To explore the distribution and polymorphisms of 23 short tandem repeat (STR) loci on the Y chromosome in the Turkish population recently settled in Sarajevo, Bosnia and Herzegovina and to investigate its genetic relationships with the homeland Turkish population and neighboring populations.
This study included 100 healthy unrelated male individuals from the Turkish population living in Sarajevo. Buccal swab samples were collected as a DNA source. Genomic DNA was extracted using the salting out method and amplification was performed using PowerPlex Y 23 amplification kit. The studied population was compared to other populations using pairwise genetic distances, which were represented with a multi-dimensional scaling plot.
Haplotype and allele frequencies of the sample population were calculated and the results showed that all 100 samples had unique haplotypes. The most polymorphic locus was DYS458, and the least polymorphic DYS391. The observed haplotype diversity was 1.0000 ± 0.0014, with a discrimination capacity of 1.00 and the match probability of 0.01. Rst values showed that our sample population was closely related in both dimensions to the Lebanese and Iraqi populations, while it was more distant from Bosnian, Croatian, and Macedonian populations.
Turkish population residing in Sarajevo could be observed as a representative Turkish population, since our results were consistent with those previously published for the homeland Turkish population. Also, this study once again proved that geographically close populations were genetically more related to each other.
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.
A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction.
A study was conducted to determine if modern forensic DNA typing methods can be properly employed throughout the world with a final goal of increasing arrests, prosecutions, and convictions of perpetrators of modern day trafficking in persons while concurrently reducing the burden of victim testimony in legal proceedings. Without interruption of investigations, collection of samples containing DNA was conducted in a variety of settings. Evidentiary samples were analyzed on the ANDE Rapid DNA system. Many of the collected swabs yielded informative short tandem repeat profiles with Rapid DNA technology.
Fine structural details of glycans attached to the conserved N-glycosylation site significantly not only affect function of individual immunoglobulin G (IgG) molecules but also mediate inflammation at the systemic level. By analyzing IgG glycosylation in 5,117 individuals from four European populations, we have revealed very complex patterns of changes in IgG glycosylation with age. Several IgG glycans (including FA2B, FA2G2, and FA2BG2) changed considerably with age and the combination of these three glycans can explain up to 58% of variance in chronological age, significantly more than other markers of biological age like telomere lengths. The remaining variance in these glycans strongly correlated with physiological parameters associated with biological age. Thus, IgG glycosylation appears to be closely linked with both chronological and biological ages. Considering the important role of IgG glycans in inflammation, and because the observed changes with age promote inflammation, changes in IgG glycosylation also seem to represent a factor contributing to aging.
Glycosylation is the key posttranslational mechanism that regulates function of immunoglobulins, with multiple systemic repercussions to the immune system. Our study of IgG glycosylation in 5,117 individuals from four European populations has revealed very extensive and complex changes in IgG glycosylation with age. The combined index composed of only three glycans explained up to 58% of variance in age, considerably more than other biomarkers of age like telomere lengths. The remaining variance in these glycans strongly correlated with physiological parameters associated with biological age; thus, IgG glycosylation appears to be closely linked with both chronological and biological ages. The ability to measure human biological aging using molecular profiling has practical applications for diverse fields such as disease prevention and treatment, or forensics.
Aging; Glycome; Glycosylation; Immunoglobulin G; Inflammation.
To analyze 8 X-linked short tandem repeat (STR) markers in the population of central Croatia and to evaluate their forensic efficiency.
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.’’ Mentype® Argus X-8 PCR amplification kit was used for typing the data of 99 unrelated healthy women and 78 men from central Croatia. Haplotype frequencies were calculated only in male samples. Arlequin 3.5 software was used to assess Hardy-Weinberg equilibrium (HWE), linkage disequilibrium (LD), observed and expected heterozygosity. Power of discrimination (PD) for men and women, polymorphism information content (PIC), power of exclusion, and mean exclusion chance for deficiency cases, normal trios, and duos were determined using online database ChrX-STR.org.
In female samples, deviations from HWE (P = 0.006) for each locus were not found. LD test performed both on female and male samples revealed no significant association between markers (P = 0.002). DXS10135 was the most polymorphic locus (PIC = 0.931). PD varied from 0.692 to 0.935 in male and from 0.845 to 0.992 in female samples. Combined PD reached 99.999999% in men and 99.9999999999% in women.
Performed analyses revealed that the studied marker set contained polymorphic markers with high power of discrimination. We can conclude that Mentype® Argus X-8 PCR kit is suitable for application in the population of central Croatia. Results of this study, together with collected allele and haplotype frequencies, are the first step in establishing a national reference X-STR database based on 8 X-STR loci.
To examine the influence of ultraviolet C (UVC) radiation on blood, saliva, semen, and naked DNA samples for preventing DNA cross-contamination on working surfaces in laboratories.
Blood, saliva, semen, and DNA isolated from buccal swab samples were obtained from a single male donor and applied to the laboratory working surfaces. UVC radiation was applied to these diluted and undiluted samples with or without previous decontamination of the working surfaces with 10% sodium hypochlorite and 20% ethanol. Genomic DNA was extracted using Chelex. After quantification, DNA was amplified using the AmpFlSTR® NGM™ PCR Amplification Kit. We tested and statistically analyzed DNA concentration, UVC dose, sample volume, radiation time, the number of correctly detected alleles on genetic loci, and the number of correctly detected alleles in four groups in which 16 loci were divided.
When working surfaces were not decontaminated and were treated only with UVC radiation in the laboratory, the genetic profile for naked DNA could not be obtained after 2 minutes of UVC radiation and for saliva after 54 hours. For blood and semen, a partial genetic profile was obtained even after 250 hours of UVC radiation in the laminar. When working surfaces were decontaminated with 10% sodium hypochlorite and 20% ethanol, genetic profile could not be obtained for naked DNA after 2 minutes, for saliva after 4 hours, for blood after 16 hours, and for semen after 8 hours of UVC radiation in the laboratory.
It is recommended to carefully and thoroughly clean working surfaces with 10% sodium hypochlorite and 20% ethanol followed by minimal 16-hour UVC exposure (dose approximately 4380 mJ/cm2) for complete and successful decontamination.
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.
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.
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.
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.
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.
The aim of this article is to offer a concise interpretation of the scientific data about the topic of Croatian genetic heritage that was obtained over the past 10 years. We made a short overview of previously published articles by our and other groups, based mostly on Y-chromosome results. The data demonstrate that Croatian human population, as almost any other European population, represents remarkable genetic mixture. More than 3/4 of the contemporary Croatian men are most probably the offspring of Old Europeans who came here before and after the Last Glacial Maximum. The rest of the population is the offspring of the people who were arriving in this part of Europe through the southeastern route in the last 10 000 years, mostly during the neolithization process. We believe that the latest discoveries made with the techniques for whole-genome typing using the array technology, will help us understand the structure of Croatian population in more detail, as well as the aspects of its demographic history.
To test the association of NOS3 gene with hypoxic-ischemic encephalopathy (HIE).
The study included 110 unrelated term or preterm born children (69 boys and 41 girls) with HIE and 128 term and preterm born children (60 boys and 68 girls) without any neurological problems after the second year of life. Children with perinatal HIE fulfilled the diagnostic criteria for perinatal asphyxia. All children were admitted to the Clinical Hospital Split between 1992 and 2008. We analyzed 6 tagging single nucleotide polymorphisms (SNP) within NOS3 gene (rs3918186, rs3918188, rs1800783, rs1808593, rs3918227, rs1799983), in addition to previously confirmed NOS3-associated SNP rs1800779. Genotyping was conducted using real-time polymerase chain reaction (PCR). Association analyses were performed according to allelic and genotypic distribution.
Allelic test did not show any SNP association with HIE. SNP rs1808593 showed genotype association (P = 0.008) and rs1800783-rs1800779 TG haplotype showed an association with HIE (P < 0.001). The study had 80% statistical power to detect (α = 0.05) an effect with odds ratio (OR) = 2.07 for rs3918186, OR = 1.69 for rs3918188, OR = 1.70 for rs1800783, OR = 1.80 for rs1808593, OR = 2.10 for rs3918227, OR = 1.68 for rs1800779, and OR = 1.76 for rs1799983, assuming an additive model.
Despite the limited number of HIE patients, we observed genotypic and haplotype associations of NOS3 polymorphisms with HIE.
Human Y-chromosome haplogroup structure is largely circumscribed by continental boundaries. One notable exception to this general pattern is the young haplogroup R1a that exhibits post-Glacial coalescent times and relates the paternal ancestry of more than 10% of men in a wide geographic area extending from South Asia to Central East Europe and South Siberia. Its origin and dispersal patterns are poorly understood as no marker has yet been described that would distinguish European R1a chromosomes from Asian. Here we present frequency and haplotype diversity estimates for more than 2000 R1a chromosomes assessed for several newly discovered SNP markers that introduce the onset of informative R1a subdivisions by geography. Marker M434 has a low frequency and a late origin in West Asia bearing witness to recent gene flow over the Arabian Sea. Conversely, marker M458 has a significant frequency in Europe, exceeding 30% in its core area in Eastern Europe and comprising up to 70% of all M17 chromosomes present there. The diversity and frequency profiles of M458 suggest its origin during the early Holocene and a subsequent expansion likely related to a number of prehistoric cultural developments in the region. Its primary frequency and diversity distribution correlates well with some of the major Central and East European river basins where settled farming was established before its spread further eastward. Importantly, the virtual absence of M458 chromosomes outside Europe speaks against substantial patrilineal gene flow from East Europe to Asia, including to India, at least since the mid-Holocene.
Y chromosome; haplogroup R1a; human evolution; population genetics
The debate concerning the mechanisms underlying the prehistoric spread of farming to Southeast Europe is framed around the opposing roles of population movement and cultural diffusion. To investigate the possible involvement of local people during the transition of agriculture in the Balkans, we analysed patterns of Y-chromosome diversity in 1206 subjects from 17 population samples, mainly from Southeast Europe. Evidence from three Y-chromosome lineages, I-M423, E-V13 and J-M241, make it possible to distinguish between Holocene Mesolithic forager and subsequent Neolithic range expansions from the eastern Sahara and the Near East, respectively. In particular, whereas the Balkan microsatellite variation associated to J-M241 correlates with the Neolithic period, those related to E-V13 and I-M423 Balkan Y chromosomes are consistent with a late Mesolithic time frame. In addition, the low frequency and variance associated to I-M423 and E-V13 in Anatolia and the Middle East, support an European Mesolithic origin of these two clades. Thus, these Balkan Mesolithic foragers with their own autochthonous genetic signatures, were destined to become the earliest to adopt farming, when it was subsequently introduced by a cadre of migrating farmers from the Near East. These initial local converted farmers became the principal agents spreading this economy using maritime leapfrog colonization strategies in the Adriatic and transmitting the Neolithic cultural package to other adjacent Mesolithic populations. The ensuing range expansions of E-V13 and I-M423 parallel in space and time the diffusion of Neolithic Impressed Ware, thereby supporting a case of cultural diffusion using genetic evidence.
Balkan Neolithic; farming transition; peopling of Europe; Y-chromosome haplogroups
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.
Bone fragments and teeth were removed from human remains found in several small and closely located hidden mass graves in the Škofja Loka area (Lovrenska Grapa and Žolšče) 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 AmpFℓSTR® 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.
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 Žološče, we were able to obtain 6 useful DNA profiles.
The results of this study, in combination with previously obtained results, demonstrate that Y-chromosome testing and miniSTR methodology can contribute to the identification of human remains of victims of revolutionary violence from World War II.
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 Škofja Loka, Slovenia.
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. QuantifilerTM 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.
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.
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.
To study the distribution of allele frequencies of 15 short tandem repeat (STR) loci in a representative sample of the Croatian population.
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 μL. The polymerase chain reaction (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.
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 the Croatian and Slovenian populations for vWA locus. There was no significant deviation from Hardy-Weinberg equilibrium for all the observed loci.
Obtained population data concurred with the expected “STR data frame” for this part of Europe.
This study illuminates the intra-nuclear fate of COL1A1 RNA in osteogenesis imperfecta (OI) Type I. Patient fibroblasts were shown to carry a heterozygous defect in splicing of intron 26, blocking mRNA export. Both the normal and mutant allele associated with a nuclear RNA track, a localized accumulation of posttranscriptional RNA emanating to one side of the gene. Both tracks had slightly elongated or globular morphology, but mutant tracks were cytologically distinct in that they lacked the normal polar distribution of intron 26. Normal COL1A1 RNA tracks distribute throughout an SC-35 domain, from the gene at the periphery. Normally, almost all 50 COL1A1 introns are spliced at or adjacent to the gene, before mRNA transits thru the domain. Normal COL1A1 transcripts may undergo maturation needed for export within the domain such as removal of a slow-splicing intron (shown for intron 24), after which they may disperse. Splice-defective transcripts still distribute thru the SC-35 domain, moving ∼1–3 μm from the gene. However, microfluorimetric analyses demonstrate mutant transcripts accumulate to abnormal levels within the track and domain. Hence, mutant transcripts initiate transport from the gene, but are impeded in exit from the SC-35 domain. This identifies a previously undefined step in mRNA export, involving movement through an SC-35 domain. A model is presented in which maturation and release for export of COL1A1 mRNA is linked to rapid cycling of metabolic complexes within the splicing factor domain, adjacent to the gene. This paradigm may apply to SC-35 domains more generally, which we suggest may be nucleated at sites of high demand and comprise factors being actively used to facilitate expression of associated loci.
osteogenesis imperfecta; RNA transport; RNA splicing; nuclear structure; collagen
Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836–0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father–son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.
Y-chromosome; Y-STRs; haplotypes; RM Y-STRs; paternal lineage; forensic
The 21st century has brought important changes in the paradigms of economic development, one of them being a shift toward recognizing knowledge and information as the most important factors of today. The European Union (EU) has been working hard to become the most competitive knowledge-based society in the world, and Croatia, an EU candidate country, has been faced with a similar task. To establish itself as one of the best knowledge-based country in the Eastern European region over the next four years, Croatia realized it has to create an education and science system correspondent with European standards and sensitive to labor market needs. For that purpose, the Croatian Ministry of Science, Education, and Sports (MSES) has created and started implementing a complex strategy, consisting of the following key components: the reform of education system in accordance with the Bologna Declaration; stimulation of scientific production by supporting national and international research projects; reversing the “brain drain” into “brain gain” and strengthening the links between science and technology; and informatization of the whole education and science system. In this comprehensive report, we describe the implementation of these measures, whose coordination with the EU goals presents a challenge, as well as an opportunity for Croatia to become a knowledge-based society by 2010.