Germline mutations in FH confer predisposition to malignant pheochromocytomas and ...

24 Pages·2013·0.3 MB·English

HumanMolecularGenetics,2014,Vol.23,No.9 2440–2446
doi:10.1093/hmg/ddt639
AdvanceAccesspublishedonDecember13,2013
Germline mutations in FH confer predisposition
to malignant pheochromocytomas and
paragangliomas
LuisJaimeCastro-Vega1,2,{,AlexandreBuffet1,2,3,{,AguirreA.DeCubas4,{,AlbertoCasco´n4,5,
Me´lanieMenara1,2,EmmanuelKhalifa1,2,3,LaurenceAmar1,2,6,SharonaAzriel7,
D
IsabelleBourdeau8,OlivierChabre9,MariaCurra´s-Freixes4,Vale´rieFranco-Vidal10, o
w
n
MarineGuillaud-Bataille11,ChristopheSimian3,Aure´lieMorin1,2,Roc´ıoLeto´n4,5, lo
a
d
A´lvaroGo´mez-Gran˜a4,PatrickJ.Pollard12,PierreRustin13,MercedesRobledo4,5,{, ed
JudithFavier1,2,{andAnne-PauleGimenez-Roqueplo1,2,3,{,∗ from
h
ttp
S1IoNrSbEonRnMe,PUaMrisRC97it0e´,,PFaarcius-ltCe´adrediMove´adseccuinlaer,RFe-7s5e0a0rc6hPCaerinst,eFr,raFn-7c5e0,13A5s,sPiastraisn,cFeraPnucbeli,q2uUen-Hivoeˆprsitiate´uPxadreisPDaerissc,aHrotˆepsit,al s://ac
a
Europe´enGeorgesPompidou,ServicedeGe´ne´tique,F-75015Paris,France,4HereditaryEndocrineCancerGroup, de
m
SpanishNationalCancerCentre(CNIO),Madrid,Spain,5CentrodeInvestigacio´nBiome´dicaenReddeEnfermedades ic
.o
Raras(CIBERER),Madrid,Spain,6AssistancePublique-HoˆpitauxdeParis,HoˆpitalEurope´enGeorgesPompidou,Unite´ up
d’HypertensionArte´rielle,F-75015Paris,France,7InfantaSof´ıaHospital,EndocrinologyService,SanSebastiandelos .co
m
Reyes,Madrid,Spain,8DivisionofEndocrinology,DepartmentofMedicine,ResearchCentre,CentreHospitalierde /h
m
l’Universite´ deMontre´al(CRCHUM),Montre´al,Que´bec,Canada,9DepartmentofEndocrinology,UniversityHospital g/a
AlbertMichallon,F-38043Grenoble,France,10DepartmentofOtolaryngologyandSkullBaseSurgery,Pellegrin rtic
UniversityHospital,UniversityBordeauxSegalen,F-33000Bordeaux,France,11ServicedeGe´ne´tique,Institutde le-a
b
Cance´rologieGustaveRoussy,94805Villejuif,France,12CancerBiologyandMetabolismGroup,EdinburghCancer stra
ResearchUKCentre,InstituteofGeneticsandMolecularMedicine,UniversityofEdinburgh,Edinburgh,UKand ct/2
13INSERMU676,HoˆpitalRobertDebre´,F-75019Paris,France 3
/9
/2
4
4
ReceivedOctober28,2013;RevisedandAcceptedDecember10,2013 0
/6
3
3
4
8
Malignantpheochromocytoma(PCC)andparaganglioma(PGL)aremostlycausedbygermlinemutationsof 0 b
SDHB,encodingasubunitofsuccinatedehydrogenase.Usingwhole-exomesequencing,werecentlyidentified y g
u
amutationintheFHgeneencodingfumaratehydratase,inaPCCwithan‘SDH-like’molecularphenotype.Here, e
s
weinvestigatedtheroleofFHinPCC/PGLpredisposition,byscreeningforgermlineFHmutationsinalargeinter- t o
n
nationalcohortofpatients.Wescreened598patientswithPCC/PGLwithoutmutationsinknownPCC/PGLsus- 1
7
ceptibilitygenes.WesearchedforFHgermlinemutationsandlargedeletions,bydirectsequencingand N
o
multiplexligation-dependentprobeamplificationmethods.GlobalalterationsinDNAmethylationandprotein ve
m
succinationwereassessedbyimmunohistochemicalstainingfor5-hydroxymethylcytosine(5-hmC)andS-(2- b
e
succinyl)cysteine(2SC),respectively.WeidentifiedfivepathogenicgermlineFHmutations(fourmissense r 2
0
andonesplicemutation)infivepatients.Somaticinactivationofthesecondallele,resultinginalossoffumarate 1
8
hydrataseactivity,wasdemonstratedintumorswithFHmutations.Lowtumorlevelsof5-hmC,resemblingthose
inSDHB-deficienttumors,andpositive2SCstainingweredetectedintumorswithFHmutations.Clinically,meta-
staticphenotype(P50.007)andmultipletumors(P50.02)weresignificantlymorefrequentinpatientswithFH
∗Towhomcorrespondenceshouldbeaddressedat:ServicedeGe´ne´tique,HoˆpitalEurope´enGeorgesPompidou,20-40rueLeblanc,75015Paris,France.
Tel:[phone];Fax:[phone];Email:[email protected]
†Theseauthorscontributedequallytothiswork.
‡Theseauthorscontributedequallytothiswork.
#TheAuthor2013.PublishedbyOxfordUniversityPress.Allrightsreserved.
ForPermissions,pleaseemail:[email protected]
HumanMolecularGenetics,2014,Vol.23,No.9 2441
mutations than those without such mutations. This study reveals a new role for FH in susceptibility to
malignantand/ormultiplePCC/PGL.Remarkably,FH-deficientPCC/PGLsdisplaythesamepatternofepi-
geneticderegulationasSDHB-mutatedmalignantPCC/PGL.Therefore,weproposethatmutationscreening
forFHshouldbeincludedinPCC/PGLgenetictesting,atleastfortumorswithmalignantbehavior.
INTRODUCTION heterozygosity(LOH)inavailabletumors.Notably,weindicate
thatFHinactivationdrivesepigeneticdysregulationsimilarto
Pheochromocytomas(PCC)andparagangliomas(PGL)arerare SDHB deficiency, as wellas widespread alterations inprotein
neuroendocrine tumors derived from chromaffin cells of the succination,bothofwhichareaccuratelydetectedbyimmuno- D
o
adrenal medulla and paraganglia from the autonomic nervous histochemistry (IHC). Therefore, we conclude that FH is a w
n
system,respectively.Overall,10–20%ofPCC/PGLaremalig- secondmajorsusceptibilitygenetomalignantPCC/PGL. lo
a
nant,andmalignancyremainsdifficulttopredictandisdefined de
d
onPlyCaCs/tPheGpLreasreenhceereodfimtaertyasinta(cid:3)se4s0in%aonfocna-scehsr,oamnadfpfirnetdiissspuoes(i1n)g. from
germlinemutationshavebeenfoundinoneoncogene(RET)and RESULTS h
ttp
10 tumor suppressor genes (SDHB, SDHC, SDHD, SDHA Alargeinternationalseriesof598patients,collectedinFrance s
(SDHx), SDHAF2, VHL, TMEM127, MAX and NF1) (2). The (n¼359) and Spain (n¼239), diagnosed with PCC/PGL ://a
c
identification of PCC/PGL susceptibility genes has made a withoutmutationsinanyofthe11knownPCC/PGLsusceptibil- ad
e
crucial contribution to our understanding of the molecular itygeneswereanalyzedforgermlinemutationsofFH(Table1). m
basis of this disease, and to the definition of markers of poor Thispopulationconsistedof151patients(25%)diagnosedwith ic.o
prognosis useful for clinical follow-up. Indeed, large geno- PCC/PGLbeforetheageof35years,85patients(14%)withmul- up
type–phenotypestudieshaveshownthatthepresenceofagerm- tiplePCC/PGLand59patients(10%)withmetastaticPCC/PGL. .co
m
lineSDHBmutationisassociatedwithahighriskofmalignancy Forty-onedifferentheterozygousvariantscoveringtheentire /h
andreducedsurvival(3,4). length of the FH gene were identified (Fig. 1; Supplementary m
g
TheSDHBgeneencodesthecatalyticsubunitofatricarboxylic Material, Table S1). We detected 18 single nucleotide poly- /a
acid(TCA)cycleenzyme,succinatedehydrogenase(SDH).SDH morphisms(SNPs):12withfrequenciessimilartothosereported rtic
le
inactivationleadstotheaccumulationofitssubstrate,succinate, inthegeneralpopulation,and6forwhichnofrequency value -a
b
whichactsasanoncometabolite(5)byinhibiting2-oxoglutarate- was available. We also identified 23 other variants: 14 (61%) s
dependent dioxygenases such as HIF prolyl-hydroxylases (6,7) were intronic, 6 (26%) were missense and 3 (13%) were syn- trac
and TET enzymes (8), promoting pseudo-hypoxic signaling (9) onymous.Thepresenceofcopynumbervariationswasexcluded t/2
3
andDNAhypermethylation(10),respectively.Theinhibitionof by multiplex ligation-dependent probe amplification (MLPA). /9
/2
thesemolecularpathwaysexplainstheextensivevascularization Five of the identified variants were classified as pathogenic, 4
4
ofSDH-relatedtumorsandmaycontributetometastaticdissemin- either because they had already been described as causal 0/6
ation,bydrivingepithelial-to-mesenchymaltransitioninSDHB- variants,oronthebasisofinsilicoprediction(Supplementary 33
4
deficienttumors(10,11). Material, Table S2). None of these variants was found in 8
0
Comprehensiveintegrativegenomicsstudieshaveimproved publicSNPdatabases. b
y
the classification of PCC/PGL (12–14). Two main clusters of We previously described the patient carrying the missense g
u
mutations have been identified in transcriptome analyses. mutation(c.349G.C;p.Ala117Pro)inexon3(10),whichhad es
Cluster1includesSDHx-(cluster1A)andVHL-relatedtumors already been identified as pathogenic by two independent t o
n
(cluster 1B) and is characterized by the pseudohypoxic signa- groups in patients with a diagnosis of multiple cutaneous and 1
7
ture.Cluster2comprisesallNF1-,RET-,MAX-andTMEM127- uterineleiomyomatosis(MCUL)(15).Asecondvariantaffect- N
o
related tumors and is associated with the activation of MAP inganessentialsplicingsite(c.268-2A.G;p.?)hadpreviously ve
m
kinase/AKT/mTOR pathways. We previously reported a been foundinapatient withFHdeficiency andreportedtobe b
e
tumorclassifiedasacluster1Atumor(14)withahypermethyla- pathogenic in the TCA Cycle Gene Mutation Database (16). r 2
torphenotype(10)butnoSDHxgenemutation.Bywhole-exome The remaining three FH missense variants identified in this 0
1
sequencing,wedetectedagermlinemutation,associatedwitha studyarepreviouslyunknownmutations,locatedwithinexon8 8
secondsomaticmutationintheFHgene,whichencodesanother (c.1142C.T; p.Thr381Ile), exon 7 (c.986A.G; p.Asn329Ser)
tricarboxyliccycleenzyme,fumaratehydratase(10).Germline andexon5(c.580G.A;p.Ala194Thr).
mutationsofFHhavepreviouslybeenreportedincasesofher- TheclinicalandgeneticfeaturesofindividualscarryingFH
editary leiomyomatosis and renal cell cancer (HLRCC) (15) mutationsarepresentedinTable2.Threeofthesepatientshad
buthadneverbeendescribedinPCC/PGL. metastatic PCC/PGL and three developed multiple tumors.
Here,weidentifyfivepathogenicgermlineFHmutationsin Thefirstpatient,describedinapreviousstudy(10),presented
five patients with PCC/PGL and without mutations in any of metastaticPCCattheageof63yearsanddiedat73yearsold.
theknownsusceptibilitygenesforthisdisease.Thesepatients Subsequent analysis of her clinical record revealed that she
present clinical characteristics compatible with malignancy. hadahistoryofhysterectomyattheageof35yearsforhemor-
Furthermore,wedeterminethefunctionalconsequencesofFH rhagic fibromas highly suggestive of uterine leiomyoma. The
inactivation by examining the enzyme activity and loss of secondpatientwasdiagnosedwithleftadrenalPCCattheage
2442 HumanMolecularGenetics,2014,Vol.23,No.9
of20yearsandwithabdominalPGL4yearslater.Patient#3had Wehadalreadyreportedthatthetumorfrompatient#1carry-
hypertensionsincechildhoodandwasdiagnosedattheageof28 ing the variant in exon 3 (c.349G.C, p.Ala117Pro) also pre-
yearswithmultiplelumbar-aorticPGLs,withevidenceofbone, sented a second somatic inactivating mutation in exon 7
liver andlymphnodemetastases. Thefourthpatient, who had (c.1043G.C,p.Gly348Ala)(10).WecheckedforLOHintwo
undergonesurgeryforZuckerkandlPGL,presentedarelapse9 other FH mutations carriers, by directly sequencing the FH
yearslater,withbilateralPCCandevidenceofbonemetastases. genefromtheavailabletumorDNA(patients#2and#3).The
Normetanephrine concentrations were high for these four mutationinexon8(c.1142C.T;p.Thr381Ile)washomozygous
patients at the time of PCC or PGL diagnosis. Only one of inpatient#3(Fig.2A),consistentwithclassicaltwo-hitgenein-
these five patients presented a single, non-secreting, benign activation.Thisinactivationwasconfirmedbyacompletelossof
tumor,locatedinthecarotidbody,whichwastreatedbyexternal fumaraseactivityinthetumor(Fig.2B).LOHwasalsoobserved
radiotherapy.Fortheentirecohort,theproportionofFHmuta- forthemutationofpatient#2(c.268-2A.G),inDNAextracted
tion carriers displaying either metastases (P¼0.007) or mul- from both her paraffin-embedded PCC and PGL tumors Do
w
tiplePCC/PGL(P¼0.02)wassignificantlyhigherthanthatin (Fig.2C). n
lo
patientsnotcarryingFHmutations.Incontrast,firstPCC/PGL WehaverecentlyshownthatSDH-relatedtumorcellsandthe a
d
diagnosisbeforetheageof35yearswasnotindicativeofFHmu- first FH-deficient PGL (patient #1) identified have low e
d
tation(P¼0.6045). 5-hydroxymethylcytosine (5-hmC), due to inhibition of TET fro
enzymes by succinate and fumarate. Moreover, it has been m
h
Table1. ClinicalandtumorcharacteristicsofPCC/PGLpatients sdtehriyonlws,gnrotShu-a(p2ts-fsutoumcgacerinantyeerl)areteaccyatsssttesapibnolenetac(nh2eeSomCui)sclay(l1wm7i)ot,hdicwfiychsatiectiihonnecsoaufnlfphrbyoe-- ttps://ac
Totalpatients n¼598 evaluatedbyIHC(18).WethusstudiedtheimpactofFHinacti- ad
e
vationonbothDNAmethylationandproteinsuccination,using m
Sex ic
Male 247(41.3%) antibodiesdirectedagainst5-hmCand2SC,respectively.These .o
u
Female 351(58.7%) experimentswereperformedinthethreeFH-mutatedsamples p
Ageatdiagnosis forwhichparaffin-embeddedtissueswere availableaswellas .co
m
Mean(range) 46.1(6–87)
in 18 other PGL/PCC for which the genetic screening of FH /h
,35years 151(25.3%) m
Tumortype was performed and was negative. Among them, there were g
/a
PPCGCL 421006((6384..64%%)) sdeisvpeonshinagrbgoerninegsg(tewrmolNinFe1o,rtwsoomRaEtiTc,mountaetTioMnsEiMnP1G27L,/PoCneCVpHreL- rticle
H&N 96(46.6%) and oneMAX). -a
TAP 114(55.3%) bs
H&N+TAP 4(1.94%) The evaluation of 5hmC levels by IHC confirmed that tra
TuPmCoCrc+haPraGcLteristics 18(3.01%) FchHa-nmgeustaatsedSDPHC-CrelaatneddtPuGmLorsd.iIsnplaadydeidtiotnh,ewesaombeserevpeidgeansepteic- ct/23
Single 513(85.8%) cific2SC-positivestaininginFH-deficientsamples,whichwas /9/2
Multiple 85(14.2%) 4
absentintumorswithoutFHmutations(Fig.3,Table2,Supple- 4
Metastatic 59(9.8%) 0
mentary Material, Table S3). Thus, the combination of low /6
3
5hmClevelsandpositiveimmunostainingfor2SCcanbeused 3
PCC,pheochromocytoma;PGL,paraganglioma;H&N,headandneck; 48
TAP,thoracic/abdominal/pelvic. topredictorvalidateFH-variantsinPCC/PGL. 0 b
y
g
u
e
s
t o
n
1
7
N
o
v
e
m
b
e
r 2
0
1
8
Figure1.GraphicalrepresentationofpathogenicFHmutationsidentifiedinthisstudyandtheresiduesoftheproteinaffected.Thevariantalreadyreportedisshownin
red,andvariantsaffectingahighlyconservedamino-acidresidueareshowninblue.Sequencealignmentsofcorrespondingresiduesindifferentspecies.
HumanMolecularGenetics,2014,Vol.23,No.9 2443
Table2. Genetic,clinicalandimmunohistologicalcharacteristicsofFHmutationcarriers
Patient Sex Age Tumortype Multiple Metastases cDNAmutation Proteinalteration LOH 5hmC 2SC
#1 F 63 PCC No Yes c.349G.C p.Ala117Pro Yes Low Positive
#2 F 20 PCC+PGL(TAP) Yes No c.268-2A.G p.? Yes Low Positive
#3 M 28 PGL(TAP) Yes Yes c.1142C.T p.Thr381Ile Yes Low Positive
#4 F 54 PCC+PGL(TAP) Yes Yes c.580G.A p.Ala194Thr NA NA NA
#5 M 70 PGL(H&N) No No c.986A.G p.Asn329Ser NA NA NA
Ageistheageatdiagnosis.Thelettersandnumbersintheproteinalterationcolumnindicatetheaminoacidfromthewild-typesequence(first)andtheaminoacidwith
whichitisreplacedinthemutatedsequence(second).Thenumberbetweenthelettersdenotesthecodonposition.Aminoacidabbreviations:Ala,alanine;Pro,proline;
Thr,threonine;Ile,isoleucine;Asn,asparagine;Ser,serine;PCC,pheochromocytoma;PGL,paraganglioma;H&N,headandneck;TAP,thoracic/abdominal/pelvic; D
LOH,lossofheterozygosity;NA,notavailable. ow
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/h
m
g
/a
rtic
le
-a
b
s
tra
c
t/2
3
/9
/2
4
4
0
Figure2.FunctionalcharacterizationofFHmutationsintumortissues.(A)LOHintumortissueforthevariantinexon8(c.1142C.T,p.Thr381Ile)identifiedin /6
3
patient#3.(B)MeasurementofFHenzymeactivityinthePGLharboringtheaforementionedvariant.(C)LOHofthesplicingvariant(c.268-2A.G)inboththe 3
4
PCCandthePGLfrompatient#2. 8
0
b
y
g
u
DISCUSSION FHmutation,revealinganewroleforthismitochondrialenzyme es
in cancer and demonstrating the functional overlap between t o
n
TCAcycleenzymesareincreasinglybeingreportedtoplayarole TCAcycleenzymesincancerpredisposition. 1
7
in the predisposition to inherited tumor syndromes and in the We searched for FH mutations in germline DNA from 598 N
o
pathogenesis of sporadic forms of cancer (19). Mutations in patientswithPCCand/orPGLwithnomutationsinknownsus- ve
m
genes encoding SDH subunits (SDHA, SDHB, SDHC and ceptibility genes. We found that germline mutations of FH b
e
SDHD) were the first to be shown to confer predisposition to accounted for 0.83% of cases, a frequency similar to that r 2
PGL and PCC, but such mutations are also responsible for reported for TMEM127 (0.9%) (26) and MAX (1.12%) (27) 0
1
gastrointestinal stromal tumors (20) and kidney cancers (21). mutations. FH mutations should therefore be considered a 8
Germline FH mutations have been identified in MCUL and new,albeitrare,sourceofpredispositiontoPCC/PGLs.
HLRCC (15) syndromes, characterized by benign smooth According to current data from the FH section of the TCA
muscle tumors, uterine leiomyoma and a highly malignant Cycle Gene Mutation Database (16), 268 mutations (154
form of papillary and collecting duct renal cancer. Finally, unique) of this gene have been reported in total, most being
somatic mutations in the genes encoding the mitochondrial found in patients with diagnoses of MCUL (112; 46%),
(IDH2)andcytosolic(IDH1)formsofisocitratedehydrogenase HLRCC (47;19%) and FH deficiency (42; 17%). Variants of
havebeenfoundingliomas(22),acutemyelogenousleukemia exon 7 (53; 19%), exon 5 (43; 16%), exon 3 (36; 13%) and
(23),incentralchondrosarcomas(24)andcholangiocarcinomas exon 8 (25; 9%) are overrepresented, suggesting that some of
(25).Followingourrecentidentificationofthefirstpatientwitha the sequences of the gene may be more prone to mutational
germlineFHmutationresponsibleforPGL,wereportherefour eventsthanothers.Consistentwiththesedata,twoofthepoten-
additionalcasesofpredispositiontoPCCand/orPGLcausedby tiallypathogenicvariantsfoundinthisstudyconcernedexon3,
2444 HumanMolecularGenetics,2014,Vol.23,No.9
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
Figure3.Expressionof5-hmCand2SCinFH-deficienttumors.(A)PCCwithRETmutation.(B)PCCwithNF1mutation.(C)PGLwithSDHBmutation.(D)PCC .o
u
frompatient#1withFHmutation.(E)PGLfrompatient#3withFHmutation.(F)PGLfrompatient#2withFHmutation.Immunohistochemicalstainingfor5-hmC p
wasstronglypositiveintumoralandendothelialcellsfromRET-andNF1-mutatedtumors.Incontrast,thisstainingwasnegativeinthetumoralcellsandpositiveinthe .co
endothelialcellsofSDHB-andFH-mutatedtumors.Immunohistochemicalstainingfor2SCwasnegativeforRET-,NF1-andSDHB-mutatedtumors,butpositivefor m
FH-mutatedtumors. /hm
g
/a
rtic
whereas the other three missense mutations were located in wide alterations in DNA methylation. These findings validate le
exons 5,7 and 8.All these variants affected highly conserved the newly identified mutations as functional. In addition, we -a
b
s
residuesoftheproteinandwerepathogenic,accordingtothebio- show for the first time that the accumulation of fumarate in tra
infTohrmreaetiocfttohoelfisv.emutationcarriersidentifiedhadeitherPCC+ PasCCre/vPeGalLedalbsoyl2eSadCsitmomgluonboasltcahinainngge.sTihnepsropteeciinficsuitcycionfat2ioSnC, ct/23
/9
PGLormultiplePGL,but,tothebestofourknowledge,nonehad staininginFH-mutatedPCC/PGLisfurthersupportedbyapre- /2
4
kidneycancerandonlyonehadhaduterinefibromas,25years viousstudy,whichfoundno2SCimmunolabelingin44PCC/ 4
0
beforePCCdiagnosis.Thereasonsforthisdifferenceinthespec- PGLsamples(17).Together,thesefindingssuggestthatfumar- /6
3
trumoftumordevelopmentremainunclearandwillneedtobe atealsoplaysakeyroleasanoncometaboliteintumorinitiation 3
4
8
addressedinfuturestudies.Nevertheless,itcouldbesuggested and metastasis in these neuroendocrine tumors. These results 0
b
that patients harboring FH mutations should be screened for suggestthattheapplicationofacombinationofthesetwoimmu- y
g
the presence of PCC or PGL. Accordingly, renal cancers and nohistochemical assays to tumor tissues from patients may be u
e
leiomyomasshouldbesearchedforinPCC/PGLpatientscarry- usefulfortheidentificationofFH-relatedPCC/PGL. st o
ingFHvariants.Threepatientspresentedevidenceofmetastatic Inconclusion,theresultsofthisstudystronglysuggestthat, n
1
disease,suggestingthatFHinactivationmayhavearolesimilar like SDHB, FH is one of the causal genes of malignant PCC/ 7
N
tothatofSDHBmutationsindrivingthemalignantphenotype. PGL.Tumoral5-hmCand2SCimmunostainingwouldbeacost- o
v
The younger patient with apparently benign PCC and PGL effectiveapproachtothedetectionofpatientsatrisk.FHshould e
m
underwentsurgery9and5yearsago,andwethereforecannot thusbe addedtothelistof PCC/PGLsusceptibilitygenes and b
e
excludethepossibilityofrecurrenceorametastaticphenotype should be considered in mutation screening, to assess the risk r 2
0
inthefuture. Giventhatsuccinateandfumarate aregenerated ofmalignantdisease. 1
8
bysequentialenzymaticreactionsinthesamemetabolicsignal-
ingpathway,SDH-andFH-deficientcellsarelikelytohave,at
least to some extent, a common tumorigenic mechanism. We MATERIALSANDMETHODS
and others have recently demonstrated that the accumulation
Clinicalsamples
of succinate and fumarate is responsible for the development
ofahypermethylatorphenotype,mediatedbytheinhibitionof Weanalyzed598unrelatedpatientsdiagnosedwithPCCand/or
DNAdemethylasesoftheTETfamilyinvolvedinthehydroxyl- PGLwithnomutationinaknownPCC/PGLsusceptibilitygene
ation of 5-methylcytosine to generate 5-hmC. Consistent with (RET, NF1, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2,
these findings, we found that FH-deficient tumors had the TMEM127orMAX).Thecharacteristicsofthetumorsandthe
same altered immunostaining pattern for 5-hmC observed for patientsarepresentedinTable1.Therelativelyhighproportion
SDHB-deficient tumors, indicating a common pathophysio- ofyoungpatientsinthiscohortmaybeexplainedbythefactthat
logicalmechanism,involvingthespecificinductionofgenome- bothdepartmentsinvolvedisthisstudyarereferralcentersfor
HumanMolecularGenetics,2014,Vol.23,No.9 2445
theseraretumors.Bloodandtumorsampleswerecollectedby MeasurementofFHenzymeactivity
the French ‘Cortico et Me´dullosurre´nale: les Tumeurs Endo-
FHenzymeactivityintumortissueswasmeasuredbyspectro-
crines’(COMETE)networkinParis(France)andbypublichos-
photometry, as previously described (10). Briefly, this assay
pitalsandtheSpanishNationalTumorBankNetworkinMadrid
monitorsthechangeinabsorbanceduetotheforward(fumarate
(Spain).Eachpatientgavewritteninformedconsentfortheana-
tomalate)andbackward(malatetofumarate)conversions.The
lysisofgermlineandsomaticDNA.Thestudywasapprovedby
enzyme activity is measured at 378C. Reagents were obtained
thelocalinstitutionalreviewboards(CPPIledeFranceII,June,
fromSigma-Aldrich(France).
2012andComite´debioe´ticaybienestaranimaldelInstitutode
SaludCarlosIII).
GermlineDNAwasextractedfromleukocytesaccordingto Statisticalanalyses
thestandardprotocols.Frozentumorsamplesweregroundtoa
StatisticalanalysiswascarriedoutwithGraphPadsoftware.Dif- D
powder in liquid nitrogen and 30–50mg of the sample was o
ferences between patients carrying and not carrying mutations w
used for DNA extraction with the AllPrep kit (Qiagen). DNA n
were analyzed for the frequency of multiple tumors, PCC plus lo
was extracted from paraffin-embedded slides according to the a
PGL, early onset of disease and malignancy, in two-tailed d
standardprotocols.DNAwasquantifiedanditspurityassessed e
d
withaNanoDropND-1000spectrophotometer(Labtech). Fisher’sexacttests.AP-value,0.05wasconsideredsignificant. fro
m
h
SUPPLEMENTARYMATERIAL ttp
Moleculargeneticanalyses s
DirectSangersequencingofthe10exonsoftheFHgene,includ- SupplementaryMaterialisavailableatHMGonline. ://aca
d
ingtheexon–intronboundaries,wasperformed.ForLOHana- e
m
lpyrsimiseornswtuemreodreDsiNgnAede,xftorarcthteedgfernoemratpiaornafofifnsmseacllteiornasm,psplieccoinfisc. ACKNOWLEDGEMENTS ic.ou
p
The primer sequences and PCR conditions are available upon WethankProfessorsPierre-Franc¸oisPlouinandX.Jeunemaitre .c
o
request. Sequencing reactions were performed with the forhelpfuldiscussionsandcontinualsupport,DrRossellaLibe, m
BigDye Terminator v3.1 kit (Life Technologies), in an ABI Dr Nathalie Sturm, Prof. Ce´cile Badoual and Dr Fre´de´rique /hm
Prism3730XLDNAAnalyzer(PerkinElmerAppliedBiosys- TissierandallmembersoftheHEGPGeneticsDepartmentfor g/a
tems,FosterCity,CA,USA).ForthedetectionoflargeFHdele- theirhelpandCatherineTritscherfortechnicalassistance.We rtic
tions,MLPAwasperformedwiththeSALSAMLPAP198-A2 thank Dr Norma Frizzell for generating the 2SC antibody. le
-a
kit and the fumarase deficiency probe mix (MRC-Holland), AlexEdelmanandAssociateseditedthetext. b
s
accordingtothemanufacturer’srecommendations. tra
c
Conflict ofIntereststatement.P.J.P.wasaco-applicantonthe t/2
filing for a patent application [USC-268-P(849)] covering 3/9
AnalysisofFHvariants immunohistochemicalscreeningwitha2SCantibodyfordeter- /2
4
We assessed the potential pathogenicity of variants with minationofFHmutations. 40/6
Alamutw-Mutation Interpretation Software version 2.3.2 33
4
(http://www.interactive-biosoftware.com/software.html). We 8
FUNDING 0
checked the frequencies of the mutations and variants in the b
y
TCACycleGeneMutationDatabase(16),theExomeSequen- ThisworkwassupportedbytheAgenceNationaledelaRecher- g
u
cingProjectand1000Genomesdatabase. che (ANR-2011-JCJC-00701 MODEOMAPP), the Fondo de es
Investigaciones Sanitarias–FIS project PI11/01359, from the t o
n
European Union Seventh Framework Programme (FP7/ 1
7
Immunohistochemistry 2007-2013)undergrantagreementno.259735,theGIS-Institut N
o
desMaladiesRaresandtheProgrammeHospitalierdeRecher- v
Formalin-fixed,paraffin-embeddedtumorsections(6mm)were e
m
cheCliniquegrantCOMETE3(AOM06179).A.B.holdsafel-
used for 5-hmC and S-(2-succinyl) cysteine (2SC) staining. b
Briefly,slidesweredeparaffinizedinxyleneandrehydratedin lowship from la Fondation ARC, and A.A.C. is a predoctoral er 2
fellowofthe‘laCaixa’/CNIOinternationalPhDprogram. 0
alcohol. Antigen retrieval was performed with a citrate-based 1
8
antigen-unmasking solution (8.2mM sodium citrate, 1.8mM
citricacid1.8mM,pH6).Thefollowingantibodiesandrespect- REFERENCES
ivespecificationswereused:polyclonalrabbit5-hmC(Active
motif catalog; Ref. 39769, 1:1200) and polyclonal rabbit 2SC 1. Lloyd,R.,Tischler,A.,Kimura,N.,McNicol,A.andYoung,J.W.(2004)In
[(28), 1:5000]. Biotinylated secondary antibodies (Vector DeLellis,R.A.L.R.,Heitz,P.U.andEng,C.(eds),WHOClassificationof
Tumours-PathologyandGenetics-TumoursofEndocrineOrgans.IARC
Laboratories) were used at a dilution of 1:400. Tumor tissues
Press.
without FH mutations were used as controls and slides were 2. Gimenez-Roqueplo,A.P.,Dahia,P.L.andRobledo,M.(2012)Anupdateon
scoredinablindedmanner.5-hmCIHCwasanalyzedbycom- thegeneticsofparaganglioma,pheochromocytoma,andassociated
paringthelevelofnuclearstainingbetweentumorcellsandad- hereditarysyndromes.Horm.Metab.Res.,44,328–333.
3. Amar,L.,Servais,A.,Gimenez-Roqueplo,A.P.,Zinzindohoue,F.,
jacentendothelialcellsasinternalcontrols.Tumorswerequoted
Chatellier,G.andPlouin,P.F.(2005)Yearofdiagnosis,featuresat
as‘low’whentumorstainingwasinferiortothatofendothelial
presentation,andriskofrecurrenceinpatientswithpheochromocytomaor
cells.2SCwasscoredaseitherpositiveornegative. secretingparaganglioma.J.Clin.Endocrinol.Metab.,90,2110–2116.
2446 HumanMolecularGenetics,2014,Vol.23,No.9
4. Amar,L.,Baudin,E.,Burnichon,N.,Peyrard,S.,Silvera,S.,Bertherat,J., MCUL(HLRCC)tumorsyndromeandcongenitalfumarasedeficiency.
Bertagna,X.,Schlumberger,M.,Jeunemaitre,X.,Gimenez-Roqueplo,A.P. BMCMed.Genet.,9,20.
etal.(2007)SuccinatedehydrogenaseBgenemutationspredictsurvivalin 17. Alderson,N.L.,Wang,Y.,Blatnik,M.,Frizzell,N.,Walla,M.D.,Lyons,
patientswithmalignantpheochromocytomasorparagangliomas.J.Clin. T.J.,Alt,N.,Carson,J.A.,Nagai,R.,Thorpe,S.R.etal.(2006)
Endocrinol.Metab.,92,3822–3828. S-(2-Succinyl)cysteine:anovelchemicalmodificationoftissueproteinsbya
5. Yang,M.,Soga,T.andPollard,P.J.(2013)Oncometabolites:linkingaltered Krebscycleintermediate.Arch.Biochem.Biophys.,450,1–8.
metabolismwithcancer.J.Clin.Invest.,123,3652–3658. 18. Bardella,C.,El-Bahrawy,M.,Frizzell,N.,Adam,J.,Ternette,N.,Hatipoglu,
6. Selak,M.A.,Armour,S.M.,MacKenzie,E.D.,Boulahbel,H.,Watson,D.G., E.,Howarth,K.,O’Flaherty,L.,Roberts,I.,Turner,G.etal.(2011)
Mansfield,K.D.,Pan,Y.,Simon,M.C.,Thompson,C.B.andGottlieb,E. Aberrantsuccinationofproteinsinfumaratehydratase-deficientmice
(2005)SuccinatelinksTCAcycledysfunctiontooncogenesisbyinhibiting andHLRCCpatientsisarobustbiomarkerofmutationstatus.J.Pathol.,225,
HIF-alphaprolylhydroxylase.CancerCell,7,77–85. 4–11.
7. Briere,J.J.,Favier,J.,Benit,P.,ElGhouzzi,V.,Lorenzato,A.,Rabier,D.,Di 19. Raimundo,N.,Baysal,B.E.andShadel,G.S.(2011)RevisitingtheTCA
Renzo,M.F.,Gimenez-Roqueplo,A.P.andRustin,P.(2005)Mitochondrial cycle:signalingtotumorformation.TrendsMol.Med.,17,641–649.
succinateisinstrumentalforHIF1alphanucleartranslocationin 20. Janeway,K.A.,Kim,S.Y.,Lodish,M.,Nose,V.,Rustin,P.,Gaal,J.,Dahia, D
SDHA-mutantfibroblastsundernormoxicconditions.Hum.Mol.Genet.,14, P.L.,Liegl,B.,Ball,E.R.,Raygada,M.etal.(2011)Defectsinsuccinate ow
3263–3269. dehydrogenaseingastrointestinalstromaltumorslackingKITandPDGFRA nlo
8. Xiao,M.,Yang,H.,Xu,W.,Ma,S.,Lin,H.,Zhu,H.,Liu,L.,Liu,Y.,Yang, mutations.Proc.NatlAcad.Sci.USA,108,314–318. ad
C.,Xu,Y.etal.(2012)Inhibitionofalpha-KG-dependenthistoneandDNA e
21. Ricketts,C.,Woodward,E.R.,Killick,P.,Morris,M.R.,Astuti,D.,Latif,F. d
doefmFHethaynldasSeDsHbytufummoarrsautpeparnedsssourcsc.iGnaetneetshDateavr.,e2a6c,c1u3m2u6l–at1e3d3i8n.mutations acnardcMinoamhear.,JE..NRa.t(l2C00a8n)ceGreIrnmstl.i,n1e0S0D,1H2B60m–u1t2at6i2o.nsandfamilialrenalcell from
9. Favier,J.andGimenez-Roqueplo,A.P.(2010)Pheochromocytomas:the 22. Parsons,D.W.,Jones,S.,Zhang,X.,Lin,J.C.,Leary,R.J.,Angenendt,P., h
10. (9LP5es7teo–uu9dz6oe8),-.Eh.y,pMoxairctihnyelploi,thCe.,siLs.oBrieostt,PCr.a,cBtu.Rrneisc.hColnin,.NE.,nAdobcerrimnoill,.NM.e,tab.,24, Mgeannokmoioc,aPn.,alCyasristeor,fHhu.,mSaiun,gIl.Miob.,laGsatollmiaa,Gm.uLl.tieftoarml.e(2.S0c0i8e)nAcen,i3n2t1eg,1ra8t0e7d– ttps://a
1812. c
Ottolenghi,C.,Janin,M.,Menara,M.,Nguyen,A.T.,Benit,P.etal.(2013) a
23. Mardis,E.R.,Ding,L.,Dooling,D.J.,Larson,D.E.,McLellan,M.D.,Chen, d
SDHmutationsestablishahypermethylatorphenotypeinparaganglioma. e
K.,Koboldt,D.C.,Fulton,R.S.,Delehaunty,K.D.,McGrath,S.D.etal. m
11. CLoarnicoet,rCC.e,lBl,u2r3n,ic7h3o9n–,7N5.2,.Gadessaud,N.,Vescovo,L.,Amar,L.,Libe,R., (2009)Recurringmutationsfoundbysequencinganacutemyeloidleukemia ic.o
genome.N.Engl.J.Med.,361,1058–1066. u
Bertherat,J.,Plouin,P.F.,Jeunemaitre,X.,Gimenez-Roqueplo,A.P.etal. p
(2012)Epithelialtomesenchymaltransitionisactivatedinmetastatic 24. Amary,M.F.,Bacsi,K.,Maggiani,F.,Damato,S.,Halai,D.,Berisha,F., .co
Pollock,R.,O’Donnell,P.,Grigoriadis,A.,Diss,T.etal.(2011)IDH1and m
pheochromocytomasandparagangliomascausedbySDHBgenemutations.
IDH2mutationsarefrequenteventsincentralchondrosarcomaandcentral /h
J.Clin.Endocrinol.Metab.,97,E954–E962. m
12. Dahia,P.L.,Ross,K.N.,Wright,M.E.,Hayashida,C.Y.,Santagata,S., andperiostealchondromasbutnotinothermesenchymaltumours. g
J.Pathol.,224,334–343. /a
(iBn2a0pr0oh5ne)toiAcnhiH,roMIFm.1,oaKclyputhnoagmr,eaAgs.u.LPla.,LtoSoraSynGlsooeo,npGelt.i.,n,Pk1os,wh7y2epr–so8,x0Ji..aFa.,nTdimscihtolecrh,oAn.dSr.iaeltsaigl.nals 25. BKo.Srg.,eSrc,hDe.nRk.,eTina,nDa.bPe.,,KH.eKze.,l,FAan.F,.K,A.Cn.,cuLkoipeewzi,cHz,.UM.,.,FLainetbinm,aVn.,RH.,.MSt.raelteayl,. rticle-a
13. Lopez-Jimenez,E.,Gomez-Lopez,G.,Leandro-Garcia,L.J.,Munoz,I., (2012)Frequentmutationofisocitratedehydrogenase(IDH)1andIDH2in bs
Schiavi,F.,Montero-Conde,C.,deCubas,A.A.,Ramires,R.,Landa,I., cholangiocarcinomaidentifiedthroughbroad-basedtumorgenotyping. tra
dLiefsfkereelna,tSp.seeutadlo.h(y2p0o1x0i)cRseigsenaartcuhrersesinouSrDceH:BtraannsdcrVipHtiLo-nraellaptreodfilingreveals 26. AObnecromloigl,isNt,.,1G7u,i7l2la–u7d9-B.ataille,M.,Burnichon,N.,Venisse,A.,Manivet,P., ct/23
pheochromocytomas.Mol.Endocrinol.,24,2382–2391. Guignat,L.,Drui,D.,Chupin,M.,Josseaume,C.,Affres,H.etal.(2012) /9
14. Burnichon,N.,Vescovo,L.,Amar,L.,Libe,R.,deReynies,A.,Venisse,A., TMEM127screeninginalargecohortofpatientswithpheochromocytoma /24
Jouanno,E.,Laurendeau,I.,Parfait,B.,Bertherat,J.etal.(2011)Integrative and/orparaganglioma.J.Clin.Endocrinol.Metab.,97,E805–E809. 40
genomicanalysisrevealssomaticmutationsinpheochromocytomaand 27. Burnichon,N.,Cascon,A.,Schiavi,F.,Morales,N.P.,Comino-Mendez,I., /6
3
paraganglioma.Hum.Mol.Genet.,20,3974–3985. Abermil,N.,Inglada-Perez,L.,deCubas,A.A.,Amar,L.,Barontini,M.etal. 3
4
15. Tomlinson,I.P.,Alam,N.A.,Rowan,A.J.,Barclay,E.,Jaeger,E.E.,Kelsell, (2012)MAXmutationscausehereditaryandsporadicpheochromocytoma 8
0
D.,Leigh,I.,Gorman,P.,Lamlum,H.,Rahman,S.etal.(2002)Germline andparaganglioma.Clin.CancerRes.,18,2828–2837. b
y
mutationsinFHpredisposetodominantlyinheriteduterinefibroids,skin 28. Nagai,R.,Brock,J.W.,Blatnik,M.,Baatz,J.E.,Bethard,J.,Walla,M.D., g
leiomyomataandpapillaryrenalcellcancer.Nat.Genet.,30,406–410. Thorpe,S.R.,Baynes,J.W.andFrizzell,N.(2007)Succinationofprotein ue
16. Bdaatyalbeays,eJ:.aPn.,oLnaliunneodnaetna,bVas.eaonfdfuTmomarlaitneshoynd,Ir.aPta.s(e20m0u8t)atTiohnesFiHnvmoluvteadtiionnthe tJh.iBoilosld.uCrhinegma.,d2ip8o2c,y3t4e2m19at–u3ra4t2io2n8:.abiomarkerofmitochondrialstress. st on
1
7
N
o
v
e
m
b
e
r 2
0
1
8