Recommendations of the Polish-speaking Working Group of the International Society for Forensic Genetics (ISFG-PL) regarding the disclosure of biological traces and the handling of evidence for identification tests. / Zalecenia Polskojezycznej Grupy Miedzynarodowego Towarzystwa Genetyki Sadowej (ISFG-PL) dotyczace ujawniania i identyfikacji sladów biologicznych przeznaczonych do badan genetycznych.

The purpose of this paper is to formulate recommendations for the disclosure of biological traces in the laboratory and the handling of forensic evidence submitted for identification tests, recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics. The paper organizes the knowledge of the most relevant stages of preliminary analysis of biological traces based on both literature sources and those resulting from years of research practice. Recommendations formulated in the course of multi-stage expert consultations contained in this study should be used in the development of laboratory procedures applied during the execution.

Examination of LT-DNA traces - literature overview and general recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics (ISFG-PL).

The available literature on traces characterised by a suboptimal amount of DNA, as well as expert research practice, show the complex nature of LT-DNA traces: from their detection and collection, through genetic analysis, up to the interpretation of final results. The aims of this paper are to systematise the current state of knowledge on handling LT-DNA traces and develop examination guidelines, as recommended by the Polish Speaking Working Group of the International Society for Forensic Genetics (ISFG-PL). The proposed guidelines should be followed by all Polish laboratories conducting forensic genetic analyses for the purpose of judicial proceedings.

Recommendations of the Polish Speaking Working Group of the International Society for Forensic Genetics on forensic Y chromosome typing.

Y chromosome typing has been performed in forensic genetic practice for more than 20 years. The latest recommendations of the DNA Commission of the International Society of Forensic Genetics (ISFG) concerning the application of Y-chromosomal markers in forensic genetics were published in 2006. The aim of this report is to recapitulate, systematise and supplement existing recommendations on the forensic analysis of polymorphism of the Y chromosome with standards already implemented in practice, new capabilities linked to the development of research techniques as well as current solutions used in statistical analysis. The recommendations have been adapted specifically to aspects related to the preparation of expert opinions in the field of forensic genetics in Poland. The Polish Speaking Working Group of the ISFG believes that the presented guidelines should become a standard implemented by all Polish laboratories performing Y chromosome typing for forensic purposes.

Dual amplification strategy for improved efficiency of forensic DNA analysis using NGM Detect™, NGM™ or Globalfiler™ kits.

The new generation of STR amplification kits with improved sensitivity and additional genetic markers is designed particularly for analyzing difficult traces with a high DNA degradation index, presence of inhibitors and low level of DNA. In the new NGM Detect™ kit, modifications including changing the primers' sequences and shortening of STR markers are introduced. The quality control system (IQCS, IQCL) used to detect DNA degradation and the presence of inhibitors in the sample is an additional feature. The purpose of this study was to compare the results of analysis of different kinds of typical casework samples obtained using NGM™ or GlobalFiler™ kits with those generated using the new NGM Detect™ kit. The results indicate that the tested kit is particularly useful for the analysis of challenging samples for which incomplete profiles are generated with the NGM™ or GlobalFiler™ kits. The increased number of positively typed alleles gives better statistical parameters in genetic identification cases. We conclude that the NGM Detect™ kit can be recommended for the double amplification protocol together with the NGM or GlobalFiler™ kits.

Development of a forensically useful age prediction method based on DNA methylation analysis.

Forensic DNA phenotyping needs to be supplemented with age prediction to become a relevant source of information on human appearance. Recent progress in analysis of the human methylome has enabled selection of multiple candidate loci showing linear correlation with chronological age. Practical application in forensic science depends on successful validation of these potential age predictors. In this study, eight DNA methylation candidate loci were analysed using convenient and reliable pyrosequencing technology. A total number of 41 CpG sites was investigated in 420 samples collected from men and women aged from 2 to 75 years. The study confirmed correlation of all the investigated markers with human age. The five most significantly correlated CpG sites in ELOVL2 on 6p24.2, C1orf132 on 1q32.2, TRIM59 on 3q25.33, KLF14 on 7q32.3 and FHL2 on 2q12.2 were chosen to build a prediction model. This restriction allowed the technical analysis to be simplified without lowering the prediction accuracy significantly. Model parameters for a discovery set of 300 samples were R(2)=0.94 and the standard error of the estimate=4.5 years. An independent set of 120 samples was used to test the model performance. Mean absolute deviation for this testing set was 3.9 years. The number of correct predictions ±5 years achieved a very high level of 86.7% in the age category 2-19 and gradually decreased to 50% in the age category 60-75. The prediction model was deterministic for individuals belonging to these two extreme age categories. The developed method was implemented in a freely available online age prediction calculator.

Examination of DNA methylation status of the ELOVL2 marker may be useful for human age prediction in forensic science.

Age estimation in forensic investigations may complement the prediction of externally visible characteristics and the inference of biogeographical ancestry, thus allowing a better description of an unknown individual. Multiple CpG sites that show linear correlation between age and degree of DNA methylation have been identified in the human genome, providing a selection of candidates for age prediction. In this study, we optimized an assay based on bisulfite conversion and pyrosequencing of 7 CpG sites located in the ELOVL2 gene. Examination of 303 blood samples collected from individuals aged 2-75 years allowed selection of the most informative site, explaining 83% of variation in age. The final linear regression model included two CpG sites in ELOVL2 and enabled age prediction with R(2)=0.859, prediction error=6.85 and mean absolute deviation MAD=5.03. Examination of a testing set of 124 blood samples (MAD=5.75) showed that 68.5% of samples were correctly predicted, assuming that chronological and predicted ages matched ± 7 years. It was found that the ELOVL2 methylation status in bloodstains had not changed significantly after 4 weeks of storage in room temperature conditions. Analysis of 45 bloodstains deposited on tissue paper after 5, 10 and 15 years of storage in room conditions indicated that although a gradual decrease of positive PCR results was observed, the general age prediction success rate remained similar and equaled 60-78%. The obtained results show that the ELOVL2 locus provides a very good source of information about human chronological age based on analysis of blood, including bloodstains, and it may constitute a powerful and reliable predictor in future forensic age estimation models.

Genetic variation of 15 autosomal STR loci in a population sample from Poland.

Fifteen autosomal STR loci included in AmpFlSTR NGM kit were analyzed in 154 unrelated individuals from Poland. This multiplex kit enables simultaneous amplification of 10 standard STR loci included in AmpFlSTR SGM Plus kit (D3S1358, vWA, D16S539, D2S1338, D8S1179, D19S433, TH01, FGA, D21S11 and D18S51) and five new mini- and midi-STR loci (D10S1248, D22S1045, D2S441, D1S1656 and D12S391). Population study was conducted to evaluate usefulness of the loci (especially the five new microsatellite systems) in forensic genetic identification examinations. All 15 markers were found to be in Hardy-Weinberg equilibrium. The combined probability of match for the 15 studied STR loci was 3.998 x 10(-19). The same parameter calculated for five new microsatellite loci equaled 8.83 x 10(-7). Discrimination power was particularly high in case of D1S1656 (0.975) and D12S391 (0.972) STR loci.

A population data for 17 Y-chromosome STR loci in South Poland population sample--some DYS458.2 variants uncovered and sequenced.

Seventeen Y-chromosomal short tandem repeat loci were analyzed in a sample of 435 unrelated healthy male individuals from Southern Poland (including highlanders from Tatra Mountains). One duplication in the locus DYS389II (29,30) and five microvariant alleles in the locus DYS458 were found. The most frequent haplotype, found in three individuals, was as follows (in the order of Yfiler loci): {16, 13, 25, 30, 15, 15, 11/14, 13, 11, 11, 10, 23, 11, 13, 14, 11, 21}. Gene diversity for South Poland population amounts close to 1.000. Performed differentiation test between all pairs of samples, based on haplotype frequencies, represented as non-differentiation exact P values indicates that there is no statistically significant differences in haplotype frequencies between South Poland and Austrian as well as South Poland and Wallachian populations.