Kinship analysis of skeletal remains from the Middle Ages.

Medieval cemeteries Klisa-Guca Gora, Alihodze and Glavica-Han Bila located in the Travnik area (Travnik, Bosnia and Herzegovina) were archaeologically examined in the period 2011-2014, revealing human skeletal remains of 11 individuals in total. Archaeological skeletal samples, previously deposited in Travnik Homeland Museum (Travnik, Bosnia and Herzegovina) were subjected to genetic analysis. The aim of this research was to test familiar relationship of 11 individuals excavated from three medieval cemeteries and to predict Y-haplogroup for male individuals. In order to perform molecular-genetic characterisation of collected human skeletal remains, two systems of genetic markers were analysed: autosomal and Y-STR loci. Complete or partial data obtained by autosomal STR typing of 11 individuals were subjected to kinship analysis. Male sex was determined in eight samples out of 11. Direct relatives of the "brother-brother" type were detected in one case with high kinship probability (KP) value of 99.99996 %. Complete or nearly complete and usable Y-STR profiles were obtained for six out of eight male individuals. The presence of identical haplotypes at Y-STR loci and results of Y-haplogroup prediction suggest that all male individuals share the same paternal lineage and belong to J2a haplogroup. Overall, this study emphasises the usefulness, efficiency and sensitivity of STR markers in the molecular-genetic characterisation of old skeletal remains as well as the importance of employing additional markers like Y-STRs in archaeogenetic studies, besides traditionally used autosomal STR markers, in order to get a comprehensive information about close and distant relatives, and ancestry.

Analysis of aborted fetal material using autosomal STR markers in forensic cases of sexual assault.

Sexual violence represents a widespread social problem associated with serious lifelong consequences. In many cases, an outcome of sexual violence is the victim's unwanted pregnancy, usually ended in an abortion. The objective of this paper is to report five rape cases, including rapes of a minor and young woman, two incest cases and a case of human trafficking for sexual exploitation, where every case resulted in the victim's pregnancy. In each case, pregnancy was terminated in the first trimester or at the beginning of the second trimester in the relevant medical center or clinic. Fresh fetal blood or aborted tissue samples were delivered to our laboratory in order to perform paternity testing for the purpose of proving the crime. DNA extraction using Qiagen Dneasy™ Tissue Kit was optimized according to the sample type. Amplification of autosomal STR (Short Tandem Repeat) markers was performed using the PowerPlex®16 System. In two cases, mixtures of maternal and fetal DNA in the aborted fetal material were found. Using the LRmix Studio v.2.1.5 Software for interpreting DNA mixtures based on a probabilistic model, the likelihoods of maternal contribution and presence of fetal allelic variants inherited from the alleged father/suspect were calculated. Based on these results, we confirmed the presence of assumed fetal fractions (determined before software analysis) in the mixtures. In all cases, positive paternity proved the crime (probabilities of paternity >99.9999%). This cases report once again pointed out the importance of DNA analysis in the process of clarifying and solving forensic cases and demonstrated that the LRmix Studio v.2.1.5 Software can deal with complex cases such as sexual assaults.

Paternal genetic structure of the Bosnian-Herzegovinian Roma: A Y-chromosomal STR study.

OBJECTIVES: Studies indicate the complex nature of the genetic structure of the European Roma which has been shaped by different effects of their demographic history, while preserving their ancestral Indian origin. The primary aims of this study were to present for the first time the paternal profiles of the Roma from Bosnia and Herzegovina based on the data from Y-chromosome STR loci, identify the components of non-Roma paternal gene flow into the Roma, and evaluate the genetic relationships with other European Roma populations. MATERIALS AND METHODS: In this study, 110 DNA samples of unrelated males from Roma populations residing in different regions of Bosnia and Herzegovina were genotyped using the 23 Y-STR loci included in the PowerPlex Y23 system. RESULTS: The analysis of the genetic structure of the Bosnian-Herzegovinian Roma revealed intra-country population substructuring and indicated differing genetic affinities between the Bosnian-Herzegovinian Roma and other European Roma populations. The paternal genetic structure of the Bosnian-Herzegovinian Roma has two components: an ancestral component represented by haplogroup H1a1a-M82, and European component presented by haplogroups I1-M253, I2a1a2b-L621, J2a1a-L26, J2a1a1a2b2a3~Z7671, J2b2a-M241, G2a2b2a1a1b-L497, and E1b1b-M215. CONCLUSION: Genetic relations between the Bosnian-Herzegovinian Roma and other European Roma are shaped by different influences on their demographic history. The data suggest that the paternal gene pool of the Roma from Bosnia and Herzegovina might be a consequence of an early separation of the proto-Roma population and the later gene flow as well as factors of the isolation that accompany the Roma populations in some Bosnian-Herzegovinian regions.

Analysis of forensic genetic parameters of 22 autosomal STR markers (PowerPlex® Fusion System) in a population sample from Bosnia and Herzegovina.

Background: Bosnia and Herzegovina is a multinational and multireligious country, located in the western part of the Balkan Peninsula. Migrations through history were a key factor in the genetic identity of the Bosnian-Herzegovinian population.Aim: To analyse genetic polymorphisms of 22 autosomal short tandem repeat (STR) loci in the population of Bosnia and Herzegovina and to compare STR allele frequencies for STR loci with the reference data for European populations.Subjects and methods: The study was conducted among 600 unrelated individuals from all regions of Bosnia and Herzegovina. Genotyping was performed using the PowerPlex® Fusion amplification kit. Allele frequencies and statistical parameters were calculated, as well as the genetic distance among analysed populations through the construction of a neighbor-joining dendrogram.Results: STR loci included in the PowerPlex® Fusion amplification kit showed high discriminatory power indicating their reliability for human identification and paternity testing. The neighbor-joining dendrogram based on the results of genetic distance analysis showed that the Bosnian and Herzegovinian population has the greatest genetic distance from Turkish and Hungarian populations and greatest similarity with Croatian, Slovenian, and Serbian populations.Conclusion: The results of this study strongly support the application of 22 autosomal genetic markers for paternity testing and personal identity testing and are in agreement with most previous human studies in the investigated human populations.

Genetic variation study on fifteen STR loci in isolated Slovenian "Inland Island" human populations of the Selska Valley Region.

This study was conducted to confirm preliminary anthropological research indicating the specificity of isolated Selska Valley populations and implement clear mapping of genetic distances between neighboring populations and similar "inland island" populations from the region. The sample consisted of 86 unrelated individuals born in the Selska Valley from the lowland villages (Bukovica, Sevlje, Dolenja Vas, Selca, Zelezniki and Zali Log) and the mountain villages (Podlonk, Prtovc, Spodnje Danje, Zgornja Sorica and Spodnja Sorica). The for mentioned 15 STR loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA) were analyzed and statistical analysis was applied to determine population-genetics and forensic parameters. The frequencies of 15 STR loci from isolated populations of Slovenian villages, Slovenia, Bosnian mountain villages and Bosnia and Herzegovina were analyzed to calculate genetic distances between them. Our results confirmed a similar genetic pattern between the Selska Valley mountain villages and Slovenian general population as well as Bosnian mountain villages and general Bosnian population. Even if the sample size was relatively small and examined populations were geographically isolated, observed genetic variation within the whole population was relatively high and comparable with neighboring populations. Additional analysis proved that the Selska Valley population is genetically closest to the Slovenian and Austrian populations. On the other hand, comparison with similarly patterned populations confirmed that this population could be recognized as "inland island" population in genetic terms.

DNA Identification of Commingled Human Remains from the Cemetery Relocated by Flooding in Central Bosnia and Herzegovina.

The floods in Bosnia and Herzegovina in May 2014 caused landslides all over the country. In the small village of Serici, near the town of Zenica, a landslide destroyed the local cemetery, relocated graves, and commingled skeletal remains. As the use of other physical methods of identification (facial recognition, fingerprint analysis, dental analysis, etc.) was not possible, DNA analysis was applied. DNA was isolated from 20 skeletal remains (bone and tooth samples) and six reference samples (blood from living relatives) and amplified using PowerPlex® Fusion and PowerPlex® Y23 kits. DNA profiles were generated for all reference samples and 17 skeletal remains. A statistical analysis (calculation of paternity, maternity, and sibling indexes and matching probabilities) resulted in 10 positive identifications. In this study, 5 individuals were identified based on one reference sample. This has once again demonstrated the significance of DNA analysis in resolving the most complicated cases, such as the identification of commingled human skeletal remains.

Allele frequencies of 15 STR loci in Bosnian and Herzegovinian population.

AIM: To determine newest the most accurate allele frequencies for 15 short tandem repeat (STR) loci in the Bosnian and Herzegovinian population, calculate statistical parameters, and compare them with the relevant data for seven neighboring populations. METHODS: Genomic DNA was obtained from buccal swabs of 1000 unrelated individuals from all regions of Bosnia and Herzegovina. Genotyping was performed using PowerPlex® 16 System to obtain allele frequencies for 15 polymorphic STR loci including D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, and FGA. The calculated allele frequencies were also compared with the data from neighboring populations. RESULTS: The highest detected value of polymorphism information content (PIC) was detected at the PentaE locus, whereas the lowest value was detected at the TPOX locus. The power of discrimination (PD) values had similar distribution, with Penta E showing the highest PD of 0.9788. While D18S51 had the highest value of power of exclusion (PE), the lowest PE value was detected at the TPOX locus. CONCLUSION: Upon comparison of Bosnian and Herzegovinian population data with those of seven neighboring populations, the highest allele frequency differentiation was noticed between Bosnian and Herzegovinian and Turkish population at 5 loci, the most informative of which was Penta E. The neighbor-joining dendrogram constructed on the basis of genetic distance showed grouping of Slovenian, Austrian, Hungarian, and Croatian populations. Bosnian and Herzegovinian population was between the mentioned cluster and Serbian population. To determine more accurate distribution of allelic frequencies and forensic parameters, our study included 1000 unrelated individuals from all regions of Bosnia and Herzegovina, and our findings demonstrated the applicability of these markers in both forensics and future population genetic studies.

Identification of human remains from the Second World War mass graves uncovered in Bosnia and Herzegovina.

AIM: To present the results obtained in the identification of human remains from World War II found in two mass graves in Ljubuski, Bosnia and Herzegovina. METHODS: Samples from 10 skeletal remains were collected. Teeth and femoral fragments were collected from 9 skeletons and only a femoral fragment from 1 skeleton. DNA was isolated from bone and teeth samples using an optimized phenol/chloroform DNA extraction procedure. All samples required a pre-extraction decalcification with EDTA and additional post-extraction DNA purification using filter columns. Additionally, DNA from 12 reference samples (buccal swabs from potential living relatives) was extracted using the Qiagen DNA extraction method. QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. PowerPlex ESI kit was used to simultaneously amplify 15 autosomal short tandem repeat (STR) loci, and PowerPlex Y23 was used to amplify 23 Y chromosomal STR loci. Matching probabilities were estimated using a standard statistical approach. RESULTS: A total of 10 samples were processed, 9 teeth and 1 femoral fragment. Nine of 10 samples were profiled using autosomal STR loci, which resulted in useful DNA profiles for 9 skeletal remains. A comparison of established victims' profiles against a reference sample database yielded 6 positive identifications. CONCLUSION: DNA analysis may efficiently contribute to the identification of remains even seven decades after the end of the World War II. The significant percentage of positively identified remains (60%), even when the number of the examined possible living relatives was relatively small (only 12), proved the importance of cooperation with the members of the local community, who helped to identify the closest missing persons' relatives and collect referent samples from them.

Determination of combined sibship indices "gray zone" using 15 STR loci for central Bosnian human population.

In our previous population studies of Bosnia and Herzegovina human population, we have used autosomal STR, Y-STR, and X-STR loci, as well as Y-chromosome NRY biallelic markers. All obtained results were included in Bosnian referent database. In order of future development of applied population molecular genetics researches of Bosnia and Herzegovina human population, we have examined the effectiveness of 15 STR loci system in determination of sibship by using 15 STR loci and calculating different cut-off points of combined sibship indices (CSI) and distribution of sharing alleles. From the perspective of its application, it is very difficult and complicated to establish strict CSI cut-off values for determination of the doubtless sibship. High statistically significant difference between the means of CSI values and in distribution of alleles sharing in siblings and non-siblings was noticed (P < 0.0001). After constructing the "gray zone", only one false positive result was found in three CSI cut-off levels with the highest percent of determined sibship/non-sibship at the CSI = 0.067, confirming its practical benefit. Concerning the distribution of sharing alleles, it is recommended as an informative estimator for its usage within Bosnia and Herzegovina human population.

Diversity of Y-short tandem repeats in the representative sample of the population of Canton Sarajevo residents, Bosnia and Herzegovina.

In our previous population study, we have used twelve Y-chromosomal short tandem repeats loci incorporated in the PowerPlex Y System to determine Y-STR diversity in B&H human population. With intent to obtain additional verification of the previously obtained results as well as to establish specific reference for a local B&H population, we have decided to test DNA samples collected from 100 unrelated healthy male Canton Sarajevo residents (from Sarajevo region) for the same twelve Y-linked short tandem repeats loci. Qiagen DNeasy Tissue Kit (Qiagen, GmbH, Hilden, Germany) was used for DNA extraction from buccal swabs and PowerPlex Y System (Promega Corp., Madison, WI) has been used to simultaneously amplify Y-STR loci by PCR. PowerPlex Y System includes 12 STR loci: DYS19, DYS385a, DYS385b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438 and DYS439. The total PCR reaction volume was 5 microL. PCR amplifications were carried out in PE GeneAmp PCR System Thermal Cycler (ABI). Electrophoresis of the amplification products was preformed on an ABI PRISM 310 genetic analyzer (ABI, Foster City, CA) according to the manufacturer's recommendations. The raw data were compiled and analyzed using the accessory software: ABI PRISM Data Collection Software and Genemapper version 3.2. In addition, we have compared the obtained "Sarajevo" dataset with the data previously generated for the entire Bosnian and Herzegovinian population, as well as with the available data on geographically close (neighboring) European populations. The results of this study will be used as guidelines in additional improving of research into genetic relationship among recent local B&H populations, both isolated and open, which is a long-term project in our country.

Identification of skeletal remains of Communist Armed Forces victims during and after World War II: combined Y-chromosome (STR) and MiniSTR approach.

AIM: To report on the use of STR, Y-STRs, and miniSTRs typing methods in the identification of victims of revolutionary violence and crimes against humanity committed by the Communist Armed Forces during and after World War II in which bodies were exhumed from mass and individual graves in Slovenia. METHODS: Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Skofja Loka area (Lovrenska Grapa and Zolsce) and 2 individual graves in the Ljubljana area (Podlipoglav), Slovenia. DNA was isolated using the Qiagen DNA extraction procedure optimized for bone and teeth. Some DNA extracts required additional purification, such as N-buthanol treatment. The QuantifilerTM Human DNA Quantification Kit was used for DNA quantification. Initially, PowerPlex 16 kit was used to simultaneously analyze 15 short tandem repeat (STR) loci. The PowerPlex S5 miniSTR kit and AmpF/STR MiniFiler PCR Amplification Kit was used for additional analysis if preliminary analysis yielded weak partial or no profiles at all. In 2 cases, when the PowerPlex 16 profiles indicated possible relatedness of the remains with reference samples, but there were insufficient probabilities to call the match to possible male paternal relatives, we resorted to an additional analysis of Y-STR markers. PowerPlex Y System was used to simultaneously amplify 12 Y-STR loci. Fragment analysis was performed on an ABI PRISM 310 genetic analyzer. Matching probabilities were estimated using the DNA-View software. RESULTS: Following the Y-STR analysis, 1 of the "weak matches" previously obtained based on autosomal loci, was confirmed while the other 1 was not. Combined standard STR and miniSTR approach applied to bone samples from 2 individual graves resulted in positive identifications. Finally, using the same approach on 11 bone samples from hidden mass grave Zolosce, we were able to obtain 6 useful DNA profiles. CONCLUSION: The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and mini-STR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II.