Forensic
Population
Genetics—Short
Communication
The
2011
GeFI
collaborative
exercise.
Concordance
study,
proficiency
testing
and
Italian
population
data
on
the
new
ENFSI/EDNAP
loci
D1S1656,
D2S441,
D10S1248,
D12S391,
D22S1045
C.
Previdere`
a,*
,
P.
Grignani
a,
F.
Alessandrini
b,
M.
Alu`
c,
R.
Biondo
d,
I.
Boschi
e,
L.
Caenazzo
f,
I.
Carboni
g,
E.
Carnevali
h,
F.
De
Stefano
i,
R.
Domenici
l,
M.
Fabbri
m,
E.
Giardina
n,
S.
Inturri
o,
S.
Pelotti
p,
A.
Piccinini
q,
M.
Piglionica
r,
N.
Resta
s,
S.
Turrina
t,
A.
Verzeletti
u,
S.
Presciuttini
v,1a
DipartimentodiSanita` Pubblica,Neuroscienze,MedicinaSperimentaleeForense,SezionediMedicinaLegaleeScienzeForensi,Universita` diPavia,Italy
b
DipartimentodiScienzeBiomedicheeSanita` Pubblica,Sez.diMedicinaLegale,Universita` PolitecnicadelleMarche,Italy
c
DipartimentoadAttivita` IntegratadiLaboratori,AnatomiaPatologica,Medicinalegale,Universita` diModenaeReggioEmilia,Italy
d
DirezioneCentralePoliziaCriminale,SSII–DirezioneCentraleAnticriminedellaPoliziadiStato,ServizioPoliziaScientifica–Roma,Italy
e
IstitutodiMedicinaLegale,Universita` Cattolica,Roma,Italy
fDipartimentodiMedicinaAmbientaleeSanita` Pubblica,Universita` degliStudidiPadova,Italy g
SODDiagnosticaGenetica,AziendaOspedaliero-UniversitariaCareggi,Firenze,Italy
h
Universita` degliStudidiPerugia,SezionediMedicinaLegale,SedediTerni,Italy
i
DipartimentodiScienzedellaSalute,SezionediMedicinaLegale,Universita` diGenova,Italy
l
SezioneDipartimentalediMedicinaLegale,Universita` diPisa,Italy
m
DipartimentodiScienzeBiomedicheeTerapieAvanzate,Sez.MedicinaLegale,Universita` diFerrara,Italy
nDipartimentodiMedicinaePrevenzione,Universita` diRomaTorVergata,Italy oDipartimentodiAnatomia,FarmacologiaeMedicinaLegale,Universita` diTorino,Italy p
DipartimentodiMedicinaeSanita` Pubblica,SezionediMedicinaLegale,Universita` diBologna,Italy
q
DipartimentodiScienzeBiomedicheperlaSalute,SezionediMedicinaLegale,Universita` diMilano,Italy
r
DipartimentoInterdisciplinarediMedicina,SezionediMedicinaLegale,Universita` diBari,Italy
s
DipartimentoInterdisciplinarediMedicina,Lab.diGeneticaMedica,Universita` diBari,Italy
t
DipartimentodiSanita` PubblicaeMedicinadiComunita`,Universita` diVerona,Italy
uDipartimentodiSpecialita` Chirurgiche,ScienzeRadiologicheeMedicoForensi,Universita` degliStudidiBrescia,Italy vDipartimentodiScienzeFisiologiche,Universita` diPisa,Italy
1. Introduction
TheISFGItalianWorkingGroupGeFIplansperiodical collabo-rativeexercisesaimedattheanalysisofgeneticmarkersrelevant fortheforensicapplication.In2011,theOrganizingCommitteehas proposedthecharacterizationofthefivenewENFSI/EDNAPloci,
D1S1656,D2S441,D10S1248,D12S391andD22S1045,inalarge
Italian population sample [1,2]. The inclusionof theseminiSTR
markers to the European Standard Set of loci (ESS) has been
established by the Council Resolution 2009-C 296-01 of 30
ForensicScienceInternational:Genetics7(2013)e15–e18
ARTICLE INFO Articlehistory: Received12May2012
Receivedinrevisedform31July2012 Accepted1August2012 Keywords: miniSTR Concordancestudy Populationdatabase GeFI ENFSI EDNAP ABSTRACT
The2011collaborativeexerciseoftheISFGItalianWorkingGroupGeFIwasaimedatvalidatingthefive ENFSI/EDNAPminiSTRlociD1S1656,D2S441,D10S1248,D12S391andD22S1045.Theprotocolrequired totypeatleast50multilocusprofilesfromlocallyresidentindividualsandtwoblindbloodstainsin duplicate(i.e.,usingatleasttwodifferentcommercialkits),andtosendtheelectropherogramstothe OrganizingCommittee.NineteenlaboratoriesdistributedacrossItalyparticipated,collectingatotalof 960samples.FullconcordancewasfoundforthefivenewminiSTRsasobservedfromthecomparisonof 13,150alleles.Theinspectionoftheelectropherogramsallowedtheidentificationofaverylimited numberofmistypingsintheminiSTRgenotypesthuscontributingtotheestablishmentofanhigh qualityItaliandatabaseoffrequencies.
ß2012ElsevierIrelandLtd.Allrightsreserved.
*Corresponding author at: Dipartimento di Sanita` Pubblica, Neuroscienze, MedicinaSperimentaleeForense,Universita` diPavia,SezionediMedicinaLegalee ScienzeForensi,viaForlanini,12,27100Pavia,Italy.Tel.:+390382987820; fax:+390382528025.
E-mailaddress:previde@unipv.it(C.Previdere`).
1
Presented,inpart,asposteratthe24thCongressoftheInternationalSocietyfor ForensicGenetics,Vienna,Austria,August29–September3,2011.
ContentslistsavailableatSciVerseScienceDirect
Forensic
Science
International:
Genetics
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 / f si g
1872-4973/$–seefrontmatterß2012ElsevierIrelandLtd.Allrightsreserved. http://dx.doi.org/10.1016/j.fsigen.2012.08.001
November2009ontheexchangeofDNAanalysisresults[3].This decisionwasbasedontheresultsofcollaborativeexercisescarried
outby ENFSI and EDNAP groups which strongly suggested the
characterizationofthesemarkersinordertoimprovethepowerof discriminationbetweenindividualsthusminimizingthe possibili-tyofadventitiousmatchesininternationalDNAdatabases[4,5].In addition,theminiSTRapproachhasbeenshowntobeveryuseful intheanalysisofdegradedandmodifiedDNAsamples.Thegenetic resultswillbecollectedinordertobuildanItaliandatabaseof frequenciesforthesenewmarkersusefulforthefollowingforensic applications.Moreover, different kits will berequested for the
genetic typing in order to verify the concordance between
genotypes.Finally,eachparticipatinglaboratorywillbeaskedto
provide information on the processing of the samples (DNA
extraction,quantification andtyping) and theevaluation of the results,bymeansofaquestionnaire.
2. Materialsandmethods
2.1. Samples
Nineteenlaboratoriesparticipatedinthecollaborativeexercise. TenlaboratorieswerelocatedinNorthItaly(Turin,Genoa,Milan, Pavia,Brescia,Verona,Padua,Modena,Bologna,Ferrara),sevenin CenterItaly(Firenze,Pisa,Ancona,Terni,Rome)andtwoinSouth Italy(Bari).
Fifteenwereforensicgeneticslabs,threemedicalgeneticslabs and onefrom a national criminaljustice service. Eachlab was requestedtotypeatleast50unrelatedItalianindividualslivingin theirareawhichprovidedtheinformedconsent.Sixlabsselected bloodsamples,ninelabssalivasamplesandtheremainingones bothbloodandsaliva.
2.2. DNAextraction
ThelabsreporteddifferentDNAextractionproceduresinthe questionnaire.TheQiagencolumns(QIAampDNAminikit;Qiagen,
Germany) andthephenol/chloroformextraction werethemost
followed(7and4labs,respectively). 2.3. DNAquantification
FourteenlabsquantifiedtheDNAextractsbeforeSTRanalysis. EightlabsusedUV-spectrophotometryandsixlabsqPCRprotocols. 2.4. DNAamplificationandconcordancestudy
Each laboratory was requested to type their samples in
duplicate, freelychoosing a combination of at leasttwo of the
following kits: PowerPlex ESX, PowerPlex ESI (Promega, USA),
AmpFlSTR NGM (Applied Biosystems, USA), and Investigator
ESSplex, Hexaplex ESS (Qiagen, Germany). Six labs used in
combination ESX+ESI, five NGM+ESSplex, three NGM+ESI, two
NGM+ESX,oneNGM+Hexaplexandtwoacombinationofthreeor
morekits.Eachlabamplified DNAamountsvaryingfrom0.2to
1.3ngfollowingthemanufacturers’recommendations. 2.5. DNAelectrophoresisandanalysis
TheamplifiedproductswererunonABI310(12labs),3130(6 labs)and3500(1lab) GeneticAnalysers(AppliedBiosystems). Mostofthelabs(16outof19)analyzedtheelectropherograms
using the GeneMapper software (Applied Biosystems) even if
three labs still used older analysis softwares (Genescan/
Genotyper).
Fig.1.TwoelectrophoreticseparationsofD1S1656alleles15.3and16.(A)Allelesnotcorrectlyseparatedandmergedinallele16.Inthiscase,thenumberofrunsperformed bythecapillarywasclosetothelimitsuggestedbythemanufacturerforthereplacement(100runs).(B)Oncereplaced,thesamesampleshowedacorrectseparationand identificationofalleles15.3and16.
C.Previdere` etal./ForensicScienceInternational:Genetics7(2013)e15–e18 e16
2.6. Labcertifications
TenlabswerecertifiedUNIENISO9001:2000andonegotthe additional ISO 15189 certification. Eleven labs participate in GEDNAPqualitycontrol/proficiencytests,forforensicDNAtyping certificates.
2.7. Proficiencytesting
TheOrganizingCommitteeprovidedtwoblindbloodstainsto eachparticipatinglaboratory.
2.8. Statisticalanalyses
StatisticalanalyseswereperformedusingArlequin3.0[6]. 2.9. Qualitycontrol
AresultsummaryintheformanExcelfile(tabularresults)was senttoeachparticipatinglaboratorytobefilledwiththegenotypes foundforeachsample.Inordertoverifythequalityandtoconfirm
the results, each laboratory was requested to send all the
electropherogramsobtainedfromthesamplestotheOrganizing Committeeeitherasprintoutsorelectronicfiles.
3. Resultsanddiscussion
Thefinaldatabaseincludedthemultilocusgeneticprofilesof 960individuals,equally distributed amongthe19 participating labs.
All the electropherograms were visually evaluated by two
independent operatorsinorder toverifythecorrectness of the genotypesassignmentsin theelectropherogramsandthe corre-spondenceinthetabularresults.
Five laboratoriesexperienced troublesin theelectrophoretic separationofallelesforsomenewminiSTRs,usingtheABIPRISM 310sequencer.Thisisthecaseofallelesdifferingfor1bp,typedfor theD1S1656andD12S391markers,inthemolecularrangeabove
200bp. This poor resolution resulted in false homozygote
genotypes (see Fig. 1) and in apparent discordances in the
genotypes when the same sample was characterized using a
differentkit showingaconfigurationof thesamemarkerswith
ampliconsbelow 200bp.This problem was resolved either
re-injecting the sample in order to get a better electrophoretic separationoroptimizingthecapillaryeletrophoresis(CE)settings
decreasing the injection time or adjusting the GeneMapper
analysisparameter‘‘peakdetection’’.
Asregardstheconcordancestudy,onlytwodifferenceswere observedoutofatotalnumberof42,188allelesinspectedinthe courseoftheexercise.Onesampleshowedanullallele(18)forthe
vWAmarker when amplified withNGM which wasconversely
heterozygoteforESSplex (17–18). Thesecondsampleexhibited theTH01genotype9–9.3forESSplexbeinghomozygote9–9for NGM.Allthedropoutswereconfirmedinasecondamplification andCE.TherateofconcordancebetweentheNGMandESSplexkits wasthuscalculatedas99.979%accordingto[7].Nodiscordance
was recorded for the new miniSTRs in the Italian population
sampleherestudied.Twodiscordancespreviouslydescribedina preliminarystudy [8]were notconfirmed bythe reportinglab whencontactedforrevision.
Theinspectionoftheelectropherogramswasmainlyfocusedon thecorrectidentificationofthegenotypesforthenewminiSTRs,as oneofthemainaimsofthiscollaborativeexercisewastobuildan highqualityItaliandatabaseoffrequenciesforthosemarkers.This processallowedtheidentificationofeightwronggenotypesforthe D1S1656andD12S391markerswhichwerecorrectlyidentifiedin
asecondamplificationandCEwhenthelabswerecontactedfor revising thesamples in question. Eight genotypesfor thenew miniSTRswere‘‘typos’’or‘‘clericalerrors’’,thatismistakesinthe courseofthetranscriptionofthetabularresults.
Inaddition,sixfurthersamplesdisplayeddiscordantgenotypes intheduplicateanalysisofD1S1656and/orD12S391.Thisisthe case,forinstance,ofasampleshowinggenotype16–16and15.3– 16,fortheD1S1656marker,whentypedwiththeESIandESXkits, respectively.Incaseofdiscrepancies,thelabswhichperformedthe experiments arbitrarily decidedto choose one genotypeas the correctonethusreportingitinthetabularresults.Whenthelabs
were contacted for revision, no more discordances were then
observedandthecorrectnessofthegenotypeassignmentinitially reportedinthetabularresultswasconfirmed.
On the opposite,after therevisionof two differentsamples showingdiscordantgenotypesfortheD2S441marker(genotypes
11–12 and 11–11.3, for ESI and ESX, respectively), a full
concordance of genotypes was established but the genotypes
initiallyreportedinthetabularresultswerethewrongones. Hardy–Weinbergequilibriumwastestedseparatelybysample (N=19 samples) and by locus (n=5 loci) with no evidence of deviation.Averylowlevelofdifferentiationamongsampleswas
found by computing pairwise F(ST) values and by partitioning
Wright’sFstatistics,locusbylocus(seeTable1).
Table1
Allele frequenciesdistributionin theItalianpopulation forthefiveminiSTRs D1S1656,D2S441,D10S1248,D12S391andD22S1045. Allele D1S1656 D2S441 D10S1248 D12S391 D22S1045 8 0.001 9 0.003 0.001 10 0.002 0.140 0.001 0.001 10.3 0.001 11 0.069 0.332 0.003 0.111 11.3 0.083 0.001 12 0.140 0.039 0.027 0.005 12.3 0.002 13 0.072 0.027 0.264 0.004 13.3 0.001 14 0.091 0.320 0.328 0.001 0.049 14.3 0.002 15 0.160 0.049 0.180 0.049 0.384 15.3 0.058 16 0.130 0.003 0.154 0.019 0.352 16.3 0.047 0.001 17 0.047 0.038 0.098 0.082 17.3 0.109 0.014 18 0.007 0.005 0.197 0.009 18.3 0.053 0.034 19 0.001 0.001 0.108 0.002 19.3 0.010 0.012 20 0.001 0.122 20.3 0.001 0.001 21 0.107 22 0.104 23 0.074 24 0.040 25 0.014 26 0.005 27 0.001 N 960 960 960 960 960 Na 18 13 12 19 10 Na>0.01 12 7 6 14 5 Hexp 0.897 0.756 0.765 0.891 0.707 FIS 0.009 0.047 0.013 0.012 0.012 MP 0.020 0.096 0.092 0.022 0.135 PE 0.791 0.544 0.547 0.781 0.467 N:numberoftypedindividuals;Na:numberofalleles,Na>0.01:numberofalleles
withp>0.01;Hexp:expectedheterozygosity;FIS:Wright’sfixationindex(=1 Hobs/
Hexp);MP:matchprobability;PE:probabilityofexcludingafalsefatherinstandard
trios.
Thus, allele frequencies were computed for all 960 typed subjects(seeTable1).Thelocus-by-locusexacttestsofpopulation differentiationwerenotsignificantofheterogeneitywith previ-ouslypublishedItaliandata[9];therefore,theconclusionofWelch
[10],thatpopulationsubstructureinEuropecanbeaccommodated using the correction factor FST=0.01, can be extended to the presentresults.
Alllabscorrectlytypedthetwoblindbloodstainsprovidedby theorganizersasproficiencytesting.
The presentGeFI collaborative exerciseshowed tobea very usefultoolformonitoringthequalityoftheparticipatinglaboratory work.Infact,thedouble-checkinspectionoftheelectropherograms allowedustoidentifyalimitednumberofmistypingandambiguous results otherwise hidden in a general population study, thus producinganhigh-qualitydatabaseoffrequencies.
Theauthorsstatethatunderstandandaccepttheguidelinesfor publicationofpopulationdatarequestedbythejournal[11]and theInternationalSocietyforForensicGenetics(ISFG)[12]. Acknowledgments
The authors wish to thank Dr. Giorgio Marrubini for his
suggestionsandcriticalreadingofthemanuscript.P.G.isafellow
of the Pathology and Medical Genetics PhD program of the
UniversityofPavia. References
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