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.

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.

Evaluation of the predictive capacity of DNA variants associated with straight hair in Europeans.

DNA-based prediction of hair morphology, defined as straight, curly or wavy hair, could contribute to an improved description of an unknown offender and allow more accurate forensic reconstructions of physical appearance in the field of forensic DNA phenotyping. Differences in scalp hair morphology are significant at the worldwide scale and within Europe. The only genome-wide association study made to date revealed the Trichohyalin gene (TCHH) to be significantly associated with hair morphology in Europeans and reported weaker associations for WNT10A and FRAS1 genes. We conducted a study that centered on six SNPs located in these three genes with a sample of 528 individuals from Poland. The predictive capacity of the candidate DNA variants was evaluated using logistic regression; classification and regression trees; and neural networks, by applying a 10-fold cross validation procedure. Additionally, an independent test set of 142 males from six European populations was used to verify performance of the developed prediction models. Our study confirmed association of rs11803731 (TCHH), rs7349332 (WNT10A) and rs1268789 (FRAS1) SNPs with hair morphology. The combined genotype risk score for straight hair had an odds ratio of 2.7 and these predictors explained ∼ 8.2% of the total variance. The selected three SNPs were found to predict straight hair with a high sensitivity but low specificity when a 10-fold cross validation procedure was applied and the best results were obtained using the neural networks approach (AUC=0.688, sensitivity=91.2%, specificity=23.0%). Application of the neural networks model with 65% probability threshold on an additional test set gave high sensitivity (81.4%) and improved specificity (50.0%) with a total of 78.7% correct calls, but a high non-classification rate (66.9%). The combined TTGGGG SNP genotype for rs11803731, rs7349332, rs1268789 (European frequency=4.5%) of all six straight hair-associated alleles was identified as the best predictor, giving >80% probability of straight hair. Finally, association testing of 44 SNPs previously identified to be associated with male pattern baldness revealed a suggestive association with hair morphology for rs4679955 on 3q25.1. The study results reported provide the starting point for the development of a predictive test for hair morphology in Europeans. More studies are now needed to discover additional determinants of hair morphology to improve the predictive accuracy of this trait in forensic analysis.

Evaluation of DNA variants associated with androgenetic alopecia and their potential to predict male pattern baldness.

Androgenetic alopecia, known in men as male pattern baldness (MPB), is a very conspicuous condition that is particularly frequent among European men and thus contributes markedly to variation in physical appearance traits amongst Europeans. Recent studies have revealed multiple genes and polymorphisms to be associated with susceptibility to MPB. In this study, 50 candidate SNPs for androgenetic alopecia were analyzed in order to verify their potential to predict MPB. Significant associations were confirmed for 29 SNPs from chromosomes X, 1, 5, 7, 18 and 20. A simple 5-SNP prediction model and an extended 20-SNP model were developed based on a discovery panel of 305 males from various European populations fitting one of two distinct phenotype categories. The first category consisted of men below 50 years of age with significant baldness and the second; men aged 50 years or older lacking baldness. The simple model comprised the five best predictors: rs5919324 near AR, rs1998076 in the 20p11 region, rs929626 in EBF1, rs12565727 in TARDBP and rs756853 in HDAC9. The extended prediction model added 15 SNPs from five genomic regions that improved overall prevalence-adjusted predictive accuracy measured by area under the receiver characteristic operating curve (AUC). Both models were evaluated for predictive accuracy using a test set of 300 males reflecting the general European population. Applying a 65% probability threshold, high prediction sensitivity of 87.1% but low specificity of 42.4% was obtained in men aged <50 years. In men aged ≥50, prediction sensitivity was slightly lower at 67.7% while specificity reached 90%. Overall, the AUC=0.761 calculated for men at or above 50 years of age indicates these SNPs offer considerable potential for the application of genetic tests to predict MPB patterns, adding a highly informative predictive system to the emerging field of forensic analysis of externally visible characteristics.

Increased risk of developing cutaneous malignant melanoma is associated with variation in pigmentation genes and VDR, and may involve epistatic effects.

Cutaneous malignant melanoma (CMM) is a malicious human skin cancer that primarily affects individuals with light pigmentation and heavy sun exposure, but also has a known familial association. Multiple genes and polymorphisms have been reported as low-penetrance susceptibility loci for CMM. Here, we examined 33 candidate polymorphisms located in 11 pigmentation genes and the vitamin D receptor gene (VDR) in a population of 130 cutaneous melanoma patients and 707 healthy controls. The genotypes obtained were evaluated for main association effects and potential gene-gene interactions. MC1R, TYR, VDR and SLC45A2 genes were found to be associated with CMM in our population. The results obtained for major function MC1R mutations were the most significant [with odds ratio (OR)=1.787, confidence interval (CI)=1.320-2.419 and P=1.715(-4)], followed by TYR (rs1393350) (with OR=1.569, CI=1.162-2.118, P=0.003), VDR (GCCC haplotype in rs2238136-rs4516035-rs7139166-rs11568820 block) (with OR=5.653, CI=1.794-17.811, P=0.003) and SLC45A2 (rs16891982) (with OR=0.238, CI=0.057-0.987, P=0.048). The study also detected significant intermolecular epistatic effects between MC1R and TYR, SLC45A2 and VDR, HERC2 and VDR, OCA2 and TPCN2, as well as intramolecular interactions between variants within the genes MC1R and VDR. In the final multivariate logistic regression model for CMM development, only the gene-gene interactions discovered remained significant, showing that epistasis may be an important factor in the risk of melanoma.

Potential association of single nucleotide polymorphisms in pigmentation genes with the development of basal cell carcinoma.

The risk of developing skin cancers is dependent on a combination of environmental factors and personal genetic predispositions. Basal cell carcinoma (BCC) has been associated with single nucleotide polymorphisms in several pigmentation genes; however, there is still controversy concerning the mechanism by which these variants may increase the risk of BCC. The pathway may lead to pigmentation alone, but evidence for their independent influence is growing. Using a single base extension protocol, candidate polymorphisms within 11 known pigment-related genes were studied for their association with BCC in a population sample consisting of 164 patients and 707 controls. The significance of variation within the MC1R gene was confirmed and, in addition, position rs12203592 within the IRF4 gene was shown to be associated with BCC. These associations remained significant after adjustment for skin color. Gene-gene interactions were found to influence susceptibility to BCC. Among interacting genes are the two above-mentioned loci with main effect on BCC risk and additionally KITLG, TYRP1, ASIP and TYR. The obtained results indicate that polymorphism at MC1R and IRF4 constitute pigmentation-independent risk factor in the development of BCC. Moreover, susceptibility to BCC may be influenced by epistatic effects between pigmentation genes.