Forensic evaluation of the 20 STR loci in the population of Croatia

Forensic evaluation of the 20 STR loci in the population of Croatia

Accepted Manuscript Title: Forensic evaluation of the 20 STR loci in the population of Croatia Author: Lucija Barbari´c PII: DOI: Reference: S1872-49...

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Accepted Manuscript Title: Forensic evaluation of the 20 STR loci in the population of Croatia Author: Lucija Barbari´c PII: DOI: Reference:

S1872-4973(17)30058-3 http://dx.doi.org/doi:10.1016/j.fsigen.2017.03.011 FSIGEN 1684

To appear in:

Forensic Science International: Genetics

Author: Petar Ozreti´c PII: DOI: Reference:

S1872-4973(17)30058-3 http://dx.doi.org/doi:10.1016/j.fsigen.2017.03.011 FSIGEN 1684

To appear in:

Forensic Science International: Genetics

Authors: Ivana Horjan, Marina Korolija, Gordan Mrˇsi´c PII: DOI: Reference:

S1872-4973(17)30058-3 http://dx.doi.org/doi:10.1016/j.fsigen.2017.03.011 FSIGEN 1684

To appear in:

Forensic Science International: Genetics

Received date:

14-2-2017

Please cite this article as: Ivana Horjan, Marina Korolija, Gordan Mrˇsi´c, Forensic evaluation of the 20 STR loci in the population of Croatia, Forensic Science International: Geneticshttp://dx.doi.org/10.1016/j.fsigen.2017.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Forensic evaluation of the 20 STR loci in the population of Croatia

Lucija Barbarića,*, Petar Ozretićb, Ivana Horjana, Marina Korolijaa, Gordan Mršića

a

Ivan Vučetić Forensic Science Center, General Police Directorate, Republic of

Croatia Ministry of Interior, Ilica 335, 10 000 Zagreb, Croatia b

Laboratory for Hereditary Cancer, Division of Molecular Medicine, Rudjer Bošković

Institute, Bijenička 54, 10002 Zagreb, Croatia

*Corresponding author. Tel:+385 14887378; fax:+385 13788051 E-mail addresses: [email protected] (L. Barbarić), [email protected] (P.Ozretić), [email protected] (I. Horjan), [email protected] (M. Korolija), [email protected] (G. Mršić)

Highlights  allele frequencies and forensic parameters for 20 STR loci were established  allele frequencies were compared with the geographically close populations  D1S1656 and D12S391 showed the highest power of discrimination  the combined probability of match was estimated to 3.708 × 10−24  statistically significant difference was noticed between Croatia and Turkey

Dear Editor

In order to improve discriminatory power of national DNA database and to facilitate DNA profiles comparison between databases within Europe, ENFSI (European Network of Forensic Science Institutes) has recommended that the European Standard Set (ESS) of loci should be expanded by additional five loci: D1S1656, D2S441, D10S1248, D12S391 and D22S1045 [1]. Croatia, as an EU member state with the established forensic DNA database, is obliged to make the database available for international comparison. Considering the ENFSI recommendations, the existing database, which previously consisted of DNA profiles produced by SGM Plus and Identifiler kits, has been expanded by the additional ESS loci. The purpose of this study was to establish allele frequencies and forensic parameters for 20 STR loci (15 established STR loci and five additional ESS STR loci), as a maximum number of loci used for the production of the DNA profiles. Additionally, results were compared to the population data of the countries geographically close to Croatia. Regarding its ethnic composition, Croatia is a homogenous country. The population of 4.3 million mostly consists of Croats (90.4%), while minorities include Serbian (4.4%), and 21 other ethnicities (less than 1% each) [2]. Genetic polymorphism of 20 autosomal STR loci (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818, FGA, D1S1656, D2S441, D10S1248, D12S391, D22S1045) amplified with the AmpFlSTR®NGMTM and AmpFlSTR® Identifiler® PCR amplification kits (Applied Biosystems) were analyzed in 500 unrelated individuals from five Croatian regions covering the entire country, after obtaining their informed consent. Genomic DNA was extracted from buccal swabs using a Chelex®100 method [3]. The concentration of extracted DNA was determined using the 7500 Real Time PCR

System (Applied Biosystems) and the HID Real-Time PCR Analysis Software, v1.2 (Applied Biosystems) according to the manufacturer’s instructions. PCR amplification was performed using the AmpFlSTR NGMTM and AmpFlSTR® Identifiler® PCR Amplification Kits in GeneAmp 9700 PCR System (Applied Biosystems), in a final reaction volume of 13 μl. Simultaneously, the positive and negative PCR controls were amplified. PCR products were detected by ABI genetic Analyzer 3130xL (Applied Biosystems) and the results were analyzed with GeneMapper ID v.3.2 software (Applied Biosystems) according to the manufacturers’ instructions. The peak hight threshold was 50 RFU for heterozygous and 200 RFU for homozygous alleles. Allele frequencies, matching probability (MP), power of discrimination (PD), typical paternity index (PI), and polymorphism information content (PIC) were calculated using PowerStats v1.2 (Promega) [4]. Arlequin version 3.5.2.2 [5] was used to calculate expected heterozygosity (He), observed heterozygosity (Ho) and probability value of Hardy-Weinberg equilibrium exact test (HWE). Allele frequencies and forensic parameters have been summarized in Supplementary Table 1 (Table S1). According to the results of the statistical analysis, no deviations from the Hardy-Weinberg equilibrium were detected, except for the locus D22S1045, which was not significant after applying the Bonferroni correction [6]. The combined power of discrimination for the 20 loci in the Croatian population was >0.9999999. The two additional ESS loci, D1S1656 and D12S391, showed the highest power of discrimination values (0.979 and 0.976, respectively) (Table S1). The combined probability of match from the 20 STR loci was estimated to be 3.708 × 10−24, which is highly informative. As shown in the Supplemetary table 2 (Table S2), out of 217 observed alleles, 25 were identified as rare alleles (frequency values below 0.006), that have not yet been published for Croatian population [7, 8]. However, for estimating statistics in the casework, the fixed minimum frequency value (0.00625) has been used for PI or MP calculations instead of the actual values of these rare alleles. In order to compare the allele frequencies calculated for the general Croatian population with the published frequencies of Austrian [9, 10], Italian [11, 12], Hungarian [13, 14], Slovenian [15-18], Vojvodina - western part of Serbia [19, 20] and Turkish [21] populations, the exact population differentiation test was performed using Arlequin version 3.5.2.2 [5]. In addition, locus-by-locus allelic frequencies were compared to the previously published Croatian population data considering the lack

of data for additional ESS loci in general Croatian population. The comparison of the allele frequency distribution is given in Supplementary Table 3 (Table S3). The difference was found in loci D16S539 and D8S1179 between Croatian and Italian populations, but was subsequently lost with the Bonferroni correction. However, statistically significant difference was noticed between populations of Croatia and Turkey for the locus D1S1656 even after Bonferroni correction (p=0.00021). As expected, the allele frequencies for 20 STR loci in Croatian population did not significantly differ from the previously published Croatian population data. The analyzed STR loci were highly polymorphic while two additional ESS loci, D1S1656 and D12S391 were the most discriminating in Croatian population, as well as in other European populations [7, 9, 13, 20-21]. In conclusion, obtained results contribute to the existing Croatian national DNA database and demonstrate usefulness for estimating match statistics in human identity testing. The expanded ESS loci improve the discriminatory potential for human identification applications and kinship analyses. Additionally, implementation of additional ESS loci in the existing Croatian national DNA database facilitates the international comparison. The DNA Analysis Unit of the Forensic Science Center “Ivan Vučetić” is accredited to ISO/IEC 17025, and regularly participates in quality control proficiency testing provided by the German DNA Profiling group (GEDNAP). This article follows the population data publication guidelines set by the journal [22].

Acknowledgements This study was funded by the Ministry of Interior, Republic of Croatia.

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