MechanismsfordifferencesinmonozygoustwinsPaulGringrasa,, WaiChenb,caTheMultipleBirthsFoundation, HammersmithHouse,Level4, QueenCharlottesandChelseaHospital,DuCaneRoad,LondonW12OHS, UKbBloomfieldClinic, DepartmentofChildandAdolescentPsychiatry, Guys, St. ThomassandKingsSchoolofMedicine, GuysHospital, St. ThomasStreet, LondonSE19RT, UKcCentreofSocial,GeneticandDevelopmentalPsychiatryResearch,InstituteofPsychiatry,KCLLondonSE5,UKReceived1July2000;receivedinrevisedform23March2001;accepted14April2001AbstractMonozygous (MZ) twins are often described as being physically and genetically identical.Clinicaldeterminationofzygosityreliesontheassumptionthatanyphysicaldifferencesbetweenapairoftwinsimplytheyaredizygous. Most twinresearchreliesontheassumptionthat dizygoustwinsshareapproximately50%ofthesamegenes,whereasmonozygoustwinsshare100%.Thereis,however,increasingevidencetochallengeboththeseassumptions.Inthisreview,wedescribeanumber of intrauterine effects and genetic mechanisms that may result in phenotypic, genotypic, andepigenetic differences betweenmonozygous twins. Newer molecular techniques are resultinginsuch differences being increasingly commonly recognised. The potential for differences inmonozygotic twin pairs is an important consideration for both clinicians and researchers involved intwinwork. D2001ElsevierScienceIrelandLtd. All rightsreserved.Keywords:Twin;Monozygous;Geneticmechanisms1. IntroductionOver 200pairs of twins are assessedeachyear at the Multiple Births Foundation,London. Despite often appearing indistinguishable to strangers, no identical twinsassessed are so alike that their mothers fail to distinguish themaccurately. Physicaldifferencesmaybeassubtleasonesmall mole, or adifferentlypositionedhair crown;0378-3782/01/$seefrontmatterD2001ElsevierScienceIrelandLtd.Allrightsreserved.PII:S0378- 3782( 01) 00171- 2*Correspondingauthor. Tel.:+44-208-383-3519.E-mailaddress:paulg@pobox.com(P.Gringras).www.elsevier.com/locate/earlhumdev*EarlyHumanDevelopment64(2001)105117but still, they exist and are unmistakable once identified. Many parents can alsodifferentiatetheir identical twinsbytheir personalities, someevenclaimfromaveryearlyage.Physical similarities betweenMZtwins are well recognised; andthese similaritieshave long formed the basis of many instruments and clinical methods designed toclassify zygosity, such as questionnaires and physical examinations. Even the mostexperiencedpractitionerscan, however, misclassifyzygosityinabout 6%ofcases[1],andmolecular geneticmethodsarenowthepreferredmethodfor establishingzygosity[2]. The termidenticalalthoughfrequentlyusedis not synonymous withmono-zygous (MZ).Most MZtwinsarephenotypicallyverysimilar, yet therearesignificant numbersofMZ pairs who are neither phenotypically nor genotypically identical. Even if oneassumesacompletelyequalapportioningofgeneticendowmentwhentwinningoccurs,thetwinpair will onlyremainidentical if post-zygoticgenetic, post-zygoticepi-geneticand post-zygotic environmental factors affect each twin equally. Given the extent ofthese influences and many potential opportunities for disruption during the long andcomplexintrauterinedevelopment, it is perhaps surprisingthat somanyMZtwins doturnouttobesoalike.Nevertheless,itistheseanomalouscasesofdiscordanttwinsthathavetaught usmuchabout humangenetics, development andtwinninginthepast. It islikely that they will continue to do so when newtechnologies are applied to futureresearchinthis area. This reviewsummarises some past findings of well establishedstudies, andalsosome frommorerecent exploratorystudies usingmoreexperimentaltechniquesanddesigns.Wewill first considertheante-natal environmental factorsandtheireffects, andthenthe genetic factors that contribute to discordance in MZ twins. Some examples ofdiscordancydonot necessarilyfit intotheaboveneat categories. Forconvenience, theyhave been grouped together and discussed in the final section on discordancies ofunknownorigin.2. TimingofmonozygoustwinningMonozygous(MZ) twinningoccurswhenonesinglefertilisedegggivesrisetotwoseparateembryos. Thetimingofthisdivisioncanbeanimportant contributoryfactorindetermining the post-zygotic discordance in MZ twins. This timing can be characterised bythe differences in amniotic sac, chorionic and placental anatomical formation [3]. Inprinciple, theearlier twinningoccurs, thelessthetwinswill sharecommonsupportivestructures; and the later, the more. The extreme example of late twinning are conjoint twinswhoevensharesomesomaticorgans.If twinning takes place prior to the first 4 days after conception, two separate placentasand sets of membranes are formed: that is, one set for each embryo. Such twins are calleddichorionic(DC)MZtwins, andtheyaccount foraboutonethirdofallMZtwins.Afterthefourthday, theprogenitorcellsoftheplacentabecomeseparatedfromtheinner cell massof theembryo. Asaresult, for twinningoccurringafter this, onlyonesingle placenta will develop. This single monochorionic (MC) placenta serves bothP. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 106embryos, and in the majority of cases, contains anastomoses of blood vessels that connecttheembryos.After about theeighthday, theMCMZpair will shareacommonamnioticsac, inaddition to the common MC placenta [4]. About 5% of MZ twins are monochorionic (MC)and monoamniotic (MA). Twinning after the second week results in the very rarephenomenonofconjoinedtwins(seeTable1).AllMCtwinsareMZbydefinition,andthisisstillthegoldstandardwhendefiningmonozygosity. Althoughoftenseeninanimals, vascularcommunicationsindichorionicplacentae in man are extremely rare [5]. The combination of monochorionicity and arterio-arterial anastomoses is a better proof of monozygositythananygenetictest currentlyavailable. If placentation has not already been established by ultrasound in the firsttrimester, it relies on placental examination by pathologists; unfortunately, this still has notbecome routine clinical practice in most hospitals, despite numerous pleas in the literature[6,7].3. Ante-natal environmentalfactors3.1. Chorionicity, twintwintransfusionsyndromeanddiscordant birthweightAnastomotic connections between foetal circulations are present in around 90% of MCplacentas. These anastomoses can result in the twin to twin transfusion syndrome (TTTS)[8]. This can result either in a chronic ante-partumtransfusion or acute intrapartumtransfusion. In the former event, growth discordance occurs and there are risks for both thedonor andrecipient. Theseincludethepossibilityof thedonor becomingmalnourishedandgrowthretarded, whiletherecipientisatriskofcardiachypertrophy,polycythaemiaand hydramnios. In general, the mortality and morbidity rate for both twins in this situationishighwithout intervention[9]. Theacutetransfusionsyndromeoccursintrapartumandcausesincreasedmortalityandmorbidity, throughbothhypovolaemiaandhypotensioninonetwin, andpolycythaemiaintheother.EvenwithoutTTTS,discordantbirthweightinMZtwinsremainscommonasaresultof: (1) unequal in-utero blood supply, and hence growth; and perhaps (2) in theory,unequaldivisionofinnercellmassattwinning.AlthoughsuchdifferencesmaydiminishTable1TimingoftwinningDayspostconceptionAmnionicity Chorionicity Frequency04 Diamniotic Dichorionic One-thirdofmonozygoustwins48 Diamniotic Monochorionic Approximatelytwo-thirdsmonozygoustwins812 Monoamniotic MonochorionictwinsFivepercentofmonozygoustwins12 +aConjoinedtwinsaTimingforconjointtwinsistheoreticalandonlysuggestedbyanimalmodels.P. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 107withage,thereisagrowingbodyofevidencethatsignificantdiscrepancyinbirthweightmayleadtolong-lastingphysiological changes inbothtwins. The concept of foetalprogramming proposes that intrauterine growth affects long-term growth and metabolismin later life. Epidemiological studies linking lowbirth weight with hypertension andcoronary artery disease in adult life suggest that undernutrition before birth programmeslater cardiovascular outcome [10]. Associations between small for dates babies with laterinsulinresistanceandcardiovasculardiseaseareconsistent withthehypothesisthat lategestationmaybeawindowof sensitivitytonutritionintermsof itsinfluenceonlatercardiovasculardisease.Intwinsdiscordantforthedevelopmentofnon-insulindependantdiabetes(NIDDM), birthweight hasbeenfoundtobelower intheaffectedtwin[11].Investigatorscontinuetousetwinswithdiscordant birthweight asameanstotest thefoetal programming hypothesis, while assumingthe twinpair wouldshare commonconfoundingvariablessuchassocial class, geneticendowment andpost-natal environ-ments. Two teams have recently reported the importance of birth weight in twins,independent of genetic differences, ininfluencingtheir bloodpressure as adults [12].Evidence for foetal programming has even been found in early infancy: in a small cohortof MZ twins, where a twintwin transfusion had occurred, differences in arterialdistensibilitywerefoundinthedonortwinwhencomparedtotherecipient[13].Appealingthoughthefindingsfromtwinstudiesmaybe,theextenttowhichtheyaregeneralisabletosingletonpopulationisunknown. It isnot fullyunderstoodifeitherthemechanism for growth discordancy or the responses to undernutrition differ between twinsandsingletons.3.2. Chorionicity, cognitionandpersonalityManytwinstudieshavedemonstratedthehighheritabilityofcognitiveabilities.Ithasbeenreportedthat MZtwins IQtest scores are almost as highlycorrelatedas thoseobtainedfromtestingandre-testingthesameperson. Thisfindingisstriking, giventheveryhightestandre-testreliabilityofIQtests[14].Whether placentationinMZtwinsaffectscognitivefunctionremainsacontroversialsubject. MCMZtwins(wheretwinningoccurslater whencomparedtoDCMZtwins)have been suggested to be more similar cognitively and behaviourally than DC MC twins.Althoughthereissomeagreement that MCMZtwinsaremorealikeinpersonality, thefindings are not consistent for concordance in intellectual abilities [15]. Chorionicity(perhapsasurrogatemarkerforthetimingoftwinning)maybeanimportant factorthatunderlies cognitive and personality differences between MZ twins, but plausible biologicalmechanismsarestillyettobeelucidated.AprospectivecohortstudyonalargeMZtwinsample, wherechorionicityareaccuratelyandreliablydetermined, wouldberequiredtofullyanswersomeofthesequestions.3.3. DifferentialinfectionsandteratogensAscendinginfections(chorioamnionitis) aremorelikelytoaffect thelowergestationsac [3]. Differential congenital infections are thus probably more common thancongenital infections, where both twins are equally infected. Infective agents includeP. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 108theusuallistoftoxoplasma,rubella,herpesandcytomegalovirus;allcanadverselyaffectMZtwins to varying degrees. Where congenital HIVinfections are concerned, birthorderemergesasamajorriskfactor.FirstborntwinstendtocontractHIV infectionfromtheir affected mothers more often than second born twins [16]. In cases where theinfection is perinatally acquired during delivery, it has been suggested that the twinclosertothecervixmayexperiencegreaterexposuretoinfectedbloodthantwinslocatedhigher intheuterus. Maybeduetoahigher rateof obstetriccomplicationsinbirthsoftwinsorreasonsnotfullyunderstood,theratesofHIV infectionintwinsarehigherthanthat of singletons[17].4. GeneticmechanismsItisawidelyheldbeliefthatMZtwinsaregeneticallyidentical; andthat subsequentphenotypical discordancesareattributabletoenvironmental influencesalone(sharedornonshared), altering and modifying the expression of the otherwise identical geneticendowment. Twinstudieshavelongbeenusedtoestimatethegeneticcomponent inarangeofconditions, frommedical andpsychiatricdisorderstovariationsinsomaticandpsychological phenotypes. The central assumption of these studies is that MZtwins,formedfromthe divisionof a single fertilisedegg, are geneticallyidentical, whereasdizygous (DZ)twins,whichresultfromtwo fertilisedovaare geneticallynomoresimilarthansiblingsbornfromseparatepregnancies.ThedegreeofsimilaritybetweenMZpairs(concordance) over that of DZpairsistakenasevidenceof geneticcontributionintheaetiologywithrespect tothephenotypestudied. Thevalidityofthisassumptionandtheabilityoftwinstudiestofullyallowbothante-natalenvironmentaleffectsandmutationalgeneticeffectsarestillbeingdebated[18].There is nowan ever-growing body of evidence that MZtwins are not alwaysgenetically identical. Anumber of phenotypic discordances in MZtwins have beendemonstrated to be caused by genetic differences alonethat is, without having to invokeante-natalenvironmentalfactorstoaccountfortheirphenotypicdifferences.Furthermore,divergent epi-genetic modifications within a MZtwin pair, can lead to differentialexpressionofinheriteddiseasedgenes.Genetically, MZtwinscanbedifferentatthelevelof(1)chromosomesor(2)DNA:1. at the level of karyotype (cytogenetics)i.e. the number or morphology ofchromosomesmayvary;and2. atthelevelofmoleculargeneticstheremaybe(i)DNAmutationsor(ii) epi-genetic modifications, such as differences in promoter regionmethylation, that suppress the expression of the DNAcoding regions, andskewedX-chromosomeinactivationbymethylationshut-downofthewholeXchromosome.It isworthbearinginmindthat theexamplescitedinthisreviewarebasedonthefindingsdetectedbylaboratorytechniquescurrentlyavailable. ThismeansthedetectedP. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 109differences are limited by the resolution power of current technologies. As more advancedtechnologies are becoming available for more detailed genomic comparison and areappliedtostudyMZdifferences, it ispossiblethat moreMZphenotypical discordancemaybefoundtohaveageneticalorepi-geneticalbasis.4.1. HeterokaryotypicaldivergenceandchromosomalmosaicismMZtwins canhave different chromosomal composition(heterokaryotypia). Awelldocumented example for this is MZ discordance for gender and UllrichTurnersyndrome (UTS)that is, one twin is phenotypically male and the co-twin, aphenotypicallyfemaleTurner [20]. This is thought tobetheconsequenceof anearlypostzygoticmitoticerror, resultinginheterokaryotictwinningthat involvesnondisjunc-tionoranaphaselagoftheYchromosome. Asaresult, onetwinis47,XYY or46,XY[20] or mosaic45,X/46,XY[22] (resultinginaphenotypical male); andtheco-twinis45,Xor mosaic46,XY/45,X(resultinginaphenotypical UllrichTurner female). Thetimingof thismitoticnondisjunctiondeterminesthepresenceanddegreeof mosaicisminoneorbothtwins. TherehavebeenmorethanninereportedcasesofsuchdiscordantMZtwin.MZ discordance for Downs syndrome (trisomy 21) or Klinefelter syndrome(47,XXY) are uncommon, though Rogers et al. [23] reported a single case of MCMZmaletwinswhowerediscordantfortrisomy21. Interestingly, eachwasnon-mosaicinfibroblast lines, but havemixtureofcellsinbloodderivedfrom46,XY/47,XY,1[21]cell lines. The theoretical explanation is that Downs and Klinefelter syndrome com-monlyresult froma pre-zygotic nondisjunction, whereas UTSfrompost-zygotic non-disjunctionpairs[21].4.2. Mosaicism, chimerismandpseudo-mosaicismFor singletons, chimera is an organism with tissues of two or more different genotypes,oftenasaresultofgraftingorintroductionofveryearlyembryostemcellsfromanothergenetically different individual(s). Achimera is often constructed experimentally inanimal studies. Incontrast tothis, mosaicismdenotes anorganismcomprisingclonesof cellswithdifferent genotypes(oftencausedbymutationsor mitoticerrors) derivingfromthesamezygote.ForMZtwins,suchadistinctionismoreproblematicasbydefinition,bothembryosarederivedfromthesamezygote.Sostrictlyspeaking,anygeneticheterogeneityinMZtwins should be classified as mosaicism. However, some authors used the termchimerism todescribetransplantationof stemcellsfromonetwintoitsMZco-twin,if genetic divergence has already occurred prior to twinning, i.e. twin A is subtly differentfromtwinBgenetically;andtwinBhassomestemcellsfromtwinAincertaintissues.Mosaicismisreservedtodescribedivergentcelllinesasaresultofmutationsoccurringwithin one twin after twinning. However, such distinction is not strictly applied within thetwinsliterature. Often, itisdifficulttomakesuchdistinctionretrospectively.In monochorionic twins who share the same placenta, cross-placental transfusionenable blood and stemcells to be transferred fromone twin to the otherdue toP. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 110placental vascular anastomoses. This results in pseudomosaicismor chimerism(i.e.transferoftheco-twinsstemcellintothebonemarrowduringfoetaldevelopment). Forgenomic comparisonstudies of monochorionic MZtwins, it is therefore important toconduct tissue(i.e. skinfibroblasts) genotyping, rather thanrelyingonbloodlympho-cytesalone[24].An example is the case report the pair of MC twins discordant for trisomy 21 by Rogerset al., ascitedabove. Theyfoundonlyonekaryotypesinfibroblast cell linesfromeachtwin(one withnormal karyotype; another withDowns karotype), but inblood, bothkaryotypes are found in both twins, indicating the presence of chimerism in blood (and inbonemarrow),butnotinsomaticcelllines.Mosaicismcanarisefrommitoticerrorinearlydevelopmentresultinginamixtureof cellswithdivergent karyotypes(e.g. amixtureof 45,Xand46,XYcell linesintheUTSco-twinofgenderdiscordantMZtwin).Insomecases,thepresenceofmosaicismmayvaryindifferent tissuesfromonesingleindividual. Insuchcases, multipletissueculturesarerequiredtoestablishmosaicism. Edwardset al. [25] reportedapair of MZheterokaryotic twins with a UTS female and a normal male phenotype. The male,despitenormal sexual development toage21, hadtwoskinfibroblast culturesandtwolymphocytecultureshowinganonmosaic45,Xkaryotype[25]. Theauthorsconcludedthat their patient wouldprobablyshowmosaicism, andif gonadal tissuewassampled,thepresenceofYchromosomalmaterialwouldbefound. Reindollaretal. [22]reportedanother pair of MZtwinswithaUTSfemaleandanormal male. Theyexaminedtwoseparateculturesofpenileskinofthemaleco-twin, showingonesingle45,Xcell; with49 cells of 46,XY; while the cultures of gonadal tissue showing 100%46,XY. Thissuggeststhat lowlevel mosaicism mayexist inthephenotypicallynormal co-twininthesecases.4.3. Epi-geneticmodification, methylationandskewedX-inactivationMethylationisanepi-geneticmechanismbywhichtheexpressionofansequenceofDNAcanbemodifiedbybecomingsilenced or switchedoff. This is achievedbyattachingamethyl grouptothecytosinenucleotideinCpGislands [26]. CpGislandsare clusters of cytosine and guanine repeats, commonly found near a gene codingDNA region. Methylation of CpG islands adjacent to a gene often leads to itsinactivation[27].Inadditiontothesilencing of aspecificgene, methylationisalsoinvolvedinthewhole-scale inactivation of the surplus Xchromosome in human femalesa processcalled X-inactivation or lyonization. In the majority of cases, X-inactivation is arandomprocess. That is, bothXchromosomes ina female have anequal chance ofbeingswitchedoff bymethylatingthe whole Xchromosome. However, for reasonsnot yet fullyunderstood, X-inactivationcanbeskewedornon-randominaminorityof individuals, that is, the Xchromosome of one parental origin is preferentiallyinactivated[28].Someauthorsreport that skewedX-activationismorecommoninfemaleMZtwinsthan predicated by chance, though this view is still subjected to debate. They propose thatskewedX-inactivationmaygiverisetoclonal differenceswithinthepost-zygoticinnerP. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 111cell massandmaytrigger theveryprocessof twinning[29,30], thusaccountingfor anexcess of females amongMZtwins, especiallyamongmonochorionicMZtwins. Thisview remains controversial. Derom et al. [31] did not find an excess of female twins in anepidemiologicalstudyofmultiplebirths.Along with the development of more robust technology in detecting skewed X-inactivation, a number of cases with non-randomX-inactivation and MZphenotypicdiscordance have been identified. They consist of expressed X-linked diseases infemales. Examples reported include fragile-Xsyndrome [32], colour blindness [24],Duchenne muscular dystrophy [33], haemophilia B, G6PDdeficiency, Aicardis syn-drome, Hunter andFabrysdiseaseindiscordant femaleMZtwins[34]. Theclinicallyaffectedtwinhasnon-randominactivationpredominantlyoftheX-chromosomecarryingthewildgene, whiletheunaffectedtwineither haspredominant inactivationof theX-chromosomecarryingthemutant geneor hasrandomX-inactivation.4.4. Post-zygoticgeneticmutationTheoretically, post-zygoticDNAmutationindiscordant MZtwins canoccur at thelevelof(1)Dominantgenes;(2)Recessivegenes;(3)Imprintablegenes;or(4)Genesinvolvedinquantitativetraitloci.Confirmedandprovenmoleculargeneticevidencearestilllacking,maybeinpartduetothe limitationof currentlyavailable genomic comparisontechniques. Anumber oftechnicalinnovationsarecurrentlyunderdevelopment, butnotyetsufficientlymatureforroutineapplication.The authors are unaware of anyreport demonstratingprovenDNAmutations thataccount for discordant phenotypes inMZtwins. However, there exist reports onMZdiscordancefor conditions transmittedbywell documentedautosomal dominant inher-itance. Someincludeconditionsinwhichimprintingisimplicated.For autosomal dominant conditions, Vaughn et al. [35] reported a pair of MZ twins withneurofibromatosis,showingmarkedly different densityof cafe-au-laitspots,assumingtheaetiology to be a de novo mutation. Easton et al. [36], however, found strikingconcordanceof0.97correlationintheexact countsofneurofibromasforfiveofthesixMZtwinpairs.Anumber of MZtwins discordant for theseverityof tuberous sclerosis havebeenreported. Amongthose documentingthe phenotypical differences, Brilliant et al. [37]demonstrateda single discordant hybridizingfragment usinghigh-resolutionSouthernblottingtechnique. However, the discordant fragment has not beenpinpointedonthespecificgenesequenceresponsiblefortuberoussclerosis. Themolecularevidencethere-foreremainsinconclusive.Anumber of reports on MZ twins concordant for Williams syndrome, also anautosomal dominant condition, founddifferencesintimingandseverityinthedevelop-P. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 112ment of abnormalities, especiallyinguinal herniaeandfacial anomaliesandother signsattributabletoconnectivetissueabnormalities[38].WiedemannBeckwithsyndrome(WBS)isacongenital anomalysyndrome, consist-ingof exomphalos, macroglossia andgigantism, withanincreasedriskof developingrare embryonal cell tumours. Though autosomal dominant inheritance (preferentiallythroughthe mother) is well recognised[39], sporadic cases withpaternal uniparentaldisomyof 11p15loci mayimplicatethat imprintingisinvolvedinWBS. Anumber ofdiscordant MZtwins for WBShave beenreported, but the precise molecular mecha-nismsfor their discordancehavenot beenclearlyestablished[40].TherearetworecentreportsexaminingMZtwinsdiscordantforschizophrenia.Tsujtaetal.[41]analysedgenomicDNAofapairofMZtwinsdiscordantforschizophreniabyusing two-dimensional electrophoresis display, following PCRandrestriction enzymedigest. Theyusedbothmethylationsensitive andnon-sensitive enzymes. Theyfounddiscrepancies of at least two spots out of approximately 2000 spots on the autoradiogramsofthepair.Bothofthespotswereestimatedtorepresent300400basepairs.Thestudycan be criticised for lacking specificity, validity as well as statistical power; andcontaminationbyerrorsor artefacts cannot beruledout. Ontheother hand, it canberegarded as providing preliminary evidence that MZ twins may differ at the levels of DNAormethylation.Smith et al. [19] used targeted genomic differential display method to evaluate,quantitatively, thelevel of genomesimilarityinagroupof twinpresentedtothemasMZtwinsconcordant anddiscordant forschizophrenia. At theDNAlevel, theyfoundthree distinct patterns. One type had DNA similarity levels equivalent to that of unselectedsibling pairs, suggesting these to be dizygous twins misidentified as monozygous twins. Atthe other extreme was a group with a very high level of similarity, suggesting these are truemonozygoustwins.Thesurprisingfindingwasanintermediategroup,lyingbetweentruemonozygous and dizygous twins. They found all concordant twin pairs were truemonozygotic. The mixeddiscordant andconcordant twinpairs belongedtothe inter-mediate group. They concluded that their findings brought into question a large number ofexperimentsthat dependedonaccuratetwinzygositydeterminationsfor measuringtheheritabilityandpenetranceofthecondition.5. Differencesofunknownorigin5.1. MirrortwinningMirroringinMZtwins is a fascinating, but poorlyunderstoodandpoorlydefinedcauseofphenotypicandperhapsbehaviouraldiscordance.Ithasbeenestimatedtooccurin25%of MZtwins [42]. Thespectrumextendsfromthosetwins withjust opposite-sided occipital hairwhorls,to thosewhere one twin has complete reversal of body organs(situs inversus). The termis perhaps most commonlyused for those MZtwins withdiscordanthandedness(rightandlefthandedness).Initself,thissuggestssomedegreeofasymmetry of cerebral hemisphere dominance. Researchers have recently confirmedusing functional magnetic resonance brain imaging on MZ twins with discordantP. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 113handedness that hemisphericlateralisationis indeedalsopresent [43]. Thesame teamsuggests that this finding may complicate interpretation of twin research in illnesseswhere cerebral lateralisation is important to their pathology (such as schizophrenia,dyslexia, autism, depression) [44].5.2. Hair, eyesandfingerprintsZygosityexaminationsandquestionnairesrelyonfindingdifferencesbetweentwins.Althoughveryminor degrees of phenotypicdiscordancehavebeenrecognisedinMZtwins, it is apparent the spectrumis widening. Different hair and eye colour wouldautomatically qualify for a DZlabel in all established questionnaires, but both haverecently been described in male MZ twins [45,46]. Putative mechanisms for suchdiscordances range from unequal division of the inner cell mass, to post-zygotic mutations.Itislikelythatcomplexgeneenvironmentinteractionsplayapartinsuchcases.Similar mechanisms where the ante-natal environment impacts on a geneticallypredeterminedtraitmayaccountforthefingerprintdifferencesseeninMZtwins. Policedepartmentshavelongbeengrateful forthisphenomenon, whichensuresthatMZtwinscannotcommittheperfectcrime sooftenpresentedinworksof fiction.Forensicstudiesin fact show that although MZ twins have different fingerprints the pattern, ridge count andother minutiaearenevertheless muchmore similar thanthose seeninDZtwins [47].Variation in vascular supply to each twin can occur during the second prenatal trimesterthecritical periodwhenfingertipdermal cellsmigratetoformridges. This is alsotheperiodwhenneural cells migrate tothe cortexandseveral investigators have triedtoexploitthistemporalrelationshipinanattempttousefingerprintsasmarkerforprenatalanatomicalinsultsthatmayhaveaffectedthetwinsdifferently.5.3. MajormalformationdiscordancesFoetalpathologistshavelongrecogniseddiscordancesformajormalformationsinMZtwins [48]. Many of these malformations do not have simple genetic origins, but are likelytobemultifactorial. Examplescommonlydescribedincludeneural tubedefects, cleft lipandpalate, andsymmelia[4]. Whileitisnot clearhowthesediscordant eventsarise, inmanycases,theapparentlyunaffectedco-twinalsohasalessseveremanifestationofthesamedisorder.Theimplicationinthesecasesisthatthepredispositionforthemalforma-tionisexpressedinbothtwins.6. SummaryThisreviewhighlightssomeoftheimportantfindingssinceFrancisGalton,thefatherof twin research, first thought of using twins to estimate the impact of genes in 1876 [49].Hispaperontheuseoftwinsasacriterionoftherelativepowersofnatureandnurturelaunched the whole field, which has since seen major advances in twin foetal developmentandtwingenetics. This reviewhas demonstratedtheproblems inassumingthat beingmonozygousissynonymouswithbeingidentical.P. Gringras,W. Chen/EarlyHumanDevelopment64(2001)105117 114Apart for academicinterest andimplicationsfor twinresearch, thesefactsalsohaveclear implications for clinical practice. In the case of a twin pair found to be discordant fora major malformation, the assumption should not be that they are automatically DZ. TheymaybeMZand, moreimportantly, MC. Inacaseof heterochromia iridum(differentcolouririses)inMZtwinsdescribedbySt. Clairetal. [45], immunosuppressivetherapyfollowing a renal transplant was needlessly continued for 15 years because dizygosity hadbeenassumed. Intheir case discussion, the authors of this paper emphasisedthat theheterochromiadiscordancemustfallwithintheacceptablespectrumofMZstatus.Thisreviewdemonstratesthat asour understandingof MZtwinningincreases, thespectrumwill continuestowiden, andthetermidentical twinsshouldbereplacedbythemoreaccuratetermmonozygoustwins.The importance of establishing the zygosity of twins has been stressed in the literature.KeithandMachin[50] havesummarisedmanyof theissues: For reasonsof personalrights to identity, medico-legal responsibility, potential for transplantation, early education,andconcordance/discordanceforgeneticdisease,manyfeelthatroutinedeterminationoftwin zygosity at birth is needed and should be implemented in the near future. Apart fromthebenefitstothetwinsandtheir parents, theconsequencesfor researchintwinsandgeneticswouldbeenormous,sincethereremainmanypuzzlingaspectsoftwinning.Tothis list is nowalso added the importance of establishing chorionicity, preferably byultrasoundinveryearlypregnancy.References[1] SegalNL.Zygositydiagnosis:laboratoryandinvestigatorsjudgement.ActaGenMedGemellol1984;33:51520.[2] KeithL,MachinG.Zygositytesting:currentstatusandevolvingissues.JReprodMed1997;11:699707.[3] MachinG, KeithL. Biologyoftwinsandothermultiplepregnancies.In:MachinG,KeithL,editors.Anatlasofmultiplepregnancy:biologyandpathology. NewYork:Parthenon;1999.p.1324.[4] ChitnisS,DeromC,VlietinckR,MonteiroJ,GregersenPK.Xchromosome-inactivationpatternsconfirmthelatetimingofmonoamniotic-MZtwinning.AmJHumGenet1999;65:5714.[5] CorneyG,MacGillivrayI,NylandersP.Placentation.In:CorneyG,editor.Humanmultiplereproduction.London:Saunders;1975.p.4076.[6] BenirschkeK. Accuraterecordingoftwinplacentation:apleatotheobstetrician. 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