Agnieszka Maciejewska

Significant genetic differentiation between Poland and Germany follows present-day political borders, as revealed by Y-chromosome analysis

To test for human population substructure and to investigate human population history we have analysed Y-chromosome diversity using seven microsatellites (Y-STRs) and ten binary markers (Y-SNPs) in samples from eight regionally distributed populations from Poland (n=913) and 11 from Germany (n=1,215). Based on data from both Y-chromosome marker systems, which we found to be highly correlated (r=0.96), and using spatial analysis of the molecular variance (SAMOVA), we revealed statistically significant support for two groups of populations: (1) all Polish populations and (2) all German populations. By means of analysis of the molecular variance (AMOVA) we observed a large and statistically significant proportion of 14% (for Y-SNPs) and 15% (for Y-STRs) of the respective total genetic variation being explained between both countries. The same population differentiation was detected using Monmonier’s algorithm, with a resulting genetic border between Poland and Germany that closely resembles the course of the political border between both countries. The observed genetic differentiation was mainly, but not exclusively, due to the frequency distribution of two Y-SNP haplogroups and their associated Y-STR haplotypes: R1a1*, most frequent in Poland, and R1*(xR1a1), most frequent in Germany. We suggest here that the pronounced population differentiation between the two geographically neighbouring countries, Poland and Germany, is the consequence of very recent events in human population history, namely the forced human resettlement of many millions of Germans and Poles during and, especially, shortly after World War II. In addition, our findings have consequences for the forensic application of Y-chromosome markers, strongly supporting the implementation of population substructure into forensic Y chromosome databases, and also for genetic association studies.

Comparison of three methods of DNA extraction from human bones with different degrees of degradation

There is a necessity for deceased identification as a result of many accidents and sometimes bones are the only accessible source of DNA. So far, a universal method that allows for extraction of DNA from materials at different stages of degradation does not exist. The aims of this study were: the comparison of three methods of DNA extraction from bones with different degree of degradation and an evaluation of the usefulness of these methods in forensic genetics. The efficiency of DNA extraction, the degree of extract contamination by polymerase chain reaction (PCR) inhibitors and the possibility of determining the STR loci profile were especially being compared. Nuclear DNA from bones at different states of degradation was isolated using three methods: classical, organic phenol–chloroform extraction, DNA extraction from crystal aggregates and extraction by total demineralisation. Total demineralisation is the best method for most cases of DNA extraction from bones, although it does not provide pure DNA. DNA extraction from aggregates removes inhibitors much better and is also a good method of choice when identity determination of exhumed remains is necessary. In the case of not buried bones (remains found outside) total demineralisation or phenol–chloroform protocols are more efficient for successful DNA extraction.

mRNA profiling for vaginal fluid and menstrual blood identification.

The detection and identification of human biological fluids, including vaginal secretions and menstrual blood, are highly important in forensic biology. Previous studies have proposed a few mRNA and bacterial markers for vaginal fluid detection, but they have not proven to be specific and reliable. The aim of this project was to develop, validate and evaluate a reliable, specific test for vaginal fluid identification that would combine detection of vaginal mRNAs and Lactobacilli. We have developed a hexaplex that detects HBD1 (human beta-defensin 1), MUC4 (mucin 4), menstrual blood marker MMP11 (matrix metalloproteinase 11), housekeeping gene G6PDH (glucose 6-phosphate dehydrogenase) and the 16S-23S rRNA intergenic spacer region of Lactobacillus crispatus and Lactobacillus gasseri/Lactobacillus johnsonii. We analysed the specificity of the markers and variations among women, as well as the sensitivity of the test and its ability to detect vaginal fluid in mixtures with semen and blood. This approach allows for the detection of vaginal fluid in stains that were up to 2 years old, if stored at room temperature and up to 18 years old if stored frozen. Through simultaneous analysis of 5 vaginal markers, the proposed hexaplex ensures high specificity and reliability in the detection of vaginal material.

Frequencies for five short tandem repeat (STR) systems in a population from North Poland

A population study of unrelated individuals from North Poland (gdansk area) was carried out to investigate the allele distributions of the five STR systems HUMCD4, HUMFES/FPS, HUMVWA31, HUMTH01 and ACTBP2. PCR products were separated on horizontal non-denaturing polyacrylamide gels followed by silver staining. For all STR systems analysed the distribution of observed phenotypes did not deviate from Hardy-Weinberg equilibrium. A comparison of allele distributions between Polish and other European Causasian population samples is presented.

mRNA heptaplex protocol for distinguishing between menstrual and peripheral blood

The identification of menstrual blood is an important issue in forensic biology, but currently, there are no confirmatory methods for its detection. Here, we demonstrate a highly reliable simple heptaplex method that allows for the discrimination between menstrual and peripheral blood. The test has been used successfully in criminal casework, in which the origin of blood on a rape victims underwear and trousers was questioned as being menstrual or traumatic peripheral blood. To solve this problem, transcripts of the following genes were used: mucin 4 (MUC4), human β-defensin 1 (HBD1), two matrix metalloproteinases (MMP7, MMP11), δ-aminolevulinate synthase 2 (ALAS2), hemoglobin alpha (HBA) and glucose 6-phosphate dehydrogenase (G6PDH). The sensitivity of the test is 0.3ng of RNA. The possibility of the detection and differentiation of menstrual and peripheral blood in mixtures that contain other body fluids was investigated. Reliable detection is possible for menstrual blood stains that are up to 1-2 years old if stored at room temperature. This easy approach, thanks to the amplification of 4 vaginal and 2 blood markers, minimizes the risk of false negative results.

Introducing novel type of human DNA markers for forensic tissue identification: DNA copy number variation allows the detection of blood and semen

Establishing the cellular or tissue-type origin of human biological traces found at crimes scenes is forensically relevant, as it allows evaluating the crime relevance of such traces and enables reconstructing the sequence of crime events. Messenger RNA and micro RNA markers are useful for forensic tissue identification, but provide challenges for linking RNA-identified cell/tissue types with DNA-identified trace donors, especially in mixed traces. DNA methylation markers overcome this problem, but provide technical challenges due to the DNA treatment required by most analysis methods. Here we introduce a novel type of DNA markers for forensic tissue identification analysed without prior DNA treatment, namely copy number variation (CNV). Using genome-wide CNV screening followed-up by targeted qPCR confirmation, and using qPCR analysis of additional CNV-like candidate DNA markers, in samples of several individuals from all commonly encountered forensically-relevant tissue types, we identified DNA markers specific for blood and semen, respectively. Preliminary forensic validation testing demonstrates that the developed qPCR assays are highly sensitive - delivering positive results down to picogram level of input DNA, specific, and can cope well with degraded DNA, providing suitable prerequisites for forensic applications. Moreover, we exemplified that using the CNV qPCR products as input material for subsequent forensic STR analysis delivered full STR profiles, opening-up new avenues of using the same DNA aliquot for both forensic purposes, tissue and individual identification. Provided additional forensic validation studies, we envision the application of these novel DNA markers for forensic tissue identification in future forensic casework. Such CNV markers are particularly useful for tissue identification in old/cold cases, where aged/old DNA extracts are available that contain no RNA and are not suitable for DNA methylation analysis due to limited DNA quantity and quality.

Changes in the Expression of MicroRNA in the Buildup Phase of Wasp Venom Immunotherapy: A Pilot Study

Background: Allergen-specific immunotherapy is the most effective method of treatment in allergy to wasp venom. However, its mechanism of action is still not fully understood. The aim of this study is to describe changes in microRNA (miRNA) expression in patients undergoing the buildup phase of venom immunotherapy. Methods: The study group comprised 7 adult patients with a history of severe systemic reactions after stinging by a wasp. In all patients, sensitization to wasp venom had been confirmed by skin tests and serum IgE. The buildup phase of wasp venom immunotherapy (VIT) was conducted according to an ultrarush protocol. In blood samples collected before and 24 h after completing the VIT buildup phase, 740 miRNAs were assessed. Results: Of the 740 miRNAs, 440 were detected in the study group, and in 5 expression was significantly changed after the buildup phase of VIT: miR-370, miR-539, miR-502-3p, miR-299, and miR-29c. Another 62 miRNAs changed 2-fold in some patients (nonsignificant), including increases in miR-143 (stimulating FOXp3 expression) and let-7d (reducing expression of IL-13, IL-6, and TLR4), and decreases in proinflammatory miR-301, miR-146b, miR-106, and miR-485. Conclusions: Several changes in miRNA expression have been found as a result of the buildup phase of wasp VIT, with lower expression of some miRNAs involved in allergic inflammation and higher expression of those possibly involved in tolerance induction. However, the role of the most significant changes is uncertain.

Polish population data on 15 autosomal STRs of AmpFlSTR NGM PCR kit.

The objective of the research was to provide a comprehensive database of autosomal microsatellite loci included in AmpFlSTR NGM PCR kit for a population of Poland considering possible genetic differentiation of a forensic interest. Fifteen STR markers were analyzed in 2041 unrelated individuals residing in eight geographically different regions. All the loci were found to be in Hardy-Weinberg equilibrium. The combined probability of match is 3.52 × 10(-19) and the combined Power of Exclusion is 0.9999998. The F(ST) estimate over all 15 STRs is 0.0051 for the Polish population. We established that a combined NGM database may be employed for a Polish population.

Different whole-genome amplification methods as a preamplification tool in Y-chromosome Loci analysis.

AbstractDegraded and low template DNA is often analyzed in forensic genetics laboratories. Reliable analysis of degraded and low template DNA is of great importance, because its results impact the quality and reliability of expert testimonies. Recently, a number of whole-genome amplification (WGA) methods have been proposed as preamplification tools improving quantity and quality of DNA. We chose, investigated, and compared 7 WGA methods to evaluate their ability to “recover” degraded and nondegraded DNA. These methods include degenerate oligonucleotide primed polymerase chain reaction, primer extension preamplification (PEP) polymerase chain reaction, GenomePlex WGA (Sigma), multiple displacement amplification, GenomiPhi Amplification Kit (Amersham Biosciences), restriction and circularization aided rolling circle amplification, and blunt-end ligation–mediated WGA. Recently, we have described the comparison of these methods’ efficiency and reliability using SGMPlus kit. However, Y-chromosome profiling is also often used in analysis of both nondegraded and degraded DNA. This includes criminal cases and investigation of kinship in male linage. Here we demonstrate the impact of WGA methods on Y-chromosome loci (Yfiler) reactivation.The best results for nondegraded DNA were obtained with GenomiPhi kit and PEP method. In the case of degraded DNA (200 base pairs), the most complete profiles were obtained with GenomePlex kit and PEP method. None of the analyzed methods allowed full reactivation of degraded (200 base pairs) DNA in terms of Y-chromosome loci profiling.

Population Studies of Two Ampflps and Two STRs Systems in a North Polish Population

Analysis of variable number of tandem repeats (VNTR) is widely used in forensic testing. PCR amplification of many polymorphic VNTR regions has been described. The most commonly amplified fragment length polymorphisms (AMPFLPs) loci, are D1S80, ApoB, D17S5 and COL2A1. Recently, a number of short tandem repeats (STRs) loci, have been described and applied to the forensic practice.