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Abstract

Sex determination is the most important step in personal identification in forensic investigations. DNA-based sex determination analysis is comparatively more reliable than the other conventional methods of sex determination analysis. Advanced technology like real-time polymerase chain reaction (PCR) offers accurate and reproducible results and is at the level of legal acceptance. But still there are situations like chimerism where an individual possess both male and female specific factors together in their body. Sex determination analysis in such cases can give erroneous results. This paper discusses the phenomenon of chimerism and its impact on sex determination analysis in forensic investigations.

Keywords: DNA, chimerism, forensic sciences, polymerase chain reaction, sex determination analysis

Introduction

Forensic science aids in the personalidentificationprocessbycomparativemethodsorbyenablingaprofileof the individual regardingage at death, and sex, in crime investigationsinvolving biological evidence, such as murder,sexualassault,anddisastervictimidentification.Investigationofviolentcrimehasalwaysbeenadrivingmotivationfor lawenforcementagenciesin any country, and more so in developingcountries. Biological specimens recoveredfrom the scene of crime provide remarkablyvaluable information about the crime and thepersonnelinvolved.Deoxyribonucleicacid(DNA)technologyhasgainedwideacceptance in crimeinvestigations involving biological evidence,such asmurder (blood evidence), sexual assault(semen evidence), murder with sexual assault(bloodandsemenevidence),orincasesofmassdisasters (saliva evidence and body tissues),and in identification of mutilated bodiesand exhumed skeletons (tissues and bones).Variabilityinbiomoleculeshasbeenexploitedforidentification of the source of the biospecimen.Earlier, identification procedures employedfor the characterization of biological specimenswere protein or ‘classical’ markers such as theABOblood group antigens, serumproteins, andRBC enzymes. However, these suffered fromlow polymorphism, poor stability and restrictedactivity of the molecules, and the limitedresolutionof thedetectionmethods.Theadvent

Brief Communication

The Impact of Chimerism in DNA-based Forensic Sex Determination AnalysisRenjith GeorGe, Preethy Mary DonalD, Sumanth Kumbargere naGraj, Jose Joy IDIculla, Rashid Hj IsmaIl

Department of Oral Medicine, Oral Diagnosis and Oral Pathology, Faculty of Dentistry, Melaka Manipal Medical College, 75150 Melaka, Malaysia

Submitted: 26Jul2012Accepted:14Nov2012

of DNA markers allowed for greater precisionand higher discriminatory power in forensictesting. DNAisthevehicleforgenerationaltransferof heritable traits. It encodes the geneticinformationinmostorganismsandisidenticalineverycellofan individual.Chemically, theDNAmoleculeisahighlystablepolymercomposedofsubunits known as nucleotides, and in humansmakes up the 22 pairs of autosomal and singlepairofsexchromosomes.Eachparentcontributesachromosometothepairanindividualinherits.The information content of DNA resides inthe sequence of bases, and although the DNAsequence in different individuals is moresimilar than different, many regions of humanchromosomes exhibit a great deal of diversity.Suchvariablesequencesaretermed‘polymorphic’(meaning‘manyforms’)andareusedforhumanidentification,paternitytesting,anddiagnosisofgeneticdiseases.Mostpolymorphismsarelocatedintheestimated95%ofthehumangenomethatdoesnotencodeforproteins(1). Various methods are employed for sexdeterminationanalysisinforensicinvestigations,and include visual, clinical, microscopic, andadvancedmethods. Visualmethods are reliable,but are subject to inter-observer variation andlack accuracy. Microscopic methods, such asidentifying sex by means of Barr bodies, showdiminishing accuracy with the time elapsedafter death (2). Amongst the above mentionedmethods,sexdeterminationanalysisbyadvanced

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methodsisfoundtobeaccuratewithreproducibleresults.Forinstance,DNAanalysisformaleandfemale specific sex-typing markers in X andY chromosomes is used in sex determinationanalysis (3). DNA is the molecule of choice forforensic analysis because of its discrimination,geneticcontinuity,sensitivity,andstability(4). The introduction of polymerase chainreactions (PCR)has helpedDNA-based analysisby amplifying identification markers even ifthe available samples are of limited quantity.Conventional PCR has its own limitations, suchas poor precision, limited dynamic range ofdetection, lowsensitivity, theneed forpost-PCRprocessing, and allowing for only size-baseddiscrimination. Further advancements such asreal-time quantitative PCR are highly sensitive,and do not require post-PCR processing. Thisallows for real-time visualisation of ampliconswith an increased dynamic range of detectionand has made DNA analysis much easier, withaccurate results. Compared to other methodsof sex determination analysis, the results aremuch more reliable and reproducible, and it isconsidered one of the gold standard techniquesfor sex identification in forensic investigations.But,aswithothermethods,therecanbesituationswhereDNA-basedsextypingcanyielderroneousresults. This paper discusses ‘chimerism’, thatis occurrence in an individual of two or morecell populations of different chromosomalconstitutions,derivedfromdifferentindividuals,which is critical inDNA-basedpersonalandsexidentification(5).

Sex typing markers

There are various sex typingmarkers usedinDNA-basedidentificationsuchasamelogenin,zincfingergenes, thesexdeterminingregionontheYchromosome,andthelike. Amelogenin is themostcommongeneusedfor sex determination analysis in the forensicfield.Inhumanstherearetwoamelogeningenes,located on the sex chromosomes (AMELX andAMELY).Thedifferenceinthelengthofthefirstintrons of the AMEL genes, located on both Xand Y chromosomes, differentiates males fromfemales. AMEL genes in females are located onboth X chromosomes and are homozygous. Inmales, theAMELgene ispresentonboth theXand Y chromosome, but they are heterozygous.If a sample yields amelogenins with the samemolecular weight, it can be concluded to be afemale sample, whereas different molecularweightsrepresentamalesample(6).

The Sex-determining Region on theY chromosome (SRY) is a gene that causesdevelopment of male characteristics. The SRYgene is located on the short (p) arm of the Ychromosomeatposition 11.3. Inhumans,wherethere are two distinct sex chromosomes, the XandY chromosomes; it is the presence of the Ychromosome that specifies male development.TheSRYgeneon theY chromosome causes theembryotodevelopasamale.Femaledevelopmentseems to be the default pathway, and in theabsenceofSRY, theurogenital tractdevelopsasthatof a female.Detectionof theSRY sequencewoulddistinguishanauthenticmaleDNAsamplefromafemaleDNAsample,asfemaleDNAlackstheSRYgene.Thedetectionof theSRYgene inDNAfromaforensicsamplecanbeconfirmatorytotypethesexasmale(7). The current methods of DNA forensic sextyping involvemultiplex X and Y STR analysis,Single Nucleotide Polymorphism (SNP) pyro-sequencing,orYSTRhaplotypeanalysis.Y-STRsare short tandem repeats found on the male-specificYchromosome.Thecodinggenes,mostlyfoundontheshortarmoftheYchromosome,arevitaltomalesexdetermination,spermatogenesis,and other male-related functions. Y-STRs arepolymorphic among unrelated males and areinherited through the paternal line with littlechangethroughgenerations. Y-STRsareusefulinexaminingsexualassaultevidence, such as vaginal swabs, that containsboth female and male DNA. It is necessary toseparate themale component (sperm cellDNA)fromthe femalecomponentDNAbydifferentialextraction, but sometimes the male and femalecomponents cannot be separated completely.As a result, the female component could existpredominantlyinthemalecomponentevenafterdifferentialseparation.ThiscanleadtomaskingofthemaleDNAbythefemaleDNAwhentheyareco-amplified,andwillleadtoerroneousresults. MaskingeffectsbyfemaleDNAdonotoccurwhenY-STRsareexamined.SincefemalesdonotpossessaYchromosome,anyY-STRdataobtainedcan only be contributed by males and will beeasilydetected,sinceonlythemalecomponentoftheDNAmixturewillbeamplified,notthefemalecomponent. The Y-STR system is especiallyhelpful when there is more than one maleassailant.Themixedmalepatternintheevidencecan help to identify thosemales responsible fortheassault.Y-STRanalysisisusefultodetectthepresenceofminimalmaleDNAwherethere isamasking effect of femaleDNAwith regular STRanalysis(8).

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Inallof theabovementionedmethods, theXorYspecificmarkerisanalysed,andbasedonthepresenceorabsenceortherelativeproportionof amplified products from the sample, the sexdeterminationisdone.However,insituationssuchasanindividualpossessingbothmaleandfemalespecific factors in their body, sex determinationmay be misleading, especially while analysingforensicsamples.

The impact of chimerism on DNA-based sex determination analysis

Aconditionwherethereismorethanonesetof cell lines with different sets of chromosomesmaking up the body is known as chimerism.A ‘chimera’ is an individual with at least twodifferentpopulationsofcells,whicharegeneticallydistinctandoriginateindifferentzygotes(5).

Chimerismcanbeclassifiedas:1.Artificial2.Transplacental3.Tetragametic

Artificial chimerism Artificial chimerism is caused by thetransplanted blood stem cells via bloodtransfusionorbonemarrow transplantation (5).After transplantation, the short tandem repeats(STRs)usedforpersonalidentificationinforensicinvestigationswillbeidenticalforthedonorandthe receiver, leading to erroneous identification.Ifthedonorisofthesamegeneticpatternasthereceiverbutofdifferentsex,thesamplesfromthereceivermaybemisidentifiedinsexidentification.Samplessuchasblood,buccalswabs,orfingernailsarenotexemptedfrominfiltrationbythedonor’scells;theexceptionishairrootcells(9).

Transplacental chimerism Afemalebearingamalefoetusisanexampleof a transplacental chimera. At a certain timeperiodinthepregnancy,thereissomecelltrafficbetween the mother and the foetus, and thesecellsfromthefoetuscanpersistinmother’sbloodfor decades (10). Microchimerism refers to asmallpopulationofcellsorDNAinoneindividualderived from another genetically distinctindividual(11). Microchimerism can occur as foetalmicrochimerism, where there is free passageof blood between mother and child, or as twinchimerism, or blood chimerism, where bloodpassesbetweentwinfoetuses.Ineithersituation,if the involved subjects are of different sex, it

canresult inchimeraswhosesamplesmayyielderroneousresultsonDNA-basedgenderanalysis.Abloodsample fromamotheror twinsbearingalleles of the opposite sex can yield ambiguousresultsinDNA-basedsexdeterminationanalysis. Costaetal.,wereabletodetectthepresenceofSRYinmaternalplasma.TheystatedthatfoetalSRY genes can be found inmaternal plasma asearlyas42daysofgestation,andthenumberoffoetalDNAincreaseswithgestationalage(12). The foetal cells canmigrate into the femalebody during pregnancy and can persist fordecades.ThebodyofawomanafterapregnancywithamaleembryocanthereforedisplayasmallfractionoffoetalcellswithYchromosomes(13). Bayes-Genis et al., stated that foetalprogenitor cells may cross the placenta duringpregnancy, persist for decades in the maternalbloodstream,andcancolonise inmaternal solidorgans.Thisstudyidentifiedmalecardiomyocytesofextra-cardiacorigin,presumablyfoetal, intheheartsoftwowomenwithmaleprogeny(14). Rao et al., reported foetal microchimerismshowingmale DNA of foetal origin inmaternalcirculation several years after delivery. TheyidentifiedSRYsequencesinnormalwomenwithamalechildasevidenceoftheexistenceoffoetalprogenitorcellsinthematernalcirculation,whichis completely absent in normal women with afemalechild(15). Hence theDNA-based analysis of a samplefromafemalewhohascarriedamalefoetusmayidentifythesourceasamale,whichcanaffecttheinvestigationinanegativemanner.Thesituationmaybesimilarinthecaseofasurrogatemotherwhoreceivesamalefoetus.

Tetragametic chimerism Fertilization of two oocytes by twospermatozoa and fusion, or the resulting twoembryosleadingtodevelopmentofoneorganism,may result in tetragametic chimerism (5). Atetragameticchimeracanhavetwogeneticprofilesin their blood, and distinct DNA markers indifferentpartsoftheirbody(11).Ahermaphroditechimera is a variant of a tetragametic chimerawhere a female embryo is merged with a maleembryo,andtheresultantchimerawillhavebothmale and female specificmarkers in their body.Toagreaterorlesserdegree,theywillalsopossessambiguous genitalia. This can occur in in vitrofertilisation where more than one fertilised eggis placed in the uterus for a better success rate.If the fertilised eggs used are of opposite sex, itcanleadtothedevelopmentofahermaphroditechimera.Twinchimerism is comparatively rarer

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thanartificialandtransplacentalchimerism. A forensic sampleobtained froma chimerathatpossessesbothmaleandfemalecelllineagescanresultinambiguousresultswithDNA-basedsex typing. Gender analysis based on detectionof male or female specific markers, will notbe applicable for a chimera that possesses cellpopulationsofbothgenders,andforthatreasonit is advisable to include differential extractionfor such samples so as to detect the ambiguousconditions.

Conclusion

Although DNA-based sex determinationmethods are a comparatively better approachforforensicsexdeterminationanalysis,thereareenigmatic conditions where the analysis can gowrong.Apart from simple procedures of genderdetermination using X or Y specific markers,detailed analysismay be necessary to identify asample from a chimera. Sex typing proceduresinvolvingasinglemarkersuchasamelogeninorSRYarenotreliable,andmultiplexSTRanalysisis preferable to single marker procedures.To reduce the risk of error in situations suchas chimerism, the suspected sample can besubjected to highly discriminating proceduressuch as multiplex Y chromosome STR typing,or SNPpyro-sequencing (16). Most of the time,however, the forensic analyst will be receivingthe samplewithout anydetails of thedonor.Asthe first and most important step in personalidentification, it is of great importance to avoidthepossiblepitfallsinforensicsexdeterminationanalysis. An investigatormust be aware of suchsituationswhere sex determination analysis canbe ambiguous and neither caused by his/hermistakenorthetechnology.

Conflict of interest

Nil.

Funds

Nil.

Authors’ Contributions

Conceptionanddesign:RGAnalysisandinterpretationofthedata:RG,PMD,SKNDraftingofthearticle:PMDCritical revision of the article for the importantintellectualcontent:SKNFinalapprovalofthearticle:JJI,ARHI

Correspondence

DrRenjithGeorgeMDS(India)AssistantProfessorDepartment of Oral Medicine, Oral Diagnosis andOralPathologyFacultyofDentistryMelakaManipalMedicalCollegeMelaka,75150,MalaysiaTel:+6010-7901963Fax:+606-2896669Email:drrenjithgeorgep@gmail.com

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