Šimun Anđelinović

Population data for 17 short tandem repeat loci on Y chromosome in northern Croatia.

Human Y-short tandem repeats (STRs) are tandem repeat arrays of two to seven base pair units on non-recombining region (NRY) of the human Y chromosome. Studies on Y-STR are interesting in both population genetics and forensics. The aim of this study was to investigate the population genetic properties of 17 STR loci on Y chromosome in the northern Croatia region. We carried out a statistical analysis of the data from previously performed genetic analysis collected during routine forensic work by the Forensic Science Centre “Ivan Vučetić”. A total of 220 unrelated healthy men from northern Croatia were selected for the purpose of this study. Genomic DNA was extracted using Chelex procedure from FTA® cards. Y-chromosomal STRs were determined using the AmpFISTR Yfiler PCR amplification kit. The haplotype frequencies were determined by direct counting and analyzed using Arlequin 3.1 and analysis of molecular variance calculated with the Y chromosome haplotype reference database online analysis tool. A total of 210 haplotypes were identified, 200 of which were unique. Total haplotype diversity was 0.995. Locus diversity varied from 0.331 for DYS392 to 0.783 for DYS385 locus. Allele frequencies diversity was 0.662. Discrimination capacity was 95.7%. The use of European minimal haplotype set indicated the most resemblance of this population to the Croatian capital of Zagreb, with modest resemblance to Bosnia and Herzegovina, Serbia and Hungary. This article provides the first overview of the Y chromosome STR variability in northern Croatia, thus providing the referent point for any future forensic and genetic epidemiology efforts in this region.

Sex determination in skeletal remains from the medieval Eastern Adriatic coast - discriminant function analysis of humeri.

Aim To investigate the usefulness of humerus measurement for sex determination in a sample of medieval skeletons from the Eastern Adriatic Coast. Additional aim was to compare the results with contemporary female population. Methods Five humerus measurements (maximum length, epicondylar width, maximum vertical diameter of the head, maximum and minimum diameter of the humerus at midshaft) for 80 male and 35 female medieval and 19 female contemporary humeri were recorded. Only sufficiently preserved skeletons and those with no obvious pathological or traumatic changes that could affect the measurements were included. For ten samples, analysis of DNA was performed in order to determine sex using amelogenin. Results The initial comparison of men and women indicated significant differences in all five measures (P < 0.001). Discriminant function for sex determination indicated that as much as 85% of cases could be properly categorized, with better results in men (86%) than women (80%). Furthermore, the comparison of the medieval and contemporary women did not show significant difference in any of the measured features. Sex results obtained by anthropological and DNA analysis matched in all 10 cases. Conclusion The results indicate that humerus measurement in Croatian medieval population may be sufficient to determine the sex of the skeleton. Furthermore, it seems that secular changes have not substantially affected contemporary population, suggesting that the results of this study are transferable to contemporary population as well.

Croatian national reference Y-STR haplotype database

A reference Y-chromosome short tandem repeat (STR) haplotype database is needed for Y-STR match interpretation as well as for national and regional characterization of populations. The aim of this study was to create a comprehensive Y-STR haplotype database of the Croatian contemporary population and to analyze substructure between the five Croatian regions. We carried out a statistical analysis of the data from previously performed genetic analyses collected during routine forensic work by the Forensic Science Centre “Ivan Vučetić”. A total of 1,100 unrelated men from eastern, western, northern, southern and central Croatia were selected for the purpose of this study. Y-STRs were typed using the AmpFISTR Yfiler PCR amplification kit. Analysis of molecular variance calculated with the Y chromosome haplotype reference database online analysis tool included 16 population samples with 20,247 haplotypes. A total of 947 haplotypes were recorded, 848 of which were unique (89.5%). Haplotype diversity was 0.998, with the most frequent haplotype found in 9 of 1,100 men (0.82%). Locus diversity varied from 0.266 for DYS392 to 0.868 for DYS385. Discrimination capacity was 86.1%. Our results suggested high level of similarity among regional subpopulations within Croatia, except for mildly different southern Croatia. Relative resemblance was found with Bosnia and Herzegovina and Serbia. Whit Atheys’ Haplogroup Predictor was used to estimate the frequencies of Y-chromosome haplogroups. I2a, R1a, E1b1b and R1b haplogroups were most frequent in all Croatian regions. These results are important in forensics and contribute to the population genetics and genetic background of the contemporary Croatian population.

Forensic DNA databases in Western Balkan region: retrospectives, perspectives, and initiatives.

The European Network of Forensic Science Institutes (ENFSI) recommended the establishment of forensic DNA databases and specific implementation and management legislations for all EU/ENFSI members. Therefore, forensic institutions from Bosnia and Herzegovina, Serbia, Montenegro, and Macedonia launched a wide set of activities to support these recommendations. To assess the current state, a regional expert team completed detailed screening and investigation of the existing forensic DNA data repositories and associated legislation in these countries. The scope also included relevant concurrent projects and a wide spectrum of different activities in relation to forensics DNA use. The state of forensic DNA analysis was also determined in the neighboring Slovenia and Croatia, which already have functional national DNA databases. There is a need for a ‘regional supplement’ to the current documentation and standards pertaining to forensic application of DNA databases, which should include regional-specific preliminary aims and recommendations.

Sex determination from femora in late antique sample from Eastern Adriatic coast (Salona necropolis)

Abstract The determination of sex is one of the first steps in anthropological analysis. When skeletal remains are fragmented, the most useful approach is application of osteometric methods. The methods are population specific, and therefore require development of discriminant functions for each population group. The aim of this study was to test sexual dimorphism of femoral measurements and to calculate discriminant functions applicable for sex determination on fragmented skeletal remains on the late antique sample from the Eastern Adriatic coast (2nd-6th century AD). 214 randomly chosen skeletons from the excavation site Solin-Smiljanovac were analyzed. Sex and age were assessed using standard anthropological methods, and skeletons were examined for pathologic and traumatic changes. In the next step, we selected 27 female and 48 male skeletons free of peri- or post-mortem changes that could affect measurements Eight standard femoral measurements were taken. Sexual dimorphism was initially compared using independent sample t test, after which discriminant functions were computed. All femoral measurements showed statistically significant sexual dimorphism (p<0.001). Ten discriminant functions for every part of femur were calculated and obtained classification accuracy of 73.1-91.8%. This study reached relatively high classification, which will improve further analysis of the skeletal remains from the Salona necropolis. Due to similar population structure in the Roman period across the Adriatic coast, the discriminant functions could be applicable for all populations from the same period and area. This study also raised a few methodological questions showing that when creating discriminant functions we should consider not only the accuracy, but also the applicability based on the experience from the anthropology laboratory that considers the state of preservation and frequent pathology.

Genetic polymorphisms of 17 short tandem repeat loci on Y chromosome in central Croatian population

In forensic casework, Y-chromosome short tandem repeat (STR) haplotyping is used in human identification, paternity testing and sexual assault cases where Y-STRs provide a male-specific DNA profile. The aim of this study was to describe the genetic structure of Y chromosome in a central Croatian population. We carried out a statistical analysis of the data from previously performed genetic analyses collected during routine forensic work by the Forensic Science Centre “Ivan Vučetić”. A total of 220 unrelated healthy men from central Croatia were selected for the purpose of this study. Genomic DNA was extracted using a Chelex procedure from FTA® cards. Y-chromosomal STRs were determined using the AmpFISTR Yfiler PCR amplification kit. The haplotype frequencies were determined by direct counting and analyzed using Arlequin 3.1 and analysis of molecular variance calculated with the Y chromosome haplotype reference database online analysis tool. A total of 212 haplotypes were identified, 204 of which were unique. Total haplotype diversity was 0.993. Locus diversity varied from 0.325 for DYS392 to 0.786 for DYS385. Discrimination capacity was 92.7%. Allele frequencies diversity was 0.615. Intermediate alleles 17.2, 18.2 and 19.2 were found at DYS458 locus. A comparison with published data for the European minimal haplotype set showed the closest relationship to the Croatian capital of Zagreb and Bosnia and Herzegovina with significant genetic distance from Slovenia and Austria. The central Croatian population is now well characterized in terms of Y-chromosome STRs, thus providing a solid basis for further forensic and genetic epidemiology studies.

Cultural inter-population differences do not reflect biological distances: an example of interdisciplinary analysis of populations from Eastern Adriatic coast

Aim To compare the population group from the Šopot graveyard with population groups from traditional Croatian medieval graveyards by using anthropological, craniometrics, and mitochondrial (mtDNA) analysis and to examine if the cultural differences between population groups reflect biological differences. Methods We determined sex, age at death, pathological, and traumatic changes of skeletal remains from the Šopot graveyard and compared them with a cumulative medieval sample from the same region. We also performed principal component analysis to compare skeletal remains from Šopot with those from Ostrovica and other Central European samples according to 8 cranial measurements. Finally, we compared 46 skeletons from Šopot with medieval (Ostrovica) and contemporary populations using mDNA haplogroup profiling. Results The remains from Šopot were similar to the cumulative sample in lifestyle and quality of life markers. Principal component analysis showed that they were closely related to Eastern Adriatic coast sites (including Ostrovica and Šopot) in terms of cranial morphology, indicating similar biological makeup. According to mDNA testing, Šopot population showed no significant differences in the haplogroup prevalence from either medieval or contemporary populations. Conclusion This study shows that the Šopot population does not significantly differ from other medieval populations from this area. Besides similar quality of life markers, these populations also had similar biological markers. Substantial archeological differences can therefore be attributed to apparent cultural influences, which in this case do not reflect biological differences.

Sex estimation standards for medieval and contemporary Croats

Aim To develop discriminant functions for sex estimation on medieval Croatian population and test their application on contemporary Croatian population. Methods From a total of 519 skeletons, we chose 84 adult excellently preserved skeletons free of antemortem and postmortem changes and took all standard measurements. Sex was estimated/determined using standard anthropological procedures and ancient DNA (amelogenin analysis) where pelvis was insufficiently preserved or where sex morphological indicators were not consistent. We explored which measurements showed sexual dimorphism and used them for developing univariate and multivariate discriminant functions for sex estimation. We included only those functions that reached accuracy rate ≥80%. We tested the applicability of developed functions on modern Croatian sample (n = 37). Results From 69 standard skeletal measurements used in this study, 56 of them showed statistically significant sexual dimorphism (74.7%). We developed five univariate discriminant functions with classification rate 80.6%-85.2% and seven multivariate discriminant functions with an accuracy rate of 81.8%-93.0%. When tested on the modern population functions showed classification rates 74.1%-100%, and ten of them reached aimed accuracy rate. Females showed higher classification rates in the medieval populations, whereas males were better classified in the modern populations. Conclusion Developed discriminant functions are sufficiently accurate for reliable sex estimation in both medieval Croatian population and modern Croatian samples and may be used in forensic settings. The methodological issues that emerged regarding the importance of considering external factors in development and application of discriminant functions for sex estimation should be further explored.

Skeleton Changes Induced by Horse Riding on Medieval Skeletal Remains from Croatia

Skeletal remains of 35 individuals from the Croatian excavation site Kamen Most – Kaldrma from the fourteenth to fifteenth centuries were analyzed anthropologically. Due to specific skeletal characteristics, eight of them were analyzed in great detail in this study. The results of additional analysis indicated that all bone elements, except pelves, of all eight persons belonged to males. All pelvic girdle have characteristics of female sex and pronounced muscle attachments that are both indicative of horse riding. All eight persons had at least two of six changes on femora indicative of horse riding. Vertebrae of seven persons (one person did not have preserved vertebrae) have Schmorls nodes. Signs of advanced osteoarthritis were found on all joints in all eight persons. All of them also have visible signs of periostitis especially on lower extremities. Pathological signs of trauma were found in five persons. Two of them had skull fractures and the rest had fractures of extremities which are also characteristic for horse riders. The average age of all eight persons was above 45 years at death. DNA analysis confirmed male sex for seven individuals. The results were additionally confirmed by later archeological findings of tombstones with illustrations of horse riders.

From biography to osteobiography: an example of anthropological historical identification of the remains of St.Paul

In the identification process of historical figures, and especially in cases of Saints bodies or mummified remains, any method that includes physical encroachment or sampling is often not allowed. In these cases, one of the few remaining possibilities is the application of nondestructive radiographical and anthropological methods. However, although there have been a few attempts of such analyses, no systematic standard methodology has been developed until now. In this study, we developed a methodological approach that was used to test the authenticity of the alleged body of Saint Paul the Confessor. Upon imaging the remains on MSCT and post‐processing, the images were analyzed by an interdisciplinary team to explore the contents beneath the binding media (e.g., the remains) and to obtain osetobiographical data for comparison with historical biological data. Obtained results: ancestry, sex, age, occupation, and social status were consistent with historical data. Although the methodological approach proved to be appropriate in this case, due to the discrepancy in the amount of data, identity could not be fully confirmed. Nonetheless, the hypothesis that the remains do not belong to St. Paul was rejected, whilst positive identification receives support. Anat Rec, 300:1535–1546, 2017.