-Essen, Essen, Germany;  3 Klinik fu¨rHa¨matologie, Universita¨tsklinikum Essen, Essen, Germany;  4 Medizinische Klinik und Poliklinik1, Universita¨tsklinikum Carl Gustav Carus, Dresden, Germany; 5 Department of Hematology, Erasmus University Medical Center, Rotterdam, The Netherlands;  6 Institut fu¨r Humangenetik, Universita¨tsklinikum Essen, Essen,Germany;  7 Institut fu¨r Pharmakogenetik, Universita¨tsklinikum Essen, Essen, Germany;  8 Abteilung fu¨r Ha¨matologie, Onkologie und Immunologie,Universita¨tsklinikum Giessen und Marburg, Marburg, Germany;  9 Abteilung fu¨r Humangenetik, Philipps-Universita¨t Marburg, Marburg, Germany;  10 Division ofHematology and Oncology, Department of Internal Medicine, Comprehensive Cancer Center, The Ohio State University, Columbus;  11 The Cancer and LeukemiaGroup B, Statistical Center, Durham, NC; 12 Department of Cytogenetics, City of Hope, Duarte, CA;  13 Laboratory of Hematology, Department of LaboratoryMedicine, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands;  14 Department of SurgicalResearch, Technische Universita¨t Dresden, Dresden, Germany; and  15 MLLMu¨nchner Leuka¨mielabor, Mu¨nchen, Germany The  GFI1  gene encodes a transcriptionalrepressor,whichregulatesmyeloiddiffer-entiation. In the mouse, Gfi1 deficiencycauses neutropenia and an accumulationof granulomonocytic precursor cells thatis reminiscent of a myelodysplastic syn-drome.Wereportherethatavariantalleleof  GFI1  ( GFI1 36N  ) is associated with acutemyeloidleukemia(AML)inwhitesubjectswith an odds ratio of 1.6 ( P  < 8  10  5 ).The  GFI1 36N  variant occurred in 1806 AMLpatients with an allele frequency of 0.055compared with 0.035 in 1691 healthy con-trol patients in 2 independent cohorts.WeobservedthatbothGFI1variantsmain-tain the same activity as transcriptionalrepressorsbutdifferintheirregulationbythe AML1/ETO (RUNX1/RUNX1T1) fusionprotein produced in AML patients with at(8;21) translocation. AML1/ETO interactsand colocalizes with the more commonGFI1 36S form in the nucleus and inhibitsits repressor activity. However, the vari-ant GFI1 36N protein has a different sub-nuclear localization than GFI1 36S . As aconsequence, AML1/ETO does not colo-calizewithGFI1 36N andisunabletoinhibitits repressor activity. We conclude thatboth variants of GFI1 differ in their abilityto be regulated by interacting proteinsand that the GFI1 36N variant form exhibitsdistinct biochemical features that mayconfer a predisposition to AML. (Blood.2010;115:2462-2472) Introduction In acute myeloid leukemia (AML), malignant blasts of myeloidsrcin accumulate in the bone marrow. 1,2 It has been shown fordifferent mouse models that deficiency of myeloid transcriptionfactors such as CCAAT/enhancer-binding protein alpha (CEBPA)and PU.1 can promote the development of AML. 3-5 In addition,mutations in certain transcription factors, such as CEBPA, arenot only causative for AML in experimental models but alsoinfluence the prognosis of patients with AML. 6,7 Similar toCEBPA and PU.1,  Growth Factor Independence 1  ( GFI1 ) is ahematopoietic transcription factor. 8,9 The GFI1 protein consistsof a N-terminal Snail/Growth factor independence 1 domain and6 C-terminal zinc fingers. 10,11 Gfi1 is involved in: T-cell lym-phomagenesis; maturation and activation of B, T, and dendriticcells; regulation of alternative splicing of the CD45 gene inT cells; development of sensory epithelial cells in the inner ear,development of neuroendocrine lung, and Purkinje cells. 10-20 Inaddition,  Gfi1   /   mice show reduced self-renewal of hematopoi-etic stem cells and a block in the development of granulocytescausing severe neutropenia. 8,9,21,22 Several patients with congenital neutropenia have mutations inthe  GFI1  gene, generating a dominant-negative loss of function. 23 The combination of a severe neutropenia and the accumulation of atypical monocytes 8 in  Gfi1 -deficient mice is reminiscent of myelodysplastic diseases and thus suggestive of a role of Gfi1 inmyeloid leukemia. These observations prompted us to investigatewhether mutations or single nucleotide polymorphisms (SNPs)exist that may play a role in the pathogenesis ofAML. In this studywe report a nonsynonymous SNP, which leads to the replacementof serine by asparagine in the N-terminal part of the coding regionof GFI1. The frequency of this SNP has been determined in2 different cohorts of patients and control patients in Germany andThe Netherlands. Our studies suggest that this variation is associ-ated with AML and has a different biochemical function andsubnuclear localization compared with the more common variant. Submitted August 26, 2009; accepted December 18, 2009. Prepublishedonline as  Blood   First Edition paper, January 14, 2010; DOI 10.1182/blood-2009-08-239822.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’in accordance with 18 USC section 1734. © 2010 by TheAmerican Society of Hematology2462 BLOOD, 25 MARCH 2010    VOLUME 115, NUMBER 12 For personal use only.on March 22, 2016. by guest www.bloodjournal.orgFrom   Methods Patients and control patients AML patients were identified on the basis of their clinical-pathologic 1,2 presentations. Peripheral blood and bone marrow samples were collectedbefore initiation of treatment. The study was approved by the InstitutionalReview Boards of the participating institutions, and all patients providedwritten informed consent in accordance with federal and institutionalguidelines and the Declaration of Helsinki. Patients  Patients from Germany.  All AML patients from Germany were recruitedby the Study Alliance Leukemia (AML96; AML2003) and by the Univer-sity Hospitals of Dresden, Marburg, and Essen between 1993 and 2003.Patients were white. The AML96 and AML2003 group recruited theirpatients from more than 80 hospitals all over Germany. The mean age(  SD) of the German patient cohort was 53.56 years (  16.57 years;range, 19-86 years) with 54% male patients. More than 90% of the eligibleAMLpatients presenting to the University Hospitals of Dresden, Essen, andMarburg were recruited for genetic studies. The study documentation didnot record what percentage of eligible patients took part in the other centers.Because most AML patients in these centers are treated according to studyprotocols, the participation rate is expected to be also greater than 90%.Smoking status, duration, and intensity of smoking were not recordedconsistently and were only available for patients from Essen and Marburg.  Patients from The Netherlands.  All AML patients from The Nether-lands were recruited from the European Organization for Treatment andResearch of Cancer (EORTC) study group by the University Hospitals atNijmegen and from the Dutch Hemato-Oncology Cooperative GroupHemato-Oncologie voor Volwassenen Nederland (HOVON) at Rotterdambetween 1989 and 2007. Patients were white. The mean age (  SD) of thispatient cohort was 50.21 years (  14.75 years; range, 14-77 years), with52% of them being male. Study protocols.  The details of the study protocols have beenpublished previously 6,24-26 or are registered at the National Cancer Institute(EORTC 06 991, NCT 00004128, AML-12/06 991). Patients first registerand are later randomized, if at all, when their cytogenetic risk profile isknown. At this time, patients had already agreed to provide material forscientific analyses. Thus, this should not affect the composition of thecohort or  GFI1  allele frequency.  Patients with t(8;21) translocations.  Besides the t(8;21) patients fromthe study groups in Germany and The Netherlands, additional white t(8;21)patients were recruited from the City of Hope (COH), the Cancer andLeukemia Group B (CALGB), and the Munich Leukemia Laboratory tocorrelate relapse-free survival with the presence of a variant  GFI1 36N  allele.These patients were recruited between 1992 and 2004. Only those patientswere taken into account for whom complete follow-up was available andwho were treated in a comparable way regarding induction, consolidation,and maintenance therapy (eg, excluding autologous or allogenic stem celltransplantation). Smoking status.  Because there is some controversy about the possiblerole of smoking in AML, which is not yet fully established, 27,28 we alsocompared the smoking status between patients and control persons. Thestudy groups do not record smoking status regularly because it is not anestablished risk factor for AML. For 80% of a control group in Germanyand for 70% and 90% of patients from Essen and Marburg, respectively, thesmoking status was determined. The odds ratio for smokers carrying the GFI1 36N  allele to develop AML was 3.4 (Table 1) and 4.3 for nonsmokers(Table 1). Thus, we conclude that smoking does not affect the associationbetween  GFI1 36N  andAML Control persons Control persons from Germany.  The control groups were recruited by theUniversity Hospitals of Essen, Marburg, and Dresden and consisted of healthy blood or stem cell donors recruited between 1996 and 2003. Allcontrol persons were white. Another control group consisted of samplesderived from the Department of Pharmacogenetics, University HospitalEssen, with known smoking status and was randomly derived frommandatory citizen registries in the Ruhr area of Germany. These partici-pants were neither physician- nor self-referred. For 80% of these controlpatients, the smoking status was known. The median age (  SD) of theGerman control group was 46.03 years (  12.27 years; range, 19-86 years)with 55% of them being male.All control persons were white. Control persons from The Netherlands.  The Dutch control patientswere recruited by the University Hospitals of Nijmegen and Rotterdam andconsisted of healthy blood donors. Control patients were recruited between1996 and 2006. The median age (  SD) of Dutch control group was53.58 years (  13.86 years; range, 19-87 years) with 50% of all partici-pants being male.All control persons were white.  Probability of control persons and patients attending the sameinstitutions.  The diagnosis and subsequent therapy (including a possiblesubsequent stem cell therapy) of AML requires a specialized hematologiclaboratory and department. This type of expertise can only be provided byuniversity hospitals or university-affiliated hospitals such as the onesproviding the samples in Germany and The Netherlands for the presentstudy. It is very likely that the vast majority of allAMLpatients would visitthe same institutions in their local area, which were taking part in our study.This implies in case of Essen, Marburg, Dresden, Rotterdam, and Nijmegen Table 1. Distribution of  GFI1 36N inAML patients and healthy control patients Population AA GA GGFrequencyallele A OR  P  *  95% CIGerman and Dutch patients and control patients Patients 1 195 1610 0.054  0.005 1.6   8  10  5 1.3-2Control patients 1 118 1572 0.035  0.003 Patients by location Germany patients 1 134 1129 0.053  0.004 1.6   2  10  3 1.2-2Germany control patients 1 87 1162 0.035  0.004The Netherlands patients 0 61 481 0.056  0.007 1.6 .02 1.1-2.6The Netherlands control patients 0 31 410 0.035  0.006 According to smoking status †Smokers AML 0 9 39 0.09  0.028 3.4 .04 1.1-11.8Smokers control patients 0 4 59 0.03  0.015Nonsmokers AML 0 13 41 0.12  0.016 4.3 .003 1.5-12.2Nonsmokers control patients 0 6 82 0.03  0.013Allele frequencies for the  GFI1 36N  variant among white AML patients and control patients in Germany and in The Netherlands.  GFI1 36N  was enriched 1.6-fold in AMLpatients ( P   8  10  5 ) compared with the control population, which was confirmed after adjusting for age and sex.AMLindicates acute myeloid leukemia; CI, confidence interval;  GFI1 ,  Growth Factor Independence 1 ; and OR, odds ratio.*The Hardy-Weinberg equilibrium was tested and fit expectation with the exception that the frequency of genotypeAAwas lower than expected (1 vs 5;  P   .01).†With reference to Essen and Marburg. GFI1 36N  ALLELE INAML 2463BLOOD, 25 MARCH 2010    VOLUME 115, NUMBER 12 For personal use only.on March 22, 2016. by guest www.bloodjournal.orgFrom   EDBA PGPDYSLRLE NVPAP N RADS human GFI1 36N PGPDYSLRLE NVPAP S RADS human GFI1 36S PGPDYSLRLE TVPAP G RAEG mouse Gfi1  30 40 92675kHypothetical Protein LOC 79781 GFI1EVI5  92565k Telomeric F   Block 5Block 6 GFI1 GAPDH  1 2 3 4 5 6 7 8 9 10 11 12 13 14 C 2464 KHANDANPOUR et al BLOOD, 25 MARCH 2010    VOLUME 115, NUMBER 12 For personal use only.on March 22, 2016. by guest www.bloodjournal.orgFrom   that the control persons would most likely visit these same institutions inthe event they developedAML. Therapeutic regimens of patients Patients with AML (except FAB M3) were treated according to thepublishedmulticenterchemotherapyprotocols(Marburg,DeutscheStudien-initiative Leuka¨mie [DSIL], EORTC, CALGB, or COH). 6,24-26 A completeremission was achieved if neutrophils (  1000 or  1500 neutrophils/   L)and platelets (  100 000 platelets/   L) recovered in peripheral blood, noblasts were detected in peripheral blood, no signs for extramedullar tumormasses were found, and less than 5% blasts were detectable in thereconstituted bone marrow. The overall survival analysis was restricted topatients younger than 65 years with de novo AML treated in the DSIL (ie,exclusion of AML cases evolving from a preceding myeloid disorder orrelated to previous anticancer therapy). Relapse-free survival refers torelapse in patients who have initially attained a complete remission.Disease-free survival is also a clinical end point that we use in completeresponders, but here the events taken into account are relapse or deathwhatever comes first. Overall survival is an analysis for all patients. Statistical analysis Odds ratios for the German and Dutch population were calculated inanalogy of the common odds ratio described by Sasieni 29 by use of the  2 test. The odds ratio for the smoker and nonsmoker cohorts (consisting of the respective control persons and patients) was calculated by use of theFisher exact test. The unit of analysis for both approaches was theindividual person. Odds ratios were adjusted for age and sex with the use of a logistic regression model with the individual person being the unit of analysis, the outcome being whether the person represents patients orcontrol persons, and the key variant being the presence or absence of the GFI1 36N  allele. The overall odds ratio for the German and Dutch popula-tions was calculated on the basis of the Mantel-Haenszel method becauseallele frequencies were almost identical and thus the individual odds ratiosfor the 2 populations also.The Fisher exact test was used with regard to the different subnuclearlocalization of GFI1 36S and GFI1 36N protein. The Hardy-Weinberg equilib-rium was calculated by log likelihood ratio  2 . For the difference betweenage, sex, platelet, and lymphocyte numbers, number of blast cells andpercentage of CD34  cells in the bone marrow, and the distribution of different cytogenetic aberrations, the Mann-Whitney  U   test was used. Forcomparing survival rates of the AML patients, the log-rank test was used.For differences in reporter-assay experiments and the mean values of AML1/ETO patients, an unpaired Student  t   test was chosen. All  P  valueswere calculated 2-sided, and values of   P  less than .05 were consideredsignificantly different. Statistical analysis was performed with Graph-PadPrism software (GraphPad software) and SysStat 12 software (SysStat). SNP, mutational analysis, and quality control Three different methods were used to genotype patients and controlpatients. GenBank accession numbers were BC074867 for the  GFI1  cDNAand NT032977 for genomic DNA.The technical quality of each sequencingresult was validated by the assessment of each individual chromatogram.Sequencing results with poor quality (low raw signal intensity, broad peaks,or high background noise) were retested and rejected if the sequencingquality was still low. As a second approach, 20 ng of genomic DNA wasused for polymerase chain reaction (PCR) amplification of Exon 2. PCRproduct was restricted with  Bfa I (New England Biolabs) for 24 hours.  Bfa Irestricts the  GFI1 36S  allele (CTA s GC), whereas the  GFI1 36N  allele (CTAAC) is not restricted. Third, genotyping was performed on an ABI PRISM7900 (ABI) or MX3005 (Stratagene) with the use of genomic  GFI1 36S  and GFI1 36N  allele-specific primers designed by ABI. Each call was verifiedwith regard to the time course of the intensity increase of the 2 fluorescencemarkers. Within this third approach,  GFI1 36N  -positive samples werereconfirmed, where possible, by the use of 20 ng of genomic DNAfor PCRamplification of Exon 2. For SNPalleles rs6662618, rs1325432, rs2031494,rs10782922, rs186682, and rs177371561, primer sets were purchased fromABI, and genotyping was performed on an ABI PRISM 7900 (ABI) or Mx3005 from Stratagene. Cell lines and cell culture Cos 7, NIH-3T3, and HeLa cells were maintained in Dulbecco modifiedEagle medium 10% fetal bovine serum (HyClone) and 1% penicillin-streptomycin (Invitrogen). Kasumi-1 cells were maintained in RPMI 1640,20% fetal bovine serum, and 1% penicillin-streptomycin. Nuclear matrix preparation, transient transfections, andreporter gene assays Nuclear matrix preparation was performed as described. 30 Cells weretransfected with 400 ng of   GFI1  binding reporter and   -Gal  reporter(400 ng) with Lipofectamine 2000 Transfection Reagent (Invitrogen). In allcases, DNA amount was added up to 1  g with empty  Flag -N3-plasmid.Promoter activity was determined 30 hours after transfection as previouslypublished. 14 All transfection settings were repeated 3 times with newplasmid preparations. For Western blot,   -GFI1 (sc-8558; Santa CruzBiotechnology) and   -ETO (sc-9737; Santa Cruz Biotechnology) wereused. Either 50 ng of or 150 ng of   GFI1 36S  or  GFI1 36N  plasmid and/or300 ng of   AML1/ETO  was used. Immunofluorescence NIH-3T3 cells were transfected with 150 ng of   GFI1 36S  , 150 ng of   GFI1 36N  ,and 50 ng of   AML1/ETO  plasmid as previously described. 14 After 30 hours,medium was removed, and cells were washed with phosphate-bufferedsaline, fixed for 10 minutes with ice-cold methanol, washed twice withphosphate-buffered saline, and equilibrated 30 minutes in solution A(10mM Tris, pH 7.5; 100mM NaCl; 0.05% Tween 20; 1% bovine serumalbumin). Cells were stained with primary antibody (Gfi1 N-20) or FlagAntibody (M2; Sigma-Aldrich), which was diluted 1:200 in solution A, fora 1-hour incubation time and secondary labeled (FITC or rhodamine)antibody (Jackson ImmunoResearch Laboratories). Nuclear staining wasdone with the use of TO-PRO-3 (Invitrogen), and cells were analyzed withthe use of confocal microscope (LSM; Zeiss) and LSM Browser 5.0software. For detecting endogenous GFI1 an   -GFI1 antibody (clone 2.5D17; Sigma-Aldrich) was used. Immunoprecipitation Cos 7 cells were electroporated with 10  g of   GFI1 36S  or  GFI1 36N  or  AML1/ETO  plasmid in Dulbecco modified Eagle medium at 500  F and Figure1.Expression,genomicrepresentation,andLDof GFI1 36N  . (A) Chromatogramofthe GFI1 36S/36N  cDNAsequenceofoneofthepatients.ChangeofGtoAatpositionc107 (c107G  A) results in the replacement of serine by asparagine. The neighboring amino acid sequences of the more common human and mouse sequences are shown.(B) Location of the  GFI1 36N  variant on genomic and protein level. The SNP is located in exon 2 and replaces a serine by an asparagine at amino acid position 36. Greenindicates N-terminal Snail/Growth factor independence 1 repressor domain of GFI1; blue, 6 C 2 H 2  zinc finger domains. (C)  GFI1  mRNAexpression in different patients.  GFI1 expression in differentAMLpatients was semiquantitatively assayed by reverse transcriptase PCR. Lanes 1 to 9 represent bone marrow and peripheral blood samples ofAMLpatients at diagnosis. Lane 10 shows cell from the t(8;21)-positive Kasumi 1 AML cell line. Lane 11 shows the control without reverse transcriptase and lane 12 a peripheralblood aphaeresis sample from an AML patient. Lane 13 shows HeLa cells (human cervical cancer) and lane 14 cells srcinating from a patient with a chronic lymphocyticleukemia (CLL). (D) Results of LD analysis in the genomic region encompassing the  GFI1  and  EVI5   loci and neighboring regions. LD block 3 spans part of  GFI1  and  EVI5  .(E) LD as determined after genotyping of 39  GFI1 36N  heterozygousAMLpatients from Essen, Marburg, and the DSILstudy group.The genotypes of 5 SNPs in the proximity ofthe  GFI1 36N  SNP were determined. The results show that  GFI1 36N  is not within the LD block that spans part of  EVI5  . (F) Results of LD of a group consisting of theaforementioned 39  GFI1 36N  heterozygousAMLpatients and 26 healthy persons homozygous for  GFI1 36S  from Essen. Similar to the analysis described previously,  GFI1 36N  isnot within the LD block that spans part of  EVI5  . GFI1 36N  ALLELE INAML 2465BLOOD, 25 MARCH 2010    VOLUME 115, NUMBER 12 For personal use only.on March 22, 2016. by guest www.bloodjournal.orgFrom   250 V.After 24 hours, cells were disrupted in Flag lysis buffer (25mM Tris,pH 7.4; 150mM NaCl; 1mM CaCl 2 ; 1%TritonX-100; and 3% bovine serumalbumin; Sigma-Aldrich). After 2 hours of incubation of either 250  g of GFI1 36S or GFI1 36N with 250  g of AML1/ETO lysate, complexes wereprecipitatedwith  - GFI1 antibodies,boundtoProtein-SepharoseG(Sigma-Aldrich), and then subjected to separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and detected by immunoblotting usingan  -ETO antibody (Santa Cruz Biotechnology). Results The SNP  GFI1 36N  predisposes toAML To determine whether GFI1 mutations or SNPs might play a role inthe pathogenesis ofAML, we sequenced genomic DNAfrom bonemarrow or blood from 92 AML patients treated at the UniversityHospital Essen. We repeatedly found in 13 cases a heterozygositycaused by a single-base substitution in the  GFI1  coding region(c.107G  A), leading to the replacement of a serine (S) by anasparagine (N) residue at amino acid position 36 (Figure 1A-B).GFI1 mRNAwas expressed in the blast cells of the patients thatwere collected at time of diagnosis (Figure 1C). The  GFI1 36N  variant allele also was present in the epithelial cell DNAof   GFI1 36N  heterozygous AML patients (data not shown), from whom epithe-lial DNA was available. Finally the  GFI1 36N  allele also could bedetected in the blood sample of normal control persons (Table 1).These findings indicate that the base substitution at c.107G  Arepresents an SNP and not a somatic acquired mutation. The factthat  GFI1 36N  is indeed a SNP was confirmed by the UCSC genomebioinformatics group denominating it as rs34631763.To determine the overall frequency of this SNP, we deter-mined its frequency in 2 independent white cohorts fromGermany and The Netherlands and their respective controlsubjects. In total, approximately 1806 AML (including theinitial 92 from Essen) patients treated at hospitals in Germanyand The Netherlands (see “Patients”), as well as 1691 healthycontrol patients from different locations in Germany and TheNetherlands, were tested for the frequency of this SNP. The GFI1 36N  variant was observed in Germany and The Netherlandswith the same elevated frequency in patients over controls(Table 1, odds ratio 1.6,  P  8  10  5 using a Mantel-Haenszelapproach). This correlation was confirmed (odds ratio 1.5, P  1  10  3 , 95% confidence interval, 1.1-2) after adjustingfor age and sex (see “Patients”) and this finding was independentof smoking status (Table 1), which is a controversial risk factorin the development of AML. 27,28 Interestingly, the number of detected homozygous carriers was lower in the control group(1 vs expected 2) and significantly lower in the patient cohort(1 observed vs expected 5;  P  .01), indicating that homozy-gous carriers might experience a selective disadvantage (Table1). We verified also the allele frequency of   GFI1 36N  in healthycontrol patients of other ethnicities such as Tanzania and Nigeria(allele frequency 0.004 in 210 samples) and Chinese (allelefrequency 0 in 205 samples). The incidence of   GFI1 36N  is lowerin these populations, as is the incidence of AML in thesepopulations. 31 Linkage disequilibrium of  GFI1 36N  A greater frequency of   GFI1 36N  among AML patients could beexplained by linkage disequilibrium (LD) of   GFI1 36N  with other,unknown, causative genetic variations. To address this, weanalyzed patterns of LD in the CEPH sample 32 using data fromthe International HapMap project. 33 In the telomeric direction,the most proximal SNP (rs11164607) to  GFI1 36N  is 1 kbp awayand belongs to the LD block 3 spanning part of   GFI1  and  EVI5 (Figure 1D; Table 2). The locus of   GFI1  is not in LD with anygenes, which lie further in the telomeric direction (Figure 1C).With regard to the centromeric direction, we tested 5 SNPsaround  GFI1 36N  in 28 healthy control patients homozygous for GFI1 36S  and 39 AML cases heterozygous for the  GFI1 36N  . Wefound complete allelic association of alleles (r 2  0.88) at locilocated in the region between rs2031494 and rs186682 but notbetween these alleles and  GFI1 36N  (Figure 1E-F). In conclusion,it is unlikely that variants in  EVI5  are associated with AML.However, a population stratification effect cannot be entirelyruled out. 34 GFI1 36N  is not associated with other establishedAML markers After determining that  GFI1 36N  predisposed to AML in these2 populations, we investigated whether  GFI1 36N  also might beassociated with prognosis or other known AML factors. Among377 de novoAML patients recruited by theAML96 study group,the presence of   GFI1 36N  did not correlate with any establishedfactors, 1,2,6,7 such as age, white blood cell count, lactatedehydrogenase level, frequency of CD34  cells, morphologicsubgroups as defined by the French-American-British classifica-tion, cytogenetic aberrations, or mutation status of   FLT  3,  NPM1 , or  PTPN11  (Tables 3-5), nor with 5-year overall survivalor relapse-free survival (Figure 2A; data not shown). Valk et al 24 have recently published a cluster analysis of genome-wide Table 2. Position of tested SNPs relative to each other Name Relative position, in bp rs6662618 0rs1325432 5533Rs4970714 5646Rs11164607 12 327Rs34631763 (rsSNP  GFI1 36N   )   13 518rs2031494 30 558rs10782922 39 184rs186682 41 864rs177371561 45 828 GFI1  indicates  Growth Factor Independence 1 ; and SNP, single nucleotidepolymorphism. Table 3. Features ofAML96  GFI1 36S  and  GFI1 36N  patients GFI1 36N  (n)  GFI1 36S  (n)  P  Number 40 337Median age, y 53.5 (40) 57 (337) .746Sex, % male 42.5 (17) 51 (172) .307Leukocytes, per fl 49 (40) 39 (337) .652Blast percentage 61 (35) 62 (303) .618LDH, IU 542 (38) 691 (323) .751Platelets, per fl 92 (40) 77 (337) .225FLT3 status negative, %* 90 (40) 88 (332) .688NPM1 mutations, % 64 (14) 50 (93) .64PTPN11 mutations, % 16 (3) 3 (5) .03MLL PTD, %† 6 (1) 7 (11) 1The characteristics of  GFI1 36  homozygous and carriers of the  GFI1 36N  allele withregard to different parameters are described.AML indicates acute myeloid leukemia;  GFI1, Growth Factor Independence 1 ;LDH,lactatedehydrogenase;MLL,mixed-lineageleukemia;andPTD,partialtandemduplication.*No internal tandem duplication mutation.†PTD mutations. 2466 KHANDANPOUR et al BLOOD, 25 MARCH 2010    VOLUME 115, NUMBER 12 For personal use only.on March 22, 2016. by guest www.bloodjournal.orgFrom 
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