A cond independent locus within DMRT1is associated with testicular germ cell tumor susceptibility
Peter A.Kanetsky 1,6,∗,Nandita Mitra 1,6,Saran Vardhanabhuti 1,David J.Vaughn 2,6,Mingyao Li 1,Stephanie L.Ciok 3,Richard Letrero 3,Kurt D’Andrea 3,Madhavi Vaddi 4,
David R.Doody 7,JoEllen Weaver 8,Chu Chen 7,10,Jacqueline R.Starr 7,10,11,Ha
˚kon Ha ˚konarson 9,Daniel J.Rader 5,Andrew K.Godwin 12,Muredach P.Reilly 5,Stephen M.Schwartz 7,10and Katherine L.Nathanson 3,6
1
Department of Biostatistics and Epidemiology,2Division of Hematology-Oncology,Department of Medicine,3Division of Medical Genetics,Department of Medicine,4Department of Pathology and Laboratory Medicine,5Cardiovascular Institute and 6Abramson Cancer Center,University of Pennsylvania School of Medicine,Philadelphia,PA 19104,USA,7
Program in Epidemiology,Division of Public Health Sciences,Fred Hutchinson Cancer Rearch Center,Seattle,WA 98109,USA,8Biosample Repository,Fox Cha Cancer Center,Philadelphia,PA 19111,USA,9Center for Applied Genomics and Division of Human Genetics,The Children’s Hospital
of Philadelphia,Philadelphia,PA 19104,USA,10
Department of Epidemiology and 11Department of Pediatrics,School of Medicine,University of Washington,Seattle,WA 98195,USA and 12Department of Pathology and Laboratory Medicine,University of Kansas Medical Center,Kansas City,KS 66160,USA
Received December 7,2010;Revid April 25,2011;Accepted May 3,2011
Susceptibility to testicular germ cell tumors (TGCT)has a significant heritable component,and genome-wide association studies (GWASs)have identified association with variants in veral genes,including KITLG ,SPRY4,BAK1,TERT ,DMRT1and ATF7IP .In our GWAS,we genotyped 349TGCT cas and 919controls and replicated top hits in an independent t of 439cas and 960controls in an attempt to find novel TGCT susceptibility loci.We identified a cond marker (rs7040024)in the doublex and mab-3-related tran-scription factor 1(DMRT1)gene that is independent of the previously described risk allele (rs755383)at this locus.In combined analysis that mutually conditions on both DMRT1single nucleotide polymorphism mar-kers,TGCT cas had elevated odds of carriage of the rs7040024major A allele [per-allele odds ratio (OR)51.48,95%confidence interval (CI)1.23,1.78;P 52.5231025]compared with controls,while the ass
ociation with rs755383persisted (per allele OR 51.26,95%CI 1.08,1.47,P 50.0036).In similar analys,the associ-ation of rs7040024among men with minomatous tumors did not differ from that among men with non-mi-nomatous tumors.In combination with KITLG ,the strongest TGCT susceptibility locus found to date,men with TGCT had greatly elevated odds (OR 514.1,95%CI 5.12,38.6;P 52.9831027)of being double homo-zygotes for the risk (major)alleles at DMRT (rs7040024)and KITLG (rs4474514)when compared with men without TGCT.Our findings continue to corroborate that genes influencing male germ cell development and differentiation have emerged as the major players in inherited TGCT susceptibility.
∗
To whom correspondence should be addresd at:University of Pennsylvania,219Blockley Hall,Philadelphia,PA 19104.Tel:+12155733282;Fax:+12155731050;Email:d.upenn.edu
#The Author 2011.Published by Oxford University Press.All rights rerved.For Permissions,plea email:journals.
Human Molecular Genetics,2011,Vol.20,No.153109–3117
doi:10.1093/hmg/ddr207超级索尼科
Advance Access published on May 6,2011
at Yale University on January 27, 2013
INTRODUCTION
In the USA,testicular germ cell tumors (TGCT [MIM 273300])are the most common cancers in young men,with a peak incidence among tho aged 25–34years.The inci-dence of TGCT among white men in the USA has incread substantially over the past 30years from 4.1per 100000in 1975to 7.0per 100000in 2007(1),and similar increas are en worldwide (2).The dia incidence varies widely across racial groups,with a 5-fold lower rate among black men in the USA (1).Differences in incidence of TGCT also exist across countries and continents,ranging from Denmark (9.2per 100000)to Algeria (0.2per 100000),which is con-sistent with racial differences and lower rates in non-white groups (3).
Familial aggregation of TGCT has been documented since the 1930s,and family history is the strong
est known risk factor for the malignancies (4,5).Risk of TGCT repeatedly has been shown to be incread among first-degree relatives of affected men,with risk to brothers (estimates range from 5-to 19-fold)being stronger than that to fathers (estimates range from 2-to 4-fold)(6–14).Both mono-and dizygotic twins of affected men have incread risk of TGCT (15,16).Genetic effects have been estimated to account for 25%of TGCT,the third highest heritability among all cancers (17).The familial data along with the racial disparity in dia occurrence provide evidence of a genetic contribution to TGCT susceptibility.
Supporting the genetic contribution to TGCT susceptibility,initial findings from genome-wide association (GWA)studies implicate variation at the KITLG ,SPRY4and BAK1as associ-ated with TGCT susceptibility (18,19).Prior to the publi-cations,no common susceptibility alleles had been validated.More recently,a follow-up GWA study from the UK also identified and replicated variation in ATF7IP ,DMRT1and TERT as associated with TGCT (20).Herein,we report the results from our follow-up GWA study in the USA,for which we augmented the number of TGCT cas ud in the discovery sample as well as TGCT cas and controls ud in the replication sample in order to identify additional TGCT susceptibility loci and to validate the new findings.
RESULTS
Information on age,risk factors and tumor characteristics for the discovery and replication sample ts are given in Table 1.The calculated genomic control inflation (l )factor in the discovery t was 0.942,and hence we report unadjusted test statistics (21,22).We again noted our previously reported statistically significant associations with markers at 12q22in KITLG (P ,5.0×1028),2p14and 5q13.3near SPRY4(P ,5.0×1026;Fig.1and Supplementary Material,Table S1)(18).Six additional markers at five additional auto-somal loci also were associated with TGCT (P ,5.0×1026).To screen out hits reprenting likely fal positive associ-ations,we imputed genotypes in the surrounding regions of the six markers using data from 1000genomes (March 2010relea)(Fig.2A and Supplementary Material,Fig.S1).After imputation,neither obrved nor imputed markers at 8q21.3and 13q12.3maintained significance at
P ,5×1026and were excluded from further study,whereas markers at 4q28.2,7q21.13and 9p24.3continued to exceed the P ,5×1026threshold.The lected markers on chromosomes 4and 7that were brought forward into replica-tion included two imputed (rs2279070,rs4834214)that mapped to 4q28.2,as the obrved single nucleotide poly-morphism (SNP)could not be designed for replication geno-typing,and two obrved (rs6951213,rs10281060)that mapped within introns 1and 2of PFTK1on 7q21.13.For chromosome 9markers,the top three imputed markers using 1000genome
s were incompatible with our replication geno-typing platform,so we re-imputed markers in this region using data from HapMap (relea 22).The top imputed marker (rs755383),which was previously showed to be associ-ated with TGCT by Turnbull et al .(20),was taken into repli-cation along with the top obrved marker (rs7040024),both of which exceeded the P ,5×1026threshold (Supplemen-tary Material,Fig.S2).
We did not replicate associations with rs2279070(P trend ¼0.62)and rs4834214(P trend ¼0.54)at 4q28.2or rs6951213(P trend ¼0.48)and rs10281060(P trend ¼0.68)at PFTK1.However,we obrved statistically significant associations with rs7040024(P trend ¼2.09×1026)and rs755383(P trend ¼2.08×1024)at DMRT1.Using a combined t of discovery and replication samples,TGCT cas had greater odds of carriage of the major A allele in rs7040024than con-trols [P ¼1.41×10211;odds ratio (OR)¼1.70,95%confi-dence interval (CI)1.46,1.99]and greater odds of carriage of the major T allele in rs755383(P ¼8.61×10210;OR ¼1.50,95%CI 1.32,1.7)(Table 2).In addition to the ca–control analysis,we performed a ca–parent analysis in 179triads and 135dyads that showed homozygous carriage of the A allele in rs7042004was strongly associated with TGCT risk (P ¼0.0033;relative risk ¼3.42,95%CI 1.50,7.76)as was homozygous carriage of the T allele in rs755383(P ¼1.20×10210;relative risk ¼4.67,95%CI 2.92,7.46).The analys support the finding of an associ-ation between the variation at the DMRT1locus and TGCT susceptibility.
To determine whether the obrved associations with rs7040024and rs755383were independent,we simultaneously modeled their main effects.Both DMRT1markers remained statistically significantly associated with TGCT status after mutual adjustment,although more moderately so compared with their respective univariate models.For rs7040024,the P -value was 2.52×1025(adjusted per allele OR ¼1.48,95%CI 1.23, 1.78),and for rs755383,the P -value was 0.0036(adjusted per allele OR ¼1.26,95%CI 1.08,1.47)(Table 2).To further investigate the independence of the two markers,we explored haplotypes in the DMRT1genomic region using obrved and imputed genotypes (bad on HapMap CEU population,relea 22)among the 919controls ud in our discovery pha.Figure 2B shows that rs7040024and rs755383do not reside in the same haplo-type block.Haplotype blocks resolved from the HapMap CEU population mirror the findings (Supplementary Material,Fig.S3).
Analys that additionally adjusted for cryptorchidism or family history of TGCT were limited to the replication sample becau information on risk factors was not collected
3110Human Molecular Genetics,2011,Vol.20,No.15
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school是什么意思英语
for discovery pha controls.Here,associations with both DMRT1markers (simultaneously considered)and TGCT were unchanged following further adjustment for either cryp-torchidism or family history.Among men without a family history of TGCT or men without cryptorchidism,the ORs were comparable with overall results.This finding suggests that the effects of DMRT1variants are not bad on the patho-logic mechanisms leading to the known strong TGCT risk factors.
In the combined discovery and replication analysis and after mutual adjustment,SNP genotypes in DMRT1were associated with both minoma and non-minomatous germ cell tumors (Table 2).While we could not reject homogeneity of the ORs comparing subtypes,odds associated with rs7040024appeared higher in men with non-minomatous germ cell tumors than among tho with minomatous tumors.
In the combined t,we explored whether the effects of DMRT1rs7040024and rs755383were modified by genotypes at KITLG rs4474514and SPRY4rs6897876,loci we previously reported as independently associated with TGCT status (18).In models that assd the joint effects of DMRT1genotypes with KITLG and SPRY4genotypes,we failed to detect departures from multiplicativity.However,we did obrve statistical evi-dence supporting departure from additivity (i.e.additive synergy)for DMRT1rs7040024and KITLG rs4474514(synergy index,S ¼2.2,95%CI 1.1,4.
2;P ¼0.02).That is to say,the OR associated with homozygous carriage of the risk allele at both loci (OR ¼14.1,95%CI 5.12,38.6;P ¼2.98×1027)was greater than the additive effect of the OR for homo-zygous carriage of the risk allele at either locus alone (OR 2.44,95%CI 0.62,9.56,P ¼0.20for DMRT1rs7040024;OR ¼5.59,95%CI 1.84,7.0,P ¼2.45×1023for KITLG rs4474514;Fig.3).Here,the comparison group was individuals who carried a total of zero or one risk allele at both loci (n ¼86),which was necessary becau only five controls and no TGCT cas were doubly homozygous for the non-risk alleles.We did not find evidence of departure from additivity for DMRT1rs7040024and SPRY4rs6897876(synergy index,S ¼2.2,95%CI 0.7,7.2,P ¼0.20).
Evidence of association with markers at ATF7IP ,BAK1and TERT was inconsistent across our sample ts.Although TGCT status was associated with rs11055991(ATF7IP ),rs210138(BAK1),rs444697(BAK1)and rs2736100(TERT )in our discovery t,no associations were noted in our replication t (Supplementary Material,Table S2).In contrast to reported findings by Turnbull et al .(20),we did not find associations at TERT to be stronger in tumors of the minoma subtype.
DISCUSSION
hall是什么意思
We verified the finding that variation at 9p24.3within DMRT1is associated with TGCT susceptibility (20)and report the
Figure 1.Manhattan plot of GWA results for 349TGCT cas and 919con-trols.SNP markers that reached significance at P ,5.0×1026bad on Fisher’s exact test are plotted above the lower line.Susceptibility loci ident-ified in our original (KITLG ,SPRY4,2p14)and current (DMRT1)analysis are indicated.
Table 1.Age,family history of TGCT and tumor type in the discovery and replication sample ts
Discovery Replication Status Ca Control Ca Control Total
n ¼349n ¼919n ¼439n ¼960n
%
小苹果英文版n
n
%
so as ton
%
Age [median,(interquartile range)]31a (25,39)57(52,62)33(27,38)34(30,39)Family history of TGCT No 31389.7–38086.686490.0Yes 32a 9.2–11b 2.510b 1.0Unknown
4 1.2–
4810.9869.0Personal history of cryptorchidism No 31088.839489.894298.1Yes
3510.0429.618 1.9Unknown 4 1.230.700Tumor type Seminoma 11432.7–26660.6––Non-minoma 22765.0–17339.4––Unknown
8
2.3
–
–
–
坚强男孩TGCT,testicular germ cell tumor.
a
市场部英文Sixteen cas were recruited bad on family history of TGCT.Among the non-lected (n ¼333)cas,the proportion reporting any family history of TGCT was 4.8%.b
Reflects reported family history of TGCT among first-degree relatives only.
Human Molecular Genetics,2011,Vol.20,No.153111
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identification of a cond risk allele (rs7040024)at this locus that is independent of the one previously reported (rs755383).In combined analysis and after mutual adjustment,TGCT cas had 50%greater odds of carriage per major A allele at rs7040024,while simultaneously having nearly 25
%greater odds of carriage per major T allele at rs755383.Of note,rs755383was the most statistically significant (imputed)marker at this locus and the marker genotyped directly by Turnbull et al .We anticipated that the markers might be independently associated with TGCT becau of their low-to-moderate linkage diquilibrium (r 2¼0.29)with each other,and the fact that the two markers do not reside in the same haplotype block.Thus,we have identified a cond SNP within DMRT1that is associated more strongly
Figure 2.Manhattan plot and linkage diquilibrium structure of the DMRT1genomic region.(A )SNP markers in the 1.2Mb region that includes DMRT1are plotted bad on Fisher’s exact test.Genotyped SNP markers are indicated in blue and imputed SNP markers are indicated in red.The two markers taken into replication are designated by triangles and labeled.(B )Linkage diquilibrium structure for the 40Kb region encompassing DMRT1bad on obrved and imputed (from HapMap CEU population,relea 22)genotypes among the 919controls in our discovery pha.Pairwi linkage diquilibrium is determined by r 2,and methods described by Gabriel et al .are ud to define haplotype blocks.Open arrow (left)indicates the position of DMRT1rs7040024;oval arrow (right)indicates position of DMRT1rs755383.
Table 2.Associations of TGCT with replicated DMRT1SNP markers Analysis group Gene marker a G
enotype count b Pha OR (95%CI)P -value e Controls Cas Per allele Heterozygote c Homozygote d Total
rs7040024A/C
493/356/68236/100/11Discovery 1.71(1.37,2.14) 1.74(0.89,3.41) 2.96(1.54,5.70) 3.57×1026484/323/57276/110/11Replication 1.69(1.36,2.10) 1.77(0.89,3.49) 2.96(1.52,5.73) 2.09×1026Combined 1.70(1.46,1.99) 1.75(1.08,2.83) 2.96(1.86,4.71) 1.41×10211Adjusted f 1.48(1.23,1.78) 1.39(0.83,2.31) 2.00(1.25,3.52) 2.52×1025rs755383g T/C
371/415/133191/133/25Discovery 1.64(1.35,1.98) 1.71(1.07,2.73) 2.74(1.73,4.35) 5.78×1027329/389/144
185/172/38Replication 1.40(1.17,1.67) 1.68(1.12,2.50) 2.13(1.43,3.18) 2.08×1024Combined 1.50(1.32,1.71) 1.68(1.24,2.28) 2.40(1.78,3.24)8.61×10210Adjusted 1.26(1.08,1.47) 1.46(1.05,2.03) 1.71(1.21,2.42)0.0036Seminomatous tumors
rs7040024A/C
78/31/4Discovery 1.75(1.22,2.51) 1.48(0.51,4.33) 2.69(0.95,7.58)0.0023156/74/8Replication 1.
46(1.13,1.88) 1.66(0.76,3.63) 2.35(1.10,5.04)0.0036
Combined 1.54(1.25,1.89) 1.59(0.84,2.98) 2.42(1.31,4.46) 4.64×1025Adjusted 1.35(1.06,1.73) 1.39(0.71,2.72) 1.87(0.95,3.69)0.015rs755383f T/C
62/40/11Discovery 1.54(1.14,2.09) 1.17(0.58,2.34) 2.02(1.03,3.95)0.0055108/104/26Replication 1.31(1.06,1.62) 1.47(0.92,2.35) 1.81(1.13,2.90)0.012
中国梦作文600字Combined 1.39(1.17,1.65) 1.37(0.93,2.03) 1.91(1.30,2.80) 2.05×1024Adjusted 1.22(1.00,1.50) 1.23(0.81,1.88) 1.49(0.95,2.33)0.055
Non-minomatous tumors
rs7040024A/C
154/67/6Discovery 1.73(1.33,2.26) 2.13(0.89,5.11) 3.54(1.51,8.31) 4.54×1025120/36/3Replication 2.17(1.53,3.09) 1.94(0.57,6.58) 4.28(1.30,14.1) 1.46×1025Combined 1.90(1.54,2.35) 2.12(1.04,4.30) 3.94(1.98,7.86) 1.60×1029Adjusted 1.64(1.29,2.10) 1.51(0.72,3.19) 2.55(1.20,5.39)7.42×1025rs755383f T/C
126/90/13Discovery 1.71(1.36,2.15) 2.22(1.20,4.10) 3.47(1.90,6.36) 4.63×102677/68/12
Replication 1.53(1.18,1.99) 2.17(1.13,4.16) 2.84(1.48,5.43)0.0015
Combined 1.63(1.37,1.94) 2.16(1.39,3.37) 3.16(2.04,4.90) 2.30×1028Adjusted
1.29(1.06,1.58)
1.78(1.11,
2.85)
2.02(1.24,
3.30)
0.012
veral的用法TGCT,testicular germ cell tumor.
a
dbSNP rsnumber and risk/non-risk alleles.b
Number of individuals genotyped as homozygous for the risk allele/heterozygous for the risk allele/homozygous for the non-risk allele.MAF for discovery pha markers given in Supplementary Material,Table S1.c
OR for heterozygous carriage of risk allele compared with homozygous carriage of non-risk allele.d
OR for homozygous carriage of risk allele compared with homozygous carriage of non-risk allele.e
Test for trend.f
OR,95%CI and P -value determined from a logistic regression model of combined data containing main effects of both DMRT1markers.g
Genotype counts,OR and 95%CI for discovery pha rs755383estimated from data imputed using HapMap (relea 22).
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with TGCT susceptibility than that previously reported.Our finding,together with others recently identified for TGCT sus-ceptibility loci,begins to unravel the complex architecture of inherited genetics of TGCT.
We acknowledge that the u of a comparison group con-sisting of men who were undergoing routine cardiac catheter-ization,of whom 76%(n ¼700)had angiographically confirmed coronary artery dia (CAD),may have impacted our findings to the degree that there are common genetic factors linking CAD and TGCT.To explore this possibility,we compared genotype frequencies of markers at BAK1,DMRT1and TERT (Table 2and Supplementary Material,Table S2)among controls with and without frank CAD and noted no statistically significant differences (0.09≤P trend ≤0.64for all comparisons).Further,we compared the obrved minor allele frequencies (MAFs)at the markers among CAD controls to tho obrved in our replication-pha controls,HapMap CEU samples and Coriell CEPH samples.We obrved slight fluctuations in allele frequencies among the four groups ranging from a 3to 8%difference;however,for each marker,the allele frequency among the CAD controls was consistent with that in at least one of the three population-bad sample ts.The comparisons lead us to believe that little bias was introduced by using CAD con-trols.As well,we recognize that 90%of CAD controls were
46years or older and had already pasd the peak age of TGCT development.Bad on available age-specific TGCT rates,we estimated that only four TGCT cas would be expected to have arin in this control group (1).It is unlikely that this potential small misclassification of phenotype would have biad results appreciably.
DMRT1is a member of a zinc finger-like DNA-binding motif (DM domain)gene family.Genes with the DM domain are highly conrved and play crucial roles in male development and x determination across the phylogenetic spectrum from flies and nematodes to birds and vertebrates (23).Dmrt1in mice is expresd only in the gonad and is esntial for postnatal germline maintenance and differen-tiation of germ cells,specifically radial migration,mitotic reactivation and survival (24,25).Dmrt1is required for normal maturation of Sertoli cells;the cells fail to polarize and stop proliferating when this gene is knocked out (24).Incread dosage of DMRT1facilitates male development,and decreasing gene dosage leads to feminization of the gonads.In humans,deletion of the region on 9p-containing DMRT1leads to male-to-female x reversal and is associated with the development of gonadoblastoma (26,27).Dmrt12/2mice also have verely dysgenetic testes,rembling tho en in XY individuals with the loss of 9p,and fail to undergo normal germ cell development,dying by P14(24,28).Moreover,a loss of Dmrt1also is associated with a high rate of
teratomas in 129Sv mice with 90%of double knockout mice developing tumors (29).In our data,variants at DMRT1may be slightly more strongly associated with non-minomatous germ cell tumors,of which teratoma is one type,than with minomas;but the difference between tumor types was not statistically significant.We do not believe our inability to detect a statistical difference in the association between common DMRT1variants by histological tumor type should be interpreted as contradicting the evidence from model systems for a role of this gene in human teratoma susceptibility.Rather,the sample size was modest,and con-quently there was limited power to detect differences between tumor types.
宫保鸡丁英文
TGCT is believed to ari from undifferentiated primordial germ cells,which progress to the precursor lesion of TGCT-termed intratubular germ cell neoplasia undifferen-tiated (ITGCNU)(30–33).All ITGCNU is thought to advance to frank here is no spontaneous regression.Consistent with this hypothesis,predisposition to TGCT is incread among patients with different types of dis-orders of xual development and delayed differentiation of primordial germ cells (34).The data from the TGCT GWA studies are strongly supportive of this model of dia devel-opment,as male germ cell differentiation is the biological pathway linking DMRT1to the TGCT susceptibility loci KITLG ,SPRY4and BAK1.
TGCT susceptibility has been linked to infertility or sub-fertility in veral studies,and the majority of evidence points to common etiologic risk factors for the conditions.The most convincing studies have followed men with known men quality for outcomes including TGCT and have exam-ined fertility in brothers of men with TGCT (35–39).Interest-ingly,Dmrt1is esntial for fertility in mice (24).Thus,the association between common variation at DMRT1
and
Figure 3.Joint effects of DMRT1genotypes with KITLG and SPRY4geno-types.OR and 95%CI for main effects adjusted for the cond SNP marker are given in the row and column headers.Within cells,frequency count (control/ca)and OR (95%CI)for joint genotypes are given with referent genotype(s)reprented as shaded cells.
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