Spitting in the wind?-The challenges of RNA sequencing for biomarker discovery from saliva.

Forensic trace contextualization, i.e., assessing information beyond who deposited a biological stain, has become an issue of great and steadily growing importance in forensic genetic casework and research. The human transcriptome encodes a wide variety of information and thus has received increasing interest for the identification of biomarkers for different aspects of forensic trace contextualization over the past years. Massively parallel sequencing of reverse-transcribed RNA ("RNA sequencing") has emerged as the gold standard technology to characterize the transcriptome in its entirety and identify RNA markers showing significant expression differences not only between different forensically relevant body fluids but also within a single body fluid between forensically relevant conditions of interest. Here, we analyze the quality and composition of four RNA sequencing datasets (whole transcriptome as well as miRNA sequencing) from two different research projects (the RNAgE project and the TrACES project), aiming at identifying contextualizing forensic biomarker from the forensically relevant body fluid saliva. We describe and characterize challenges of RNA sequencing of saliva samples arising from the presence of oral bacteria, the heterogeneity of sample composition, and the confounding factor of degradation. Based on these observations, we formulate recommendations that might help to improve RNA biomarker discovery from the challenging but forensically relevant body fluid saliva.

Forensically relevant anatomical brain regions cannot be sub-differentiated by RNA expression analysis.

The contextualization of biological traces generated by severe head injuries can be beneficial for criminal investigations. Here we aimed to identify and validate mRNA candidates for a robust sub-differentiation of forensically and traumatologically relevant brain regions. To this purpose, massively parallel sequencing of whole transcriptomes in sample material taken from four different areas of the cerebral cortex (frontal, temporal, parietal, occipital lobe) was performed, followed by bioinformatical data analysis, classification, and biostatistical candidate selection. Candidates were evaluated by Multiplex-RT-PCR and capillary electrophoresis. Only a weak relative upregulation and solely for candidates expressed in the parietal lobe was observed. Two candidates with upregulation in the cerebellar region (PVALB and CDR2L) were chosen for further investigation; however, PVALB could not reliably and repeatedly be detected in any lobe whereas CDR2L was detectable in all lobes. Consequently, we suggest that differences in mRNA expression between four regions of the cerebral cortex are too small and less pronounced to be useful for and applicable in forensic RNA analysis. We conclude that sub-differentiation of these brain regions via RNA expression analysis is generally not feasible within a forensic scope.

Ten years of molecular ballistics-a review and a field guide.

Molecular ballistics combines molecular biological, forensic ballistic, and wound ballistic insights and approaches in the description, collection, objective investigation, and contextualization of the complex patterns of biological evidence that are generated by gunshots at biological targets. Setting out in 2010 with two seminal publications proving the principle that DNA from backspatter collected from inside surfaces of firearms can be retreived and successfully be analyzed, molecular ballistics covered a lot of ground until today. In this review, 10 years later, we begin with a comprehensive description and brief history of the field and lay out its intersections with other forensic disciplines like wound ballistics, forensic molecular biology, blood pattern analysis, and crime scene investigation. In an application guide section, we aim to raise consciousness to backspatter traces and the inside surfaces of firearms as sources of forensic evidence. Covering crime scene practical as well as forensic genetic aspects, we introduce operational requirements and lay out possible procedures, including forensic RNA analysis, when searching for, collecting, analyzing, and contextualizing such trace material. We discuss the intricacies and rationales of ballistic model building, employing different tissue, skin, and bone simulants and the advantages of the "triple-contrast" method in molecular ballistics and give advice on how to stage experimental shootings in molecular ballistic research. Finally, we take a look at future applications and prospects of molecular ballistics.

Nothing but hot air?-On the molecular ballistic analysis of backspatter generated by and the hazard potential of blank guns.

Blank cartridge guns are prevalent especially in countries with laws restricting access to conventional firearms, and it is a common misconception that these weapons are harmless and only used as toys or for intimidation. However, although their harming potential is well-documented by numerous reports of accidents, suicides, and homicides, a systematic molecular biological investigation of traces generated by shots from blank cartridges at biological targets has not been done so far. Herein, we investigate the occurrence and analyzability of backspatter generated by shots of different types of blank cartridge guns firing different types of blank ammunition at ballistic gelatin model cubes doped with human blood and radiological contrast agent soaked into a spongious matrix and covered with three different variants of skin simulants. All skin simulants were penetrated, and backspatter was created in 100% of the shots in amounts sufficient for forensic short tandem repeat (STR) typing that resulted in the correct identification of the respective blood donor. Visible backspatter was documented on the muzzle and/or inside the barrel in all cases, and in 75% of cases also on the outer surfaces and on the shooter's hand(s). Wound cavities were measured and ranged between 1 and 4.5 cm in depth. Discussing our findings, we provide recommendations for finding, recovering, and analyzing trace material from blank guns, and we demonstrate the considerable hazard potential of these devices, which is further emphasized by the presentation of a comprehensive overview of the pertinent literature on injuries inflicted by blank guns.

A distant relationship?-investigation of correlations between DNA isolated from backspatter traces recovered from firearms, wound profile characteristics, and shooting distance.

In molecular ballistics, where traces originating from the use of firearms against biological targets are investigated, "backspatter" traces are of particular importance. This biological material comprising blood and tissue from the victim is propelled back from the bullet entry site towards the direction of the shooter and can consolidate and persist on the inner and outer surfaces of the firearm, from where it can be collected and analyzed. Thus, a connection between the weapon and the victim can be established solely by molecular biological trace analysis. For the criminalistic investigation of gun-related crimes, the determination of the distance between the weapon and the victim can be of critical importance in reconstructing the circumstances of a crime. In this study, we investigated possible correlations between the shooting distance and the amount of backspatter in/on the used firearm. To this purpose, we employed a previously established skull model and performed shootings in triplicates from various distances up to 50 cm with two types of handguns (pistol and revolver). Backspatter was collected from various sampling locations, and DNA contents were quantified. A post-shooting wound channel evaluation was conducted by optical and radiological evaluation. The obtained DNA yields varied considerably between replicates from the same and from different distances. In contrast, apart from contact shots, no meaningful differences were observable in wound channel evaluations. In summary, no meaningful correlation between backspatter distribution and DNA yields, the shooting distance and the condition of the wound channel could be established.

All mixed up?-genotype change after stem cell transplantation impeded verification of 21-year-old semen sample-a case report.

We report a case of identity testing in which a patient charged us with the verification of a semen sample that he had donated and cryopreserved more than 20 years ago and now was suspecting of having been inadvertently interchanged. We found a non-match of the DNA profiles of the patient's blood and the semen samples but could show that this was due to the patient having received a stem cell transplantation of his full brother as part of a cancer therapy in 1997 which was not known to us when the samples were first tested. Also, the blood and semen samples exhibited a low probability of full sibship at first supporting the patient's suspicion that his semen sample might indeed have been interchanged. By also testing Y-STRs and including hair roots in the DNA analysis, we could show that the transplant did indeed originate from the patient's brother and that the semen sample did indeed originate from the patient.

Evaluation of the backspatter generation and wound profiles of an anatomically correct skull model for molecular ballistics.

Molecular ballistics connects the molecular genetic analysis of biological traces with the wounding events and complex forensic traces investigated in terminal ballistics. Backspatter, which originates from a projectile hitting a biological target when blood and/or tissue is propelled back into the direction of the gun, is of particular interest; those traces can consolidate and persist on the outer and inner surfaces of firearms and serve as evidence in criminal investigations. Herein, we are the first to present an anatomically correct head model for molecular ballistic research based on a polyurethane skull replica enclosing tissue-simulating sponge material that is doped with "triple-contrast" mixture (EDTA-blood, acrylic paint, and an x-ray contrast agent). Ten percent ballistic gelatin was used as brain simulant. We conducted contact and intermediate-range shots with a Glock 19 pistol (9 mm Luger), a pump-action shotgun (12/70 slugs), and blank cartridge handguns. Each shot was documented by a high-speed camera at 35,000 fps. Apart from the blank cartridge guns, all gunshots penetrated the skull model and created backspatter, which was recovered from the distal part of the barrels and analyzed. The pistol contact shots and one of three shotgun shots yielded full STR profiles. While the shotgun slugs destroyed the skulls, the remaining models could be used for radiological and optical fracture and wound channel evaluation. Known backspatter mechanisms and their respective timing could be confirmed visually by video analysis. Our complete model setup proved to be well applicable to molecular ballistic research as well as wound channel and fracture pattern investigation.

RNA/DNA co-analysis from bloodstains on aged polyvinyl-alcohol gloves prepared for securing evidence from the hands of victims of fatal gunshot injuries.

In contrast to cumulative techniques (e.g., tape-lift) for qualitative gunshot residues (GSR) analysis, topographic methods are commonly applied to preserve the integrity of evidence from a shooter's or victim's hand in cases of gun-related crimes. Topographic sampling techniques employing adhesive foils, latex, or the polyvinyl alcohol (PVAL) method enable unambiguous sampling of biological and non-biological trace material while preserving its spatial distribution and relation to each other. The PVAL method in particular allows for a topographically veridic and quantitative conservation of traces of GSR and biological stains that are embedded in the PVAL glove, because it completely removes these traces from the hand. The present study investigated the success rates of STR profiling and the detection of blood and brain-specific gene expression from minimal traces of blood splatter as well as parallel to the positive detection of gunshot residues embedded in 17 PVAL gloves taken from the hands of deceased persons in the context of homicide cases in the period between 1996 and 2003. The water-soluble PVAL matrix is shown to be fully compatible with successful STR profiling and the detection of blood- and brain-specific miRNA expression, even after up to 20 years of storage, demonstrating that this sampling technique offers advantages compared to other more simplistic sampling methods like taping.

Alterations in gene expression after gamma-hydroxybutyric acid intake-A pilot study.

Gamma-hydroxybutyric acid (GHB) acts as an agonist of the GABAB receptor, where GHB induces a depressant effect in the central nervous system. Besides its therapeutic application, GHB is also used as a date rape drug. However, the detection of GHB ingestion proves to be difficult due to its narrow detection window. The aim of this pilot study was to assess differential gene expressions after GHB intake to identify potential biomarkers for the detection of GHB intake. To this aim, alteration in gene expression of ALDH5A1, AKR7A2, EREG, and PEA15 was investigated via quantitative PCR (qPCR). Data normalization was based on a previously established and empirically derived normalization strategy. Blood samples of patients (n = 3) therapeutically taking sodium oxybate solution (GHB) and of donors without GHB intake (n = 49) were analyzed and compared. All qPCR procedures and results are reported according to the MIQE guidelines. Investigation of suitable reference genes using established algorithms suggested PPIB and FPGS as best-suited normalizers. Alterations in gene expression relating to GHB intake could not be confirmed to a forensically sufficient degree. However, significant differences in expression of EREG in the control group were observed, when time-point of sample collection was considered, indicating circadian rhythm. The study's main limitation is the small number of study subjects. Herein, we are first to present an empirically derived strategy for a robust normalization of qPCR data from the analysis of GHB-induced gene expression in human blood. We present results of the analysis of differential expression of ALDH5A1, AKR7A2, EREG, and PEA15 in the GHB-negative population. Finally, we report our findings on the effect of GHB intake on the expression of these genes and their presumable potential as GHB biomarkers.

RNA/DNA co-analysis on aged bloodstains from adhesive tapes used for gunshot residue collection from hands.

In cases of firearm related fatalities a systematic investigation at the scene of death is indispensable to differentiate between self-inflicted and homicidal gunshot injuries. A common method to preserve gunshot residues (GSR) is their collection using adhesive tapes. However, the biological material gathered at the same time by the tapes would be of special interest if backspatter, ejected from the entrance wound against the direction of fire, could be detected. In the present study we examined the success rate of co-analysis of RNA and DNA recovered from biological traces sampled with adhesive tapes. The material originated from eight cases of fatal gunshots, taken from the hands of suspects or victims, examined 5 to 19 years ago for GSR. For all types of adhesive tapes tested, quantity and quality of the co-extracted nucleic acids was insufficient for successful DNA profiling, but was sufficient for the detection of blood-specific micro RNA (miRNA). In summary, sampling trace evidence from the hands of persons involved in fatal gunshots with adhesive tapes has a long-term detrimental effect on biological traces.

Staurosporine analogs promote distinct patterns of process outgrowth and polyploidy in small cell lung carcinoma cells.

We have recently shown that staurosporine mediates the conversion of small cell lung carcinoma (SCLC) cells into a neuron-like process-bearing phenotype. Here, we have extended these studies to the staurosporine analogs K252a, lestaurtinib, PKC412, stauprimide, and UCN-01 and analyzed their influence on process extension, cell cycle distribution, and induction of polyploidy in four SCLC cell lines. In GLC-2 cells, all compounds provoked extensive process formation with the exception of PKC412 that showed no response. In H1184 cells, process formation was predominantly induced by staurosporine and, to lesser extent, in lestaurtinib-, stauprimide-, and UCN-01-treated cells. In the presence of K252a or PKC412, cells became bipolar and spindle shaped or showed pronounced cell flattening. In GLC-36 and SCLC-24H cells, only cell flattening was detectable. Process formation was reversible upon drug removal as shown for GLC-2 and H1184 cells. Fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) analysis indicated the induction of polyploidy in all staurosporine and in two out of four stauprimide-treated SCLC cell lines. For other staurosporine analogs, polyploidy was observed only in UCN-01-treated GLC-36 cells and in K252a-treated H1184 and GLC-36 cells. The presence of staurosporine or its analogs did not alter the constitutive activation pattern of the canonical Akt/PI3K or MEK/extracellular signal-regulated kinase (ERK)1/2 signaling pathways nor could we detect an influence of stauprimide application on the expression level of the c-Myc oncogene. These data demonstrate that in SCLC cells, albeit a higher substrate specificity, staurosporine analogs can induce staurosporine-comparable effects.

The 'triple contrast' method in experimental wound ballistics and backspatter analysis.

In practical forensic casework, backspatter recovered from shooters' hands can be an indicator of self-inflicted gunshot wounds to the head. In such cases, backspatter retrieved from inside the barrel indicates that the weapon found at the death scene was involved in causing the injury to the head. However, systematic research on the aspects conditioning presence, amount and specific patterns of backspatter is lacking so far. Herein, a new concept of backspatter investigation is presented, comprising staining technique, weapon and target medium: the 'triple contrast method' was developed, tested and is introduced for experimental backspatter analysis. First, mixtures of various proportions of acrylic paint for optical detection, barium sulphate for radiocontrast imaging in computed tomography and fresh human blood for PCR-based DNA profiling were generated (triple mixture) and tested for DNA quantification and short tandem repeat (STR) typing success. All tested mixtures yielded sufficient DNA that produced full STR profiles suitable for forensic identification. Then, for backspatter analysis, sealed foil bags containing the triple mixture were attached to plastic bottles filled with 10% ballistic gelatine and covered by a 2-3-mm layer of silicone. To simulate backspatter, close contact shots were fired at these models. Endoscopy of the barrel inside revealed coloured backspatter containing typable DNA and radiographic imaging showed a contrasted bullet path in the gelatine. Cross sections of the gelatine core exhibited cracks and fissures stained by the acrylic paint facilitating wound ballistic analysis.

Simultaneous analysis of nuclear and mitochondrial DNA, mRNA and miRNA from backspatter from inside parts of firearms generated by shots at "triple contrast" doped ballistic models.

When a firearm projectile hits a biological target a spray of biological material (e.g., blood and tissue fragments) can be propelled from the entrance wound back towards the firearm. This phenomenon has become known as "backspatter" and if caused by contact shots or shots from short distances traces of backspatter may reach, consolidate on, and be recovered from, the inside surfaces of the firearm. Thus, a comprehensive investigation of firearm-related crimes must not only comprise of wound ballistic assessment but also backspatter analysis, and may even take into account potential correlations between these emergences. The aim of the present study was to evaluate and expand the applicability of the "triple contrast" method by probing its compatibility with forensic analysis of nuclear and mitochondrial DNA and the simultaneous investigation of co-extracted mRNA and miRNA from backspatter collected from internal components of different types of firearms after experimental shootings. We demonstrate that "triple contrast" stained biological samples collected from the inside surfaces of firearms are amenable to forensic co-analysis of DNA and RNA and permit sequence analysis of the entire mtDNA displacement-loop, even for "low template" DNA amounts that preclude standard short tandem repeat DNA analysis. Our findings underscore the "triple contrast" method's usefulness as a research tool in experimental forensic ballistics.

RNA Analysis in Forensic Molecular Biology.

BACKGROUND: Different types of RNA take on multiple crucial functions in living cells and tissues. Messenger RNA (mRNA) is a temporary molecular carrier of genetic information. Analysis of the composition of all mRNA contained in a cell at a given moment, the so-called transcriptome, enables the determination of the type of cell and its condition, e.g., in pathologically altered states. METHODS: This review is based on pertinent publications retrieved by a selective literature search. RESULTS: The analysis of differential gene expression has already been used in forensic molecular biology to determine the type of tissue contained in biological specimens. It is also being used in criminal investigations to determine the composition of mixed traces of various bodily fluids and/or organ tissues. The method is limited by degradation of the mRNA molecules through environmental influences. The use of newly developed molecular biological methods such as massive parallel sequencing can expand the information obtainable by this investigative method. Current research also addresses the forensic potential of deriving relevant information about the crime-e.g., its timing, or the condition of the involved persons-from the totality of mRNA species present in the specimens. CONCLUSION: Forensic RNA analysis can yield a great deal of relevant information. It is likely to be applicable in a much wider variety of forensic situations in the near future.

Comprehensive body fluid identification and contributor assignment by combining targeted sequencing of mRNA and coding region SNPs.

Forensic genetic analyses aim to retrieve as much information as possible from biological trace material recovered from crime scenes. While standard short tandem repeat (STR) profiling is essential to individualize biological traces, its significance is diminished in crime scenarios where the presence of a suspect's DNA is acknowledged by all parties. In such cases, forensic (m)RNA analysis can provide crucial contextualizing information on the source level about a trace's composition, i.e., body fluids/tissues, and has therefore emerged as a powerful tool for modern forensic investigations. However, the question which of several suspects contributed a specific component (body fluid) to a mixed trace cannot be answered by RNA analysis using conventional methods. This individualizing information is stored within the sequence of the mRNA transcripts. Massively parallel sequencing (MPS) represents a promising alternative, offering not only higher multiplex capacity, but also the typing of individual coding region SNPs (cSNPs) to enable the assignment of contributors to mixture components, thereby reducing the risk of association fallacies. Herein, we describe the development of an extensive mRNA/cSNP panel for targeted sequencing on the IonTorrent S5 platform. Our panel comprises 30 markers for the detection of six body fluids/tissues (blood, saliva, semen, skin, vaginal and menstrual secretion), along with 70 linkage-controlled cSNPs for contributor assignment. It exhibited high reliable detection sensitivity with RNA inputs down to 0.75 ng and a conservatively calculated probability of identity of 0.03 - 6 % for individual body fluid-specific cSNP profiles. Limitations and areas for future work include RNA-related allele imbalances, inclusion of markers to correctly identify rectal mucosa and the optimization of specific markers. In summary, our new panel is intended to be a major step forward to interpret biological evidence at sub-source and source level based on cSNP attribution of a body fluid component to a suspect and victim, respectively.

Selecting mRNA markers in blood for age estimation of the donor of a biological stain.

RNA has gained a substantial amount of attention within the forensic field over the last decade. There is evidence that RNAs are differentially expressed with biological age. Since RNA can be co-extracted with DNA from the same piece of evidence, RNA-based analysis appears as a promising molecular alternative for predicting the biological age and hence inferring the chronological age of a person. Using RNA-Seq data we searched for markers in blood potentially associated with age. We used our own RNA-Seq data from dried blood stains as well as publicly available RNA-Seq data from whole blood, and compared two different approaches to select candidate markers. The first approach focused on individual gene analysis with DESeq2 to select the genes most correlated with age, while the second approach employed lasso regression to select a set of genes for optimal prediction of age. We present two lists with 270 candidate markers, one for each approach.

Monoamine oxidase A gene polymorphism and the pathogenesis of sudden infant death syndrome.

OBJECTIVES: To test the hypothesis that there is a significant association between functionally relevant allelic variants of the monoamine oxidase A (MAO-A) polymorphism and sudden infant death syndrome (SIDS). STUDY DESIGN: In a case-control study of 142 cases of SIDS and 280 sex-matched control cases, the distribution of allelic and genotype variants of a promoter polymorphism of the MAO-A gene was examined using polymerase chain reaction locus amplification and fluorescence based fragment length analysis. RESULTS: There was a significantly differential distribution of allelic and genotype variants between females with SIDS and controls. Moreover, there was a significant association between SIDS in females and allelic and genotype variants, each related to a higher transcriptional activity at the MAO-A locus. CONCLUSIONS: Our results suggest a role of MAO-A in female SIDS pathogenesis exerted by functionally relevant allelic and genotype variants of the MAO-A polymorphism. However, with the complex and inconsistent evidence available to date, the impact of the MAO-A promoter polymorphism on SIDS etiology remains unclear.

Functional single-nucleotide variant of HSPD1 in sudden infant death syndrome.

BACKGROUND: An insufficient stress response due to a genetically impaired heat shock protein (Hsp) could play a role in the pathogenesis in a subgroup of sudden infant death syndrome (SIDS) cases. Herein, we are the first to investigate whether a functionally impairing and thus pathogenic variant of the gene for Hsp60, encoded by HSPD1 (rs72466451), is correlated with the occurrence of SIDS. METHODS: In a case-control study of a series of 133 cases of SIDS and 192 gender-matched German Caucasian control cases, the occurrence and distribution of the HSPD1 single-nucleotide variant (SNV) was analyzed using SNV genotyping by minisequencing. RESULTS: The results show significantly increased frequency of the pathogenic variant of the HSPD1 SNV in a subgroup (4.5%) of SIDS cases. CONCLUSION: The results suggest that the pathogenic variant of rs72466451 may play a role in a subgroup of SIDS cases with impaired Hsp60-mediated stress response.

Persistence of biological traces in gun barrels--an approach to an experimental model.

Traces of backspatter in gun barrels after homicidal or suicidal contact shots may be a valuable source of forensic evidence. Yet, a systematic investigation of the persistence and durability of DNA from biological traces in gun barrels is lacking. Our aim was to generate a realistic model to emulate blood and tissue spatters in gun barrels generated by contact gunshots at biological targets and to analyse the persistence and typability of DNA recovered from such stains. Herein, we devise and evaluate three different models for the emulation of backspatter from contact shots: a gelatine-based model with embedded blood bags, a model based on a spongious matrix soaked with blood and covered with a thin plastic membrane and a head model consisting of an acrylic half sphere filled with ballistic gelatine and with blood bags attached to the sphere under a 3-mm silicone layer. The sampling procedure for all three models: a first shot was fired with several types of guns at each model construction and subsequently a second shot was fired at a backstop. Blood samples were collected after each shot by probing the inner surface of the front and rear end of the respective gun barrel with a sterile swab. DNA was then extracted and quantified and up to 20 different short tandem repeat (STR) systems were amplified to generate DNA profiles. Although DNA quantity and STR typing results were heterogenous between the models, all models succeeded in delivering full STR profiles even after more than one shot. We conclude that biological traces in gun barrels are robust and accessible to forensic analysis and that systematic examination of the inside of gun barrels may be advisable for forensic casework.