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Background: Sudden unexplained death (SUD) is a devastating event in the young. Despite efforts to identify causal genetic variants, many cases remain unexplained after genetic screening. This study aimed to investigate an alternative potential contributor to SUD by studying the human methylome using the MethylationEPIC v2.0 BeadChip kit in heart tissue from SUD cases. The genome-wide methylation results of the SUD cases were compared to the results of a control cohort. The SUD cases were divided into three main groups based on their autopsy reports, heart morphology and histopathology (primaryN: macroscopically and histologically normal heart; primaryCM: macroscopically or histologically abnormal heart, suspected cardiomyopathies; and secondary: myocardial damage due to other underlying conditions). The main focus of this study was to identify differentially methylated regions (DMRs) between the case groups and the control cohort., Results: We identified DMRs for both the primaryN and primaryCM groups, whereas the secondary group yielded no such results. In the primaryN cases, the corresponding genes for each DMR led to the identification of genes with common biological pathways. Some were associated with heart morphology (e.g. heart outflow tract morphogenesis or trabecular morphogenesis), but the majority belonged to more general cellular regulatory pathways (e.g. transcription coactivator activity, long non-coding RNAs, etc.). Although no common pathways were found for the primaryCM group, some common regulatory molecular functions were identified, such as p53 binding and transcription coactivator activity., Conclusions: Our study is the first to investigate the whole human methylome in heart tissue of SUD cases. We propose that there are observable differences in the methylation patterns of the case groups that may have contributed to SUD. Still, further studies are required to improve our understanding of the impact of methylation levels on SUD risk and to pinpoint methylation-based screening opportunities for SUD relatives., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical approval for this study was provided by the local ethics committee in Zurich (KEK-No. 2013-0086), and the study was conducted in full conformance with Swiss laws and regulations. The requirements of the local ethics committee included written informed consent of family members. If no family members were available, the cases were anonymized. Consent for publication: All authors of the manuscript have read and agreed to its content and consent to publish it. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cardiac arrhythmia is currently considered to be the direct cause of death in a majority of sudden unexplained death (SUD) cases, yet the genetic predisposition and corresponding endophenotypes contributing to SUD remain incompletely understood. In this study, we aimed to investigate the involvement of Coenzyme Q (CoQ) deficiency in SUD. First, we re-analyzed the exome sequencing data of 45 SUD and 151 sudden infant death syndrome (SIDS) cases from our previous studies, focusing on previously overlooked genetic variants in 44 human CoQ deficiency-related genes. A considerable proportion of the SUD (38%) and SIDS (37%) cases were found to harbor rare variants with likely functional effects. Subsequent burden testing, including all rare exonic and untranslated region variants identified in our case cohorts, further confirmed the existence of significant genetic burden. Based on the genetic findings, the influence of CoQ deficiency on electrophysiological and morphological properties was further examined in a mouse model. A significantly prolonged PR interval and an increased occurrence of atrioventricular block were observed in the 4-nitrobenzoate induced CoQ deficiency mouse group, suggesting that CoQ deficiency may predispose individuals to sudden death through an increased risk of cardiac arrhythmia. Overall, our findings suggest that CoQ deficiency-related genes should also be considered in the molecular autopsy of SUD., (© 2024. The Author(s).)

Sudden unexplained death (SUD) can affect apparently healthy adolescents and young adults with no prior clinical symptoms and no clear diagnostic findings at autopsy. Although primary cardiac arrhythmias have been shown to be the direct cause of death in the majority of SUD cases, the genetic predisposition contributing to SUD remains incompletely understood. Currently, molecular autopsy is considered to be an effective diagnostic tool in the multidisciplinary management of SUD, but the analysis focuses mainly on the coding region and the significance of many identified variants remains unclear. Recent studies have demonstrated the strong association between human disease and genetic variants in untranslated regions (UTRs), highlighting the potential role of UTR variants in the genetic predisposition to SUD. In this study, we searched for UTR variants with likely functional effects in the exome data of 45 SUD cases. Among 244 genes associated with cardiac diseases, three candidate variants with high confidence of pathogenicity were identified in the UTRs of SCO2, CALM2 and TBX3 based on a rigorous filtering strategy. A functional assay further validated the effect of these candidate variants on gene transcriptional activity. In addition, the constraint metrics, intolerance indexes, and dosage sensitivity scores of genes affected by the candidate variants were considered when estimating the consequence of aberrant gene expression. In conclusion, our study presents a practical strategy for UTR variant prioritization and functional annotation, which could improve the interpretation of molecular autopsy findings in SUD cohorts., (© 2024. The Author(s).)

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., (© 2023. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Genetic studies in sudden infant death syndrome (SIDS) and sudden unexplained death (SUD) cohorts have indicated that cardiovascular diseases might have contributed to sudden unexpected death in 20-35 % of autopsy-negative cases. Sudden unexpected death can also occur in people with epilepsy, termed as sudden unexpected death in epilepsy (SUDEP). The pathophysiological mechanisms of SUDEP are not well understood, but are likely multifactorial, including seizure-induced hypoventilation and arrhythmias as well as genetic risk factors. The sudden death of some of the SIDS/SUD victims might also be explained by genetic epilepsy, therefore this study aimed to expand the post-mortem genetic analysis of SIDS/SUD cases to epilepsy-related genes., Methods: Existing whole-exome sequencing data from our 155 SIDS and 45 SUD cases were analyzed, with a focus on 365 epilepsy-related genes. Nine of the SUD victims had a known medical history of epilepsy, seizures or other underlying neurological conditions and were therefore classified as SUDEP cases., Results: In our SIDS and SUD cohorts, we found epilepsy-related pathogenic/likely pathogenic variants in the genes OPA1, RAI1, SCN3A, SCN5A and TSC2., Conclusion: Post-mortem analysis of epilepsy-related genes identified potentially disease-causing variants that might have contributed to the sudden death events in our SIDS/SUD cases. However, the interpretation of identified variants remains challenging and often changes over time as more data is gathered. Overall, this study contributes insight in potentially pathophysiological epilepsy-related mechanisms in SIDS, SUD and SUDEP victims and underlines the importance of sensible counselling on the risk and preventive measures in genetic epilepsy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Background: Binge drinking is a widespread health compromising behavior among adolescents and young adults, leading to significant health problems, injuries and mortality. However, data on alcohol consumption is often unreliable, as it is mainly based on self-reporting surveys. In this five-year study (2014-2019) at the University Children's Hospital Zurich, we analyzed blood samples from adolescent binge drinking patients to investigate blood alcohol concentrations (BACs), co-ingestion of drugs, assess compliance between self-reported and measured substance use, and test for genetic components of innate alcohol tolerance. Furthermore, hair analysis was performed to retrospectively access drug exposure and to evaluate the potential of hair analysis to assess binge drinking., Methods: In a prospective, single-center study, patients with alcohol intoxications aged 16 years and younger were included. Blood and hair samples were analyzed by sensitive liquid chromatography - tandem mass spectrometry drug analysis. HTTLPR genotyping was performed with PCR and fragment analysis., Results: Among 72 cases, 72 blood and 13 hair samples were analyzed. BACs ranged from 0.08-3.20‰ (mean 1.63‰, median 1.60‰), while a mean concentration of 3.64 pg/mg hair (median 3.0 pg/mg) of the alcohol marker ethyl glucuronide (EtG) was detected in eleven hair samples, providing no evidence of chronic excessive drinking. In 47% of the cases, co-ingested drugs were qualitatively detected next to ethanol, but only 9% of the detected drugs had blood concentrations classified as pharmacologically active. Cannabis consumption (22%) and stimulant intake (16%) were the most frequently observed drugs. Compliance between patients' statements and measured substances matched well. Although we investigated the genetic contribution to innate alcohol tolerance via the 5-HTTLPR polymorphism, the diverse genetic background of the cohort and small sample size did not allow any conclusions to be drawn., Conclusion: Almost half of our binge drinking patients tested positive for other substances, primarily cannabis. We anticipate that our study enhances understanding of consumption behavior of young people and encourage continued efforts to address the harmful effects of binge drinking and co-occurring substance use., (© 2023. BioMed Central Ltd., part of Springer Nature.)

An accurate method to estimate the age of a stain or the time since deposition (TsD) would represent an important tool in police investigations for evaluating the true relevance of a stain. In this study, two laboratories reproduced an mRNA-based method for TsD estimation published by another group. The qPCR-based assay includes four transcripts (B2M, LGALS2, CLC, and S100A12) and showed preferential degradation of the 5' end over the 3' end. In this study, the blood-specific marker ALAS2 was added to examine whether it would show the same degradation pattern. Based on our qPCR data several elastic net models with different penalty combinations were created, using training data from the two laboratories separately and combined. Each model was then used to estimate the age of bloodstains from two independent test sets each laboratory had prepared. The elastic net model built on both datasets with training samples up to 320 days old displayed the best prediction performance across all test samples (MAD=18.9 days). There was a substantial difference in the prediction performance for the two laboratories: Restricting TsD to up to 100 days for test data, one laboratory obtained an MAD of 2.0 days when trained on its own data, whereas the other laboratory obtained an MAD of 15 days., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Biogeographical ancestry (BGA) inference from ancestry-informative markers (AIMs) has strong potential to support forensic investigations. Over the past two decades, several forensic panels composed of AIMs have been developed to predict ancestry at a continental scale. These panels typically comprise fewer than 200 AIMs and have been designed and tested with a limited set of populations. How well these panels recover patterns of genetic diversity relative to larger sets of markers, and how accurately they infer ancestry of individuals and populations not included in their design remains poorly understood. The lack of comparative studies addressing these aspects makes the selection of appropriate panels for forensic laboratories difficult. In this study, the model-based genetic clustering tool STRUCTURE was used to compare three popular forensic BGA panels: MAPlex, Precision ID Ancestry Panel (PIDAP), and VISAGE Basic Tool (VISAGE BT) relative to a genome-wide reference set of 10k SNPs. The genotypes for all these markers were obtained for a comprehensive set of 3957 individuals from 228 worldwide human populations. Our results indicate that at the broad continental scale (K=6) typically examined in forensic studies, all forensic panels produced similar genetic structure patterns compared to the reference set (G'≈90%) and had high classification performance across all regions (average AUC-PR > 97%). However, at K= 7 and K= 8, the forensic panels displayed some region-specific clustering deviations from the reference set, particularly in Europe and the region of East and South-East Asia, which may be attributed to differences in the design of the respective panels. Overall, the panel with the most consistent performance in all regions was VISAGE BT with an average weighted AUC̅ W score of 96.26% across the three scales of geographical resolution investigated., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The ability to associate a contributor with a specific body fluid in a crime stain can aid casework investigation. The detection of body fluids combined with DNA analyses may supply essential information, but as the two tests are independent, they may not be associated. Recently, the analysis of coding region SNPs (cSNPs) within the RNA transcript has been proven to be a promising method to face this challenge. In this study, we performed targeted RNA sequencing of 158 samples (boxershorts, fingernail swabs and penile swabs) collected from 12 couples at different time points post-intimate contact and after non-intimate contact, using the Ion S5™ System and BFID-cSNP-6F assay. The aim of the study was to compare the performance of the MPS and CE methods in the detection of mRNA markers, and to associate body fluids with contributors by their cSNP genotypes. The results of the study show a lower success rate in the detection of vaginal mucosa by the MPS compared to the CE method. However, the additional information obtained with the cSNP genotypes could successfully associate body fluids with contributors in most cases.

The evidentiary value of DNA profiles varies depending upon the context in which the DNA was found. Linking a DNA profile to a particular cellular phenotype in mixtures may aid in assessing its evidentiary relevance and value. We report the development of two dual-function high-resolution messenger RNA (mRNA) sequencing assays that can each identify the presence of 6 body fluids/tissues (blood, semen, saliva, vaginal secretions, menstrual blood, skin) and, via coding region SNPs (cSNPs) present in the body fluid-specific mRNA transcripts, directly associate particular body fluids with their specific DNA donors in mixtures. The original blood, semen, and saliva (BSS) assay contains 23 cSNPs for blood, semen, and saliva, while the expanded 6F (all 6 fluids/tissues) assay encompasses the BSS assay and also contains 23 additional cSNPs for vaginal secretions, menstrual blood, and skin. Software tools were developed to infer the identity of the body fluids present as well as providing the corresponding cSNP genotypes. Concomitant genomic DNA assays (BSS-d and 6F-d), required to genotype the same cSNPs from persons of interest/inferred contributors to the body fluid mixture, were also developed. Body fluid specificity was demonstrated by the ability to identify the body fluid origin of single-source and two-fluid admixtures. The discriminatory power (European Caucasians) for all body fluids is 0.957-0.997, with linkage disequilibrium considered. Reciprocal body fluid admixtures (mixture pairs with the same two donors but reversed body fluid types) were used to demonstrate the ability to identify the body fluid source of origin as well as associate the donor of each of the two fluids., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Sudden infant death syndrome (SIDS) is still one of the leading causes of postnatal infant death in developed countries. The occurrence of SIDS is described by a multifactorial etiology that involves the respiratory control system including chemoreception. It is still unclear whether genetic variants in genes involved in respiratory chemoreception might play a role in SIDS., Methods: The exome data of 155 SIDS cases were screened for variants within 11 genes described in chemoreception. Pathogenicity of variants was assigned based on the assessment of variant types and in silico protein predictions according to the current recommendations of the American College of Medical Genetics and Genomics., Results: Potential pathogenic variants in genes encoding proteins involved in respiratory chemoreception could be identified in 5 (3%) SIDS cases. Two of the variants (R137S/A188S) were found in the KNCJ16 gene, which encodes for the potassium channel Kir5.1, presumably involved in central chemoreception. Electrophysiologic analysis of these KCNJ16 variants revealed a loss-of-function for the R137S variant but no obvious impairment for the A188S variant., Conclusions: Genetic variants in genes involved in respiratory chemoreception may be a risk factor in a fraction of SIDS cases and may thereby contribute to the multifactorial etiology of SIDS., Impact: What is the key message of your article? Gene variants encoding proteins involved in respiratory chemoreception may play a role in a minority of SIDS cases. What does it add to the existing literature? Although impaired respiratory chemoreception has been suggested as an important risk factor for SIDS, genetic variants in single genes seem to play a minor role. What is the impact? This study supports previous findings, which indicate that genetic variants in single genes involved in respiratory control do not have a dominant role in SIDS., (© 2022. The Author(s).)

The association of body fluids/cell types and donors in mixed biological traces is an important, but challenging task required to evaluate the value of evidence given forensic propositions concerning the source of the DNA. The linking of a DNA profile with evidence from presumptive tests or RNA analysis is not straightforward. Coding region SNPs (cSNPs) are a novel type of evidential markers that are both cell type specific and individual specific. They thereby provide a direct link between a donor and a body fluid in mixed biological stains. In this proof-of-concept paper we consider the evaluation of cSNP profiles given source level propositions and explore the use of the open-source software EuroForMix to compute likelihood ratios. The discrimination power of the cSNPs for various body fluids is investigated with simulations. We provide case examples where the type of biological material is questioned and where cSNP profiles can be used to assign a donor to a body fluid, and discuss how the results can be reported in court., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Sudden cardiac death (SCD) is an important public health issue. In young persons aged between 1 and 40 years, most SCDs are caused by potentially inherited cardiac diseases, often not detectable during conventional medico-legal investigations and therefore termed as sudden unexplained deaths (SUD). In this study, we describe the implementation, feasibility and importance of a standardized procedure to investigate SUD cases within the forensic framework at the Zurich Institute of Forensic Medicine in Switzerland. This new approach involves a multidisciplinary collaboration including forensic autopsy, second pathology expert opinion, post-mortem molecular genetic testing, cardiac counselling of relatives, and a tentative financing. This procedure is in line with the published Swiss and European recommendations on the management of SCDs. During a two-year pilot project, 39 sudden and unexpected death cases were collected, whereof 10 deceased remained without any identifiable cause of death after medico-legal investigation and second expert evaluation. Molecular autopsy, including 393 genes involved in cardio-vascular and metabolic diseases, identified eight pathogenic or likely pathogenic genetic variants in five out of the 10 deceased (50%). Cardio-genetic follow-up investigations in the families of the 10 deceased revealed phenotype-positive relatives in four families and required specific therapies, including an implantable cardioverter defibrillator (ICD) for primary prevention. Multidisciplinary collaboration is crucial for an optimal management of sudden unexplained death cases, to identify additional relatives at risk, and to prevent other tragic deaths within a family., Competing Interests: Declaration of Competing Interest Ardan M. Saguner received educational grants through his institution from Abbott, Bayer Healthcare, Biosense Webster, Biotronik, Boston Scientific, BMS/Pfizer, and Medtronic; and speaker fees from Bayer Healthcare, BMS/Pfizer and Daiichii Sankyo. The other authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

DNA methylation has become one of the most useful biomarkers for age prediction and body fluid identification in the forensic field. Therefore, several assays have been developed to detect age-associated and body fluid-specific DNA methylation changes. Among the many methods developed, SNaPshot-based assays should be particularly useful in forensic laboratories, as they permit multiplex analysis and use the same capillary electrophoresis instrumentation as STR analysis. However, technical validation of any developed assays is crucial for their proper integration into routine forensic workflow. In the present collaborative exercise, two SNaPshot multiplex assays for age prediction and a SNaPshot multiplex for body fluid identification were tested in twelve laboratories. The experimental set-up of the exercise was designed to reflect the entire workflow of SNaPshot-based methylation analysis and involved four increasingly complex tasks designed to detect potential factors influencing methylation measurements. The results of body fluid identification from each laboratory provided sufficient information to determine appropriate age prediction methods in subsequent analysis. In age prediction, systematic measurement differences resulting from the type of genetic analyzer used were identified as the biggest cause of DNA methylation variation between laboratories. Also, the use of a buffer that ensures a high ratio of specific to non-specific primer binding resulted in changes in DNA methylation measurement, especially when using degenerate primers in the PCR reaction. In addition, high input volumes of bisulfite-converted DNA often caused PCR failure, presumably due to carry-over of PCR inhibitors from the bisulfite conversion reaction. The proficiency of the analysts and experimental conditions for efficient SNaPshot reactions were also important for consistent DNA methylation measurement. Several bisulfite conversion kits were used for this study, but differences resulting from the use of any specific kit were not clearly discerned. Even when different experimental settings were used in each laboratory, a positive outcome of the study was a mean absolute age prediction error amongst participant's data of only 2.7 years for semen, 5.0 years for blood and 3.8 years for saliva., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Microscopic sperm detection is an important task in sexual assault cases. In some instances, the samples contain no or only low amounts of semen. Therefore, the biological material is transferred onto a glass slide and needs to be manually scanned using an optical microscope. This work can be very time consuming, especially when no spermatozoa is present. In such a case, the result needs to be validated. In this article we show how convolutional neural networks can perform this task and how they can reduce the scanning time by locating the sperm cells on images taken under the microscope. For this purpose, we trained a VGG19 network and a VGG19 variation with 1942 images, some containing sperm cells and some not., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The importance of RNA evidence is growing with new developments in RNA profiling methods and purposes. As forensic samples often can be of small quantity, extraction methods with high yields of both DNA and RNA are desirable. In order to identify the optimal DNA/RNA co-extraction workflow for forensic samples, we evaluated the performance of three frequently-used methods, two new approaches for DNA/RNA co-extraction and a manual phenol/chloroform RNA-only extraction method on blood and saliva samples. Based on a comprehensive analysis of the RNA and DNA quantities, as well as the STR genotyping and mRNA profiling results, we conclude that the two frequently-used co-extraction methods, combining commercially available DNA and RNA extraction kits, achieved the best performance. However, not any combination of commercially available DNA and RNA extraction kits works well and extensive optimization is necessary, as seen in the poor results of the two new approaches., Competing Interests: Conflicts of interest The authors declare that they have no conflict of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Collection of touch DNA from an offender on the victim's skin can provide relevant evidence for investigations of criminal cases. Therefore, the choice of the optimal sample collection method is crucial. In this study, we investigated the recovery of STR profiles from touch DNA on human skin by comparing nine different collection methods: the dry and wet cotton swabs in three different movements, the double-swab (wet-dry) method, the wet and dry Copan FLOQSwabs™, and the Scene Safe FAST™ minitapes. Mock assault scenarios were conducted with a male offender grasping the forearms of a female victim. Samples were collected from the assaulted area of the victim's skin, and the recovery of the offender's STR profile was evaluated. Our results indicate that the different swabs and swabbing techniques did not have a distinct impact on the STR recovery; however, the lowest STR recovery was achieved with Scene Safe FAST™ minitapes. In addition, we compared the double-swab method to the single-swab method by analyzing the DNA quantity of the wet and dry swabs separately. We found on average 13.7% more offender DNA using the double-swab method, but this did not translate into higher STR recovery. Our findings indicate that several methods perform equally well when collecting touch DNA from human skin, although SceneSafe FAST™ minitapes seem to be the least adequate for this purpose., (© 2021 American Academy of Forensic Sciences.)

Sudden unexplained death (SUD) takes up a considerable part in overall sudden death cases, especially in adolescents and young adults. During the past decade, many channelopathy- and cardiomyopathy-associated single nucleotide variants (SNVs) have been identified in SUD studies by means of postmortem molecular autopsy, yet the number of cases that remain inconclusive is still high. Recent studies had suggested that structural variants (SVs) might play an important role in SUD, but there is no consensus on the impact of SVs on inherited cardiac diseases. In this study, we searched for potentially pathogenic SVs in 244 genes associated with cardiac diseases. Whole-exome sequencing and appropriate data analysis were performed in 45 SUD cases. Re-analysis of the exome data according to the current ACMG guidelines identified 14 pathogenic or likely pathogenic variants in 10 (22.2%) out of the 45 SUD cases, whereof 2 (4.4%) individuals had variants with likely functional effects in the channelopathy-associated genes SCN5A and TRDN and 1 (2.2%) individual in the cardiomyopathy-associated gene DTNA. In addition, 18 structural variants (SVs) were identified in 15 out of the 45 individuals. Two SVs with likely functional impairment were found in the coding regions of PDSS2 and TRPM4 in 2 SUD cases (4.4%). Both were identified as heterozygous deletions, which were confirmed by multiplex ligation-dependent probe amplification. In conclusion, our findings support that SVs could contribute to the pathology of the sudden death event in some of the cases and therefore should be investigated on a routine basis in suspected SUD cases.

Importance: Identification of geographic population-based differences in genotype and phenotype heterogeneity are important for targeted and patient-specific diagnosis and treatment, counseling, and screening strategies., Objective: To report disease-causing variants and their detailed phenotype in patients with bilateral congenital cataract from a single center in Switzerland and thereby draw a genetic map and perform a genotype-phenotype comparison of this cohort., Design, Setting, and Participants: This clinical and molecular-genetic cohort study took place through the collaboration of the Department of Ophthalmology at the University Hospital Zurich and the Institute of Medical Molecular Genetics, University of Zurich, Schlieren, Switzerland. Thirty-seven patients from 25 families with different types of bilateral congenital cataract were included. All participating family members received a comprehensive eye examination. Whole exome sequencing was performed in the index patients, followed by a filtering process to detect possible disease-associated variants in genes previously described in association with congenital cataract. Probable disease-causing variants were confirmed by Sanger sequencing in available family members. All data were collected from January 2018 to June 2020, and the molecular-genetic analyses were performed from January 2019 to July 2020., Main Outcomes and Measures: Identification of the underlying genetic causes of bilateral congenital cataract, including novel disease-causing variants and phenotype correlation., Results: Among the 37 patients (18 [49%] male and 19 [51%] female; mean [SD] age, 17.3 [15.9] years) from 25 families, pathogenic variants were detected in 20 families (80% detection rate), which included 13 novel variants in the following genes: BCOR, COL4A1, CRYBA2, CRYBB2, CRYGC, CRYGS, GJA3, MAF, NHS, and WFS1. Putative disease-causing variants were identified in 14 of 20 families (70%) as isolated cases and in 6 of 20 families (30%) with syndromic cases. A recessive variant in the CRYBB2 gene in a consanguineous family with 2 affected siblings showing a nuclear and sutural cataract was reported in contrast to previously published reports. In addition, the effect on splicing in a minigene assay of a novel splice site variant in the NHS gene (c.[719-2A>G]) supported the pathogenicity of this variant., Conclusions and Relevance: This study emphasizes the importance of genetic testing of congenital cataracts. Known dominant genes need to be considered for recessive inheritance patterns. Syndromic types of cataract may be underdiagnosed in patients with mild systemic features.

Ancient DNA (aDNA) analysis is a powerful tool in multidisciplinary research on human remains, potentially leading to kinship scenarios and historical identifications. In this study, we present a genetic investigation of three noble families from the 17th to 19th centuries AD entombed in burial crypts at the cloister church of Riesa (Germany). Tests were aimed at identifying anticipated and incidental genetic relationships in our sample and the implications thereof for the assumed identity of the deceased. A total of 17 individuals were investigated via morphological, radiographic and aDNA analysis, yielding complete and partial autosomal and Y-STR profiles and reliable mtDNA sequences. Biostatistics and lineage markers revealed the presence of first to third degree relationships within the cohort. The pedigrees of the families Hanisch/von Odeleben and von Welck were thereby successfully reproduced, while four previously unknown individuals could be linked to the von Felgenhauer family. However, limitations of biostatistical kinship analysis became evident when the kinship scenario went beyond simple relationships. A combined analysis with archaeological data and historical records resulted in (almost) unambiguous identification of 14 of the 17 individuals., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Knowledge about the age of a stain, also termed as time since deposition (TsD), would provide law-enforcing authorities with valuable information for the prosecution of criminal offenses. Yet, there is no reliable method for the inference / assessment of TsD available. The aim of this study was to gain further insight into the RNA degradation pattern of forensically relevant body fluids and to find candidate markers for TsD estimation. Blood, menstrual blood, saliva, semen and vaginal secretion samples were exposed to indoor (dark, room temperature) and outdoor (exposed to sun, wind, etc. but protected from rain) conditions for up to 1.5 years. Based on expression and degradation analyses, we were able to identify body fluid specific signatures and RNA degradation patterns. The indoor samples showed a marked drop in RNA integrity after 6 months, while the outdoor samples were difficult to interpret and therefore excluded for some of the analyses. Up to 4 weeks, indoor samples showed more stable and less degrading transcripts than outdoor samples. Stable transcripts tended to be significantly shorter than degrading ones or transcripts, which are neither degrading nor stable. We reinforced the body fluid specific and the housekeeping gene nature of previously reported markers. With an unbiased approach, we selected stable and degrading genes for each body fluid in the short term and assessed their integrity during extended storage. We identified several stable and degrading gene transcripts, which could be tested in a targeted assay to assess the TsD interval e.g. by analyzing the ratio of degrading vs stable transcripts. In conclusion, we were able to detect RNA degradation patterns in samples being aged up to 1.5 years and identified several candidate markers, which could be evaluated for TsD estimation., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Current body fluid identification methods do not reveal any information about the time since deposition (TsD) of biological traces, even though determining the age of traces could be crucial for the investigative process. To determine the utility of microbial RNA markers for TsD estimation, we examined RNA sequencing data from five forensically relevant body fluids (blood, menstrual blood, saliva, semen, and vaginal secretion) over seven time points, ranging from fresh to 1.5 years. One set of samples was stored indoors while another was exposed to outdoor conditions. In outdoor samples, we observed a consistent compositional shift, occurring after 4 weeks: this shift was characterized by an overall increase in non-human eukaryotic RNA and an overall decrease in prokaryotic RNA. In depth analyses showed a high fraction of tree, grass and fungal signatures, which are characteristic for the environment the samples were exposed to. When examining the prokaryotic fraction in more detail, three bacterial phyla were found to exhibit the largest changes in abundance, namely Actinobacteria, Proteobacteria and Firmicutes. More detailed analyses at the order level were done using a Lasso regression analysis to find a predictive subset of bacterial taxa. We found 26 bacterial orders to be indicative of sample age. Indoor samples did not reveal such a clear compositional change at the domain level: eukaryotic and prokaryotic abundance remained relatively stable across the assessed time period. Nonetheless, a Lasso regression analysis identified 32 bacterial orders exhibiting clear changes over time, enabling the prediction of TsD. For both indoor and outdoor samples, a larger number (around 60%) of the bacterial orders identified as indicative of TsD are part of the Actinobacteria, Proteobacteria and Firmicutes. In summary, we found that the observed changes across time are not primarily due to changes associated with body fluid specific bacteria but mostly due to accumulation of bacteria from the environment. Orders of these environmental bacteria could be evaluated for TsD prediction, considering the location and environment of the crime scene. However, further studies are needed to verify these findings, determine the applicability across samples, replicates, donors, and other variables, and also to further assess the effect of different seasons and locations on the samples., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The application of transcriptome analyses in forensic genetics has experienced tremendous growth and development in the past decade. The earliest studies and main applications were body fluid and tissue identification, using targeted RNA transcripts and a reverse transcription endpoint PCR method. A number of markers have been identified for the forensically most relevant body fluids and tissues and the method has been successfully used in casework. The introduction of Massively Parallel Sequencing (MPS) opened up new perspectives and opportunities to advance the field. Contrary to genomic DNA where two copies of an autosomal DNA segment are present in a cell, abundant RNA species are expressed in high copy numbers. Even whole transcriptome sequencing (RNA-Seq) of forensically relevant body fluids and of postmortem material was shown to be possible. This review gives an overview on forensic transcriptome analyses and applications. The methods cover whole transcriptome as well as targeted MPS approaches. High resolution forensic transcriptome analyses using MPS are being applied to body fluid/ tissue identification, determination of the age of stains and the age of the donor, the estimation of the post-mortem interval and to post mortem death investigations., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

In recent years, forensic mRNA profiling has increasingly been used to identify the origin of human body fluids. By now, several laboratories have implemented mRNA profiling and also use it in criminal casework. In 2018 the FoRNAP (Forensic RNA Profiling) group was established among a number of these laboratories with the aim of sharing experiences, discussing optimization potential, identifying challenges and suggesting solutions with regards to mRNA profiling and casework. To compare mRNA profiling methods and results a collaborative exercise was organized within the FoRNAP group. Seven laboratories from four countries received 16 stains, comprising six pure body fluid / tissue stains and ten mock casework samples. The laboratories were asked to analyze the provided stains with their in-house method (PCR/CE or MPS) and markers of choice. Five laboratories used a DNA/RNA co-extraction strategy. Overall, up to 11 mRNA markers per body fluid were analyzed. We found that mRNA profiling using different extraction and analysis methods as well as different multiplexes can be applied to casework-like samples. In general, high input samples were typed with high accuracy by all laboratories, regardless of the method used. Irrespective of the analysis strategy, samples of low input or mixed stains were more challenging to analyze and interpret since, alike to DNA profiling, a higher number of markers dropped out and/or additional unexpected markers not consistent with the cell type in question were detected. It could be shown that a plethora of different but valid analysis and interpretation strategies exist and are successfully applied in the Forensic Genetics community. Nevertheless, efforts aiming at optimizing and harmonizing interpretation approaches in order to achieve a higher consistency between laboratories might be desirable in the future. The simultaneous extraction of DNA alongside RNA showed to be an effective approach to identify not only the body fluid present but also to identify the donor(s) of the stain. This allows investigators to gain valuable information about the origin of crime scene samples and the course of events in a crime case., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Artificial mummification has been used since antiquity and is best known from ancient Egypt. Despite ancient Egyptian mummies being studied for several decades, the mummification techniques of that time are not well understood. Modern mummification experiments involving animal and human tissues have contributed additional insights relevant to a broad field of research. In the current study, we present follow-up results of an experiment on artificial mummification, which began in 2009. A human leg was artificially mummified and monitored for almost a year with histological, molecular, and radiological techniques. Since then, it has remained in a dry, natron salt blend for 9 years. The current analyses show further progression of dehydration and tissue alterations, as well as DNA degradation, suggesting an ongoing process. Our results add new insights into the mechanisms of tissue mummification. Taking into account that the process is still ongoing, further research is required, including a re-evaluation of the human leg in the future., (© 2019 American Association for Anatomy.)

Human blood traces are amongst the most commonly encountered biological stains collected at crime scenes. Identifying the body site of origin of a forensic blood trace can provide crucial information in many cases, such as in sexual and violent assaults. However, means for reliably and accurately identifying from which body site a forensic blood trace originated are missing, but would be highly valuable in crime scene investigations. With this study, we introduce a taxonomy-independent deep neural network approach based on massively parallel microbiome sequencing, which delivers accurate body site of origin classification of forensically-relevant blood samples, such as menstrual, nasal, fingerprick, and venous blood. A total of 50 deep neural networks were trained using a large 16S rRNA gene sequencing dataset from 773 reference samples, including 220 female urogenital tract, 190 nasal cavity, 213 skin, and 150 venous blood samples. Validation was performed with de-novo generated 16S rRNA gene massively parallel sequencing (MPS) data from 94 blood test samples of four different body sites, and achieved high classification accuracy with AUC values at 0.992 for menstrual blood (N = 23), 0.978 for nasal blood (N = 16), 0.978 for fingerprick blood (N = 30), and 0.990 for venous blood (N = 25). The obtained highly accurate classification of menstrual blood was independent of the day of the menses, as established in additional 86 menstrual blood test samples. Accurate body site of origin classification was also revealed for 45 fresh and aged mock casework blood samples from all four body sites. Our novel microbiome approach works based on the assumption that a sample is from blood, as can be obtained in forensic practise from prior presumptive blood testing, and provides accurate information on the specific body source of blood, with high potentials for future forensic applications., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Forensic DNA Phenotyping (FDP) provides the ability to predict externally visible characteristics from minute amounts of crime scene DNA, which can help find unknown perpetrators who are typically unidentifiable via conventional forensic DNA profiling. Fundamental human genetics research has led to a better understanding of the specific DNA variants responsible for physical appearance characteristics, particularly eye, hair, and skin color. Recently, we introduced the HIrisPlex-S system for the simultaneous prediction of eye, hair, and skin color based on 41 DNA variants generated from two forensically validated SNaPshot multiplex assays using capillary electrophoresis (CE). Here we introduce massively parallel sequencing (MPS) solutions for the HIrisPlex-S (HPS) system on two MPS platforms commonly used in forensics, Ion Torrent and MiSeq, that cover all 41 DNA variants in a single assay, respectively. Additionally, we present the forensic developmental validation of the two HPS-MPS assays. The Ion Torrent MPS assay, based on Ion AmpliSeq technology, illustrated the successful generation of full HIrisPlex-S genotypic profiles from 100 pg of input control DNA, while the MiSeq MPS assay based on an in-house design yielded complete profiles from 250 pg of input DNA. Assessing simulated forensic casework samples such as saliva, hair (bulb), blood, semen, and low quantity touch DNA, as well as artificially damaged DNA samples, concordance testing, and samples from numerous species, all illustrated the ability of both versions of the HIrisPlex-S MPS assay to produce results that motivate forensic applications. By also providing an integrated bioinformatics analysis pipeline, MPS data can now be analyzed and a file generated for upload to the publically accessible HIrisPlex online webtool (https://hirisplex.erasmusmc.nl). In addition, we updated the website to accept VCF input data for those with genome sequence data. We thus provide a user-friendly and semi-automated MPS workflow from DNA sample to individual eye, hair, and skin color prediction probabilities. Furthermore, we present a 2-person mixture separation tool that not only assesses genotype reliability with regards genotyping confidence but also provides the most fitting mixture scenario for both minor and major contributors, including profile separation. We envision this MPS implementation of the HIrisPlex-S system for eye, hair, and skin color prediction from DNA as a starting point for further expanding MPS-based forensic DNA phenotyping. This may include the future addition of SNPs predictive for more externally visible characteristics, as well as SNPs for bio-geographic ancestry inference, provided the statistical framework for DNA prediction of these traits is in place., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sudden arrhythmic death syndrome (SADS) in young individuals is a devastating and tragic event often caused by an undiagnosed inherited cardiac disease. Although post-mortem genetic testing represents a promising tool to elucidate potential disease-causing mechanisms in such autopsy-negative death cases, a variant interpretation is still challenging, and functional consequences of identified sequence alterations often remain unclear. Recently, we have identified a novel heterozygous missense variant (N1774H) in the Na v 1.5 channel-encoding gene SCN5A in a 19-year-old female SADS victim. The aim of this study was to perform a co-segregation analysis in family members of the index case and to evaluate the functional consequences of this SCN5A variant. Functional characterization of the SCN5A N1774H variant was performed using patch-clamp techniques in TsA-201 cell line transiently expressing either wild-type or variant Na v 1.5 channels. Electrophysiological analyses revealed that variant Na v 1.5 channels show a loss-of-function in the peak current densities, but an increased late current compared to the wild-type channels, which could lead to both, loss- and gain-of-function respectively. Furthermore, clinical assessment and genetic testing of the relatives of the index case showed that all N1774H mutation carriers have prolonged QT intervals. The identification of several genotype and phenotype positive family members and the functional implication of the SCN5A N1774H variant support the evidence of the in silico predicted pathogenicity of the here reported sequence alteration.

Analysis of biological evidence typically begins with a preliminary screening for the presence of biological fluids, traditionally with enzymatic or immunologic tests and of late by RNA profiling. The goal of this study was to create a whole transcriptome protocol, to view potential degradation effects in forensically relevant body fluids. Total RNA from fresh and aged blood, menstrual blood, saliva, semen, skin and vaginal secretion was analyzed with a massively parallel sequencing method. Two RNA-Seq library protocols with and without rRNA depletion were tested and compared. The rRNA depletion step had a negative influence on the sequencing quality and on the downstream analyses. From the human and bacterial RNA sequences, source-specific signatures could be identified. Aged samples showed in general a higher level of RNA degradation and decreased bacterial diversity. In summary, we could show that transcriptional profiling and metagenome analysis are powerful tools to provide additional information about the trace evidence., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Correct identification of different human epithelial materials such as from skin, saliva and vaginal origin is relevant in forensic casework as it provides crucial information for crime reconstruction. However, the overlap in human cell type composition between these three epithelial materials provides challenges for their differentiation and identification when using previously proposed human cell biomarkers, while their microbiota composition largely differs. By using validated 16S rRNA gene massively parallel sequencing data from the Human Microbiome Project of 1636 skin, oral and vaginal samples, 50 taxonomy-independent deep learning networks were trained to classify these three tissues. Validation testing was performed in de-novo generated high-throughput 16S rRNA gene sequencing data using the Ion Torrent ™ Personal Genome Machine from 110 test samples: 56 hand skin, 31 saliva and 23 vaginal secretion specimens. Body-site classification accuracy of these test samples was very high as indicated by AUC values of 0.99 for skin, 0.99 for oral, and 1 for vaginal secretion. Misclassifications were limited to 3 (5%) skin samples. Additional forensic validation testing was performed in mock casework samples by de-novo high-throughput sequencing of 19 freshly-prepared samples and 22 samples aged for 1 up to 7.6 years. All of the 19 fresh and 20 (91%) of the 22 aged mock casework samples were correctly tissue-type classified. Moreover, comparing the microbiome results with outcomes from previous human mRNA-based tissue identification testing in the same 16 aged mock casework samples reveals that our microbiome approach performs better in 12 (75%), similarly in 2 (12.5%), and less good in 2 (12.5%) of the samples. Our results demonstrate that this new microbiome approach allows for accurate tissue-type classification of three human epithelial materials of skin, oral and vaginal origin, which is highly relevant for future forensic investigations., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In this study, we have screened the six most relevant forensic body fluids / tissues, namely blood, semen, saliva, vaginal secretion, menstrual blood and skin, for miRNAs using a whole miRNome massively parallel sequencing approach. We applied partial least squares (PLS) and linear discriminant analysis (LDA) to predict body fluids based on the expression of the miRNA markers. We estimated the prediction accuracy for models including different subsets of miRNA markers to identify the minimum number of markers needed for sufficient prediction performance. For one selected model consisting of 9 miRNA markers we calculated their importance for prediction of each of the six different body fluid categories., (Copyright © 2019 Elsevier B.V. All rights reserved.)

In the forensic reconstruction of crime scene activities, the identification of biological traces and their bodily origin are valuable evidence that can be presented in court. While several presumptive and confirmatory tests are currently available, the limitations in specificity and sensitivity have instigated a search for alternative methods. Bacterial markers have been proposed as a novel approach for forensic body fluid/tissue identification. Bacteria are not only ubiquitous throughout the human body, but also, as shown by recent microbiome sequencing studies of the 16S rRNA gene, bacterial community structures are distinct across body sites. Traces and stains at crime scenes are, however, often exposed to the environment outside the human body for variable periods of time before laboratory processing. Thus, it is not clear whether exposed samples continue to harbor microbial signatures characteristic of their body site of origin. In this proof-of-concept study we collected samples from six different body sites: saliva, skin, peripheral blood, vaginal fluid, menstrual blood and semen. We exposed a subset of these samples to indoor conditions for 30 days while the remaining samples were processed directly after extraction. Our analyses of 16S rRNA gene sequence data for a total of 46 control and exposed samples show that both types of samples group by body site, although a few outliers are observed. Based on our results, vaginal and menstrual samples share their microbial signatures, and cannot be distinguished using bacterial markers. Overall, our findings indicate that bacterial markers are a promising avenue for forensic body fluid/tissue identification., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human head hair shape, commonly classified as straight, wavy, curly or frizzy, is an attractive target for Forensic DNA Phenotyping and other applications of human appearance prediction from DNA such as in paleogenetics. The genetic knowledge underlying head hair shape variation was recently improved by the outcome of a series of genome-wide association and replication studies in a total of 26,964 subjects, highlighting 12 loci of which 8 were novel and introducing a prediction model for Europeans based on 14 SNPs. In the present study, we evaluated the capacity of DNA-based head hair shape prediction by investigating an extended set of candidate SNP predictors and by using an independent set of samples for model validation. Prediction model building was carried out in 9674 subjects (6068 from Europe, 2899 from Asia and 707 of admixed European and Asian ancestries), used previously, by considering a novel list of 90 candidate SNPs. For model validation, genotype and phenotype data were newly collected in 2415 independent subjects (2138 Europeans and 277 non-Europeans) by applying two targeted massively parallel sequencing platforms, Ion Torrent PGM and MiSeq, or the MassARRAY platform. A binomial model was developed to predict straight vs. non-straight hair based on 32 SNPs from 26 genetic loci we identified as significantly contributing to the model. This model achieved prediction accuracies, expressed as AUC, of 0.664 in Europeans and 0.789 in non-Europeans; the statistically significant difference was explained mostly by the effect of one EDAR SNP in non-Europeans. Considering sex and age, in addition to the SNPs, slightly and insignificantly increased the prediction accuracies (AUC of 0.680 and 0.800, respectively). Based on the sample size and candidate DNA markers investigated, this study provides the most robust, validated, and accurate statistical prediction models and SNP predictor marker sets currently available for predicting head hair shape from DNA, providing the next step towards broadening Forensic DNA Phenotyping beyond pigmentation traits., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Establishing the cellular or tissue-type origin of human biological traces found at crimes scenes is forensically relevant, as it allows evaluating the crime relevance of such traces and enables reconstructing the sequence of crime events. Messenger RNA and micro RNA markers are useful for forensic tissue identification, but provide challenges for linking RNA-identified cell/tissue types with DNA-identified trace donors, especially in mixed traces. DNA methylation markers overcome this problem, but provide technical challenges due to the DNA treatment required by most analysis methods. Here we introduce a novel type of DNA markers for forensic tissue identification analysed without prior DNA treatment, namely copy number variation (CNV). Using genome-wide CNV screening followed-up by targeted qPCR confirmation, and using qPCR analysis of additional CNV-like candidate DNA markers, in samples of several individuals from all commonly encountered forensically-relevant tissue types, we identified DNA markers specific for blood and semen, respectively. Preliminary forensic validation testing demonstrates that the developed qPCR assays are highly sensitive - delivering positive results down to picogram level of input DNA, specific, and can cope well with degraded DNA, providing suitable prerequisites for forensic applications. Moreover, we exemplified that using the CNV qPCR products as input material for subsequent forensic STR analysis delivered full STR profiles, opening-up new avenues of using the same DNA aliquot for both forensic purposes, tissue and individual identification. Provided additional forensic validation studies, we envision the application of these novel DNA markers for forensic tissue identification in future forensic casework. Such CNV markers are particularly useful for tissue identification in old/cold cases, where aged/old DNA extracts are available that contain no RNA and are not suitable for DNA methylation analysis due to limited DNA quantity and quality., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sudden cardiac death (SCD) is one of the major causes of mortality worldwide, mostly involving coronary artery disease in the elderly. In contrary, sudden death events in young victims often represent the first manifestation of undetected genetic cardiac diseases, which remained without any symptoms during lifetime. Approximately 30% of these sudden death cases have no definite cardiac etiology after a comprehensive medicolegal investigation and are therefore termed as sudden unexplained death (SUD) cases. Advances in high-throughput sequencing approaches have provided an efficient diagnostic tool to identify likely pathogenic variants in cardiovascular disease-associated genes in otherwise autopsy-negative SUD cases. The aim of this study was to genetically investigate a cohort of 34 unexplained death cases by focusing on candidate genes associated with cardiomyopathies and channelopathies. Exome analysis identified potentially disease-causing sequence alterations in 29.4% of the 34 SUD cases. Six (17.6%) individuals had variants with likely functional effects in the channelopathy-associated genes AKAP9, KCNE5, RYR2, and SEMA3A. Interestingly, four of these six SUD individuals were younger than 18 years of age. Since the total SUD cohort of this study included five children and adolescents, post-mortem molecular autopsy screening indicates a high diagnostic yield within this age group. Molecular genetic testing represents a valuable approach to uncover the cause of death in some of the SUD victims; however, 70-80% of the cases still remain elusive, emphasizing the importance of additional research to better understand the pathological mechanisms leading to a sudden death event.

We used our previously published NGS mRNA approach for body fluid identification to analyse 183 body fluids/tissues, including mock casework samples. The resulting data set was used to build a probabilistic model that predicts the origin of a stain. Our approach uses partial least squares followed by linear discriminant analysis to classify samples into six commonly occurring forensic body fluids. The model differs from the ones previously suggested in that it incorporates quantitative information (NGS read counts) rather than just presence/absence of markers. The suggested approach also allows for visualisation of important markers and their correlation with the different body fluids. We compared our model to previously published methods to show that the inclusion of read count information improves the prediction. Finally, we applied the model to mixed body fluid samples to test its ability to identify the individual components in a mixture., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Acrocallosal syndrome (ACLS) is an autosomal recessive neurodevelopmental disorder caused by KIF7 defects and belongs to the heterogeneous group of ciliopathies related to Joubert syndrome (JBTS). While ACLS is characterized by macrocephaly, prominent forehead, depressed nasal bridge, and hypertelorism, facial dysmorphism has not been emphasized in JBTS cohorts with molecular diagnosis. To evaluate the specificity and etiology of ACLS craniofacial features, we performed whole exome or targeted Sanger sequencing in patients with the aforementioned overlapping craniofacial appearance but variable additional ciliopathy features followed by functional studies. We found (likely) pathogenic variants of KIF7 in 5 out of 9 families, including the original ACLS patients, and delineated 1000 to 4000-year-old Swiss founder alleles. Three of the remaining families had (likely) pathogenic variants in the JBTS gene C5orf42, and one patient had a novel de novo frameshift variant in SHH known to cause autosomal dominant holoprosencephaly. In accordance with the patients' craniofacial anomalies, we showed facial midline widening after silencing of C5orf42 in chicken embryos. We further supported the link between KIF7, SHH, and C5orf42 by demonstrating abnormal primary cilia and diminished response to a SHH agonist in fibroblasts of C5orf42-mutated patients, as well as axonal pathfinding errors in C5orf42-silenced chicken embryos similar to those observed after perturbation of Shh signaling. Our findings, therefore, suggest that beside the neurodevelopmental features, macrocephaly and facial widening are likely more general signs of disturbed SHH signaling. Nevertheless, long-term follow-up revealed that C5orf42-mutated patients showed catch-up development and fainting of facial features contrary to KIF7-mutated patients.

TASK-1 potassium channels have been implicated in central and peripheral chemoreception; however, the precise contribution of TASK-1 for the control of respiration is still under debate. Here, we investigated the respiration of unrestrained adult and neonatal TASK-1 knockout mice (TASK-1 -/- ) using a plethysmographic device. Respiration in adult female TASK-1 -/- mice under control (21% O 2 ), hypoxia and hypercapnia was unaffected. Under acute hypoxia male TASK-1 -/- mice exhibited a reduced increase of the respiratory frequency (f R ) compared to wildtypes. However, the tidal volume (V T ) of male TASK-1 -/- mice was strongly enhanced. The volatile anesthetic isoflurane induced in male TASK-1 -/- and male wild type mice (TASK-1 +/+ ) a similar respiratory depression. Neonatal TASK-1 -/- mice demonstrated a 30-40% decrease of the minute volume, caused by a reduction of the f R under control condition (21% O 2 ). Under hypoxia, neonatal TASK-1 -/- mice more frequently stopped breathing (apnea>3s) suggesting an increased hypoxia-sensitivity. As reported before, this increased hypoxia sensitivity had no influence on the survival rate of neonatal TASK-1 -/- mice. In adult and neonatal mice, TASK-1 gene deletion induced a significant prolongation of the relaxation time (R T ), which is a parameter for expiration kinetics. Additionally, screening for mutations in the human TASK-1 gene in 155 cases of sudden infant death syndrome (SIDS) was inconclusive. In conclusion, these data are suggestive for an increased hypoxia-sensitivity of neonatal TASK-1 -/- mice, however, without causing an increase in neonatal lethality. In adult female TASK-1 -/- mice respiration was unaffected, whereas adult male TASK-1 -/- mice showed a modified breathing pattern. These results are suggestive for sex-specific mechanisms for compensating the inactivation of TASK-1 in mice., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Objectives: The island of Sardinia has one of the highest incidence rates of β-thalassemia in Europe due to its long history of endemic malaria, which, according to historical records, was introduced around 2,600 years ago by the Punics and only became endemic around the Middle Ages. In particular, the cod39 mutation is responsible for more than 95% of all β-thalassemia cases observed on the island. Debates surround the origin of the mutation. Some argue that its presence in the Western Mediterranean reflects the migration of people away from Sardinia, others that it reflects the colonization of the island by the Punics who might have carried the disease allele. The aim of this study was to investigate β-globin mutations, including cod39, using ancient DNA (aDNA) analysis, to better understand the history and origin of β-thalassemia and malaria in Sardinia., Materials and Methods: PCR analysis followed by sequencing were used to investigate the presence of β-thalassemia mutations in 19 individuals from three different Roman and Punic necropolises in Sardinia., Results: The cod39 mutation was identified in one male individual buried in a necropolis from the Punic/Roman period. Further analyses have shown that his mitochondrial DNA (mtDNA) and Y-chromosome haplogroups were U5a and I2a1a1, respectively, indicating the individual was probably of Sardinian origin., Conclusions: This is the earliest documented case of β-thalassemia in Sardinia to date. The presence of such a pathogenic mutation and its persistence until present day indicates that malaria was likely endemic on the island by the Roman period, earlier than the historical sources suggest., (© 2017 Wiley Periodicals, Inc.)

Sudden infant death syndrome (SIDS) is described as the sudden and unexplained death of an apparently healthy infant younger than one year of age. Genetic studies indicate that up to 35% of SIDS cases might be explained by familial or genetic diseases such as cardiomyopathies, ion channelopathies or metabolic disorders that remained undetected during conventional forensic autopsy procedures. Post-mortem genetic testing by using massive parallel sequencing (MPS) approaches represents an efficient and rapid tool to further investigate unexplained death cases and might help to elucidate pathogenic genetic variants and mechanisms in cases without a conclusive cause of death. In this study, we performed whole-exome sequencing (WES) in 161 European SIDS infants with focus on 192 genes associated with cardiovascular and metabolic diseases. Potentially causative variants were detected in 20% of the SIDS cases. The majority of infants had variants with likely functional effects in genes associated with channelopathies (9%), followed by cardiomyopathies (7%) and metabolic diseases (1%). Although lethal arrhythmia represents the most plausible and likely cause of death, the majority of SIDS cases still remains elusive and might be explained by a multifactorial etiology, triggered by a combination of different genetic and environmental risk factors. As WES is not substantially more expensive than a targeted sequencing approach, it represents an unbiased screening of the exome, which could help to investigate different pathogenic mechanisms within the genetically heterogeneous SIDS cohort. Additionally, re-analysis of the datasets provides the basis to identify new candidate genes in sudden infant death.

Trace DNA is often found in forensic science investigations. Experience has shown that it is difficult to retrieve a DNA profile when trace DNA is collected from clothing. The aim of this study was to compare four different DNA collection techniques on six different types of clothing in order to determine the best trace DNA recovery method. The classical stain recovery technique using a wet cotton swab was tested against dry swabbing, scraping and a new method, referred to as the mini-tape lifting technique. Physical contact was simulated with three different "perpetrators" on 18 machine-washed garments. DNA was collected with the four different DNA recovery methods and subjected to standard PCR-based DNA profiling. The comparison of STR results showed best results for the mini-tape lifting and scraping methods independent of the type of clothing. The new mini-tape lifting technique proved to be an easy and reliable DNA collection method for textiles., (© 2016 American Academy of Forensic Sciences.)

Jörg Jenatsch, a leading freedom fighter during the Thirty Year's War in Graubünden, Switzerland, was assassinated on carnival 1639. Jenatsch's controversial biography and the unclear circumstances of his death inspired the formation of various legends, novels and films. In 1959, a skeleton discovered in the cathedral of Chur with remains of wealthy baroque clothing was tentatively attributed to Jenatsch. Here, we reassess the skeleton based on a new exhumation. Our multidisciplinary analysis and the head injuries are consistent with reports of the eyewitnesses of the crime, demonstrating that Jenatsch was killed from behind with a semi-sharp implement, supposedly an axe, as well as by a blow with a broad-surfaced object. Moreover, our facial reconstruction closely matches an oil portrait of Jenatsch, and the HIrisPlex system applied to DNA-extracts from the femoral bone reveals brown eye and dark brown hair colour, which coincides well with the portrait, too. Finally, isotope analysis of the femoral bone and a molar support Jenatsch's high social status, luxury diet and a high mobility in the last decade of his life. This multidisciplinary approach thus reinforces personal identification and provides additional insight into the life of this important historic person beyond written resources., Competing Interests: The authors have declared that no competing interests exist.

Sudden death of healthy young adults in the absence of any medical reason is generally categorised as autopsy-negative sudden unexplained death (SUD). Approximately 30 % of all SUD cases can be explained by lethal sequence variants in cardiac genes causing disturbed ion channel functions (channelopathies) or minimal structural heart abnormalities (cardiomyopathies). The aim of this study was to perform whole-exome sequencing (WES) in five young SUD cases in order to identify potentially disease-causing mutations with a focus on 184 genes associated with cardiac diseases or sudden death. WES analysis enabled the identification of damaging-predicted cardiac sequence alterations in three out of five SUD cases. Two SUD victims carried disease-causing variants in long QT syndrome (LQTS)-associated genes (KCNH2, SCN5A). In a third case, WES identified variants in two genes involved in mitral valve prolapse and thoracic aortic aneurism (DCHS1, TGFβ2). The genome of a fourth case carried several minor variants involved in arrhythmia pointing to a multigene influence that might have contributed to sudden death. Our results confirm that post-mortem genetic testing in SUD cases in addition to the conventional autopsy can help to identify familial cardiac diseases and can contribute to the identification of genetic risk factors for sudden death.

Elderly patients may be different from the average population in regard to the treatment of shoulder disorders. Challenges are the decreased quality of bone, tendons and cartilage, decreased blood perfusion and a generally aged biology. The advantages however are the often more realistic expectations and more cautious use of the extremity, and the limited life expectancy of prosthetic implants is a less pressing issue. Local pathologies such as in the AC-joint or long head of the biceps may also in the aged patient be treated with infiltration or arthroscopic means. If however large rotator cuff tears and osteoarthritis are present, (reverse) total shoulder implants are the treatment of choice due to the high reliability and uncomplicated rehabilitation.

RNA analysis is a valuable tool for the identification of the forensically relevant body fluids, saliva, blood, menstrual blood, cervicovaginal fluid, and semen. Multiple human mRNA and bacterial RNA markers have been identified for each of these body fluids. RNA and DNA can be coextracted from the same portion of a sample and RNA markers for different body fluids can be multiplexed in a single PCR, thereby maximizing the number of analyses that can be performed with limited sample material.