Experiences in successive forensic analysis of GSR and DNA traces collected on a single tape lift stub.

Gunshot residue (GSR) and deoxyribonucleic acid (DNA) analyses are used in the reconstruction of shooting incidents involving firearms. In both fields, adhesive tapes are commonly applied to recover traces. Using a simultaneous sampling approach for retrieving both types of traces from objects related to a shooting can be powerful. We present the results for DNA testing and GSR analysis using "DNA stubs" to recover both types of traces in one sampling. The analyses are successive: stubs are first examined with scanning electron microscopy for the presence of GSR and next subjected to DNA extraction and short tandem repeat (STR) typing. The results show no negative effect on the DNA and GSR results. The risk of DNA contamination during GSR analysis is addressed, showing that it is negligible. Our experiences in casework show that the approach can be successfully implemented.

Extended specificity studies of mRNA assays used to infer human organ tissues and body fluids.

Messenger RNA (mRNA) profiling is a technique increasingly applied for the forensic identification of body fluids and skin. More recently, an mRNA-based organ typing assay was developed which allows for the inference of brain, lung, liver, skeletal muscle, heart, kidney, and skin tissue. When applying this organ typing system in forensic casework for the presence of animal, rather than human, tissue is an alternative scenario to be proposed, for instance that bullets carry cell material from a hunting event. Even though mRNA profiling systems are commonly in silico designed to be primate specific, physical testing against other animal species is generally limited. In this study, human specificity of the organ tissue inferring system was assessed against organ tissue RNAs of various animals. Results confirm human specificity of the system, especially when utilizing interpretation rules considering multiple markers per cell type. Besides, we cross-tested our organ and body fluid mRNA assays against the target types covered by the other assay. Marker expression in the nontarget organ tissues and body fluids was observed to a limited extent, which emphasizes the importance of involving the case-specific context of the forensic samples in deciding which mRNA profiling assay to use and when for interpreting results.

Advancing forensic RNA typing: On non-target secretions, a nasal mucosa marker, a differential co-extraction protocol and the sensitivity of DNA and RNA profiling.

The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen for skin and variable results occur for vaginal and nasal mucosa. Lastly, we show that replicates are useful for interpretation of RNA data, as variations can be found even for true technical replicates. Increased numbers of replicates (over four) do, however, not cancel out the impact of this variation on data interpretation. Overall, the results of this study further forensic RNA profiling.

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.

Development of a mRNA profiling multiplex for the inference of organ tissues.

Forensic characterisation of organ tissue generally occurs through histological and immunological assays of limited sensitivity. Here, we explore an alternative approach and examine a total of 41 candidate mRNA markers for their ability to differentiate between brain, lung, liver, skeletal muscle, heart, kidney and skin. Various selection rounds are applied involving 85 organ tissues (36 excised autopsy specimens and 49 frozen tissue sections, with at least ten specimens for each organ type), 20 commercially available RNAs from different human tissues and at least two specimens of blood, saliva, semen, vaginal mucosa, menstrual secretion or touch samples. Finally, 14 markers are regarded tissue-specific and included in an endpoint RT-PCR multiplex together with one general muscle, one blood and one housekeeping marker. This 17-plex is successfully used to analyse a blind test set of 20 specimens including mixtures, and samples derived from stabbing of organ tissues. With the blind test set samples, it is shown that an earlier described interpretation strategy for RNA cell typing results [1] is also effective for tissue inference. As organ-typing is embedded in a procedure of combined DNA/RNA extraction and analysis, both donor and organ type information is derived from the same sample. Some autopsy specimens presented DNA profiles characteristic for degraded DNA. Nevertheless, the organ-typing multiplex could generate full RNA profiles, which is probably due to small sizes of the amplicons. This assay provides a novel tool for analysis of samples from violent crimes.