Role of CYP1A1, CYP17, and CYP1B1 Mutations in Girls with Precocious Puberty: A Pilot Study

Are CYP1A1, CYP17 and CYP1B1 mutation genes involved on girls with precocious puberty? A pilot study

Cezar Noboru Matsuzaki

^a

, Jose´ Maria Soares Ju´nior

^a,

*, Durval Damiani

^b

, Raymundo Soares de Azevedo Neto

^c

, Ka´tia Caˆndido Carvalho

^a

,

Felisbela Soares de Holanda

^a

, Eline Maria Stafuzza

^a

, Jose´ Alcione Macedo Almeida

^a

, Edmund Chada Baracat

^a

aDisciplinadeGinecologiadoDepartamentodeObstetrı´ciaeGinecologiadaFaculdadedeMedicinadaUniversidadedeSa˜oPaulo,05403000,Sa˜oPaulo,SP, Brazil

bDepartamentodePediatriadaFaculdadedeMedicinadaUniversidadedeSa˜oPaulo,05403000,Sa˜oPaulo,SP,Brazil

cDepartamentodePatologiadaFaculdadedeMedicinadaUniversidadedeSa˜oPaulo,05403000,Sa˜oPaulo,SP,Brazil

Introduction

Pubertytimingisstillunknown.Itsmechanismisthesourceof debateinvolvinggeneticsusceptibilityalongwithnutritionaland environmental factors. The genetic factors influencing puberty timingareyettobedetermined[1].Moststudiesonthetimingof earlypubertycorrelatecentralfactors,whichareassociatedwith neurotransmitters,suchasglutamate andthekisspeptin/GPR54 complex[2].

Therearefewstudiesassessingtheperipheralfactorswhichmay influenceearlyactivationofthehypophyseal–hypothalamicaxis.

Forsomeresearchers,estradiolwouldbenotonlytheconsequence ofpubertyonsetbutalsoacontributoryfactorinthedevelopmentof the hypophyseal–hypothalamic axis [3–5]. In a few of the prospectivegenes,suchasthosecodifyingtheCYPenzymecomplex,

whichcontrolthebiosynthesis,action,andmetabolismofthesex steroids,polymorphismshavebeen foundassociatedwith varia- tionsonpubertytiming[6].Suchgenesarealsoassociatedwith othergynecologicaldysfunctionsinvolvingestrogenaction,suchas leiomyoma,endometriosis,andbreastcancer[7,8].TheCYP17gene isbelievedtobeinvolvedinincreasedproductionofsexsteroids[9].

Ontheotherhand,theCYPA1andCYPB1polymorphismsmaybe related to more active metabolites (4-OH estradiol and 16-OH estradiol)actingontheestrogenreceptorandonestrogen-signaling pathways [10]. Therefore, our study aimed at investigating the possibilitythattheassociationbetweensuchpolymorphismsand thecentralprecociouspubertyprocessisbroughtonbyincreased estrogenactionandattendantGnRHrelease.

Materialsandmethods

A total of 177 patients wasselected from a groupof 1685 patientsbeingtreatedattheSectorofGynecologyofChildhoodand ARTICLE INFO

Articlehistory:

Received24June2014

Receivedinrevisedform23July2014 Accepted29July2014

Keywords:

Geneticpolymorphism Estrogens

Precociouspuberty

ABSTRACT

Objectives:Toinvestigatethreegenesassociatedwithpubertytimingingirlswithcentralprecocious pubertybyevaluatingthe associationbetweenpolymorphism inthegene sequence codifyingthe enzymesparticipatinginsteroidogenesis,CYP1A1,CYP17,andCYP1B1andcentralprecociouspuberty.

Studydesign: Atotalof177patientswasincludedanddividedintotwogroups:Casegroupwith73girls diagnosedwithcentralprecociouspuberty;Controlgroupwith104girlswithpubertyonsetafter8years ofagewhowerefollowedat theSectorof GynecologyofChildhood andAdolescence, Division of GynecologyClinic,HC-FMUSP.Polymorphismpresencewasassessedinthegenesinvolvedinestrogen metabolism(CYP1A1,CYP17,and CYP1B1)bytherestrictionfragmentlength polymorphism(RFLP) techniqueusingDNAfromperipheralblood.

Results:NosignificantdifferenceinthedistributionoftheCYP1A1Mspl(p=0.86)andCYP17(p=0.12) genotypeswasdetectedbetweenthetwostudygroups.AsforCYP1B1Eco571,themutatedC/Cgenotype wasfoundtobemorefrequentinthecontrolgroupthaninthecasegroup(p=0.03).

Conclusion:OurdatasuggesttheCYP1B1Eco571genevariantisassociatedwithpubertytiming.

ß2014ElsevierIrelandLtd.Allrightsreserved.

* Correspondingauthor.Tel.:+551126617838;fax:+551126617838.

E-mailaddress:jsoares415@hotmail.com(J.M.S.Ju´nior).

ContentslistsavailableatScienceDirect

European Journal of Obstetrics & Gynecology and Reproductive Biology

j o urn a l hom e pa ge : ww w. e l s e v i e r. c om/ l o ca t e / e j ogr b

http://dx.doi.org/10.1016/j.ejogrb.2014.07.042

0301-2115/ß2014ElsevierIrelandLtd.Allrightsreserved.

Adolescence, Gynecology Discipline, Department of Obstetrics and Gynecology, School of Medicine of the University of Sa˜o Paulo(FMUSP).The177patientswereallocatedtotwogroups:

Casegroupwith73girlswithacurrentorpreviousdiagnosisof precocious puberty and Control group with 104 girls who showed no signsof puberty untilthey were 8 years old. The diagnosisofcentralprecociouspubertywasbasedontheclinical features,laboratoryexams(LH,FSH,GnRHstimulatedgonado- tropinlevels,estradiol levels),boneage,pelvicultrasoundand brainMRI.Theexclusioncriteriacomprisedpresenceofovarian or adrenal neoplasms, Cushing syndrome, McCune–Albright syndrome,eyedisorders,clinicalorlaboratoryhypothyroidism, use of hormone medications prior to diagnosis, and use of psychotropic or antiepileptic medication. The project was approved by the Ethics Committee for Analysis of Research Projects atHC-FMUSP andFMUSP (CAPPesq–researchprotocol No.1120/09).

Thevolunteerswereincludedaftertheirlegalguardiansfreely signedaninformedconsentstatement.

Polymorphonuclearleukocytesseparatedfrombloodsamples wereutilizedfor thepatients’totalDNAextraction, carriedout withtheQIAmp¹DNAminiBlood(Qiagen)kit.

The CYP1A1polymorphism derives fromthe replacementof cytosinewiththymineinthe3⁰noncodingregion,whichcreatesan Msplrestrictionsite allowingtheidentificationof thefollowing genotypes:CYP1A1wt/wt(wild homozygote), CYP1A1wt/vt(het- erozygote),andCYP1A1vt/vt(mutanthomozygote)[10].

The5⁰region,untranslatedfromCYP17,becomespolymorphic withthesubstitution ofthymine forcytosine,creatinganMspl restriction site, which identifies two alleles, A1 (wild) and 2 (mutant).ThelatterincreasesCYP17expression,boostingestradiol biosynthesis[9].

Replacingaguaninebasewithcytosineinexon3oftheCYP1B1 geneleadstothesubstitutionofleucineforvalineincodon432, creatinganEco571restrictionsite,enablingidentificationoftheG/

G (wild homozygote), G/C (heterozygote), and C/C (mutant homozygote)genotypes[8].

Analysis of the polymorphisms in the CYP1A1, CYP17, and CYP1B1 genes was performed using the PCR-RFLP (restriction fragment length polymorphism) technique. The PCR reactions wereproducedwith50ngoftotalDNAin20mMTris–HCl(pH 8.4),50mMKCl,0.2mMdNTP,0.1mMofeachinitiator,1.5mMof MgSO2,and0.4UofPlatinumTaqDNAPolymeraseHighFidelity (Life Biosciences).The cycling conditionswere asfollows: pre- heatingto958Cfor10min,followedby40cyclesat958Cfor15s, at558Cfor1min,at728Cfor1min,followedbyafinalextension stepat708Cfor7min.Aspecificinitiatoroligonucleotidewasused foreachofthepolymorphismsthatwerebeinginvestigated.The oligonucleotidesaredescribedinTable1.

Followingamplification,thePCRfragmentscorresponding to the CYP1A1, CYP17, and CYP1B1 genes were digested with the restrictionenzymes(Promega,Brazil)specificforeachpolymor- phism as reported in the literature (Table 1). Fragments were analyzed in 1% to 3% agarose gel and stained with ethidium

bromide. The PCR and RFLP conditions were those already standardizedforeachgeneasdescribedintheliterature[8–10].

Afterdatacollection,themean,median,andstandarddeviation werecalculated.Next,samplehomogeneitywasassessedbythe Bartletttest.Resultsweredisplayedasmeanstandarderrorofthe mean.Whenthetwogroupswereinvolvedinacomparison,thechi- squaretestwasemployed.Forproportionanalysis,theFishertestwas used,andforpolymorphisms,atestofHardy–Weinbergequilibrium.

Analyses were carried out with the SPSS version 16.0 software program. In all tests, the significance level for rejecting the null hypothesiswassetat5%or0.05(p<0.05).

Results

Theclinicalandepidemiologicalcharacteristicsofthe73girls withcentralprecociouspuberty(Casegroup)andofthe104girls withnormalpuberty(Controlgroup)makingupourstudysample areshowninTable2.

Attheonsetofpuberty,themeanageofthegirlsintheControl groupshowedtheywereolder(114.919.3months)thanthosein theCasegroup(75.814.9months,p<0.01).Also,atmenarche,the meanageofthecontrolgroup (143.915.5months)wassignifi- cantlygreater than thatofthe casegroup(128.731.6months).

Therewasnodifferenceinthemother’sageatmenarchebetweenthe twogroups,i.e.,theControlgroup(147.917.7months)andtheCase group (146.219.3months,p<0.01).Boththecontrolgroup(19 girls) and the case group (14 girls) had the same proportion of ancestorswithprecociouspuberty(p=0.90).Therewasnodifference betweenthetwogroupsintermsofAfricandescent,socioeconomic strata and nutritional intakes (p>0.05). The GnRH stimulated gonadotropinleveltestwasperformedin20patientswithprecocious puberty.

Figs. 1,2and 3showthePCR-RFLP reactionstotheCYP1A1, CYP17, and CYP1B1 genes from the study participants. Table 3 displaysthegenotypingresults.There wasno difference inthe distributionoftheCYP1A1Mspl (p=0.86)and CYP17genotypes (p=0.12)betweenthetwogroups.TheC/Cmutantgenotypefrom CYP1B1Eco571wasmorefrequentintheControlgroupthaninthe Casegroup(p=0.03).Theoddsratioswas2.55[95%CI—1.24–5.24]

ofthecomparisons.

Comments

Investigationofthegenemechanismspossiblyinvolvedinearly onset puberty might help todetermine geneticmarkers for an earlierdiagnosisandtodevelopnew(pharmacogenetic)therapies with improvement in prognosis. However, little is still known aboutthegeneticchangesunderlyingsexualdevelopmentatan earlyage[1].

Most studies assessing the influence of genetic factors on precocious puberty timing correlate central factors, which are associated with neurotransmitters [11,12]. Few studies have sought to evaluate a peripheral component as an early onset trigger [3,13]. Many researchers debatetherole ofestrogen in

Table1

OligonucleotidesforPCRreactions.

Gene Initiators Polymorphism/Enzyme Reference

CYP1B1 S—5⁰-GTGGTTTTTGTCAACCAGTGG-3⁰ AS—5⁰-GCCTCTTGCTTCTTATTGGCA-3⁰

TransitionVal/Leucodon432/Eco571 Zhenget al.[8]

CYP1A1 S—5⁰-TAGGAGTCTTGTCTCATGCCT-3⁰ AS—5⁰-CAGTGAAGAGGTGTAGCCGCT-3⁰

TransitionTA/CGregion3⁰UTR/MspI Huanget al.[10]

CYP17 S—5⁰-CATTCGCACTCTGGAGTC–3⁰

AS—5⁰-AGGCTCTTGGGGTACTTG–3⁰

AdditionalpromotorSP1(CCACCbox)/Msp1A Feigelsonet al.[9]

S–Senseinitiator.

AS—Antisenseinitiator.

gynecologicaldisorders,suchasendometriosis,leiomyoma,breast cancer, and precocious puberty [14]. In the latter, estradiol is believedtobenotonlyaconsequencebutpossiblyacontributory factorintheearlydevelopmentofthehypothalamic–hypophyse- al–ovarian axis as well [3–5]. This is the reason we chose polymorphisms which might modify steroidogenesis and thus serumestrogenlevelsand/orincreasethemetaboliteswithgreater actiononthesteroidreceptor.

To date there are no reports on patients with precocious pubertywho havebeen evaluated in relationtothe polymor- phisms of the CYP1A1, CYP17, and CYP1B1 genes. There are, however, studies that show the correlation between the polymorphismsof thesegenes andotherdysfunctions suchas endometriosis [7] and breast cancer [8,10,15]. A risk factor associatedwithbreastcancerisknowntobeearlymenarche.As shownabove,theenzymesoftheCYPfamilymightberelatedto precocious puberty due to their interference in estrogen metabolism.

TheenzymesoftheCYPfamilywhichparticipateinsteroido- genesis areas importantin estrogen production as theyarein estrogen bioavailability and degradation. Several studies have identified mutations in the enzyme complex that may be influential in theonset and prognosis of diseases [16]. This is whywechosetostudytheCYP1A1,CYP17,andCYP1B1genes.

TheCYP1A1geneparticipatesinestrogenhydroxylationactivity generating its hydroxy estradiol metabolites, (HE2), 4-HE2, and 16

a

-HE₂, which have very potent hormone action [10] on the estrogen receptor. Gorai et al.[3] studied317postmenopausal Japanesewomenandconcludedtherewasnosignificantdifference intermsofCYP1A1mutationandageatmenarchebetweenthe study groups. Nor did we observe a higher frequency of such polymorphism in the CYP1A1 gene in the precocious puberty patientsinourstudy.

TheCYP17istheenzymethatcatalyzes17

a

-hydroxylaseand 17–20-lyase activity,increasing androgen and estrogen biosyn- thesis [17]. In studies examining the CYP17 polymorphism in womenwithbreastcancer,Feigelsonetal.[15]detectedtheA2 alleleinwomenofayoungerageatmenarche,whileHaimanetal.

[18]foundnosignificantdifference.Ourdatasuggestthegenedoes notparticipateinthetimingofprecociouspuberty.

TheCYP1B1geneislocatedonchromosome2,region2p21–22, contains three exons,and codes the P450-1B1cytochrome,the enzymeresponsiblefortheoxidativemetabolismofestradiol.Its polymorphismresultsfromthereplacementofaguaninebase(G) withacytosine(C)inexon3,which triggersthesubstitution of leucineforvalineincodon432,creatinganEco571restrictionsite.

TheCYP1B1geneplaysanimportantroleinthehydroxylation andconjugationprocessescatalyzingtheformationof4-HE2[8].

CYP1B1 expressionis regulated byestradiol and it binds to its receptor.ChangesattheCYP1B1expressionlevelnotonlymodify theintensityofestrogenaction,butmayalsomodifytheprofileof itsphysiologicaleffectontheliverandtargettissues.

Fig.1.RepresentativegelofthepolymorphismpatternobtainedfromMsplrestrictionofenlargedsamplesforCYP1A1.P,standardmolecularweightof100basepairs(bp).

Sampleswithonlyonebandindicateawildprofile,(340bp,e.g.,lanes12–14),whilesampleswithtwo(210and130bp,e.g.,lane37)andthreebands(340, 210e130bp,e.g.,lanes4–5)indicatethehomozygousandheterozygousprofileformutation.Thearrowspointatmigrationofthemolecularweightstandardin500bp.

Table2

Clinicaldatafrompatientsparticipatinginthestudy.

Controlgroup (n=104)

Casegroup (n=73)

p Ageatpuberty

onset(months)

114.919.3 75.814.9 <0.01 Menarche(months) 143.915.5 128.731.6 <0.01 Mother’smenarche

(months)

147.917.7 146.219.3 0.88

Ancestor^*(number) 19 14 0.90

EthnicityofAfrican descent

77 56 0.87

NotofAfricandescent 26 16

* Motherwithprecociouspuberty.

Intargettissuessuchasthemyometrium,inleiomyomas,and inbenignbreasttumors,anincreasein4-HE2hasbeendetected.At theselocalities,a riseinCYP1B1expressionlevel hasalsobeen confirmed, and the predominant route is 4-hydroxylation of

estrogen. Besides, the CYP1B1 expression level is abundant in tumortissues,moresothaninnormaltissues[19].

ItappearsthattheassociationbetweenCYP1B1andmammary andendometrialcarcinogenesisisformedprimarilyviageneration Fig.2.RepresentativegelofthepolymorphismpatternobtainedfromMsp1ArestrictionofenlargedsamplesforCYP17.P,standardmolecularweightof100basepairs.

Sampleswithonlyonebandindicateawildprofile,(A1/A1,e.g.lanes13–17),whilesampleswiththree(A1/A2,e.g.,lanes26–39)indicatetheheterozygousprofile.Thearrows pointattheapproximatemigrationheightofthethreefragmentsthatwereobtained.

Fig.3.RepresentativegelofthepolymorphismpatternobtainedfromEco571restrictionofenlargedsamplesforCYP1B1.P,standardmolecularweightof100basepairs.

Sampleswithonlyonebandindicateawildprofile,(G/G,e.g.lanes19–21),whilesampleswiththree(A1/A2,e.g.,lanes26–39)andtwobands(C/C,e.g.,lanes61–64)indicate theheterozygousandthehomozygousprofile,respectively.Thearrowspointattheapproximatemigrationheightofthethreefragmentsthatwereobtained.

Table3

Resultsfromgenotyping.

Genotyping Controlgroup(n=104) Casegroup(n=73) p

Wild Heterozygote Homozygote Wild Heterozygote Homozygote

CYP1A1(n) 60(W/W) 41(W/VT) 3(VT/VT) 40(W/W) 30(W/VT) 3(VT/VT) 0.86

CYP17(n) 35(A1/A) 69(A1/A2) 0(A2/A2) 33(A1/A) 40(A1/A2) 0(A2/A2) 0.12

CYP1B1(n) 29(G/G) 38(G/C) 37(C/C) 23(G/G) 37(G/C) 13(C/C) 0.03

of toxic metabolites, the so-called pro-carcinogens and pro- mutagens,ratherthanhormonepotency,giventhat4-hydroxyl- ation of estrogen by CYP1B1 leads to a reduction in estrogen activity [19]. It should be pointed out that increased SHBG productioninthelivermaydiminishtheavailabilitynotonlyofthe metabolitebut ofestrogenitself[20].Anotherhypothesisis the non-participationofthispolymorphisminpubertaldevelopment.

Infact,Mitchelletal.[21]studied152whitewomenandconcluded thatthe CYP1B1polymorphism wasnot associated withageat menarche.

Finally,wefoundaninverserelationshipbetweenCYP1B1and precociouspuberty. Our data suggest the CYP1B1Eco571 gene variantisassociatedwithpubertytiming.Hence,furtherresearch should focus on the functional investigation of this particular polymorphism.

Condensation

The CYP1B1 Eco571 gene variant may be associated with pubertytiminginpatientswithprecociouspuberty.

Acknowledgement

WewouldliketothankProfMariaLuciaFlynnformanuscript revision.

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