Forensic parameters based on 15 AmpFlSTR Identifiler short tandem repeat (STR) loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818 and FGA) were evaluated in the sample of 122 unrelated, autochthonous, adult individuals from the Island of Cres (Croatia). PCR amplification was performed with the AmpFlSTR Identifiler PCR Amplification Kit and the amplified products were separated and detected using the ABI 3130 DNA genetic analyzer. The agreement with Hardy Weinberg Equilibrium (HWE) was confirmed for all loci (p>0.05). The combined power of discrimination (PD) and the combined power of exclusion (PE) for the 15 tested STR loci were 0.99999999999999997988728679 and 0.999997397, respectively. According to the presented data, D18S51 proved to be the most informative marker followed by markers D2S1338 and D21S11. Interpopulation comparisons in allele frequencies with other East Adriatic Islands revealed significant differences for all analyzed population pairs ranging from 4 loci (Cres vs. Hvar) to 1 locus (Cres vs. Krk). Furthermore, allele frequencies comparisons of Cres and Croatian mainland revealed the lack of statistically significant differences at all studied loci. The results of the current study indicate that the examined fifteen STR loci are useful genetic markers for individual identification and paternity testing in Croatian population from the Island of Cres

Blaženka Grahovac

Bojana Mohar

Damir Marjanović

Gordan Lauc

Miljenko Kapović

Natalija Novokmet

Pavao Rudan

Petar Projić

Vedrana Škaro

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Coll. Antropol. 33 (2009) 4: 1319–1322Original scientific paperForensic Efficiency Parameters for the 15 STRLoci in the Population of the Island of Cres(Croatia)Natalija Novokmet1, Damir Marjanovi}2,3, Vedrana [karo2, Petar Proji}2, Gordan Lauc4,5,Bla`enka Grahovac6, Bojana Mohar6, Miljenko Kapovi}7 and Pavao Rudan11 Institute for Anthropological Research, Zagreb, Croatia2 Genos, DNA Laboratory, Zagreb, Croatia3 Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina4 DNA Laboratory, School of Medicine, »J. J. Strossmayer« University of Osijek, Osijek, Croatia5 Genos, DNA Laboratory, Osijek, Croatia6 Department of Pathology and Pathological Anatomy, School of Medicine, University of Rijeka, Rijeka Croatia7 Department of Biology and Medical Genetics, School of Medicine, University of Rijeka, Rijeka CroatiaA B S T R A C TForensic parameters based on 15 AmpFlSTR Identifiler short tandem repeat (STR) loci (D8S1179, D21S11, D7S820,CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818 and FGA) wereevaluated in the sample of 122 unrelated, autochthonous, adult individuals from the Island of Cres (Croatia). PCR am-plification was performed with the AmpFlSTR Identifiler PCR Amplification Kit and the amplified products were sepa-rated and detected using the ABI 3130 DNA genetic analyzer. The agreement with Hardy Weinberg Equilibrium (HWE)was confirmed for all loci (p>0.05). The combined power of discrimination (PD) and the combined power of exclusion(PE) for the 15 tested STR loci were 0.99999999999999997988728679 and 0.999997397, respectively. According to thepresented data, D18S51 proved to be the most informative marker followed by markers D2S1338 and D21S11. Inter-population comparisons in allele frequencies with other East Adriatic Islands revealed significant differences for all an-alyzed population pairs ranging from 4 loci (Cres vs. Hvar) to 1 locus (Cres vs. Krk). Furthermore, allele frequenciescomparisons of Cres and Croatian mainland revealed the lack of statistically significant differences at all studied loci.The results of the current study indicate that the examined fifteen STR loci are useful genetic markers for individualidentification and paternity testing in Croatian population from the Island of Cres.Key word: STRS, AmpFlSTR identifiler, forensic parameters, the Island of Cres, CroatiaIntroductionShort tandem repeats (STRs) are loci with allelescomposed of tandemly repeated short DNA sequences of2–7 base pair in length1. There are hundreds of theseSTR regions in the human genome, but 10 to 15 regionsare sufficient to give high levels of discrimination be-tween individuals2. Numerous previous studies have de-monstrated that STRS have become the choice of loci fordetermination of biological and parentage relationship ofindividuals as well as for forensic analysis2–10.The island of Cres is the second largest island in theCroatian section of the Adriatic sea (Figure 1). The lastofficial population census (2001) lists a total populationof 2946 inhabitants living in 26 settlements. Cres is ahilly island, 66 km long and ranging in width from 2 to12 km.The current population structure of Cres was formedthrough several immigratory episodes of genetically dis-tant populations. The earliest available archeological da-ta show that this area was continuously inhabited sincethe middle Paleolithic. The oldest western Balkan popu-lation of a clearly Indo European-speaking Illyrians re-mained the basic population after which other popula-1319Received for publication November 6, 2009tion followed such as Greeks (4th century B.C.), Romans(3rd to 6th centuries A.D.) and the Venetians (from 1409to 1797)11. Apart from this, the current population struc-ture is also shaped by a Slavic element, which was intro-duced through a first major immigration of Croats (peo-ple of Slavic origin) by the early 6th century AD in theregion that is today’s continental Croatia. In the 6th and8th century A.D. Croats reached the Adriatic Sea coastwhere they became a predominant population. The sec-ond large immigration wave of Croats occurred duringthe Turkish wars between the 16th and 18th centuries,when population groups from the mountainous region ofthe Adriatic hinterland settled in the Adriatic islands12.In this study, we present the allele distribution and fo-rensic parameters at fifteen highly polymorphic tetranu-cleotide STR loci included in the AmpFlSTR IdentifilerPCR amplification kit (D8S1179, D21S11, D7S820,CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338,D19S433, VWA, TPOX, D18S51, D5S818 and FGA) in apopulation sample of the island of Cres (Croatia).Sample and MethodsSampleOne hundred and twenty two unrelated adult individ-uals from the Island of Cres (Croatia) participated in thestudy, after giving the informed consent. Whole bloodsamples were obtained by venipuncture, collected intoEDTA tubes and stored at –20 °C.Genomic DNA was extracted from whole blood sam-ples using the NucleoSpin Blood kit (Macherey-Nagel,Germany) according to the manufacturer’s instructions.DNA analysisAmpFlSTR Identifiler PCR Amplification Kit (Ap-plied Biosystems, Foster City, CA, USA) was applied to si-multaneously amplify 15 STR loci. The PCR amplifica-tion was carried out in AB Gene Amp PCR System 9700Thermal Cycler (Applied Biosystems) according to themanufacturer’s recommended protocol. DNA typing wasperformed on the ABI PRISM 3130 Genetic Analyzer(Applied Biosystems) with accessory software – ABI DataCollection Software and GeneMapperTM 3.2 (AppliedBiosystems).Data analysisAllele frequencies were computed by the gene count-ing method using the software package SPSS 7.5 forWindows. The agreement with the Hardy-Weinberg ex-pectations (HWE) of genotype frequencies was deter-mined by the chi-square test based on the number of ob-served and expected heterozygotes and the exact testbased on the number of observed and expected genoty-pes13. Forensic efficiency parameters such as Heterozy-gosity (H), Polymorphism Information Content (PIC),Power of Discrimination (PD), Probability of Match (PM)and Power of Exclusion (PE) were calculated by Power-Stats v 1.2 software package14. The exact tests of popula-tion differentiation were computed according to the me-thods implemented in Arlequin version 2.000 softwareadapted for STR loci (Schneider 2000)15.Results and DiscussionForensic statistical parameters for forensic testing atthe 15 AmpFlSTR Identifiler Loci in the Cres populationare presented in Table 1. According to the measures ofheterozygosity and the polymorphism information con-tent (PIC), as measures of informativeness, D18S51,D2S1338 and D21S11 loci are the most variable and themost informative markers for population genetic analy-ses and forensic testing showing the highest level of ob-served (0.844–0.877) and expected (0.841–0.871) heterozy-gosity and polymorphism information content (0.82–0.86).On the other hand, loci TPOX and D16S539 proved to bethe least variable according to the lowest observed (0.590and 0.672, respectively) and expected (0.626 and 0.744,respectively) heterozygosity, and the lowest value of poly-morphism information content (0.57 and 0.70 respectively).The probability that two randomly chosen personshave the same unspecified genotype at a locus is designa-ted as probability of match (PM) or individualization poten-tial of a locus2, and is used to calculate the power of discri-mination (PD) of a locus, 1-PM. The probability of match(PM) for the 15 studied loci ranged from 0.035 (D18S51)to 0.195 (TPOX). Even relatively reduced levels of geneticdiversity at some of the loci provide discrimination powerbetween 80.5% (TPOX) and 96.5% (D18S51) (average of91.5%). The combined power of discrimination (PD) forthe 15 studied loci was 0.99999999999999997988728679,which means when used together these fifteen loci candistinguish samples from different individuals with aprobability of 99.99%.Forensic parameter, namely, the power of exclusion(PE) is used to evaluate the strength of a locus to excludefaulty accused individuals. It represents the percentageof individuals in the relevant population who would notN. Novokmet et al.: 15 STR Loci in the Population of the Island of Cres (Croatia), Coll. Antropol. 33 (2009) 4: 1319–13221320Fig. 1. Geographical position of the Island of Cres (Croatia).share the same DNA profile presented in a paternitycase16. Single locus PE values ranged from 0.279 (TPOX)to 0.749 (D18S51), whereas combined value using all fif-teen loci has increased the forensic utility to 0.999997397(99.99%).The agreement with Hardy-Weinberg equilibrium(Table 1), tested by the c2-test based on the number ofobserved and expected heterozygotes and the exact testbased on the number of observed and expected geno-types, is confirmed for all tested loci (p>0.05).In this study we also compared allele frequencies offifteen loci of the Cres population with the same numberof loci obtained from Croatian mainland17, whereas nineavailable loci were compared with East Adriatic Islan-ds18–22. These exact tests of population differentiation23show statistically significant differences (significance le-vel was 0.05) in allele frequencies between Cres and Hvarat 4 loci (D7S820, CSF, TPOX and D5S818), followed byCres and Kor~ula at 3 loci (D7S820, CSF and D3S1358),Cres and Vis at 3 loci (D3S1358, D13S317 and FGA),Cres and Bra~ at 2 loci (TH01 and FGA). There are sig-nificant differences at 1 locus (D7S820) only between twogeographically closest islands Cres and Krk. Significantdifferences in allele frequencies between the Island ofCres and Croatian mainland were not observed at any ofthe studied loci.In conclusion, the analysis of fifteen AmpFlSTR Iden-tifiler short tandem repeat (STR) loci yielded reliable fo-rensic parameters and was highly differentiating in studiedisolated rural populations from Eastern Adriatic islands,thus proving the effectiveness of multiple STR locus pro-files even in small rural populations.AcknowledgementsThis study was supported by the Ministry of Science,Education and Sports of the Republic of Croatia (Grantsno. 196-1962766-2751 to P.R., 062-19627660309 to M.K.and 309-1300855-2738 to D.M.). We would like to thankNina Jeran, B.Sc. for valuable comments. We are gratefulto all inhabitants of the Island of Cres who participatedin this project.R E F E R E N C E S1. WEBER JL, MAY PE, Am J Hum Genet, 44 (1989) 388. — 2.HAMMOND HA, JIN L, ZHONG Y, CASKEY CT, CHAKRABORTYS R,Am J Hum Genet, 55 (1994) 175. — 3. SÃO-BENTO M, CARVALHO M,ANDRADE L, LOPES V, SERRA A, GAMERO JJ, CORTE-REAL F, VIDEMC, ANJOS MJ, Forensic Sci Int: Genetics Supplement Series, 1 (2008)367. — 4. BUTLER JM, J Forensic Sci, 51 (2006) 253. — 5. SHIMADA I,BRINKMANN B, TUYEN NQ, HOHOFF C, Int J Legal Med, 116 (2002)246. — 6. BUDOWLE B, CHAKRABORTY R, Legal Medicine, 3 (2001)29. — 7. SPARKES R, KIMPTON C, GILBARD S, CARNE P, ANDER-SON J, OLDROYD N, THOMAS D, URQUHART A, GILL P, Int J LegalMed, 109 (1996) 195. — 8. HAGELBERG E, GRAY IC, JEFFREYS AD,Nature, 352 (1991) 427. — 9. JEFFREYS AJ, ALLEN MJ, HAGELBERGE, SONNBERG A, Forensic Sci Int, 56 (1992) 65. — 10. MARJANOVI]D, KAPUR L, DROBNI^ K, BUDOWLE B, POJSKI], HAD@ISELIMO-VI] R, Human Biology, 76 (2004) 15. — 11 FORENBAHER S, CollAntropol, 23 (1999) 521. — 12. RUDAN P, FINKA B, JANI]IJEVI] B,JOVANOVI] V, KU[EC V, MILI^I] J, MI[IGOJ-DURAKOVI] M, ROB-ERTS DF, SCHMUTZER LJ, SMOLEJ NARAN^I] N, SUJOLD@I] A,SZIROVICZA L, [IMI] D, [IMUNOVI] P, [POLJAR-VR@INA SM, An-thropological investigations of Eastern Adriatic. Biological and culturalmicrodifferentiation of the rural populations on the island of Hvar (in Cro-atian) 1990, Zagreb, HAD. — 13. GUO SW AND THOMPSON EA, Bio-metrics, 48 (1992) 361. — 14. TEREBA A, Tools for analysis of populationstatistics, Profiles in DNA 3, Promega Corporation, 1999, Available from:URL: http://promega.com/ geneticidtools/powerstats/. — 15. SCHNEI-DER S, ROESSLI SD, AND EXCOFFIER L, A software for population ge-N. Novokmet et al.: 15 STR Loci in the Population of the Island of Cres (Croatia), Coll. Antropol. 33 (2009) 4: 1319–13221321TABLE 1FORENSIC STATISTICAL PARAMETERS AT THE 15 STR LOCI IN THE POPULATION FROM THE ISLAND OF CRES (CROATIA)Allele D8S1179 D21S11 D7S820 CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51 D5S818 FGAHobsa 0.795 0.844 0.786 0.721 0.819 0.762 0.795 0.672 0.868 0.745 0.819 0.59 0.877 0.713 0.795Hexpb 0.808 0.841 0.817 0.7 0.779 0.758 0.796 0.744 0.863 0.78 0.81 0.626 0.871 0.737 0.8362-test 0.064 0.001 0.565 0.17 0.917 0 0.005 2.985 0.002 0.67 0.02 0.556 0.003 0.267 1.218Exacttest(p)0.524 0.592 0.107 0.274 0.45 0.647 0.099 0.06 0.352 0.29 0.579 0.348 0.662 0.073 0.723PMc 0.071 0.048 0.068 0.145 0.089 0.098 0.079 0.108 0.040 0.076 0.071 0.195 0.035 0.105 0.049PDd 0.929 0.952 0.932 0.855 0.911 0.902 0.921 0.892 0.960 0.924 0.929 0.805 0.965 0.895 0.951PEe 0.590 0.684 0.575 0.462 0.636 0.531 0.590 0.386 0.732 0.503 0.636 0.279 0.749 0.449 0.590PICf 0.78 0.82 0.79 0.65 0.75 0.72 0.77 0.70 0.85 0.75 0.78 0.57 0.86 0.70 0.82aHobs – observed heterozygositybHexp – expected heterozygositycPM – probability of matchdPD – power of discriminationePE – power of exclusionfPIC – polymorphism information contentnetics data analysis, Arlequin version 2.0, Genetics and Biometry Labora-tory, University of Geneva, Geneva, Switzerland, 2000. — 16. BRENNERC, MORCIS JW, Paternity index calculations in single locus hypervariableDNA probes: validation and other studies. The International Symposiumon Human Identification, (Promega Corporation, Madison, WI, 1990). —17. PROJI] P, [KARO V, [AMIJA I, POJSKI] N, DURMI]-PA[I] A, KO-VA^EVI] L, BAKAL N, PRIMORAC D, MARIJANOVI] D, Croat Med J,48 (2007) 473. — 18. MARTINOVI] I, BARA] L, FURA^ I, JANI]IJE-VI] B, KUBAT M, PERI^I] M, VIDOVI] B, RUDAN P, Human Biology,71 (1999) 341. — 19. MARTINOVI] KLARI] I, BARA] L, BUKOVI] D,FURA^ I, GEBER G, JANI]IJEVI] B, KUBAT M, PERI^I] M, VIDO-VI] PUPI] B, RUDAN P, Annals of Human Biology, 28 (2001) 281. — 20.MARTINOVI] KLARI] I, BARA] L, BUKOVI] D, FURA^ I, GEBER G,HOMO – Journal of Comparative Human Biology, 51 (2000) 141. — 21.MARTINOVI] KLARI] I, PERI^I] M, BARA] LAUC L, JANI]IJEVI]B, KUBAT M, PAVI^I] D, RUDAN I, WANG N, JIN L, CHAKRABORTYR, DEKA R, RUDAN P, Human Biology, 77 (2000) 471. — 22. MARTI-NOVI] KLARI] I, American Journal of Human Biology, 12 (2000) 509.— 23. RAYMOND M, ROUSSET F, Evolution, 49 (1995) 1280.N. NovokmetInstitute for Anthropological Research, Gajeva 32, 10000 Zagreb, Croatiae-mail: nat@inantro.hrU^INKOVITOST FORENZI^KIH PARAMETARA 15 STR LOKUSA U POPULACIJI OTOKA CRESA(HRVATSKA)S A @ E T A KForenzi~ki parametri odre|eni su na uzorku od 122 nesrodne, odrasle osobe podrijetlom sa otoka Cresa, na temelju 15AmpFlSTR Identifiler (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433,VWA, TPOX, D18S51, D5S818 and FGA) kratkih uzastopno ponavljaju}ih sljedova DNK (STR). Lan~ana reakcija poli-merazom provedena je upotrebom AmpFlSTR Identifiler PCR Amplification Kit sustava. Umno`eni produkti razdvo-jeni su i odre|eni pomo}u instrumenta ABI 3130 DNK Genetic Analyzer. Slaganje s Hardy-Weinbergovom ravnote`om(HWE) potvr|eno je za sve analizirane lokuse (p>0,05). Zajedni~ka snaga diskriminacije (PD) i zajedni~ka snaga isklju-~ivanja (PE) za 15 analiziranih lokusa iznosila je 0,99999999999999997988728679 odnosno 0,999997397. Prema dobi-venim rezultatima, lokus D18S51 je najinformativniji, a zatim slijede lokusi D2S1338 i D21S11. Na temelju usporedbeu~estalosti alela izme|u populacije otoka Cresa i ostalih istra`ivanih otoka isto~nog Jadrana, utvr|ene su zna~ajnerazlike izme|u svih analiziranih oto~nih parova. Najve}a razlika utvr|ena je izme|u otoka Cresa i Hvara (na 4 lokusa),dok je najmanja razlika utvr|ena izme|u otoka Cresa i Krka (na 1 lokusu). Nadalje, usporedba otoka Cresa i konti-nentalne Hrvatske nije pokazala statisti~ki zna~ajnu razliku niti na jednom od analiziranih lokusa. Rezultati prove-denog istra`ivanja ukazali su na zna~ajnu u~inkovitost 15 analiziranih STR lokusa kao va`nih geneti~kih biljega zautvr|ivanje identiteta osoba i dokazivanje o~instva u populaciji otoka Cresa.N. Novokmet et al.: 15 STR Loci in the Population of the Island of Cres (Croatia), Coll. Antropol. 33 (2009) 4: 1319–13221322

English

Forensic Efficiency Parameters for the 15 STR Loci in the Population of the Island of Cres (Croatia)

Novokmet, Natalija

Marjanović, Damir

Škaro, Vedrana

Projić, Petar

Lauc, Gordan

Grahovac, Blaženka

Mohar, Bojana

Kapović, Miljenko

Rudan, Pavao

Repository of the University of Rijeka

Coll. Antropol. 33 (2009) 4: 1319–1322Original scientific paperForensic Efficiency Parameters for the 15 STRLoci in the Population of the Island of Cres(Croatia)Natalija Novokmet1, Damir Marjanovi}2,3, Vedrana [karo2, Petar Proji}2, Gordan Lauc4,5,Bla`enka Grahovac6, Bojana Mohar6, Miljenko Kapovi}7 and Pavao Rudan11 Institute for Anthropological Research, Zagreb, Croatia2 Genos, DNA Laboratory, Zagreb, Croatia3 Institute for Genetic Engineering and Biotechnology, Sarajevo, Bosnia and Herzegovina4 DNA Laboratory, School of Medicine, »J. J. Strossmayer« University of Osijek, Osijek, Croatia5 Genos, DNA Laboratory, Osijek, Croatia6 Department of Pathology and Pathological Anatomy, School of Medicine, University of Rijeka, Rijeka Croatia7 Department of Biology and Medical Genetics, School of Medicine, University of Rijeka, Rijeka CroatiaA B S T R A C TForensic parameters based on 15 AmpFlSTR Identifiler short tandem repeat (STR) loci (D8S1179, D21S11, D7S820,CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, VWA, TPOX, D18S51, D5S818 and FGA) wereevaluated in the sample of 122 unrelated, autochthonous, adult individuals from the Island of Cres (Croatia). PCR am-plification was performed with the AmpFlSTR Identifiler PCR Amplification Kit and the amplified products were sepa-rated and detected using the ABI 3130 DNA genetic analyzer. The agreement with Hardy Weinberg Equilibrium (HWE)was confirmed for all loci (p>0.05). The combined power of discrimination (PD) and the combined power of exclusion(PE) for the 15 tested STR loci were 0.99999999999999997988728679 and 0.999997397, respectively. According to thepresented data, D18S51 proved to be the most informative marker followed by markers D2S1338 and D21S11. Inter-population comparisons in allele frequencies with other East Adriatic Islands revealed significant differences for all an-alyzed population pairs ranging from 4 loci (Cres vs. Hvar) to 1 locus (Cres vs. Krk). Furthermore, allele frequenciescomparisons of Cres and Croatian mainland revealed the lack of statistically significant differences at all studied loci.The results of the current study indicate that the examined fifteen STR loci are useful genetic markers for individualidentification and paternity testing in Croatian population from the Island of Cres.Key word: STRS, AmpFlSTR identifiler, forensic parameters, the Island of Cres, CroatiaIntroductionShort tandem repeats (STRs) are loci with allelescomposed of tandemly repeated short DNA sequences of2–7 base pair in length1. There are hundreds of theseSTR regions in the human genome, but 10 to 15 regionsare sufficient to give high levels of discrimination be-tween individuals2. Numerous previous studies have de-monstrated that STRS have become the choice of loci fordetermination of biological and parentage relationship ofindividuals as well as for forensic analysis2–10.The island of Cres is the second largest island in theCroatian section of the Adriatic sea (Figure 1). The lastofficial population census (2001) lists a total populationof 2946 inhabitants living in 26 settlements. Cres is ahilly island, 66 km long and ranging in width from 2 to12 km.The current population structure of Cres was formedthrough several immigratory episodes of genetically dis-tant populations. The earliest available archeological da-ta show that this area was continuously inhabited sincethe middle Paleolithic. The oldest western Balkan popu-lation of a clearly Indo European-speaking Illyrians re-mained the basic population after which other popula-1319Received for publication November 6, 2009tion followed such as Greeks (4th century B.C.), Romans(3rd to 6th centuries A.D.) and the Venetians (from 1409to 1797)11. Apart from this, the current population struc-ture is also shaped by a Slavic element, which was intro-duced through a first major immigration of Croats (peo-ple of Slavic origin) by the early 6th century AD in theregion that is today’s continental Croatia. In the 6th and8th century A.D. Croats reached the Adriatic Sea coastwhere they became a predominant population. The sec-ond large immigration wave of Croats occurred duringthe Turkish wars between the 16th and 18th centuries,when population groups from the mountainous region ofthe Adriatic hinterland settled in the Adriatic islands12.In this study, we present the allele distribution and fo-rensic parameters at fifteen highly polymorphic tetranu-cleotide STR loci included in the AmpFlSTR IdentifilerPCR amplification kit (D8S1179, D21S11, D7S820,CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338,D19S433, VWA, TPOX, D18S51, D5S818 and FGA) in apopulation sample of the island of Cres (Croatia).Sample and MethodsSampleOne hundred and twenty two unrelated adult individ-uals from the Island of Cres (Croatia) participated in thestudy, after giving the informed consent. Whole bloodsamples were obtained by venipuncture, collected intoEDTA tubes and stored at –20 °C.Genomic DNA was extracted from whole blood sam-ples using the NucleoSpin Blood kit (Macherey-Nagel,Germany) according to the manufacturer’s instructions.DNA analysisAmpFlSTR Identifiler PCR Amplification Kit (Ap-plied Biosystems, Foster City, CA, USA) was applied to si-multaneously amplify 15 STR loci. The PCR amplifica-tion was carried out in AB Gene Amp PCR System 9700Thermal Cycler (Applied Biosystems) according to themanufacturer’s recommended protocol. DNA typing wasperformed on the ABI PRISM 3130 Genetic Analyzer(Applied Biosystems) with accessory software – ABI DataCollection Software and GeneMapperTM 3.2 (AppliedBiosystems).Data analysisAllele frequencies were computed by the gene count-ing method using the software package SPSS 7.5 forWindows. The agreement with the Hardy-Weinberg ex-pectations (HWE) of genotype frequencies was deter-mined by the chi-square test based on the number of ob-served and expected heterozygotes and the exact testbased on the number of observed and expected genoty-pes13. Forensic efficiency parameters such as Heterozy-gosity (H), Polymorphism Information Content (PIC),Power of Discrimination (PD), Probability of Match (PM)and Power of Exclusion (PE) were calculated by Power-Stats v 1.2 software package14. The exact tests of popula-tion differentiation were computed according to the me-thods implemented in Arlequin version 2.000 softwareadapted for STR loci (Schneider 2000)15.Results and DiscussionForensic statistical parameters for forensic testing atthe 15 AmpFlSTR Identifiler Loci in the Cres populationare presented in Table 1. According to the measures ofheterozygosity and the polymorphism information con-tent (PIC), as measures of informativeness, D18S51,D2S1338 and D21S11 loci are the most variable and themost informative markers for population genetic analy-ses and forensic testing showing the highest level of ob-served (0.844–0.877) and expected (0.841–0.871) heterozy-gosity and polymorphism information content (0.82–0.86).On the other hand, loci TPOX and D16S539 proved to bethe least variable according to the lowest observed (0.590and 0.672, respectively) and expected (0.626 and 0.744,respectively) heterozygosity, and the lowest value of poly-morphism information content (0.57 and 0.70 respectively).The probability that two randomly chosen personshave the same unspecified genotype at a locus is designa-ted as probability of match (PM) or individualization poten-tial of a locus2, and is used to calculate the power of discri-mination (PD) of a locus, 1-PM. The probability of match(PM) for the 15 studied loci ranged from 0.035 (D18S51)to 0.195 (TPOX). Even relatively reduced levels of geneticdiversity at some of the loci provide discrimination powerbetween 80.5% (TPOX) and 96.5% (D18S51) (average of91.5%). The combined power of discrimination (PD) forthe 15 studied loci was 0.99999999999999997988728679,which means when used together these fifteen loci candistinguish samples from different individuals with aprobability of 99.99%.Forensic parameter, namely, the power of exclusion(PE) is used to evaluate the strength of a locus to excludefaulty accused individuals. It represents the percentageof individuals in the relevant population who would notN. Novokmet et al.: 15 STR Loci in the Population of the Island of Cres (Croatia), Coll. Antropol. 33 (2009) 4: 1319–13221320Fig. 1. Geographical position of the Island of Cres (Croatia).share the same DNA profile presented in a paternitycase16. Single locus PE values ranged from 0.279 (TPOX)to 0.749 (D18S51), whereas combined value using all fif-teen loci has increased the forensic utility to 0.999997397(99.99%).The agreement with Hardy-Weinberg equilibrium(Table 1), tested by the c2-test based on the number ofobserved and expected heterozygotes and the exact testbased on the number of observed and expected geno-types, is confirmed for all tested loci (p>0.05).In this study we also compared allele frequencies offifteen loci of the Cres population with the same numberof loci obtained from Croatian mainland17, whereas nineavailable loci were compared with East Adriatic Islan-ds18–22. These exact tests of population differentiation23show statistically significant differences (significance le-vel was 0.05) in allele frequencies between Cres and Hvarat 4 loci (D7S820, CSF, TPOX and D5S818), followed byCres and Kor~ula at 3 loci (D7S820, CSF and D3S1358),Cres and Vis at 3 loci (D3S1358, D13S317 and FGA),Cres and Bra~ at 2 loci (TH01 and FGA). There are sig-nificant differences at 1 locus (D7S820) only between twogeographically closest islands Cres and Krk. Significantdifferences in allele frequencies between the Island ofCres and Croatian mainland were not observed at any ofthe studied loci.In conclusion, the analysis of fifteen AmpFlSTR Iden-tifiler short tandem repeat (STR) loci yielded reliable fo-rensic parameters and was highly differentiating in studiedisolated rural populations from Eastern Adriatic islands,thus proving the effectiveness of multiple STR locus pro-files even in small rural populations.AcknowledgementsThis study was supported by the Ministry of Science,Education and Sports of the Republic of Croatia (Grantsno. 196-1962766-2751 to P.R., 062-19627660309 to M.K.and 309-1300855-2738 to D.M.). We would like to thankNina Jeran, B.Sc. for valuable comments. We are gratefulto all inhabitants of the Island of Cres who participatedin this project.R E F E R E N C E S1. WEBER JL, MAY PE, Am J Hum Genet, 44 (1989) 388. — 2.HAMMOND HA, JIN L, ZHONG Y, CASKEY CT, CHAKRABORTYS R,Am J Hum Genet, 55 (1994) 175. — 3. SÃO-BENTO M, CARVALHO M,ANDRADE L, LOPES V, SERRA A, GAMERO JJ, CORTE-REAL F, VIDEMC, ANJOS MJ, Forensic Sci Int: Genetics Supplement Series, 1 (2008)367. — 4. BUTLER JM, J Forensic Sci, 51 (2006) 253. — 5. SHIMADA I,BRINKMANN B, TUYEN NQ, HOHOFF C, Int J Legal Med, 116 (2002)246. — 6. BUDOWLE B, CHAKRABORTY R, Legal Medicine, 3 (2001)29. — 7. SPARKES R, KIMPTON C, GILBARD S, CARNE P, ANDER-SON J, OLDROYD N, THOMAS D, URQUHART A, GILL P, Int J LegalMed, 109 (1996) 195. — 8. HAGELBERG E, GRAY IC, JEFFREYS AD,Nature, 352 (1991) 427. — 9. JEFFREYS AJ, ALLEN MJ, HAGELBERGE, SONNBERG A, Forensic Sci Int, 56 (1992) 65. — 10. MARJANOVI]D, KAPUR L, DROBNI^ K, BUDOWLE B, POJSKI], HAD@ISELIMO-VI] R, Human Biology, 76 (2004) 15. — 11 FORENBAHER S, CollAntropol, 23 (1999) 521. — 12. RUDAN P, FINKA B, JANI]IJEVI] B,JOVANOVI] V, KU[EC V, MILI^I] J, MI[IGOJ-DURAKOVI] M, ROB-ERTS DF, SCHMUTZER LJ, SMOLEJ NARAN^I] N, SUJOLD@I] A,SZIROVICZA L, [IMI] D, [IMUNOVI] P, [POLJAR-VR@INA SM, An-thropological investigations of Eastern Adriatic. Biological and culturalmicrodifferentiation of the rural populations on the island of Hvar (in Cro-atian) 1990, Zagreb, HAD. — 13. GUO SW AND THOMPSON EA, Bio-metrics, 48 (1992) 361. — 14. TEREBA A, Tools for analysis of populationstatistics, Profiles in DNA 3, Promega Corporation, 1999, Available from:URL: http://promega.com/ geneticidtools/powerstats/. — 15. SCHNEI-DER S, ROESSLI SD, AND EXCOFFIER L, A software for population ge-N. Novokmet et al.: 15 STR Loci in the Population of the Island of Cres (Croatia), Coll. Antropol. 33 (2009) 4: 1319–13221321TABLE 1FORENSIC STATISTICAL PARAMETERS AT THE 15 STR LOCI IN THE POPULATION FROM THE ISLAND OF CRES (CROATIA)Allele D8S1179 D21S11 D7S820CSF1PO D3S1358 TH01 D13S317 D16S539 D2S1338 D19S433 VWA TPOX D18S51 D5S818 FGAHobsa 0.795 0.844 0.786 0.721 0.819 0.762 0.795 0.672 0.868 0.745 0.819 0.59 0.877 0.713 0.795Hexpb 0.808 0.841 0.817 0.7 0.779 0.758 0.796 0.744 0.863 0.78 0.81 0.626 0.871 0.737 0.8362-test 0.064 0.001 0.565 0.17 0.917 0 0.005 2.985 0.002 0.67 0.02 0.556 0.003 0.267 1.218Exacttest(p)0.524 0.592 0.107 0.274 0.45 0.647 0.099 0.06 0.352 0.29 0.579 0.348 0.662 0.073 0.723PMc 0.071 0.048 0.068 0.145 0.089 0.098 0.079 0.108 0.040 0.076 0.071 0.195 0.035 0.105 0.049PDd 0.929 0.952 0.932 0.855 0.911 0.902 0.921 0.892 0.960 0.924 0.929 0.805 0.965 0.895 0.951PEe 0.590 0.684 0.575 0.462 0.636 0.531 0.590 0.386 0.732 0.503 0.636 0.279 0.749 0.449 0.590PICf 0.78 0.82 0.79 0.65 0.75 0.72 0.77 0.70 0.85 0.75 0.78 0.57 0.86 0.70 0.82aHobs – observed heterozygositybHexp – expected heterozygositycPM – probability of matchdPD – power of discriminationePE – power of exclusionfPIC – polymorphism information contentnetics data analysis, Arlequin version 2.0, Genetics and Biometry Labora-tory, University of Geneva, Geneva, Switzerland, 2000. — 16. BRENNERC, MORCIS JW, Paternity index calculations in single locus hypervariableDNA probes: validation and other studies. The International Symposiumon Human Identification, (Promega Corporation, Madison, WI, 1990). —17. PROJI] P, [KARO V, [AMIJA I, POJSKI] N, DURMI]-PA[I] A, KO-VA^EVI] L, BAKAL N, PRIMORAC D, MARIJANOVI] D, Croat Med J,48 (2007) 473. — 18. MARTINOVI] I, BARA] L, FURA^ I, JANI]IJE-VI] B, KUBAT M, PERI^I] M, VIDOVI] B, RUDAN P, Human Biology,71 (1999) 341. — 19. MARTINOVI] KLARI] I, BARA] L, BUKOVI] D,FURA^ I, GEBER G, JANI]IJEVI] B, KUBAT M, PERI^I] M, VIDO-VI] PUPI] B, RUDAN P, Annals of Human Biology, 28 (2001) 281. — 20.MARTINOVI] KLARI] I, BARA] L, BUKOVI] D, FURA^ I, GEBER G,HOMO – Journal of Comparative Human Biology, 51 (2000) 141. — 21.MARTINOVI] KLARI] I, PERI^I] M, BARA] LAUC L, JANI]IJEVI]B, KUBAT M, PAVI^I] D, RUDAN I, WANG N, JIN L, CHAKRABORTYR, DEKA R, RUDAN P, Human Biology, 77 (2000) 471. — 22. MARTI-NOVI] KLARI] I, American Journal of Human Biology, 12 (2000) 509.— 23. RAYMOND M, ROUSSET F, Evolution, 49 (1995) 1280.N. NovokmetInstitute for Anthropological Research, Gajeva 32, 10000 Zagreb, Croatiae-mail: nat@inantro.hrU^INKOVITOST FORENZI^KIH PARAMETARA 15 STR LOKUSA U POPULACIJI OTOKA CRESA(HRVATSKA)S A @ E T A KForenzi~ki parametri odre|eni su na uzorku od 122 nesrodne, odrasle osobe podrijetlom sa otoka Cresa, na temelju 15AmpFlSTR Identifiler (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433,VWA, TPOX, D18S51, D5S818 and FGA) kratkih uzastopno ponavljaju}ih sljedova DNK (STR). Lan~ana reakcija poli-merazom provedena je upotrebom AmpFlSTR Identifiler PCR Amplification Kit sustava. Umno`eni produkti razdvo-jeni su i odre|eni pomo}u instrumenta ABI 3130 DNK Genetic Analyzer. Slaganje s Hardy-Weinbergovom ravnote`om(HWE) potvr|eno je za sve analizirane lokuse (p>0,05). Zajedni~ka snaga diskriminacije (PD) i zajedni~ka snaga isklju-~ivanja (PE) za 15 analiziranih lokusa iznosila je 0,99999999999999997988728679 odnosno 0,999997397. Prema dobi-venim rezultatima, lokus D18S51 je najinformativniji, a zatim slijede lokusi D2S1338 i D21S11. Na temelju usporedbeu~estalosti alela izme|u populacije otoka Cresa i ostalih istra`ivanih otoka isto~nog Jadrana, utvr|ene su zna~ajnerazlike izme|u svih analiziranih oto~nih parova. Najve}a razlika utvr|ena je izme|u otoka Cresa i Hvara (na 4 lokusa),dok je najmanja razlika utvr|ena izme|u otoka Cresa i Krka (na 1 lokusu). Nadalje, usporedba otoka Cresa i konti-nentalne Hrvatske nije pokazala statisti~ki zna~ajnu razliku niti na jednom od analiziranih lokusa. Rezultati prove-denog istra`ivanja ukazali su na zna~ajnu u~inkovitost 15 analiziranih STR lokusa kao va`nih geneti~kih biljega zautvr|ivanje identiteta osoba i dokazivanje o~instva u populaciji otoka Cresa.N. Novokmet et al.: 15 STR Loci in the Population of the Island of Cres (Croatia), Coll. Antropol. 33 (2009) 4: 1319–13221322

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Forensic Efficiency Parameters for the 15 STR Loci in the Population of the Island of Cres (Croatia)

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