Molecular pathology in forensic medicine—Introduction

Forensic Science International 203 (2010) 3–14 Contents lists available at ScienceDirect Forensic Science International journal homepage: www.elsevier.com/locate/forsciint Molecular pathology in forensic medicine—Introduction§ Burkhard Madea a,*, Pekka Saukko b, Antonio Oliva c, Frank Musshoff a a b c Institute of Forensic Medicine, University of Bonn, Bonn, Germany Institute of Forensic Medicine, University of Turku, Turku, Finland Institute of Forensic Medicine, Catholic University Rome, Rome, Italy A R T I C L E I N F O A B S T R A C T Article history: Available online 21 August 2010 Techniques of molecular biology have improved diagnostic sensitivity, accuracy and validity in forensic medicine very much, especially in the field of identification (paternity testing, stain analysis). Since more than 10 years these techniques – meanwhile well established in clinical disciplines – are used also for other applications in forensic medicine: determination of cause and manner of death, tissue identification by mRNA and microRNA, examination of gene expression levels (survival time, time since death, cause of death), toxicogenetics. ß 2010 Elsevier Ireland Ltd. All rights reserved. Keywords: Molecular biology Cause of death Tissue identification Gene expression Evidential value 1. Introduction Molecular biology techniques have not only improved but also revolutionized diagnostics in forensic medicine—especially in the field of identification. Paternity testing, immigration cases and analysis of biological stains in criminal cases were the first fields in forensic medicine, which have benefited since 1985 from the new techniques. Since then many improvements have been achieved concerning technology, methods, DNA-systems and fields of application. Regarding methods and DNA-systems there was a quick follow-up from multilocus probes to singlelocus probes, RFLP systems over PCR-based systems, mt-DNA to SNP’s, concerning fields of application from paternity testing, stain analysis, determination of geographic origin to molecular photofitting. The main aim of all these efforts was more or less identification. However, meanwhile the methods of molecular biology are used also within clinical pathology and the application there is named molecular pathology. Molecular diagnostics has more or less infiltrated all branches of anatomic and clinical pathology. Molecular pathology in clinical practice assists in the diagnosis of disease, therapeutic choice, therapeutic outcome, monitoring, prognosis prediction of disease risk, directing preventive strategies, beginning of live choices, § This paper is part of the special issue entitled: Molecular Pathology in Forensic Medicine, Guest-edited by Burkhard Madea and Pekka Saukko. * Corresponding author at: Institute of Forensic Medicine, University of Bonn, Stiftsplatz 12, 53111 Bonn, Germany. Tel.: +49 228 738315; fax: +49 228 738368. E-mail address: [email protected] (B. Madea). 0379-0738/$ – see front matter ß 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2010.07.017 etc. [13,17,27,48,65,70]. Meanwhile every organ in clinical pathology has its own branch of molecular pathology, especially in neoplastic diseases. Molecular biological techniques are also used in forensic medicine and forensic pathology with its completely different tasks compared to clinical pathology (Fig. 1). One task has already been mentioned – identification – not only regarding paternity testing and stain analysis, but also specimen identification which is carried out on tissue [36,37,63]: – for instance identification of biological samples in cases of contamination of cytological slides with cells from different persons [36]; – do different body parts in criminal dismembering of bodies belonging to the same person [63]; – do biopsies belong to one or different persons. 1.1. Illustrative case examples 1.1.1. Case 1 In a case of criminal dismemberment of a body several body parts were found over a period of 7 years (Figs. 2 and 3). At first, 1987, the lower legs were found in plastic bags, 1 month later the thighs (Fig. 2a and b). That these body parts belong together was already evident from anatomical reasons. Two years later an advanced skeletonised skull was found with rests of degraded tissue and hair (Fig. 2c). Again 4 years later body parts of the trunk were found in a great plastic tub (Fig. 3). It was still possible to find out the probable cause of death (stab wounds of the heart) as to determine characteristics of the injuries (stabbing with a single edged knife). DNA typing was successful on all body parts and with the STR-systems used at that time it was [(Fig._1)TD$IG] 4 B. Madea et al. / Forensic Science International 203 (2010) 3–14 Fig. 1. Applications of molecular pathology in forensic medicine. [(Fig._2)TD$IG] possible to determine that all body parts belong to the same person (Fig. 4). 1.1.2. Case 2 A discrete section of the cytological slide of a female individual was obviously contaminated with pleura liquid of a female tumour patient (Fig. 5). Analysis of the cancerous pleura liquid and the healthy cells of the slide preparation showed different DNA profiles, indicating that the material originated from two different female individuals (Fig. 6). The DNA profile of the cell mixture revealed a heterogeneous pattern whereby the alleles could be assigned to the healthy and the tumour patient. Loss of heterozygosity was observed in 4 of 8 short tandem repeat systems for the pleura liquid and the cell mixture. Despite a low amount of DNA on the slide preparation and the occurrence LOH it was possible to clarify the case and to support the assumption that a drop of cancerous pleura liquid contaminated the cytological slide. 1.1.3. Case 3 In another case several biopsies from the large bowel were taken during colonoscopy. All biopsies were embedded into the same paraffin block (Fig. 7). While 7 of the biopsies were histologically normal, 1 showed a beginning infiltrating adenocarcinoma (Fig. 7). Unfortunately the gastroenterologist did not mark in which position in the gut the biopsies were taken. Due to the diagnosis of an infiltrating adenocarcinoma in one biopsy an operation of the gut was necessary. However, before the operation it had to be cleared if really all biopsies belonged to the same patient. The DNA profile of all biopsies was identical (Fig. 8). However, molecular pathology in forensic medicine meanwhile covers besides identification a broad field of applications and basic research which is more or less characteristic for this discipline: – determination of cause and manner of death [1–4,12,14,15,18– 21,31,38,39,56,57,60,67–69]; – determination of the time since death or the age of bloodstains [8,9]; – tissue identification [5–7,10,73,74]; – wound age estimation; – toxicogenetics [25,26,34,35,41,42,49,55,62,66,71]. In all of these fields basic research is carried out and practical applications have been developed [28–30,32,33,58,72–76]. More than 10 years ago Tester and Ackermann [67–69] were the first to report on a post-mortem molecular diagnosis of an arrhythmia (inherited LQTS in a 19-year-old woman who died after near drowning). For this post-mortem diagnosis they used the term ‘‘molecular autopsy’’. The main point in forensic pathology unlike clinical disciplines is of course that we often have to do with tissues, especially putrefied tissue (in contrast to clinical pathology), or formalinfixed paraffin-embedded tissue. Within this special issue it is not possible to address all aspects of molecular pathology in forensic Fig. 2. Body parts found over a period of 3 years (lower leg, thigh, skeletonised skull). [(Fig._3)TD$IG] B. Madea et al. / Forensic Science International 203 (2010) 3–14 5 Fig. 3. Body parts found 4 years after the skull in a plastic tub. medicine in detail. It was our aim to address some fundamental principles, frequent questions and future developments. 2. Cause of death The determination of the cause of death is carried out on different levels, using different substrates with differing level of evidence (Table 1). The lowest level is the external examination of a body taking into account the history of the deceased. When autopsies are used [(Fig._4)TD$IG] as control for the diagnosis of the cause of death the diagnosis is wrong in about 20–30% of cases, in 10% with therapeutic consequences for the patient (Class I error) [51,64]. On the level of autopsies, especially when ancillary investigations like histology, post-mortem biochemistry and toxicology are applied, more than 90–95% of cases can be solved [51,52]. However, there remains a small proportion of cases where the cause of death remains unclear after a thorough investigation, e.g. in cardiac arrhythmias. In many sudden deaths of children and young adults the cause of death remains unexplained and from epidemiologic studies it has been concluded that nearly 30% of sudden cardiac deaths in young people are autopsy negative. Obviously many of these cases have a genetic background [1,3,4,15,19,20,56,57,67– 69]. In these cases the detection of mutations giving rise to genetic alterations that may constitute the cause of death or predispose to death in critical situations may be important. However, the diagnostic significance of mutations has to be cleared on two levels: – What is the functional significance of the mutation? – Is this functional deviation sufficient to be the cause of death in the respective situation? The functional significance of mutations, e.g. for cardiac arrhythmias can be assessed by using molecular biology and electrophysiological techniques such as site-directed mutagenesis and whole-cell voltage patch clamp analysis. If the mutation can be accepted as cause of death depends on Fig. 4. PCR-results from the ‘‘stone-age’’ of the DNA era with identical typings for DNA extractions of various body parts. – what other causes of death have been excluded and – if the circumstances of death are in accordance with the functional deviations of the mutation. [(Fig._5)TD$IG] 6 B. Madea et al. / Forensic Science International 203 (2010) 3–14 Fig. 5. Cytological slide with tumour cells (a) and typical superficial squamous epithelium (b). The polymorphism or mutation may not be itself the cause of death but a predisposition in critical situations, e.g. physical stress, medication, etc. Before a mutation is accepted as reasonable cause of death all other causes of death should be excluded. The diagnosis is by exclusion after elimination of other causes and the validity is dependent on accuracy and completeness of the investigation. This requires that all ancillary investigations after autopsy are carried out. Autopsy findings and results of further investigations which are thought to be or contribute to the cause of death can be graded as follows (Table 2). This grading has already successfully been used since 100 years in forensic medicine concerning the significance of morphological findings [51]. In the majority of sudden cardiac deaths a clear pathological cause can be identified, although with a varying degree of confidence. While some findings are without any doubt the cause of death, others have a lower degree of certainty (from highly probable to uncertain; Table 3). [(Fig._6)TD$IG] Most cases of sudden death after the age of 35–40 are due to coronary arteriosclerosis. In contrast most cases of sudden death before the age of 35 are due to coronary anomalies (anomalous origin and course of coronary arteries), ruptured vessels (aorta), cardiac hypertrophy, cardiomyopathies or arrhythmias, the latter having a strong genetic background (structural cardiomyopathies like hypertrophy, arrhythmogenic, dilated, restrictive, non compaction cardiomyopathy; channelopathies like Brugada, Long QT, Short QT, WPW-syndrome (Fig. 9)). In a large molecular autopsy series of sudden unexplained death (n = 49) Tester and Ackermann were able to show that more than one-third of cases hosted a presumably pathogenic cardiac channel mutation [68]. In a cohort of SIDS in 5–10% of cases mutations were found after post-mortem cardiac channel genetic testing [67]. With mutation detection screening the working group of Carracedo was also successful in identifying several mutations both in adults and in children [14,60]. Fig. 6. Electropherogram with DNA profile of the pleural liquid of the female tumour patient. [(Fig._7)TD$IG] B. Madea et al. / Forensic Science International 203 (2010) 3–14 7 [(Fig._8)TD$IG] Fig. 7. Normal mucosa with part of a tubulo-villous adenoma (a); in another section invasive adenocarcinoma (b). Although the incidence of the sudden infant death syndrome declined in the last two decades it is still the leading cause of death for infants aged between 1 month and 1 year in developed countries [16]. After the identification of avoidable risk factors such as the prone sleeping position, bottle-feeding, smoking and overheating, subsequent reduction-of-the-risk campaigns have been initiated which led to a decline of the prevalence of SIDS. However, the pathophysiology is still unknown. Since hypertrophic cardiomyopathy (HCM) is one of the most prevalent causes of sudden cardiac death in younger adults (below the age of 35) it was hypothesized that HCM genes may be implicated in the development of sudden death in infants. The working group in Santiago de Compostela developed a MassArray genotyping platform analysing more than 600 HCM mutations. Meanwhile 140 cases of SIDS have been studied by using Sequenom MassArrayTM system and a total of 14 samples were detected as carrying genetic variants in 4 different genes [14]. Based on these preliminary results HCM mutations may have a relation with SIDS; however, functional studies have to be carried out to get information on the functional significance of these mutations in structural normal hearts. Fig. 8. Amplification results of DNA extraction from tubulo-villous adenoma and adenocarcinoma. 8 B. Madea et al. / Forensic Science International 203 (2010) 3–14 Table 1 Diagnostic levels for determining the cause of death. Diagnostic level Substrate of examination External examination Autopsy Intact body Organ systems, organs, injuries, macroscopic visible organ changes, diseases, disease related changes of organ systems Changes on tissue and cellular level Subcellular level Viral DNA Mutations Ion channel defects Slow metabolizers Disturbances of homeostasis, diabetes mellitus, uraemia, water-electrolyte-metabolism Substrates, metabolites, quantitation, distribution Histology including immunohistochemistry Molecular pathology Post-mortem biochemistry Toxicology Table 2 Grading of findings concerning the evidential value of cause of death. Group 1 Group 2 Group 3 Findings which are without any doubt the cause of death (e.g. ruptured myocardial infarction) Findings which may be sufficient to explain the cause of death but not sudden death. Further triggering events are necessary (e.g. acute coronary insufficiency in physical stress) Cause of death remains unclear after thorough investigation 2.1. Evidential value of new mutations An interesting case concerning the evidential value of new mutations was reported by Rognum (Bjarkoy case) [16,61]. In Norway a mother lost between 1992 and 1997 four subsequent infants. In the first two infants the diagnosis of the cause of death was sudden infant death syndrome. In the third child cause of death was pneumonia due to aspiration of amniotic fluid. In the first three children no death scene investigation was carried out. After the fourth child had died, suspicion was raised that all deaths were probably due to Munchausen-by-proxy-syndrome since the mother had always been the only person present. Many pathologists worldwide have seen similar cases with subsequent deaths of children. After the death of the fourth child a plastic bag was found in the kitchen garbage with the lip mark and DNA from the baby and fingerprints of the mother. The mother admitted having had the bag over the babies’ head but only two days before death. Munchausen-by-proxy was suspected and the cause of death probably suffocation supported by the finding of massive intra-alveolar haemorrhage in all lung sections. The mother was convicted for homicide of the fourth child, however, subsequent genetic testing revealed a previously not described mutation in the LQTS associated KCNH2 gene. Although the functional significance of this particular mutation was not known – obviously expression Table 3 Certainty of diagnosis of cause of death in sudden cardiac death autopsies (from Basso et al.). Certain Highly probable Uncertain Massive pulmonary embolism Stable atherosclerotic plaque with luminal stenosis >75% with or without healed myocardial infarction Haemopericardium due to aortic or cardiac rupture Anomalous origin of the LCA from the right sinus and inter-arterial course Cardiomyopathies (hypertrophic, arrhythmogenic right ventricular, dilated, others) Myxoid degeneration of the mitral valve with prolapse, with atrial dilatation or left ventricular hypertrophy and intact chordae Aortic stenosis with left ventricular hypertrophy ECG documented ventricular pre-excitation (Wolff–Parkinson–White syndrome, Lown-Ganong-Levine syndrome) ECG documented sinoatrial or AV block Minor anomalies of the coronary arteries from the aorta (RCA from the left sinus, LCA from the right without inter-arterial course, high take-off from the tubular portion, LCx originating from the right sinus or RCA, coronary ostia plication, fibromuscular dysplasia, intramural small vessel disease) Intra-myocardial course of a coronary artery (myocardial bridge) Mitral valve papillary muscle or chordae tendineae rupture with acute mitral valve incompetence and pulmonary edema Acute coronary occlusion due to thrombosis, dissection or embolism Anomalous origin of the coronary artery from the pulmonary trunk Neoplasm/thrombus obstructing the valvular orifice Thrombotic block of the valvular prosthesis Laceration/dehiscence/poppet escape of the valvular prosthesis with acute valvular insufficiency Massive acute myocarditis Congenital heart diseases, operated Focal myocarditis, hypertensive heart disease, idiopathic left ventricular hypertrophy Myxoid degeneration of the mitral valve with prolapse, without atrial dilatation or left ventricular hypertrophy and intact chordae Dystrophic calcification of the membranous septum (mitral annulus/aortic valve) Atrial septum lipoma AV node cystic tumour without ECG evidence of AV block, conducting system disease without ECG documentation Congenital heart diseases, un-operated with or without Eisenmenger syndrome Source: Data from Basso et al. AV: atrioventricular; ECG: electrocardiogram; LCA: left coronary artery; LCx: left circumflex branch; RCA: right coronary artery. [(Fig._9)TD$IG] B. Madea et al. / Forensic Science International 203 (2010) 3–14 9 Fig. 9. Genetic cause of sudden cardiac death (according to Rodrı́guez-Calvo et al. 2008). analysis was not carried out – the jury found the finding sufficient to raise reasonable doubt to the question of guilt. 2.2. Diagnosis of infection Molecular pathological techniques are also of great importance in the diagnosis of infectious diseases and have been successfully applied also on very old tissue, e.g. mummy tissue. In forensic medicine these techniques have been applied for the diagnosis of e.g. myocarditis [2,24,44–46]. Frequent causes of viral myocarditis are especially adenoviruses, enteroviruses, Epstein–Barr-virus, influenza virus, parvovirus B19, and cytomegalovirus. The diagnosis of myocarditis is besides clinical history and clinical examination based on the investigation of myocardial tissue, in living on endomyocardial biopsies. The endomyocardial biopsies are searched for viruses by PCR, Rt-PCR and in situ hybridization, furthermore immunohistological investigations are used. There is, however, no correlation between molecular pathological and immunohistochemical techniques, i.e. positive virus findings may be seen in morphological normal hearts and vice versa [50]. Positive virus findings in the myocardium can of course explain the cause of death, especially if signs of myocarditis like myocyte necrosis, myocytolysis and interstitial lymphomonocytic infiltrates are visible. There are several case reports in the literature on positive myocardial virus findings in cases of sudden death. For SIDS it was even claimed that positive virus findings (especially enteroviruses but also adenoviruses or parvovirus B19) might be the cause of death in nearly 30% of cases. However, these findings could not be confirmed by succeeding investigations and may be due to methodical problems [43,53]. 3. Manner of death There is no doubt that pharmacogenetics as a diagnostic tool has the potential to improve patient therapy, especially individualized drug therapy. Especially genomic variants of phase I and phase II drug metabolizing enzymes play an important role in an individual dose adjustment according to a patient’s genotype. Polymorphisms of the cytochrome P4502D6 have great influence on the metabolism of many drugs, some being of great forensic relevance as the analgesics codeine, tramadol, hydrocodone and oxycodone. Further candidate genes are e.g. m-opioid-receptors. Meanwhile a considerable amount on genetic variants and their impact on pain and analgesia has been published which is not only of great interest concerning individualized medication, side-effects of drug therapy but may also be of forensic interest concerning the interpretation of serum levels. Molecular pathological methods are not only of help in determining the cause but also the manner of death, e.g. in the differential diagnosis of accident/suicide or even accident/homicide. This differential diagnosis may be of utmost importance also for the regulation of insurance compensations. Koski et al. reported the case of a 43-year-old male alcoholic with suicidal tendencies who was found dead; the cause of death was a doxepin intoxication, however, the manner of death remained unclear (suicide/accident). The post-mortem DNA analysis revealed two CYP2D6 alleles, which were both, not functional (CYP2D6*3/*4). Therefore doxepin could only be metabolized to nordoxepin, but not to 2-hydroxydoxepin or 2hydroxy-nordoxepin. Correspondingly high doxepin and especially high nordoxepin concentrations were found with a ratio of 0.83. In suicidal poisonings the ratio is much higher, therefore an accidental chronic intoxication could be assumed. In another case reported by Koren et al. [40] post-mortem DNA analysis was important to exclude homicidal morphine intoxication. Concerned was a full term healthy male infant with intermittent difficulties in breast-feeding and lethargy, starting on day 7. On day 12 it presented with grey skin and milk intake decreased on day 12, on day 13 it was found dead. The autopsy revealed no anatomical cause of death, however, a blood morphine level of 70 ng/ml was found. Neonates breastfed by mothers receiving codeine typically have morphine serum concentrations of 0– 2.2 ng/ml. The mother received a combination preparation of codeine 30 mg and paracetamol, which she took for two weeks. She stored milk at day 10 which had a morphine concentration of 87 ng/ml (normally 1.9–20.5 ng/ml). Of course the question was for the reason of the intoxication (accidental/homicidal). Genotype analysis revealed that the mother was an ultra-rapid metabolizer (CYP2D6*2A/*2 2) consistent with an increased formation of morphine from codeine. Thus, cause of death was accidental morphine intoxication. Molecular biological methods are also of importance for the assessment of side-effects during medical treatment. Different CYP2D6 genotypes may determine in comparison to the wild type [(Fig._10)TD$IG] 10 B. Madea et al. / Forensic Science International 203 (2010) 3–14 Fig. 10. Milwaukee pharmacogenomic algorithm for forensic toxicology (from Wong et al., 2006). (extensive metabolizer = EM) a missing (poor metabolizer = PM) deteriorated (intermediate metabolizer = IM) or an increased enzyme function (ultra-rapid metabolizer = UM). This means that CYP2D6 genetic variations determine for instance plasma levels of tramadol and its metabolites, the o-desmethyltramadol enantiomers. At the m-opiate receptor active M1 metabolite (+) o- desmethyltramadol is of great importance for analgesia. If plasma concentrations of tramadol and M1-metabolites depend on the CYP2D6 genotypes, especially ultra-rapid metabolizers may suffer from respiratory depression in postoperative analgesia [55,66]. Meanwhile algorithms have been developed in which cases pharmacogenomic investigations should be carried out (Fig. 10). [(Fig._1)TD$IG] B. Madea et al. / Forensic Science International 203 (2010) 3–14 11 Fig. 11. Collaboration between pathologist, toxicologist and geneticist in pharmacogenetics (from Sajantila et al. [42,62]). 4. RNA analysis molecular identification of body fluids by analysis of cell-specific mRNA expression already represents a new technique supplementing DNA analysis in forensic cases [5,10]. The detection of epithelial cells in dry bloodstains by reverse transcriptasepolymerase chain reaction (Rt-PCR) is based on cell- and tissuespecific gene expression. For instance matrix metalloproteinase (MMP) mRNA could be detected in endometrium but not in blood and other epithelia. This was confirmed in further studies with artificial menstrual blood stains indicating that the detection of MMP expression in blood stains may serve as a forensic marker for menstrual blood [5]. Cell-specific gene expression cannot only be used to identify menstrual blood but also the basic nuclear proteins protamine 1 and 2 are suitable markers for spermatozoa identification because they are exclusively expressed in the haploid genome. Protamine mRNA can be detected in semen stains by the highly sensitive Rt-PCR. With seminested PCR, 10– 100 spermatozoa are theoretically sufficient to provide positive amplification results, with hot-start PCR at least 100–1000 cells are required corresponding to an average semen volume of 0.01– 0.1 ml [7]. 4.1. Identification of body fluids 4.2. Age of bloodstains, post-mortem interval Post-mortem RNA profiling plays an increasing role in forensic medicine, e.g. concerning human post-mortem tissue identification or examination of gene expression levels at the time of death. The examination of gene expression at the time of death may be of great importance concerning the determination of cause and circumstances of death, survival time, pathophysiological conditions of disease and injury, duration of terminal episode, etc. [28– 30,32,33,72–76]. Although RNA is known to be quite unstable, RNA could be extracted in adequate quality from tissue samples collected post-mortem. However, a main point in the interpretation of gene expression levels regarding post-mortem degradation is the standardization on the expression of endogenous control genes. RNA analysis offers insight into diseases and mechanisms leading to death and could develop into a valuable tool for diagnosis of the cause of death in forensic pathology. Other possible applications include the determination of the age of wounds and injuries and the post-mortem interval [8,9]. The In vitro RNA degradation is a complex and non-linear process which can serve as indicator for the quality and age of stains. Bauer et al. developed a semiquantitative duplex Rt-PCR assay, which in combination with competitive Rt-PCR using an external standard allows quantification of RNA degradation levels. Using this method they investigated 106 bloodstains stored up to 15 years. The distribution of peak area quotients of standard and target messenger mRNA as measured by laser induced fluorescence capillary electrophoresis was closely correlated with the age of samples. Bloodstains with age difference of 5 years and more exhibits statistical significant variances in peak area quotients. Bauer et al. were able to show that FASN multiplex PCR is a suitable method to quantify the degree of RNA fragmentation, which is significantly correlated with the post-mortem interval. Overall RNA degradation is a slow process under the conditions investigated experimentally and significant differences, which might be used for determination of the PMI, do not occur prior to 3–4 days post-mortem. However, as with other laboratory As always in forensic medicine a close collaboration between pathologist, toxicologist and geneticist are necessary for the interpretation of the results (Fig. 11). In the literature several cases have been described with a difficult differential diagnosis between homicide and poisoning and very high pethidine doses for palliative reasons. Daldrup [22] and Lehmann [47] reported the case of a 88-year-old woman who died in the house of one of her physicians. She was treated with very high doses of pethidine (300 mg) in an infusion, additionally promethacin. She died within a short time after beginning of the infusion. Suspect was raised since the doctor presented a forged testament. In venous blood of the deceased high concentrations of pethidine (6.4 mg/l) were found. In a first trial the doctor was convicted of murder and sentenced to long lasting imprisonment (11 years). In a second trial he plead to have administered a high dose of pethidine for pain relief and was acquitted. This case illustrates that in similar cases of claimed palliative care genetic studies may be of importance, too [23]. 12 B. Madea et al. / Forensic Science International 203 (2010) 3–14 methods for the determination of the time since death rather large confidence intervals have to be considered so that the use of this method in forensic casework is limited [8,9]. 4.3. Wound age estimation Vital reactions are defined as local reaction of tissue at the site of damage. Typical vital reactions are e.g. an inflammatory reaction or expression patterns of adhesion molecules. Today, based on systematic post-mortem quantitative analysis of mRNA transcripts molecular biological methods are applied for wound age estimation. Animal experimental studies have already suggested time dependent expressions of cytokine and chemokine mRNAs after skin injury. However, for the interpretation of the validity of these changes – besides the problems of quantitative analysis of mRNA transcripts – the manifestation time of these local vital reactions has to be considered in comparison to the duration of the agonal period. From many studies on immunohistochemical parameters of vitality it is well known that there is a correlation between the manifestation time of various criteria of vitality and their validity as vital parameters. This means, the shorter the manifestation time, the lower the validity as vital parameter since induction of supravital reactions seems to be possible even post-mortem. All investigations on vitality and wound age estimation based on molecular pathological methods have to keep this in mind [50,54]. [(Fig._12)TD$IG] 4.4. MOR1-receptor mRNA expression in human brains of drugrelated fatalities Novel RNA technology involving real-time reverse transcription polymerase chain reaction has provided a practical procedure for quantitative post-mortem mRNA analysis. Post-mortem mRNA quantification is an important tool in the study of the pathophysiology of diseases and traumas leading to death. Further applications are wound age estimation, wound healing processes, but also systemic responses to violence or environmental hazards. In opiate addiction the m-opiate receptor (MOR1) is the primary site of action for morphine in the most commonly used opioids. The MOR1-receptor expression in animal and human brain has intensively been studied. The expression of the human m-opiate receptor (MOR1) in post-mortem human brain tissue was examined using real-time PCR technology. Tissue samples from 11 fatalities due to opiate overdose and five normal subjects with different causes of death were analysed in order to elucidate whether chronic opiate abuse is followed by a regulation of MOR1 expression. In each case nine selected brain regions (thalamus, caudate nucleus, hypothalamus, ventral tegmentum, hippocampus, amygdale, frontal cortex, nucleus accumbens, putamen) were evaluated. The MOR1-mRNA level was determined relative to the housekeeping gene b2-microglobulin. While in most regions the MOR-mRNA levels in the brain of addicts were not different from the control group – with varying levels between 0% and 15% of housekeeping gene level – in the brains of three drug-related fatalities an enormous increase was encountered in the thalamus where the MOR-mRNA level amounted for up to 10,000% of the measured housekeeping gene level. The results obtained by toxicological hair analysis in the group of drug-related fatalities indicate that the enormous thalamic MOR1 expression is primarily found in individuals who died from acute heroin overdose but did not show signs of a substantial chronic administration of the drug. Further studies have to be performed to evaluate if the observed MOR1-mRNA up-regulation in the thalamus in a subpopulation of acute lethal intoxications mirrors a state of functional hypersensitivity associated with the occurrence of death [11]. Fig. 12. Flow chart of care of relatives of deceased in cases of young or adult sudden cardiac death with genetic background (from Oliva et al.). B. Madea et al. / Forensic Science International 203 (2010) 3–14 5. MicroRNA Today, body fluid identification via specific mRNA quantification is an established standard technique in many forensic laboratories. Much work has been done to improve single aspects of this method and to detect and validate RNA signatures for different kinds of biological stains. However, mRNA stability and susceptibility to degradation has always been an issue for mRNA based gene expression analysis and it was shown that impaired mRNA integrity, represented by the RNA integrity number (RIN), has an influence on the reproducibility of results by introducing a variable extent of bias. This is particularly intricate for forensic routine applications using mRNA, because biological stains from casework are often challenged by moisture, UV light, temperature, suboptimal environmental pH, etc., potentially degrading mRNA beyond usability. Although evidence concerning mRNA stability in forensic settings is controversial, there can be no doubt that single stranded RNA transcripts of considerable length are less stable and more susceptible to degradation by physical and chemical strain and especially by ubiquitous RNAses than for instance a DNA molecule of comparable length. miRNA profiling has serious advantages as compared to mRNA profiling. Firstly, due to their tiny size of about 22 nt mature miRNAs are much more stable than mRNAs which is of paramount importance in forensic settings as it renders mature miRNAs decidedly less susceptible against fractionation by chemical or physical strain. This also applies for formalin-fixed paraffinembedded (FFPE) tissue samples, which can be of pivotal importance to forensic casework but in which intense nucleic acid fractionation occurs. Not only has miRNA recovery from FFPE tissues been shown to be feasible and to lead to valid profiling results but it even outperforms FFPE tissue mRNA expression profiling by achieving a higher degree of resemblance to fresh tissue profiles and therefore significantly better correlation. 6. Pathologists responsibilities regarding relatives of the deceased With the post-mortem detection of mutations the forensic pathologist has also medical responsibilities regarding a potential risk of family members of the deceased and an appropriate genetic counselling is necessary [56,57,59,60]. As far as with molecular autopsies a reasonable cause of death has been identified the forensic pathologist also has the duty to inform the family of all autopsy results (Fig. 12). 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