A recent bottleneck of Y chromosome diversity coincides with a global change in culture.

It is commonly thought that human genetic diversity in non-African populations was shaped primarily by an out-of-Africa dispersal 50-100 thousand yr ago (kya). Here, we present a study of 456 geographically diverse high-coverage Y chromosome sequences, including 299 newly reported samples. Applying ancient DNA calibration, we date the Y-chromosomal most recent common ancestor (MRCA) in Africa at 254 (95% CI 192-307) kya and detect a cluster of major non-African founder haplogroups in a narrow time interval at 47-52 kya, consistent with a rapid initial colonization model of Eurasia and Oceania after the out-of-Africa bottleneck. In contrast to demographic reconstructions based on mtDNA, we infer a second strong bottleneck in Y-chromosome lineages dating to the last 10 ky. We hypothesize that this bottleneck is caused by cultural changes affecting variance of reproductive success among males.

Next-generation sequencing of the BRCA1 and BRCA2 genes for the genetic diagnostics of hereditary breast and/or ovarian cancer.

Genetic testing for hereditary breast and/or ovarian cancer mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the BRCA1 and BRCA2 genes. We explored a more efficient genetic screening strategy based on next-generation sequencing of the BRCA1 and BRCA2 genes in 210 hereditary breast and/or ovarian cancer patients. We first validated this approach in a cohort of 115 samples with previously known BRCA1 and BRCA2 mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq BRCA1 and BRCA2 panel. The DNA Libraries were pooled, barcoded, and sequenced using an Ion Torrent Personal Genome Machine sequencer. The combination of different robust bioinformatics tools allowed detection of all previously known pathogenic mutations and polymorphisms in the 115 samples, without detecting spurious pathogenic calls. We then used the same assay in a discovery cohort of 95 uncharacterized hereditary breast and/or ovarian cancer patients for BRCA1 and BRCA2. In addition, we describe the allelic frequencies across 210 hereditary breast and/or ovarian cancer patients of 74 unique definitely and likely pathogenic and uncertain BRCA1 and BRCA2 variants, some of which have not been previously annotated in the public databases. Targeted next-generation sequencing is ready to substitute classic molecular methods to perform genetic testing on the BRCA1 and BRCA2 genes and provides a greater opportunity for more comprehensive testing of at-risk patients.

Standing at the gateway to Europe--the genetic structure of Western balkan populations based on autosomal and haploid markers.

Contemporary inhabitants of the Balkan Peninsula belong to several ethnic groups of diverse cultural background. In this study, three ethnic groups from Bosnia and Herzegovina - Bosniacs, Bosnian Croats and Bosnian Serbs - as well as the populations of Serbians, Croatians, Macedonians from the former Yugoslav Republic of Macedonia, Montenegrins and Kosovars have been characterized for the genetic variation of 660 000 genome-wide autosomal single nucleotide polymorphisms and for haploid markers. New autosomal data of the 70 individuals together with previously published data of 20 individuals from the populations of the Western Balkan region in a context of 695 samples of global range have been analysed. Comparison of the variation data of autosomal and haploid lineages of the studied Western Balkan populations reveals a concordance of the data in both sets and the genetic uniformity of the studied populations, especially of Western South-Slavic speakers. The genetic variation of Western Balkan populations reveals the continuity between the Middle East and Europe via the Balkan region and supports the scenario that one of the major routes of ancient gene flows and admixture went through the Balkan Peninsula.

Maternal genetic heritage of Southeastern Europe reveals a new Croatian isolate and a novel, local sub-branching in the x2 haplogroup.

High mtDNA variation in Southeastern Europe (SEE) is a reflection of the turbulent and complex demographic history of this area, influenced by gene flow from various parts of Eurasia and a long history of intermixing. Our results of 1035 samples (488 from Croatia, 239 from Bosnia and 130 from Herzegovina, reported earlier, and 97 Slovenians and 81 individuals from Zumberak, reported here for the first time) show that the SEE maternal genetic diversity fits within a broader European maternal genetic landscape. The study also shows that the population of Zumberak, located in the continental part of Croatia, developed some unique mtDNA haplotypes and elevated haplogroup frequencies due to distinctive demographic history and can be considered a moderate genetic isolate. We also report seven samples from the Bosnian population and one Herzegovinian sample designated as X2* individuals that could not be assigned to any of its sublineages (X2a'o) according to the existing X2 phylogeny. In an attempt to clarify the phylogeny of our X2 samples, their mitochondrial DNA has been completely sequenced. We suppose that these lineages are signs of local microdifferentiation processes that occurred in the recent demographic past in this area and could possibly be marked as SEE-specific X2 sublineages.

Haplotype data for 23 Y-chromosome markers in a reference sample from Bosnia and Herzegovina.

AIM: To detect polymorphisms of 23 Y-chromosomal short tandem repeat (STR) loci, including 6 new loci, in a reference database of male population of Bosnia and Herzegovina, as well as to assess the importance of increasing the number of Y-STR loci utilized in forensic DNA analysis. METHODS: The reference sample consisted of 100 healthy, unrelated men originating from Bosnia and Herzegovina. Sample collection using buccal swabs was performed in all geographical regions of Bosnia and Herzegovina in the period from 2010 to 2011. DNA samples were typed for 23 Y STR loci, including 6 new loci: DYS576, DYS481, DYS549, DYS533, DYS570, and DYS643, which are included in the new PowerPlex® Y 23 amplification kit. RESULTS: The absolute frequency of generated haplotypes was calculated and results showed that 98 samples had unique Y 23 haplotypes, and that only two samples shared the same haplotype. The most polymorphic locus was DYS418, with 14 detected alleles and the least polymorphic loci were DYS389I, DYS391, DYS437, and DYS393. CONCLUSION: This study showed that by increasing the number of highly polymorphic Y STR markers, to include those tested in our analysis, leads to a reduction of repeating haplotypes, which is very important in the application of forensic DNA analysis.

No evidence from genome-wide data of a Khazar origin for the Ashkenazi Jews.

The origin and history of the Ashkenazi Jewish population have long been of great interest, and advances in high-throughput genetic analysis have recently provided a new approach for investigating these topics. We and others have argued on the basis of genome-wide data that the Ashkenazi Jewish population derives its ancestry from a combination of sources tracing to both Europe and the Middle East. It has been claimed, however, through a reanalysis of some of our data, that a large part of the ancestry of the Ashkenazi population originates with the Khazars, a Turkic-speaking group that lived to the north of the Caucasus region ~1,000 years ago. Because the Khazar population has left no obvious modern descendants that could enable a clear test for a contribution to Ashkenazi Jewish ancestry, the Khazar hypothesis has been difficult to examine using genetics. Furthermore, because only limited genetic data have been available from the Caucasus region, and because these data have been concentrated in populations that are genetically close to populations from the Middle East, the attribution of any signal of Ashkenazi-Caucasus genetic similarity to Khazar ancestry rather than shared ancestral Middle Eastern ancestry has been problematic. Here, through integration of genotypes from newly collected samples with data from several of our past studies, we have assembled the largest data set available to date for assessment of Ashkenazi Jewish genetic origins. This data set contains genome-wide single-nucleotide polymorphisms in 1,774 samples from 106 Jewish and non-Jewish populations that span the possible regions of potential Ashkenazi ancestry: Europe, the Middle East, and the region historically associated with the Khazar Khaganate. The data set includes 261 samples from 15 populations from the Caucasus region and the region directly to its north, samples that have not previously been included alongside Ashkenazi Jewish samples in genomic studies. Employing a variety of standard techniques for the analysis of population-genetic structure, we found that Ashkenazi Jews share the greatest genetic ancestry with other Jewish populations and, among non-Jewish populations, with groups from Europe and the Middle East. No particular similarity of Ashkenazi Jews to populations from the Caucasus is evident, particularly populations that most closely represent the Khazar region. Thus, analysis of Ashkenazi Jews together with a large sample from the region of the Khazar Khaganate corroborates the earlier results that Ashkenazi Jews derive their ancestry primarily from populations of the Middle East and Europe, that they possess considerable shared ancestry with other Jewish populations, and that there is no indication of a significant genetic contribution either from within or from north of the Caucasus region.

Genetic polymorphisms of 15 STR loci within Turkish student population living in Sarajevo, Bosnia and Herzegovina.

Allele frequencies of 15 STRs included in the PowerPlex 16 System (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, VWA, D8S1179, TPOX and FGA) were calculated from the referent sample of 100 unrelated individuals of both sexes from Turkish student population living in Sarajevo, Bosnia and Herzegovina. Buccal swab, as a source of DNA, was collected from the volunteers from whom the informed consent form was obtained. DNA extraction was performed using QIAamp DNA Micro kit by Qiagen. DNA template ranging from 0.5 to 2 ng was used to amplify 15 STR loci by PCR multiplex amplification which was performed by using the PowerPlex 16 kit (Promega Corp., Madison, WI, USA) according to the manufacturer's protocol. The amplifications were carried out in a PE Gene Amp PCR System thermal cycler (Applied Biosystems) and capillary electrophoresis was carried out in an ABI PRISM 310 Genetic Analyzer (Applied Biosystems) in accordance with the manufacturer's recommendations. The frequency of each locus was calculated from the numbers of each observed genotype. Deviation from Hardy-Weinberg equilibrium and observed heterozygosity were calculated. Data were analyzed by using Microsoft Excel workbook template--Powerstats V12 and the power of discrimination (PD), power of exclusion (PE), as well as other population genetic indices for the 15 STR loci were calculated. Obtained results contribute to existing Turkish DNA database, as well as insight of differences and similarities in comparison to population of Bosnia and Herzegovina. In addition, 13 autosomal STR loci frequencies (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSFIPO, Penta D, VWA, D8S1 179, TPOX, and FGA) were studied in 15 different worldwide populations (Turkish, Bosnian, Croatian, Serbian, Montenegrin, Macedonian, Albanian, Kosovan, Greek, Russian, Japanese, Korean, Lithuanian, Iraqi, Belarusian). For the proof of corresponding data, two different Turkish population STR data obtained from previously published articles were compared with our data and this showed that our data correspond to these 2 previously published data. Further, STR allele frequency data for 13 loci for each population were obtained from previous scientific articles and the allele frequencies and genetic diversity among the 15 sample populations were compared. In addition, even though the populations are from different nationalities, the STR data are similar among the geographically close populations. The phylogenetic tree established among worldwide populations and genetic distance values show a great affinity among the 15populations. Our data is useful for anthropological and further comparative genetic studies of populations.

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.

Prediction of eye and skin color in diverse populations using seven SNPs.

An essential component in identifying human remains is the documentation of the decedent's visible characteristics, such as eye, hair and skin color. However, if a decedent is decomposed or only skeletal remains are found, this critical, visibly identifying information is lost. It would be beneficial to use genetic information to reveal these visible characteristics. In this study, seven single nucleotide polymorphisms (SNPs), located in and nearby genes known for their important role in pigmentation, were validated on 554 samples, donated from non-related individuals of various populations. Six SNPs were used in predicting the eye color of an individual, and all seven were used to describe the skin coloration. The outcome revealed that these markers can be applied to all populations with very low error rates. However, the call-rate to determine the skin coloration varied between populations, demonstrating its complexity. Overall, these results prove the importance of these seven SNPs for potential forensic tests.

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.

Allele frequencies for 15 short tandem repeat loci in representative sample of Croatian population.

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.

DNA identification of skeletal remains from the World War II mass graves uncovered in Slovenia.

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.

Population data at two short tandem repeat loci D2S1338 and D19S433 in the sample of multinational Bosnia and Herzegovina residents.

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%]).

Optimisation of forensic genetics procedures used in disputed paternity testing: adjustment of the PCR reaction volume.

Standard molecular techniques, with only a slight modification, are very useful in obtaining and interpreting the final results in the field of forensic genetic. Data obtained through such analysis are highly reliable and can be used as a very powerful tool that produces valuable results. However, success and swiftness of DNA typing of biological evidence either that found at a crime scene or used in disputed paternity testing, depends on the optimization of numerous factors. One of the most important and critical phases that ensures reliability of the whole procedure is the choice of the most suitable volume for the amplification protocol. Buccal swabs were collected from volunteers. DNA was extracted by Qiagen Dnaeasy Tissue Kit. PowerPlex 16 kit was used to simultaneously amplify 15 STR loci by PCR. Amplification was carried out as described previously. The tested total working reaction volumes were 5, 10 and 25 microl. The PCR amplification was carried out in PE Gene Amp PCR System Thermal Cycler (ABI, Foster City, CA). Amplification products were analyzed on an ABI PRISM 377 instrument (ABI, Foster City, CA) in 5% bis-acrilamide gel. Amplification was generally successful for all the tested reaction volumes. Lower partial to complete DNA profiles ratio, the quality of obtained STR profiles, significantly reduced amount of reaction's components give advantage to 5 microl reaction volume over other two tested volumes in this case.