AssociationofCYP19andESR1PleiotropicGenesWith HumanLongevity

RosaMariaCorbo,1,2LauraUlizzi,1LidiaPositano,3andRenatoScacchi2

2CNRInstituteofMolecularBiologyandPathology,Rome,Italy. 3CORISAConsortium,Salerno,Italy.

Rome,Italy.Email:rosamaria.corbo@uniroma1.it

Aromatase(CYP19)andestrogenreceptor-alpha(ESR1)areinvolvedinthemetabolismofestrogens,whichhavearele- vantroleinfemaleandmaleaging.Moreover,duetotheirinfluenceonfertility,bothgenesmaybepartofthelongevity– associatedwithreducedfertilityinthesamesample,wemayinferthatESR1genotypescouldexertanantagonisticpleio- tropiceffectonlongevityandfertility.

Key Words: ESR1—CYP19—Polymorphism—Longevity—Longevity–fertilitytrade-off.

Received June 24, 2010; Accepted August 16, 2010

Decision Editor: Rafael de Cabo, PhD

Research on the longevity–fertility trade-off in humans has nearly always been done using a historic– demographicapproach(11–12).Despitecertaindiscrepan- longevity–fertility trade-off. Studies on pleiotropic shown that some longevity candidate genes can reproductive function as well, thus exerting a actionindifferentperiodsoflife.

and nongenetic factors. Great efforts have been ex- pendedbyresearchersintheattempttoidentifythegenes involvedinlife-spandetermination(1–4).Variationinsex hormone(estrogensandandrogens)levelisatypicalfeature ofaging,butthepossibleimpactonthehumanlifespanof geneticvariationofthegenesregulatingsexhormonepro- ductionandactionhasneverbeeninvestigatedbefore.As astartingpoint,weexaminedgenesinvolvedinestrogen metabolism.

physiology,boneandlipidmetabolism,andmaintenanceand protectionofthecardiovascularandnervoussystems(5–7). Morerecently,similarestrogenfunctionshavebeenidenti- fiedinmenaswell(8).Thedrasticreductioninestrogen levelatmenopausemarksthebeginningofagingforwomen asitaffectsthemultiplemetabolicprocessesinfluencedby estrogenearlyinlife.Althoughnotsoabrupt,thedeclinein estrogenisresponsiblefornumeroussignsofagingalsoin men(8).Afurtherpointofinterestinexaminingestrogen metabolismgenesisthat,beinginvolvedinthedetermination offertility,theycouldbepartofthelongevity–fertilitytrade- off mechanism. It has been postulated that a trade-off betweenresourcesavailableforreproductionoccursonthe onehandandatrade-offbetweenmaintenanceandlifespan ontheother.Becausenaturalselectiontendstofavoranindi- vidual’s reproductive ability over longevity, it is plausible that an antagonistic relationship exists between these two

ovaryandtheplacentaaswellasinanumberofextrago- nervoustissue(17).ThissuggeststhatCYP19,asitisin- volved in the production of estrogen, also participates regulatingreproductivefunctionsandgrowthordifferentia- with a regulatory role (17). Recently, CYP19 polymor- cancer, and Alzheimer’s disease (18–23). The

andthoseaged90yearsorolderin2009.Thecontrolsam- plewasmadeupofindividualswhohaddiedbetween2000 and2009.

ER-beta encoded by the ESR2 gene on chromosome foundinallhumanreproductivetissues.Itisexpressedpri- withER-beta,iscoexpressedinanumberoftissues,includ- with female and male infertility, spontaneous andAlzheimer’sdisease(5,25–27).

Fertilityrateswerederivedfromresponsestoaquestion- naireitemaskingaboutthenumberofchildrenthepartici- pantsreportedtheyhadhad.Theprotocolforcollectionof biologicmaterialforthescientificstudieswasapprovedby theinstitutionalethicscommittees.Informedconsentwas obtainedfromallparticipants.

Venousbloodwasdrawninethylenediaminetetraacetic acid from all participants. Genomic DNA was extracted fromwholebloodaccordingtotheproceduredescribedby Miller and colleagues (30). Two ESR1 polymorphisms (PvuII,or–401T/C,orrs2234693andXbaI,or–354A/G, or rs9340799) were examined. Both polymorphisms are locatedinintron1oftheESR1geneintheregionofthe transcriptionfactor–bindingsiteandarecloselylinked(8). Ithasbeenspeculatedthattheychangethegene’senhancer activity.Thereareindicationsthatsupportthishypothesis, especially for rs2234693 (31–32), although slight differ- encesinenhanceractivitywerereportedforrs9340799as well(33).Bothpolymorphismsweregenotypedaccording tothetechniqueofYaichandcolleagues(34)withslight modifications.Specifically,adifferentprimercouplewas designed: 5′-AGGGTTATGTGGCAATGACG-3′ as for- ward primer and 5′-CCTGCACCAGAATATGTTACCT-3′ asreverseprimer.Thepolymerasechainreactionproduct wasdigestedovernightbyPvuIIandXbaIandthenvisual- ized on agarose gel. ESR1*P and *X alleles indicate the absenceofacuttingsiteforthePvuIIandXbaIrestriction enzymes, respectively, whereas ESR1*p and *x alleles indicatethepresenceofacuttingsite.TheCYP19rs4646 singlenucleotidepolymorphismislocatedat3′-UTRofthe CYP19gene.Itisatagsinglenucleotidepolymorphismof haplotype Block 4 of CYP19 containing all the CYP19 coding exons (exons 2–10) and part of the 3′-UTR (21). There is statistical evidence for a significant relationship betweenCYP19singlenucleotidepolymorphismsinlinkage disequilibriumBlocks3and4,includingrs4646,andcircu- lating estrogen levels in postmenopausal women (35). rs4646wasgenotypedaccordingtothetechniquesreported byPetryandcolleagues(36).

The association of ESR1 (PvuII, rs2234693 and rs9340799)andCYP19(rs4646)polymorphismswithlon- age: 83.1 ± 5.7 years) recruited in 2000.After thecontrols.

studyonESR1polymorphismsinthesamestudypopula- tionwascarriedoutpreviously(28).

Materials and Methods

study (LONCILE study) on the anthropological and bio- areainthedistrictofSalerno,southernItaly(29).Itcon- sisted of 258 unrelated individuals (43.3% men) between1900and1930(meanage:83.1±5.7years±stan- dard deviation), consecutively enrolled without criteria. The mortality information on these oldormore.Duringthe9-yearfollow-upperiod,themor- tality rate was 63.7% (51.9% men): 58.2% 6 years for men (ISTAT, .html?lingua=ita),weconsideredaslonglivedthosepar- years).Thesampleofthelong