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.

Modern Bosnia and Herzegovina is a multi-ethnic and multi-religion country, with a very stormy history. Certain archaeological findings indicate continuous population of its territory since the Paleolithic. In time, vast number of different factors jointly influenced fascinating diversity of local human populations. A great number of small, more or less isolated, indigenous populations, make this area quite attractive for population-genetic surveys of different levels and approaches. Austro-Hungarian military physicians conducted the very first known bio-anthropological analyses of Bosnia-Herzegovina population at the end of the 19th century. Thus, the first step towards resolving the genetic structures of local B&H human populations was made. The studies that followed (conducted throughout most of the 20th century) were primarily based on the observation of various phenotypic traits. This stage was followed by the examination of various cytogenetic and fundamental DNA based molecular markers. The efforts undertaken over the last three centuries revealed "human genetic treasure" in Bosnia and Herzegovina. However, even now, after all the studies that were conducted, many interesting features remain to be discovered and described within the existing local human populations.

Among explored speleological objects near the Krupa village, close to Gornji Vakuf, the most interesting cave from paleocultural aspect, is the cave named “Cave number 5”. In the internal part of that cave, besides ceramic fragments and one metal needle, rich anthropological material have been found, compounds of numerous humans bones and teeth. Anato-morphological analysis of the one part of material showed that this site is a prehistoric crypt with remains of at least 13 people. Radiocarbon 14C bone analysiss results showed that those people lived 2765 ± 75 before presence. The fact that anthropological material includes some remains which belonged to children shows some characteristics of burial culture of that period on area of Uskoplje. This cave crypt is the one of the biggest on this region. Paleostomatological analysis of teeth and jaw fragments gave us clear evidence about therapeutic interventions performed in this period. X-rays analysis showed presence of the metal body inside the bone in one mandibular fragment. It’s supposed that this metal is broken part of instrument used for tooth extraction.

AIM To present the joint effort of three institutions in the identification of human remains from the World War II found in two mass graves in the area of Skofja Loka, Slovenia. METHODS The remains of 27 individuals were found in two small and closely located mass graves. The DNA was isolated from bone and teeth samples using either standard phenol/chloroform alcohol extraction or optimized Qiagen DNA extraction procedure. Some recovered samples required the employment of additional DNA purification methods, such as N-buthanol treatment. Quantifiler Human DNA Quantification Kit was used for DNA quantification. PowerPlex 16 kit was used to simultaneously amplify 15 short tandem repeat (STR) loci. Matching probabilities were estimated using the DNA View program. RESULTS Out of all processed samples, 15 remains were fully profiled at all 15 STR loci. The other 12 profiles were partial. The least successful profile included 13 loci. Also, 69 referent samples (buccal swabs) from potential living relatives were collected and profiled. Comparison of victims' profile against referent samples database resulted in 4 strong matches. In addition, 5 other profiles were matched to certain referent samples with lower probability. CONCLUSION Our results show that more than 6 decades after the end of the World War II, DNA analysis may significantly contribute to the identification of the remains from that period. Additional analysis of Y-STRs and mitochondrial DNA (mtDNA) markers will be performed in the second phase of the identification project.

AIM To study the distribution of allele frequencies of 15 short tandem repeat (STR) loci in a representative sample of Croatian population. METHODS A total of 195 unrelated Caucasian individuals born in Croatia, from 14 counties and the City of Zagreb, were sampled for the analysis. All the tested individuals were voluntary donors. Buccal swab was used as the DNA source. AmpFlSTR Identifiler was applied to simultaneously amplify 15 STR loci. Total reaction volume was 12.5 microL. The PCR amplification was carried out in PE Gene Amp PCR System Thermal Cycler. Electrophoresis of the amplification products was preformed on an ABI PRISM 3130 Genetic Analyzer. After PCR amplification and separation by electrophoresis, raw data were compiled, analyzed, and numerical allele designations of the profiles were obtained. Deviation from Hardy-Weinberg equilibrium, observed and expected heterozygosity, power of discrimination, and power of exclusion were calculated. Bonferroni's correction was used before each comparative analysis. RESULTS We compared Croatian data with those obtained from geographically neighboring European populations. The significant difference (at P<0.01) in allele frequencies was recorded only between Croatian and Slovenian populations for vWA locus. There was no significant deviation from Hardy-Weinberg equilibrium for all the observed loci. CONCLUSION Obtained population data concurred with the expected "STR data frame" for this part of Europe.

The validation of a Powerplex 16 (PP16) database for southern Croatians provided the opportunity to address questions raised by Birus et al (1) regarding the occurrence of linkage disequilibrium and false matches. The PP16 database had no significant deviations from HWE and were not significantly different from the previous study of the 13 shared CODIS loci (2) indicating that the two data sets could be pooled. A test for two locus linkage disequilibrium did not yield any unexpected significant correlations precluding any higher order disequilibrium. The power of exclusion (RPNE) (3) in a parentage test assumes that a single comparison is being performed. When using parents or children to identify remains the exclusionary power using either allele is very low compared to using separate alleles, e.g. using the pooled Croatian database with the two most common alleles at each locus the combined RPNE for 15 loci when using two alleles is only 0.011, compared to an RPNE of 3.24E-11 when using separate alleles, or a random match likelihood using the same alleles of 9.3E-14. Thus, the likelihood of false matches when making large numbers of comparisons is very high, making it necessary to either compare multiple relatives to profiles, when a hit is made, or correct the expected RPNE for the number of comparisons made to get an estimate of the true RPNE.

During the 1992-1995 siege, as well as after the war activities, citizens of Sarajevo were most probably exposed to various potential genotoxic agents. The effects of those potential genotoxins were evaluated by micronucleus-cytokinesis blocked assay. The study included 30 individuals who resided in the area of Sarajevo during the war and the postwar period. Point bi-serial coefficient analysis did not reveal any relationship between the frequencies of binuclear cells with micronuclei as well as total number of micronuclei and smoking habits or gender. Simple linear regression revealed statistically significant positive correlation between the age and micronuclei formation. Due to the war related environmental contamination more extensive study is recommended.

POPULATION: We have analyzed the distribution of allele frequencies at two short tandem repeats loci (D2S1338 and D19S433) in a multinational sample of Bosnia and Herzegovina (B&H) residents. A total of 110 unrelated male and female individuals (Caucasians) from different regions of B&H were sampled for the analysis. We ensured that the sample reflected approximate proportional participation of the three main ethnic groups in the population of B&H (Bosniacs‐Muslim [45%], Serbs [34%], Croats [21%]).