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Germline Mutations in MSR1, ASCC1

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Germline Mutations in MSR1,ASCC1,and CTHRC1in Patients With Barrett Esophagus and Esophageal Adenocarcinoma

Mohammed Orloff,PhD

Charissa Peterson,MS

Xin He,MD,PhD

Shireen Ganapathi

Brandie Heald,MS,CGC

Yi-ran Yang,MD

Gurkan Bebek,PhD

Todd Romigh,MS

Jee Hoon Song,BS

Wenjing Wu,BS

Stefan David,MD

Yulan Cheng,MD

Stephen J.Meltzer,MD

Charis Eng,MD,PhD

T HE INCIDENCE OF ESOPHAGEAL

adenocarcinoma(EAC)in the

United States and Europe has

increased350%since1970,

with uncertain etiology.1Although

early-stage EAC is curable,most cases

are detected at an advanced stage with

poor survival.Esophageal adenocarci-

noma is believed to be preceded by

Barrett esophagus(BE),a premalig-

nant metaplasia caused by chronic

gastroesophageal reflux disease

(GERD).2-6GERD-related inflamma-

tion and the transforming growth fac-

tor?(TGFB)pathway have been implicated in sporadic BE and EAC, just as the role of inflammation has become prominent in a range of human cancers.7Although acknowl-edged,the role of inflammation in BE and EAC has not been thoroughly Author Affiliations:Genomic Medicine Institute(Drs Or-

loff,He,Yang,Bebek,and Eng and Mss Peterson,Ga-

napathi,and Heald and Mr Romigh),Taussig Cancer

Institute(Drs Orloff and Eng and Ms Heald),and Stan-

ley Shalom Zielony Institute for Nursing Excellence

(Dr Eng),Cleveland Clinic,Cleveland,Ohio;Center

for Proteomics and Bioinformatics(Dr Bebek),Depart-

ment of Genetics(Dr Eng),and CASE Comprehensive

Cancer Center(Drs Bebek and Eng),Case Western

Reserve University School of Medicine,Cleveland,Ohio;

and Department of Medicine and Sidney Kimmel Com-

prehensive Cancer Center,Johns Hopkins University

School of Medicine,Baltimore,Maryland(Drs David,

Cheng,and Meltzer and Messrs Song and Wu).

Corresponding Author:Charis Eng,MD,PhD,Ge-

nomic Medicine Institute,Cleveland Clinic,9500

Euclid Ave,Mailstop NE-50,Cleveland,OH44195

(engc@https://www.wendangku.net/doc/3c18747837.html,).

Context Barrett esophagus(BE)occurs in1%to10%of the general population and is believed to be the precursor of esophageal adenocarcinoma(EAC).The incidence of EAC has increased350%in the last3decades without clear etiology.Finding pre-disposition genes may improve premorbid risk assessment,genetic counseling,and management.Genome-wide multiplatform approaches may lead to the identification of genes important in BE/EAC development.

Objective To identify risk alleles or mutated genes associated with BE/EAC. Design,Setting,and Patients Model-free linkage analyses of21concordant-affected sibling pairs with BE/EAC and11discordant sibling pairs(2005-2006).Sig-nificant germline genomic regions in independent prospectively accrued series of176 white patients with BE/EAC and200ancestry-matched controls(2007-2010)were validated and fine mapped.Integrating data from these significant genomic regions with somatic gene expression data from19BE/EAC tissues yielded12“priority”can-didate genes for mutation analysis(2010).Genes that showed mutations in cases but not in controls were further screened in an independent prospectively accrued vali-dation series of58cases(2010).

Main Outcome Measures Identification of germline mutations in genes associ-ated with BE/EAC cases.Functional interrogation of the most commonly mutated gene.

Results Three major genes,MSR1,ASCC1,and CTHRC1were associated with BE/ EAC(all P?.001).In addition,13patients(11.2%)with BE/EAC carried germline mu-tations in MSR1,ASCC1,or CTHRC1.MSR1was the most frequently mutated,with 8of116(proportion,0.069;95%confidence interval[CI],0.030-0.130;P?.001)cases with c.877C?T(p.R293X).An independent validation series confirmed germline MSR1 mutations in2of58cases(proportion,0.035;95%CI,0.004-0.120;P=.09).MSR1 mutation resulted in CCND1up-regulation in peripheral-protein lysate.Immunohis-tochemistry of BE tissues in MSR1-mutation carriers showed increased nuclear expres-sion of CCND1.

Conclusion MSR1was significantly associated with the presence of BE/EAC in deri-vation and validation samples,although it was only present in a small percentage of the cases.

JAMA.2011;306(4):https://www.wendangku.net/doc/3c18747837.html,

studied.8Barrett esophagus is common in the general population,estimated to occur in1%to10%6;it develops in 12%to15%of patients with GERD.5 The risk of EAC in patients with BE is approximately0.4%per year.9 Although most BE and EAC are be-lieved to be sporadic,genetic(heri-table)etiologies have been supported by observation of familial clustering of cases noted over several decades,although few large families co-segregating BE/EAC have been reported.10,11In1referral se-ries,clinical epidemiologic analyses sug-gest7%of individuals with BE,EAC,or both have at least1affected blood rela-tive.12Although shared environmental factors may contribute to such familial aggregation,an autosomal dominant mode of inheritance with incomplete penetrance is consistent with most pub-lished studies,and rare reported cases are consistent with autosomal recessive in-heritance.10

The discovery of germline muta-tions in a gene or genes that predis-pose to BE/EAC may have ramifica-tions regarding cancer risk assessment, genetic counseling,premorbid diagno-sis,and targeted surveillance and management,and also add to the fun-damental understanding of the patho-physiology of sporadic BE and EAC.We therefore sought to identify a gene or genes associated with BE/EAC predis-position.

METHODS

Our study(2005-2010),approved by respective institutions’review board for research participants,involved pro-spective recruitment of all298con-senting adults with histologically proven BE,EAC,or both,as well as families with2or more cases with BE, EAC,or both from16academic and community hospitals and clinics na-tionally(two-thirds originated from Cleveland Clinic,Cleveland,Ohio,and Johns Hopkins Medical Institutions, Baltimore,Maryland;?1%of re-search participants declined participa-tion).All BE cases were long segment (eMethods;available at http://www https://www.wendangku.net/doc/3c18747837.html,).For discordant sibling pair studies,the nonaffected sibling had en-

doscopy documented unaffected sta-

tus.Only white participants of north-

ern or western European descent were

selected and sex-matched in cases and

controls.

Identification of Loci Using

Genome-Wide Mapping Methods

Model-Free Linkage Analysis.

Twenty-one concordant-affected sib-

ling pairs(42individuals with

BE/EAC)and11discordant sibling

pairs(11with BE/EAC and11with-

out BE/EAC)(2005-2006)were geno-

typed using Affymetrix GeneChip

Human Mapping100K SNP set(Af-

fymetrix,Santa Clara,California)

(F IGURE1).Significant linkage to

chromosomal regions found by1

model-free linkage analysis method

was self-replicated by a second

model-free linkage analysis approach,

which is used for small sample-sized

data sets.13-15Genomic regions were

considered potentially interesting

when?log10P value(pP)?2.2by

SIBPAL analysis13had logarithm of

odds?3.2by LODPAL analysis.13

These regions from this pilot linkage-

association analysis were considered

“potentially interesting”and served

as regions to be validated(eMethods

and Figure1).

Independent Validation and Fine

Mapping Significant Regions.It is

standard in this field to single out sig-

nificant genomic regions from pilot

analysis to follow up with increased

sample sizes from independent cases

(validation),more genetic markers

(fine mapping),16-19or both in a sec-

ond validation stage(Figure1).We

followed this strategy of independent

validation and fine mapping of the

“potentially interesting”regions iden-

tified by the pilot linkage-association

analysis.We also paid particular

attention to2additional regions

(1q23and8p22),because these

regions were found previously to be

frequently somatically lost(by array

comparative genomic hybridization)

in EAC or gastroesophageal junction

cancers.20

Population substructure of cases and

controls was determined by PLINK

and EIGENSTRAT(eMethods).21,22

Analysis using EIGENSTRAT soft-

ware21,22and principal component

analysis identified the top eigenvalues

from the376available eigenvalues.

Regression analyses were used to al-

low for potential population substruc-

ture by2separate analyses(PLINK-

derived and EIGENSTRAT-derived

analyses).After population substruc-

ture was assessed to be similar

(?85%),21,22single single-nucleotide

polymorphism(SNP)association analy-

ses of the above targeted genomic re-

gions were performed with an indepen-

dent validation series totaling176

patients with BE/EAC and200ancestry-

matched population controls(2007-

2010)whose SNP data were derived

from the denser Illumina Human610-

Quad BeadChips(Illumina Inc,Hay-

ward,California).Although we only

were validating specific regions,we rea-

soned that it would be more cost-

efficient to genotype all markers in a

commercially available Chip instead of

creating a new automation process for

a reduced marker set.If the underly-

ing genetic effect had been negligible,

we would not have expected to see any

savings on the average sample size,but

fortunately the underlying genetic effect

was large enough to warrant savings on

sample size.

Statistical simulations have indi-

cated that haplotype analysis with mul-

tiple SNPs may be more powerful than

single SNP analysis,23-25because mul-

tiple alleles at different loci on the same

chromosome that are in linkage dis-

equilibrium(LD)are likely to interact

with each other to result in a pheno-

type.Thus,haplotype analysis was per-

formed using PLINK26to predict the

most likely haplotypes and those that

were significantly associated with the

BE/EAC phenotype.T o account for type

I error,an empirical P value corrected

for testing multiple markers was ob-

tained by permuting(10000permuta-

tions)the affectation status across the

individual genotypes,as described in

PLINK.26

Integrating Information From Significant Regions

With Publicly Available Somatic Gene Expression Data Sets

To narrow in on one or a subset of genes within and in proximity to the significant SNPs/haplotypes germane to BE/EAC (by tissue-specific expres-sion in oncologic pathways and for functional-genomic validation),we integrated our significant regions with publicly available somatic gene expression data derived from 19patients with BE/EAC (GDS3472or GSE13083),27followed up by unsu-pervised hierarchical clustering 28of genes within 250kb flanking the sig-nificant SNPs and haplotypes across BE/EAC and unaffected individuals (eFigure 1,T ABLE 1,and T ABLE 2).

Prioritized Candidate Gene Analysis

A final list of biologically plausible can-didate genes (“priority”candidate genes)was then scanned for germline mutations in BE/EAC cases and com-pared with ancestry-matched popula-tion controls (Figure 1).Genes with mutations in cases but not in controls were screened in an independent vali-dation series of 58cases prospectively accrued from outpatient endoscopy units (2010)(Figure 1and eTable 1).

MSR1and CCND1Protein Levels and Cell Lines

Proteins were extracted from immortal-ized lymphoblastoid cells obtained from patients with BE/EAC and normal con-trols.After processing,protein lysates were loaded onto sodium dodecyl sul-fate-polyacrylamide gel electrophore-sis gels.Antibodies specific to CCND1(Cell Signaling Technology Inc,Dan-vers,Massachusetts),MSR1(Abcam,Cambridge,Massachusetts),and ?-tu-bulin (Sigma-Aldrich,St Louis,Mis-souri)were used for Western blotting.Wild-type MSR1or pCMV-FLAG empty vector were transiently trans-fected into MSR1-null HEK293cells using Lipofectamine LTX and Plus Re-agent (Invitrogen,Carlsbad,Califor-nia).Cells were harvested after 24hours

Figure 1.Schema of Strategy for Mapping BE/EAC Loci and Candidate Gene Selection

D I S C O V

E R Y

BE/EAC indicates Barrett esophagus/esophageal adenocarcinoma.The multistage strategy used to identify BE/EAC susceptibility genes via a genome-wide combined linkage-association analysis,followed up by an independent genome-wide single-nucleotide polymorphism (SNP)-based case-control validation.A series of multiple,including functional,platform integration resulted in a prioritized candidate gene list,with the final 12top priority candidates brought forward to candidate gene mutation analysis in a case-control series followed up by validation in an independent series of patients and by functional interrogation.GERMLINE MUTATIONS IN MSR1,ASCC1,AND CTHRC1IN PATIENTS WITH BARRETT ESOPHAGUS

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JAMA,July 27,2011—Vol 306,No.4

and lysates (30μg of protein)were ana-lyzed by Western blotting using anti-bodies against FLAG (Sigma-Aldrich,1:1000),CCND1(Thermo-Fisher Sci-entific Inc,Waltham,Massachusetts,1:200),and ?-tubulin (Sigma-Aldrich,1:5000).

CCND1Immunohistochemical Analysis

CCND1immunohistochemical analysis was performed using an avidin-biotin complex immunoperoxidase technique.RESULTS

Linkage and Association Analyses

A pilot combined linkage-association analysis based on modification of es-tablished criteria 29revealed 5candi-date regions (1q24.1-25.3,1q41,

8q21.11-22,10q21-22,and 11q21)(Table 1and Figure 1).

Subsequently,we performed a val-idation study in an independent series of 176cases and 200controls (Figure 1),usingadenserSNP-markersetbutfocus-ingonlyonthegermlineregionsofinter-est and the 2somatically lost hot spot re-gions (1q23and 8p22).We were able to validate 4(1q24.1-25.3,1q41,8q21.11-22,and10q21-22)ofthese5pilot-derived germlinecandidateregions,whileexclud-ing one (11q21).Three additional loci at locations remote from the pilot linkage peaks(1q21.2,8p22,and11q25)werealso found (Table 1).The most significant SNPs from this validative association analysis were located within or in the vi-cinityofthemostpromisingpilot-derived linkage peaks (Table 1).

Moving-Window Haplotype Analysis

Haplotype and LD analysis conducted on the 176cases and 200controls confirmed our findings—any single SNP that was significant in the above single SNP analysis always revealed a haplotype block containing significant SNPs within the haplotypes (at least in LD)(P ?.005)(Table 2).In the haplotype analysis,we considered regions of highest priority as those haplotypes exhibiting significance across multiple SNPs.The combined P values from the significant single SNPs and the significant haplotypes facilitated prioritization of regions of interest for further follow-up.There were 4significant regions that over-lapped from the linkage,single SNP

Table 1.Significant Single SNP Association Results From Pilot-Combined Linkage-Association and Independent Validation Case-Control Analyses in Patients With BE/EAC Region db SNP Location,Build 36.1P Value Association Analysis,FDR-Corrected

Linkage Analysis,LOD Scores (pP SP )a

Single SNP Association,pP Asscn b Gene at the Significant SNP

1q21.2

rs2809811100,805,788?.0010.0185

4.80

1q24.1-25.3

rs10494465164,810,325.007 4.31(2.17)rs950302165,350,678.004 4.38(2.40)rs10489191165,772,769.004 3.68(2.36)rs10489211166,579,946.004 3.32(2.35)

rs6659944167,046,343?.0010.0175

5.03rs10494476167,267,368.009 3.10(2.06)

rs3853181169,241,785?.0010.0198 4.47C1orf129rs6661125178,493,273?.0010.0175 5.06LHX41q41

rs10209401217,912,676?.0010.0214 4.07DIRC3rs12070516218,894,580?.0010.0199 4.75MARK1

rs12062054229,020,356?.0010.0199

4.93

rs2355230237,851,538.007 4.12(2.18)

rs4498839243,586,955?.0010.0173 5.22KIF26B 8p22

rs38111116,090,070?.0010.0253

3.29

MSR1

8q21.11-22

rs446944875,457,415.01 3.19(2.01)rs473975581,665,497.01 3.39(2.00)

rs309741894,851,657?.0010.0253 3.33TMEM678q22.1-24.22

rs3098224104,515,622?.0010.0253 3.24WDSOF1rs3098233104,463,670?.0010.0253 3.24CTHRC1rs4388439133,277,554?.0010.0253 3.10KCNQ310q21-22

rs1100105653,599,547?.0010.0463

3.04

PRKG1

rs205038155,029,991?.001 3.604(3.52)rs1050902156,518,547?.001 2.566(4.00)

rs1100019073,577,964?.0010.0262

3.46

ASCC1

11q21rs710718594,342,447?.001 4.588(3.40)rs125553794,958,558?.001 4.54(3.52)

11q25

rs11223500

132,917,451

?.001

0.0500

3.23

OPCML

Abbreviations:BE,Barrett esophagus;EAC,esophageal adenocarcinoma;FDR,false discovery rate;LOD,logarithm of odds;SNP,single-nucleotide polymorphism.

a LOD score was derived from LODPAL.15-18pP SP indicates ?log 10(P value)derived from SIBPAL (considered ?log 10

(P value)?2.00),from analysis of the 32sibling pairs (21concordant-affected sibling pairs and 11discordant sibling pairs).15-18

b pP Asscn indicates ?log 10

(P value)derived from the validation case-control association analysis using independent n=376(comprising 176cases and 200controls).GERMLINE MUTATIONS IN MSR1,ASCC1,AND CTHRC1IN PATIENTS WITH BARRETT ESOPHAGUS

GERMLINE MUTATIONS IN MSR1,ASCC1,AND CTHRC1IN PATIENTS WITH BARRETT ESOPHAGUS

Table2.Haplotypes Significantly Associated With BE/EAC Cases vs Controls a

Chromosome Region Haplotype SNPs P Value Significant Genes 1q21.212212rs3806237,rs12060945,rs2270694,rs12722868,rs2809811b?.001

22121rs12060945,rs2270694,rs12722868,rs2809811b,rs34552536?.001

221rs2270694,rs12722868,rs2809811b?.001

212rs12722868,rs2809811,rs34552536?.001

1q24.2122rs6659944b,rs12067866,rs12069349?.001

222rs6659944b,rs12067866,rs12069349?.001

1q24.3222rs16828284,rs3853181b,rs1800822?.001C1orf129

21212rs16828284,rs3853181b,rs1800822,rs2066530,rs2066536?.001C1orf129

222rs3853181b,rs1800822,rs2066530?.001C1orf129

1q25.2-25.322112rs6661125,rs17300107,rs6670868,rs16856123,rs17302632?.001LHX4

122rs6661125b,rs17300107,rs6670868?.001LHX4

1q4111112rs1338775,rs6694126,rs17007991,rs12070516b,rs17008285?.001MARK1

11122rs6694126,rs17007991,rs12070516,rs17008285,rs17008643?.001MARK1

11222rs17007991,rs12070516b,rs17008285,rs17008643,rs17008806?.001MARK1

12222rs12070516b,rs17008285,rs17008643,rs17008806,rs3806325?.001MARK1

22222rs6694126,rs17007991,rs12070516b,rs17008285,rs17008643?.001MARK1

1122rs17007991,rs12070516b,rs17008285,rs17008643?.001MARK1

2222rs17007991,rs12070516,rs17008285,rs17008643?.001MARK1

1112rs6694126,rs17007991,rs12070516b,rs17008285?.001MARK1

1222rs12070516b,rs17008285,rs17008643,rs17008806?.001MARK1

2222rs6694126,rs17007991,rs12070516,rs17008285?.001MARK1

2222rs1338775,rs6694126,rs17007991,rs12070516b?.001MARK1

1111rs1338775,rs6694126,rs17007991,rs12070516b?.001MARK1

222rs6694126,rs17007991,rs12070516?.001MARK1

112rs17007991,rs12070516b,rs17008285?.001MARK1

222rs17007991,rs12070516b,rs17008285?.001MARK1

122rs12070516b,rs17008285,rs17008643?.001MARK1

222rs12070516b,rs17008285,rs17008643?.001MARK1

111rs12070516,rs17008285,rs17008643,rs17008806,rs3806325?.001MARK1

8p2222221rs4265186,rs268387,rs354521,rs354517,rs381111b.002MSR1

22112rs354521,rs354517,rs381111b,rs2959634,rs2959631.004MSR1

8q22.121222rs3097422,rs3097418,rs6989157,rs6987276,rs4392869.002TMEM67

12222rs3097418b,rs6989157,rs6987276,rs4392869,rs987036.002TMEM67

212rs3097422,rs3097418b,rs6989157?.001TMEM67

221rs3097418,rs6989157,rs6987276?.001TMEM67

8q22.1-23.122122rs3098233b,rs3098224b,rs3098218,rs3098212,rs2959025?.001CTHRC1,WDSOF1 11211rs3098233b,rs3098224b,rs3098218,rs3098212,rs2959025?.001CTHRC1,WDSOF1

22112rs6988793,rs6987078,rs3098233b,rs3098224b,rs3098218.001CTHRC1,WDSOF1

12212rs6987078,rs3098233b,rs3098224b,rs3098218,rs3098212.002CTHRC1,WDSOF1

21121rs6987078,rs3098233,rs3098224,rs3098218,rs3098212.002CTHRC1,WDSOF1

22211rs2959644,rs6988793,rs6987078,rs3098233b,rs3098224b.002CTHRC1,WDSOF1

12112rs3098224b,rs3098218,rs3098212,rs2959025,rs2957452.002WDSOF1

8q24.2-24.2222222rs6989059,rs6986982,rs6988942,rs6989209,rs4388439b?.001KCNQ3

12212rs6986982,rs6988942,rs6989209,rs4388439b,rs3843561.002KCNQ3

10q21.112212rs11000400,rs11000436,rs11000798,rs11001056b,rs11001210.003PRKG1

22222rs11001056,rs11001210,rs11001213,rs11001447,rs11001702.002PRKG1

2212rs11000436,rs11000798,rs11001056b,rs11001210.002PRKG1

2122rs11000798,rs11001056b,rs11001210,rs11001213.003PRKG1

221rs11000436,rs11000798,rs11001056?.001PRKG1

212rs11000798,rs11001056b,rs11001210?.001PRKG1

222rs11001056b,rs11001210,rs11001213?.001PRKG1

association,and haplotype-LD analy-ses(1q24.1-25.3[encompassing 1q24.2,1q24.3,and1q25.2-25.3fine-mapped regions],1q41,8q21.11-22 [encompassing8q21.11-22and 8q22.1-24.22fine-mapped regions], and10q21-22).Additionally,there were3significant regions that over-lapped in the single SNP association and haplotype analyses(1q21.2,

8p22,and10q22.1).Thus,we

selected these regions,shown in

Table2,as“regions of interest”

(Figure1,eTable2,and eTable3).Each

of these regions contained SNPs that

were statistically significant at P?.005

and also had multiple haplotype

windows showing significance(P?.01).

Functional-Genomic Validation

Integration of our significant SNP

and haplotypes with publicly avail-

able so matic BE/EAC transcriptome

data(Figure1)yielded38genes

located within250kb flanking each

significant SNP,within significant

haplotypes,or both that accurately

clusteredBE/EAC cases from controls

Table2.Haplotypes Significantly Associated With BE/EAC Cases vs Controls a(continued)

Chromosome Region Haplotype SNPs P Value Significant Genes 10q22.122221rs11000101,rs11000108,rs11000122,rs11000152,rs11000190b.003ASCC1

22211rs11000108,rs11000122,rs11000152,rs11000190b,rs11000202.002ASCC1

12222rs11000122,rs11000152,rs11000190,rs11000202,rs11000348.009ASCC1

21122rs11000152,rs11000190,rs11000202,rs11000348,rs11000828?.001ASCC1

1122rs11000190b,rs11000202,rs11000348,rs11000828?.001ASCC1

2211rs11000122,rs11000152,rs11000190b,rs11000202.002ASCC1

2221rs11000108,rs11000122,rs11000152,rs11000190b.002ASCC1

221rs11000122,rs11000152,rs11000190b.003ASCC1

211rs11000152,rs11000190,rs11000202.001ASCC1

112rs11000190,rs11000202,rs11000348.003ASCC1

11q1422112rs1381720,rs12146457,rs3924745,rs665153,rs2926467?.001

11221rs1381722,rs1381720,rs12146457,rs3924745b,rs665153?.001

12212rs1381720,rs12146457,rs3924745b,rs665153,rs2926467.001

21122rs12146457,rs3924745b,rs665153,rs2926467,rs1871684.001

12211rs1381722,rs1381720,rs12146457,rs3924745,rs665153.003

2112rs12146457,rs3924745,rs665153,rs2926467?.001

1122rs3924745b,rs665153,rs2926467,rs1871684.001

1122rs1381722,rs1381720,rs12146457,rs3924745b.002

221rs1381720,rs12146457,rs3924745b?.001

211rs12146457,rs3924745b,rs665153?.001

112rs3924745b,rs665153,rs2926467?.001

122rs1381720,rs12146457,rs3924745.002

Abbreviations:BE,Barrett esophagus;EAC,esophageal adenocarcinoma;SNPs,single-nucleotide polymorphisms.

a These results were obtained from the haplotype analysis of the independent validation data set comprising176cases and200controls.In the“Haplotype”column,1represents the major allele and2represents the minor allele at each respective marker.

b Represent SNP associations significant in both the single SNP analysis and the haplotype analysis.

Table3.Germline Mutations in3Candidate Genes in BE/EAC Cases

Gene Variant Total No.

No./Total(%)

With Mutations

Proportion of Cases

With Variant(95%CI)

Value Cases Controls

MSR1(mutation analysis)a c.877C?T,p.R293X2558/116(6.9)0/1390.069(0.030-0.130)?.001 MSR1(validation)b c.877C?T,p.R293X1972/58(3.4)0/1390.034(0.004-0.120).09 MSR1(pooled) c.877C?T,p.R293X32310/184(5.4)0/1390.054(0.026-0.098).006 MSR1(mutation analysis)a c.760C?G,p.L254V2552/116(1.7)0/1390.017(0.021-0.061).19 ASCC1(mutation analysis)a c.869A?G,p.N290S2202/95(2.1)0/1250.021(0.003-0.074).18 CTHRC1(mutation analysis)a c.131A?C,p.Q44P2141/89(1.1)0/1250.011(0.0003-0.061).42 CTHRC1(validation)b c.131A?C,p.Q44P1831/58(1.7)0/1250.017(0.0004-0.092).32 CTHRC1(pooled)c c.131A?C,p.Q44P2722/147(1.4)0/1250.014(0.0009-0.026).50 Abbreviations:ASCC1,activating signal cointegrator1complex subunit1;BE,Barrett esophagus;CI,confidence interval;CTHRC1,collagen triple-helix repeat-containing1;EAC,esoph-ageal adenocarcinoma;MSR1,macrophage scavenger receptor1.

a Candidate gene mutation analysis in BE/EAC cases and controls.

b MSR1and CTHRC1mutations validated in small independent series of BE/EAC cases.

c Poole

d series comprising series of cases and controls used for candidat

e gene mutation analysis and independent validation series.

(eFigure 2).An additional filtering step based on known organ-specific func-tions resulted in a final short list of 12priority candidate genes (LHX4,DIRC3,MARK1,KIF26B ,MSR1,TMEM67,WD-SOF1,CTHRC1,KCNQ3,PRKG1,ASCC1,and OPCML ),which were also functionally plausible,within our re-gions of interest (Table 1and Table 2).

Mutational Analyses

of Priority Candidate Genes

Mutational analyses of these 12pri-ority candidate genes in BE/EAC cases and controls revealed germ-line mutations in 3genes (MSR1[macrophage scavenger receptor 1][MIM153622],ASCC1[activating signal co-integrator 1complex subunit 1][NC_000010.10],or CTHRC1[collagen triple-helix repeat-containing 1][MIM610635])in 13of 116patients (11.2%)with BE/EAC (T ABLE 3and eTable 1).No sequence variants were found in the remaining 9genes that were not also present to the same degree in controls.

Among the 116patients with BE/EAC,8patients (proportion,0.069;95%confidence interval [CI],0.030-0.130;P ?.001)had a germline trun-cating mutation in MSR1c.877C ?T,re-sulting in p.R293X (F IGURE 2and Table 3),and 2additional patients (pro-portion,0.017;95%CI,0.021-0.061;P =.19)with BE/EAC carried germline MSR1p.L254V (c.760C ?G)in exon 5(Table 3and eFigure 3A).These mu-tations were not found in 139ancestry-matched population controls.Addi-tionally,we identified 2germline missense mutations—c.869A ?G in exon 8of ASCC1,resulting in p.N290S in 2patients (proportion,0.021;95%CI,0.003-0.074;P =.18);and c.131A ?C in exon 1of CTHRC1,re-sulting in p.Q44P in 1patient (propor-tion,0.011;95%CI,0.0003-0.061;P =.42),neither of which were found among 125controls (Table 3,eFigure 3B,and eFigure 3C).

Independent Validation of Germline MSR1,ASCC1,and CTHRC1Mutations

To confirm the mutations found in the 3candidate genes (Table 3),mutational analyses were then per-formed in an independent series of 58cases obtained from outpatient endoscopy units.These samples con-firmed the presence of germline MSR1c.877C ?T,p.R293X mutation in 2of 58cases (proportion,0.034;95%CI,0.004-0.120;P =.09)and CTHRC1c.131A ?C,p.Q44P muta-tion in 1of 58cases (1.7%).After pooling the original 116cases with the validation series of 58,a total of 10cases with BE/EAC carried p.R293X (proportion,0.054;95%CI,0.026-0.098;P =.006)(Table 3).

MSR1and CCND1Protein Levels

Western blotting of germline protein lysates from 5MSR1mutation-

positive patients with BE/EAC and 7controls revealed variable decreases in MSR1protein levels in 3cases (F IGURE 3).All 5MSR1-mutation positive patients had increased CCND1levels compared with con-trols (Figure 3).Barrett esophagus tissues from patients who were mutation-positive showed increased nuclear expression of CCND1by immunohistochemistry compared with control esophageal specimens (F IGURE 4).We then proceeded with the converse experiment by overex-pressing wild-type MSR1in HEK293cells,resulting in decreased CCND1protein (Figure 3).

COMMENT

Barrett esophagus is prevalent in the general population and has the potential to progress to EAC.Because late-stage EAC carries a poor out-come,it is desirable to identify pre-disposition or risk alleles that will eventually allow premorbid risk assessment and affect subsequent management.Herein,we have identi-fied germline mutations in 3candi-date genes in approximately 11%of our series of patients with BE/EAC,with the most commonly affected being MSR1(approximately 7%),fol-lowed by ASCC1and CTHRC1.Find-ings of germline MSR1and CTHRC1mutations were replicated in an inde-pendent validation series.

MSR1on 8p22encodes the class A macrophage scavenger receptor,which are macrophage-specific trimeric inte-gral membrane glycoproteins impli-cated in many macrophage-associ-ated,hormonal,and pathological processes,including inflammation,in-nate and adaptive immunity,oxida-tive stress,and apoptosis.30-32The MSR1c.877C ?T sequence variant,resulting in p.R293X,located within a highly conserved collagen-like domain of the MSR1protein,33would be expected to disrupt function.The MSR1p.R293X was previously shown to associate with prostate cancer in specific ancestries,although this association is controver-sial.30,33-36Given our observations and

Figure 2.Chromatogram of Germline MSR1Mutation

G G

B MSR1 mutation c.877C>T carrier

A,Wild-type sequence.B,Representative example of chromatogram showing MSR1exon 6c.877C ?T (p.R293X)mutation that was observed in approxi-mately 5%(10of 184)of Barrett esophagus and esophageal adenocarcinoma cases,but not in any of 139controls (wild-type sequence,control).The het-erozygous single-nucleotide variant is indicated by the arrow.

GERMLINE MUTATIONS IN MSR1,ASCC1,AND CTHRC1IN PATIENTS WITH BARRETT ESOPHAGUS

416

JAMA,July 27,2011—Vol 306,No.4

taking the p.R293X-prostate cancer as-sociation at face value,one possible ex-planation is that this particular muta-tion is associated with both BE/EAC and prostate cancer predisposition,with the latter at lower penetrance.In many in-herited neoplasia syndromes,single gene mutations predispose to cancers in more than 1organ.37We found both p.R293X and p.L254V (also within the conserved coiled-coil domain and near the glycosylation site at the 249th amino acid)in the germline of our BE/EAC cases,but not in the ancestry-matched population controls,which strongly suggest that these mutations contrib-ute to BE/EAC risk,or at least are nec-essary for BE/EAC predisposition.Whether they are also sufficient is cur-rently unknown.

Accumulating evidence links MSR1to inflammatory events.38Barrett esopha-gus/EAC may also be associated with in-flammatory events,7thus supporting our observations that MSR1is a plausible candidate susceptibility gene for BE/EAC.The molecular mechanisms un-derlying the pathogenesis of inflamma-tion-associated cancer are complex and involve both the innate and adaptive im-mune systems.39-42

More and more examples linking in-flammatory and carcinogenic path-

ways,such as the cell cycle,are surfac-ing.For example,signal transducer and activator of transcription 3and nuclear factor kappa-B link phosphatase and tensin homologue,deleted on chromo-some 10to inflammatory pathways.43Beyond genetic evidence,we have ad-ditionally shown up-regulation of key cell cycle molecule CCND1by both Western blotting of germline proteins and immunohistochemistry of MSR1mutation -related BE tissue,in which CCND1is overexpressed in the nucleus.MSR1p.R293X results in a truncated protein (affecting cytoplas-mic topology,the transmembrane and parts of the collagen-like motifs),which still expresses,but variably.We ob-

Figure 3.Western Blot Detection of MSR1and CCND1Protein Levels

0187X X

CCND1Germline protein lysates from BE lymphoblastoid cells

MSR1

Tubulin

CCND1

FLAG-MSR1

Tubulin

0189X X

0192X X

0197X X

2841X X

Wild-type MSR1(n=7 controls)MSR1 mutation c.877C>T

(n=5 cases)

2844X X

845X

0401G S

1888N C

2855J B

3189S B

4063F

p C M V -F L A G (e m p t y v e c t o r )p C M V -F L A G -M S

R 1

Transfection of MSR1-null HEK293 cells

A,Representative Western blot showing CCND1protein levels from lymphoblastoid cells derived from pa-tients with Barrett esophagus (BE)(n=5)and from population controls (n=7).The Western blot shown is rep-resentative of 2independent experiments.Note variably decreased MSR1accompanied by increased CCND1protein expression in patients with BE compared with controls.B,Representative Western blot of MSR1and CCND1protein levels after HEK293cells were transiently transfected with empty vector or wild-type MSR1constructs.Tubulin was used as a loading control for both A and B.

Figure 4.Immunohistochemistry Detection of CCND1in Esophageal Specimen From a Patient With BE

200 μm 200 μm A Hematoxylin-eosin stain

B Immunostaining for CCND1

200 μm

A,Hematoxylin-eosin staining of an esophageal lesion from biopsy specimen displaying characteristic goblet cells from a representative patient with Barrett esophagus (BE).B,CCND1-positive staining (brown by immunoperoxidase)in the nuclei of BE lesion cells from a patient who was germline MSR1-mutation positive.Hematoxylin counterstain.Detail at higher magnification.

GERMLINE MUTATIONS IN MSR1,ASCC1,AND CTHRC1IN PATIENTS WITH BARRETT ESOPHAGUS

served germline MSR1mutation,with variably decreased MSR1protein lev-els,was associated with overexpres-sion of nuclear CCND1in BE tissues in MSR1-mutation carriers(but not in control normal epithelium).This ob-servation suggests a linkage of inflam-mation to the cell cycle and a poten-tial etiology for BE,via loss of control of the G1-S transition consistent with checkpoint-mediated cell cycle de-lays.44,45CCND1elevation in both spo-radic and heritable BE/EAC as an im-portant final common pathway has precedent,46-49linking the WNT and ad-enomatous polyposis coli protein(APC) cascades,and lending credence to our observations.It remains to be deter-mined whether increased expression of CCND1in the setting of germline MSR1mutation can by itself,or in combination with other oncogenic events,lead to neoplastic transforma-tion in BE.

We also found that ASCC1germ-line p.N290S,affecting a region con-served across species,occurred in2.1% of BE/EAC cases,but not in ancestral-matched population controls.The can-didacy of ASCC1as a risk allele for BE/ EAC is supported by somatic expression array data comparing normal esopha-geal epithelium,premalignant BE,and EAC samples.50ASCC1enhances nuclear factor kappa-B and activator protein1transactivation by directly binding to JUN kinase.51Although little else is known about ASCC1/TRIP4 function,its putative cross-talk with JUN and nuclear factor kappa-B again links inflammatory to tumor suppres-sive/oncogenic pathways.Its role in po-tentially co-regulating the androgen re-ceptor52may also begin to explain the known epidemiologic increased BE risk in men.In addition,although we found only1germline missense mutation in CTHRC1,this gene is intriguing in its cross-talk with2established path-ways in sporadic BE/EAC pathogen-esis,TGFB,and WNT53via APC. Although this sequence alteration is a rare variant,and this study may not be powered to differentiate between1 of89and0of125,a change from glu-tamine to proline results in an altera-

tion from a single-branch polar(nega-

tive)amino acid to a small hydrophobic

cyclic amino acid.This change is pre-

dicted to disrupt the protein’s ability to

form secondary structure and the helix-

loop-helix structure necessary for col-

lagen deposition,fibrosis,and involve-

ment in the WNT and APC pathways.

CTHRC1is expressed in tissue repair

processes and may be important in the

host’s response to GERD.Given its role

in collagen matrix deposition and its ex-

pression in myofibroblasts,53an altera-

tion of CTHRC1might predispose to

decreased lower esophageal sphincter

tone and consequent tendency toward

GERD and BE.

Because we sought to identify risk

alleles that contribute to a reasonable

subset of heritable BE/EAC that also

yield clinically useful attributable risks

(or protection),we consciously used a

relatively small series.We did this be-

cause the larger the series,the more

likely the genetic effect sizes shrink,

such that case-control series in the

thousands result in odds ratios in the

1.1to1.3range.We added rigor by

using a second-stage independent vali-

dation series of cases and controls that

were sufficiently powered based on our

pilot data.In addition,rather than per-

forming brute-force sequencing of at

least38genes in just our9top prior-

ity regions(which would have in-

creased the likelihood of finding

“noise”),we used a systems-biology ap-

proach of statistically prioritizing re-

gions of interest by integrating mul-

tiple platforms to finally come up with

12top priority genes,which eventu-

ally yielded germline mutations in

MSR1,ASCC1,and CTHRC1in pa-

tients with BE/EAC but not in popula-

tion controls.These3genes together

accounted for11%of our cases,reflect-

ing what is normally considered a mod-

erate-to high-penetrance genetic load

for a disease.The functional interro-

gation and immunohistochemistry re-

sults support the pathogenicity of these

germline MSR1mutations.Nonethe-

less,future independent studies are

needed to replicate our data in other pa-

tient populations to confirm the con-

clusions.

In summary,germline mutations in

MSR1,ASCC1,and CTHRC1in pa-

tients with BE/EAC appear physiologi-

cally relevant to BE,encoding pro-

teins involved in apoptosis,innate

immunity,polarity,and mobility that

affect inflammatory and TGFB/WNT

signaling https://www.wendangku.net/doc/3c18747837.html,rger cohort stud-

ies may be necessary to determine the

usefulness of these genes and their vari-

ants in risk assessment and premorbid

diagnosis.

Author Contribution:Dr Eng had full access to all of

the data in the study and takes responsibility for the

integrity of the data and the accuracy of the data analy-

sis.Drs Meltzer and Eng contributed equally to the

manuscript.

Study concept and design:Orloff,He,Heald,Romigh,

Meltzer,Eng.

Acquisition of data:Peterson,He,Ganapathi,

Heald,Yang,Bebek,Song,Wu,David,Cheng,

Meltzer.

Analysis and interpretation of data:Orloff,He,

Ganapathi,Bebek,Romigh,Meltzer,Eng.

Drafting of the manuscript:Orloff,He,Bebek,David,

Meltzer,Eng.

Critical revision of the manuscript for important in-

tellectual content:Orloff,Peterson,He,Ganapathi,

Heald,Yang,Bebek,Romigh,Song,Wu,David,Cheng,

Meltzer,Eng.

Statistical analysis:Orloff,He,Bebek,Eng.

Obtained funding:Meltzer,Eng.

Administrative,technical,or material support:

Peterson,He,Ganapathi,Heald,Yang,Romigh,Song,

Wu,David,Cheng,Meltzer,Eng.

Study supervision:Meltzer,Eng.

Accruing research subjects documented and verified

phenotypes and/or participated in material acquisi-

tion:Heald,Song,Wu,David,Cheng,Meltzer,Eng.

Genetic counselor coordinator:Heald.

Conflict of Interest Disclosures:All authors have com-

pleted and submitted the ICMJE Form for Disclosure

of Potential Conflicts of Interest.Dr Bebek reported

receiving grant funding from the National Institutes

of Health(NIH).Dr Meltzer reported receiving grants

from the National Cancer Institute(NCI)and the Na-

tional Institute of Diabetes and Digestive and Kidney

Diseases;and travel,accommodations,and meeting

expenses from Hong Kong University.Dr Eng re-

ported receiving grants from the NCI and Rudy En-

dowment.All other authors reported no financial dis-

closures.

Funding/Support:This work was funded in part by the

M.Frank and Margaret Domiter Rudy Endowment of

the Cleveland Clinic Taussig Cancer Institute and grants

CA30722,DK087454,CA146799,and CA133012

from the NCI/NIH(S.J.M.and C.E.).Dr Meltzer is the

Harry&Betty Myerberg/Thomas R.Hendrix Profes-

sor of Gastroenterology and was funded in part by the

Esophageal Cancer Research Fund at the Johns Hop-

kins University School of Medicine.Dr Eng was a re-

cipient of the Doris Duke Distinguished Clinical Sci-

entist Award,is an American Cancer Society Clinical

Research Professor generously funded in part by the

F.M.Kirby Foundation,holds the Sondra J.&Ste-

phen R.Hardis Chair in Cancer Genomic Medicine at

the Cleveland Clinic,and is an honorary fellow of the

Cancer Research UK Human Cancer Genetics Re-

search Group,University of Cambridge,Cambridge,

England.

GERMLINE MUTATIONS IN MSR1,ASCC1,AND CTHRC1IN PATIENTS WITH BARRETT ESOPHAGUS

Role of the Sponsors:The funding organizations had no role in the design and conduct of the study,in the collection,analysis,and interpretation of the data,or in the preparation,review,or approval of the manu-script.

Online-Only Material:eMethods,3eTables,3eFig-ures,and eReferences are available at http://www https://www.wendangku.net/doc/3c18747837.html,.

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