Occurrence of metopic suture in modern and archaeological Croatian population.

AIM: To determine the incidence of metopism in the modern and archaeological Croatian population. METHODS: A total of 800 specimens (454 modern multi-slice computed tomography [MSCT] scans and 346 dry archaeological skulls) were visually examined for metopic suture presence. The metopic suture was deemed complete when aligned nasion to bregma. RESULTS: In the overall sample, the metopic suture was observed in 36 of 800 subjects (4.5%): 19 of 424 (4.5%) men and 17 of 370 (4.6%) women. A significant difference was not observed between modern and archaeological samples (χ2=3.219, P=0.359) or between the sexes (χ2=0.006, P=0.939). The frequency of metopism varied from 3.5% in the modern population to 7.04% in the samples from the Roman period. CONCLUSION: There are no visible secular changes on metopic suture in the Croatian population through time. Some variations can be the result of differences in sample size in different time periods.

Identification of skeletal remains in Croatia and Bosnia and Herzegovina, including the homeland war - a 30-year review.

Over the past 30 years, forensic experts from Croatia and Bosnia and Herzegovina have embraced advanced technologies and innovations to enable great efficacy and proficiency in the identification of war victims. The wartime events in the countries of former Yugoslavia greatly influenced the application of the selected DNA analyses as routine tools for the identification of skeletal remains, especially those from mass graves. Initially, the work was challenging because of the magnitude of the events, technical aspects, and political aspects. Collaboration with reputable foreign forensic experts helped tremendously in the efforts to start applying DNA analysis routinely and with increasing success. In this article, we reviewed the most significant achievements related to the application of DNA analysis in identifying skeletal remains in situations where standard identification methods were insufficient.

ABO and Rh blood groups, demographics, and comorbidities in COVID-19 related deaths: A retrospective study in Split-Dalmatia County, Croatia.

BACKGROUND: Blood group phenotypes have been associated with COVID-19 susceptibility and severity. This study aimed to examine ABO/Rh blood group distribution in COVID-19-related deaths considering demographics and pathological conditions. METHODS: We conducted a retrospective study at the University Hospital Center Split, Croatia, that included 245 COVID-19 positive individuals that died from April 8, 2020, to January 25, 2021. We extracted data on their blood groups, demographics, and pre-existing comorbidities and compared findings with general population data from blood group donations (n = 101,357) and non-COVID-19 deaths from 2019 (n = 4968). RESULTS: The proportion of dead males was significantly higher than in non-COVID-19 cases (63.7% vs. 48.9%, P < 0.001), while the proportion of older individuals did not differ. The prevailing pre-existing diseases were hypertension (59.6%), diabetes (37.1%), heart failure (28.8%), digestive disorder (26.5%), and solid tumor (21.6%). The ABO distribution in the deceased and donors' group showed significant differences, with the higher prevalence of A/AB group and lower prevalence of 0, but with individual differences significant only for AB and non-AB groups. There was a reduced proportion of females within the deceased with group 0 (P = 0.014) and a higher proportion of AB individuals with coronary heart disease (P = 0.024). CONCLUSION: The study confirmed a higher risk of death in males. The lower proportion of type 0 in deceased individuals was greater in females, implying that group 0 is not necessarily an independent protective factor. Coronary heart disease was identified as a potential risk factor for AB individuals.

Croatian genetic heritage: an updated Y-chromosome story.

AIM: To analyze an additional set of ˝Y-chromosome genetic markers to acquire a more detailed insight into the diversity of the Croatian population. METHODS: A total of 518 Yfiler Plus profiles were genotyped. Allele frequencies, haplotype frequencies, and haplotype diversity were calculated by using the STRAF software v. 2.0.4. Genetic distances were quantified by Rst with AMOVA online tool from the YHRD. The evolutionary history was inferred with the neighbor-joining method of phylogenetic tree construction in the MEGAX software. Whit Athey's Haplogroup Predictor v. 5 was used for additional comparison with regional and other European populations. RESULTS: A total of 507 haplotypes were used for genetic STR analysis. An interpopulation study on 17 Y-STR markers showed the lowest genetic diversity between the Croatian and Bosnian-Herzegovinian populations and the highest between the Croatian and Irish populations. Additional interpopulation comparison with the original 27 Y-STR markers (for the population with available data) was also performed. A total of 518 haplotypes were used in the determination of haplogroup diversity. Haplogroup I with its sublineage I2a expressed the highest prevalence. The second most prevalent haplogroup was R, with its major sublineage R1a, except for the subpopulation of Hvar, where E1b1b was the second most prevalent haplogroup. Rare haplogroups also confirmed in this study were L, T, and Q. G1 was detected for the first time in the Croatian population. CONCLUSION: We obtained a new insight into the differences between examined subpopulations of Croatia and their possible (dis)similarities with neighboring and distant populations.

BUILDING THE COVID-19 TESTING CAPACITIES IN CROATIA: ESTABLISHING THE INTERDEPARTMENTAL COVID-19 UNIT AT THE SPLIT UNIVERSITY HOSPITAL CENTRE.

This paper presents the chronology, experiences, and challenges in introducing COVID-19 RT-PCR testing in Split, Croatia. We describe the processes from March 12, 2020 to May 26, 2020, starting from the initial knowledge transfer, expert team formation and management, testing implementation, and concluding with the standalone testing facilities, which used automated processes sufficient to meet testing requirements at that time. In the case presented, the COVID-19 unit was organized by joining human and laboratory resources from five clinical departments at the Split University Hospital Centre. Sample preparation procedures and analyses were launched within the restricted time frame while simultaneously training and organizing new laboratory staff and completing equipment requirements. As a result, the process that started with 30 tests per day was constantly improved over time and reached up to 160 tests per day when MagNA Pure was added to automatize RNA extraction at the end of April. At that pace, the cumulative number of samples soon exceeded the first thousand, and by the end of May it exceeded 4000. The case presented provides an example of good practice for crisis response and organization that successfully enabled sufficient COVID-19 testing capacities within the restricted time frame, human and technical resources. Despite limited understanding of COVID-19 at that time, appropriate management, transfer of knowledge, previous experiences in related laboratory and diagnostic work, as well as interdisciplinary and interdepartmental cooperation proved appropriate to overcome the above limitations and ensure adequate healthcare response.

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 or 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 classified in the mediaeval populations, whereas males were better classification rates 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.

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 Sopot 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 Sopot graveyard and compared them with a cumulative medieval sample from the same region. We also performed principal component analysis to compare skeletal remains from Sopot with those from Ostrovica and other Central European samples according to 8 cranial measurements. Finally, we compared 46 skeletons from Sopot with medieval (Ostrovica) and contemporary populations using mDNA haplogroup profiling. RESULTS: The remains from Sopot 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 Sopot) in terms of cranial morphology, indicating similar biological makeup. According to mDNA testing, Sopot population showed no significant differences in the haplogroup prevalence from either medieval or contemporary populations. CONCLUSION: This study shows that the Sopot 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 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.

Prevalence and genotyping of the human papillomavirus in the cervical specimens among women of Southern Croatia (Dalmatia County).

AIM: The aim of this study was to evaluate the prevalence of HPV types in the female population with either normal or abnormal Pap smears in Southern Croatia (Dalmatia County) and to evaluate the distribution of HPV types and their association with different cytological findings. METHODS: During one year period, we routinely screened 1,160 women for HR HPV at the Educational Public Health Institute of Split and Dalmatia County, by using Hybrid Capture 2 HPV DNA test. Only HR HPV positive specimens determined by HC2 were further genotyped by polymerase chain reaction (PCR) based assay at the Department of Pathology and Forensic Medicine, Split. RESULTS: Out of the total number of tested women, 406 (35%) of them were positive to HR HPV. The following frequency was observed: HPV 16 in 10.8%, HPV 18 in 7.8%, HPV 31 in 2.3%, HPV 33 in 1.1%, HPV 52 in 0.9%, HPV 45 in 0.6%, and HPV 59 in 0.4% specimens. CONCLUSION: The data obtained from our study indicate that HPV 16 is the most common HPV type found in the cervical specimens among women of Southern Croatia, as in most studies worldwide, followed by HPV 18 and HPV 31. The positivity rates in our study slightly differed for HPV types 18 and 31 from those found in the other studies conducted in Northern Croatia. These suggesting geographic and environmental factors are related to the prevalence of HPV types. We consider it very important to gather information on regional variations in the HPV type spectrum.

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 Vucetic". 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.

Efficiency of the Adjusted Binary Classification (ABC) Approach in Osteometric Sex Estimation: A Comparative Study of Different Linear Machine Learning Algorithms and Training Sample Sizes.

The adjusted binary classification (ABC) approach was proposed to assure that the binary classification model reaches a particular accuracy level. The present study evaluated the ABC for osteometric sex classification using multiple machine learning (ML) techniques: linear discriminant analysis (LDA), boosted generalized linear model (GLMB), support vector machine (SVM), and logistic regression (LR). We used 13 femoral measurements of 300 individuals from a modern Turkish population sample and split data into two sets: training (n = 240) and testing (n = 60). Then, the five best-performing measurements were selected for training univariate models, while pools of these variables were used for the multivariable models. ML classifier type did not affect the performance of unadjusted models. The accuracy of univariate models was 82−87%, while that of multivariate models was 89−90%. After applying ABC to the crossvalidation set, the accuracy and the positive and negative predictive values for uni- and multivariate models were ≥95%. Sex could be estimated for 28−75% of individuals using univariate models but with an obvious sexing bias, likely caused by different degrees of sexual dimorphism and between-group overlap. However, using multivariate models, we minimized the bias and properly classified 81−87% of individuals. A similar performance was also noted in the testing sample (except for FEB), with accuracies of 96−100%, and a proportion of classified individuals between 30% and 82% in univariate models, and between 90% and 91% in multivariate models. When considering different training sample sizes, we demonstrated that LR was the most sensitive with limited sample sizes (n < 150), while GLMB was the most stable classifier.

The repeatability of standard cranial measurements on dry bones and MSCT images.

This study examined if the cranial measurements from Data Collection Procedures for Forensic Skeletal Material 2.0 are repeatable when measured in dry bones and MSCT images and if the virtual measurements correspond to the physical ones. The sample included 33 dry crania imaged by MSCT. Two observers measured dry bones, two placed landmarks on 2D and 3D MSCT reconstructions, and one conducted measurements/landmarking on both media. One of the observers for each media repeated the measurements. Technical and relative technical error of measurement (TEM and rTEM) and percentage differences were calculated to examine the repeatability of measurements and compare measuring modalities. Intraobserver rTEM was above 1.5% for six bone measurements: FOB, ZOB, OBB, NLH, DKB, MDH (1.51%-4.87%) and for seven MSCT measurements: OBH, FOB, OBB, MDH, NLB, ZOB, DKB (1.57%-5.55%). The interobserver rTEM was above the acceptable level (>2%) for 11 measurements: PAC, NLH, OBB, EKB, MAL, FOB, NLB, OBH, ZOB, DKB, and MDH (2.01%-9.34%). The percentage differences were not systematically larger for measurements taken by the same user on both modalities than those obtained by different users on the same modality. When physical and MSCT measurements were tested on sex classification standards, the proportion of crania classified as male or female did not significantly differ (p > 0.05). The study showed that physical and virtual cranial measurements could be interchangeable for developing or applying sex estimation standards. However, clarifications and adaptations are necessary for measurements of mastoid, nasal, and orbital regions that did not meet the standard criteria.

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 Vucetic". 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.

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 Vucetic". 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.

Adjusted binary classification (ABC) model in forensic science: An example on sex classification from handprint dimensions.

Binary classification techniques are commonly used in forensic examination to test if a specimen belongs to a particular group and base the expert opinion on the questioned evidence. However, most of the currently used methods do not achieve sufficient accuracy due to the ignoring of the specimens classified in the overlapping area. To address the issue, we proposed a novel Adjusted binary classification (ABC) algorithm that automatically adjusts posterior probabilities to reach classification accuracy and positive/negative predicted values (PPV, NPV) of 95 %. In the presented example, we used three handprint measurements from 160 participants (80 males and 80 females) to develop models that would classify sex from their dimensions. The sample was split into the training/cross-validated (70 %) and testing sample (30 %). We developed four classification models using linear discriminant analysis (LDA) by employing traditional single cut-off values and ABC approach that for each group provides a specific posterior probability cut-off threshold. In the cross-validated sample, the accuracy of traditional models was 78.7-92.5 %, while PPVs/NPVs ranged between 78.2 and 93 %. ABC models provided 95 % accuracy, PPV, and NPV, and could classify 35.5-88.1 % of specimens. In the testing sample, ABC models achieved accuracy of 97.3-100 %, PPV/NPV 95.4-100 %, and could be applied to 29.1-87.5 % of specimens. The study demonstrated that the ABC approach could adjust classification models to reach predefined values of accuracy, PPV, and NPV. Therefore, it could be an efficient tool for conducting a binary classification in forensic settings and minimizing the possibilities of incorrect classifications.

SARS-CoV-2 Antibody Seroprevalence in Industry Workers in Split-Dalmatia and Sibenik-Knin County, Croatia.

OBJECTIVES: To examine seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies in industry workers population sample. METHODS: From 23 to April 28, 2020, we conducted serological testing for antibodies (Immunoglobulin G (IgG) and Immunoglobulin M (IgM)) on 1494 factory employees living in the Split-Dalmatia and Sibenik-Knin County (Croatia). RESULTS: We detected antibodies in 1.27% of participants (95% confidence interval [CI] 0.77-1.98%). In Split facility 13/1316 (0.99%, 95% CI 0.53-1.68%) of participants were tested positive, of which 13/1079 (1.20%, 95% CI 0.64-2.05%) of those living outside the facility and 0/237 (0%, 95% CI 0-1.26%) of those living inside the facility. In Knin facility, 6/178 (3.37%, 95% CI 1.25-7.19%) participants were tested positive for antibodies. CONCLUSIONS: The study showed relatively small SARS-CoV-2 antibody seroprevalence in the DIV Group population sample.

Population genetic analysis of haplotypes based on 17 short tandem repeat loci on Y chromosome in population sample from eastern Croatia.

AIM: To investigate the population genetics of 17 short tandem repeat (STR) loci on the Y chromosome in the population of eastern Croatia. METHODS: 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 Vucetic". A total of 220 unrelated healthy men from eastern Croatia were selected for the purpose of this study. Genomic DNA was extracted by Chelex 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. RESULTS: A total of 207 haplotypes were recorded, 197 of which were unique (90%). Haplotype diversity was 0.9993, with the most frequent haplotype found in 4 of 220 men (1.8%). Average locus diversity was 0.600, and it ranged from 0.256 for DYS392 to 0.780 for DYS458. Our results were compared with the pattern of Y-chromosome variability in publicly available population samples based on a minimal European haplotype set of 9 STRs and the greatest resemblance was found with samples from the Croatian capital of Zagreb, from Bosnia and Herzegovina, and from Serbia. CONCLUSION: This is the first description of Y chromosome haplotyping of the population of eastern Croatia, which may serve as a basis for genetic epidemiology and forensic studies. Further studies are needed for characterization of the genetic structure of the Y-chromosome in the modern Croatian population.

Future perspectives of personalized oncology.

Based upon an individual's molecular make-up, personalized molecular medicine provides information regarding the origin of disease, its treatment and progression, while personalized molecular pharmacology advises on drug prescription and patient response to it, thus ensuring drug effectiveness and preventing drug toxicity or lack of response. Interindividual differences in drug responses are mostly due to structural variation in parts of genome, e.g. in genes participating in drug metabolism, transport or targeting. However, a wide variety of diseases and accompanying health conditions, including patient's therapy or drug response, also have epigenetic or epigenomic etiology. High priority for personalized oncologic research stems from inter/intraindividual tumor heterogeneity provoked by gradual acquisition of multiple random, or programmed mutations and rearrangements as well as epigenetic alterations or by stochastic fluctuations in cell components, all in tight feedback interaction with tumor's environmental or therapy conditions. Natural selection subsequently shapes inter/intraindividual tumor heterogeneity by promoting clonal expansion of cells that have acquired advantageous mutations for tumor population. Hence, the main rationale of personalized molecular oncology should focus on treating disease by relying on relevant structure and state of patient's whole molecular network (genome/transcriptome/RNome/proteome/metabolome/metabonome) in interaction with its unique environmental conditions, thus implying right therapy for the right patient at the right dose and time. The future of personalized oncology should therefore rely on the methods of systems biology applied in cytology and pathology in order to develop and utilize the efficient and effective diagnostic, prognostic and predictive biomarkers, consequently providing the molecular information on tumor origin, its potential for metastasis, adequate therapy, tumor specific therapy responsiveness, and the probability of its recurrence.

Adjusting posterior probabilities to meet predefined accuracy criteria: A proposal for a novel approach to osteometric sex estimation.

The osteometric methods are the most reliable way to estimate the sex of skeletons when DNA analysis is not used. However, as osteometric studies usually ignore the overlap in male and female skeletal dimensions, they rarely achieve accuracy sufficient for forensic application. To resolve this issue, recent studies suggest sex estimation only when posterior probability (pp) is greater than 0.95, but that approach does not always provide sufficient accuracy and creates a large proportion of unsexed skeleton. Thus, our study aimed to explore whether it is possible to adjust pp on skeletal measurements with pronounced sexual dimorphism to meet 95% accuracy and to enable sex estimation on a reasonable proportion of individuals. From 207 skeletons, we included 65 postcranial measurements and selected 10% of variables with the highest sexual dimorphism. We computed univariate and bivariate discriminant functions using pp threshold of 0.5, 0.95, and the threshold required to achieve accuracy of ≥ 95%. Discriminant functions with pp=0.5 obtained accuracy of 85%-93%, while those with pp≥0.95 and adjusted posterior probabilities obtained 94%-99%. However, we showed that by selecting a particular threshold, sex could be estimated on a greater proportion of individuals than for pp≥0.95: 42%-86% vs. 24%-62% for univariate and 69%-95% vs. 49%-78% for bivariate functions. Therefore, when developing sex estimation models, we suggest not to use fixed pp level, but to adjust pp to achieve 95% accuracy and to minimize the percentage of unsexed skeletons.

A Forensic Genomics Approach for the Identification of Sister Marija Crucifiksa Kozulic.

Sister Marija Krucifiksa Kozulic (1852-1922) was a Croatian nun who is in consideration for beatification by the Vatican, which is facilitated by the identification of her 20th-century remains. Sister Marija was buried in a tomb in Rijeka, Croatia, along with other nuns including her biological sister, Tereza Kozulic (1861-1933). When the remains were exhumed in 2011, they were found in a deteriorated state and commingled with several other sets of remains. Thus, mitochondrial genome sequencing of the long bones was performed to sort the remains by mitochondrial haplotype. Two similar but unique haplotypes belonging to haplogroup H1bu were identified, and samples from these bones were subjected to autosomal short tandem repeat (STR) and single nucleotide polymorphism (SNP) sequencing. Although only partial profiles were obtained, the data were sufficient for kinship analysis with the profile of a paternal niece of Sister Marija (Fides Kozulic). The data indicate that it is 574,195-fold more likely that the two sets of skeletal remains represent 2nd-degree relatives of Fides than sisters who are unrelated to Fides. Although it is impossible to discern which set of remains belongs to Marija and which belongs to Tereza, forensic genomics methods have enabled identification of the sisters.