Unpredicted Aberrant Splicing Products Identified in Postmortem Sudden Cardiac Death Samples.

Molecular screening for pathogenic mutations in sudden cardiac death (SCD)-related genes is common practice for SCD cases. However, test results may lead to uncertainty because of the identification of variants of unknown significance (VUS) occurring in up to 70% of total identified variants due to a lack of experimental studies. Genetic variants affecting potential splice site variants are among the most difficult to interpret. The aim of this study was to examine rare intronic variants identified in the exonic flanking sequence to meet two main objectives: first, to validate that canonical intronic variants produce aberrant splicing; second, to determine whether rare intronic variants predicted as VUS may affect the splicing product. To achieve these objectives, 28 heart samples of cases of SCD carrying rare intronic variants were studied. Samples were analyzed using 85 SCD genes in custom panel sequencing. Our results showed that rare intronic variants affecting the most canonical splice sites displayed in 100% of cases that they would affect the splicing product, possibly causing aberrant isoforms. However, 25% of these cases (1/4) showed normal splicing, contradicting the in silico results. On the contrary, in silico results predicted an effect in 0% of cases, and experimental results showed >20% (3/14) unpredicted aberrant splicing. Thus, deep intron variants are likely predicted to not have an effect, which, based on our results, might be an underestimation of their effect and, therefore, of their pathogenicity classification and family members' follow-up.

Post-mortem toxicology analysis in a young sudden cardiac death cohort.

Risk of sudden cardiac death (SCD) increases with age, and several studies have examined the impact of different drugs on cardiovascular function. However, few studies have integrated epidemiological drug consumption data and genetic background in the context of cardiac death. We performed a retrospective population-based study in forensic sudden death cases from a 9-year period in Catalonia. The young cohort included 924 cases 18-50 years old, 566 of which had a cardiac cause of death. Complete autopsy, toxicological, and histopathological studies were performed. Molecular autopsy using next-generation sequencing was performed in nearly 400 cardiac cases. Cases related with fatal acute intoxication were excluded. Drug consumption prevalence was similar between forensic cases of cardiac and non-cardiac origin (62.5% versus 69.5%), with the exception of alcohol, which was more prevalent in the cardiac group than in the non-cardiac group (23.3% versus 17.1%). Individuals in the toxicology-positive group were carriers of more rare genetic variants and were significantly younger than the toxicology-negative group. Psychopharmacological drugs were identified in 22.3% of cardiac cases, and molecular autopsy identified an association between antiepileptic drugs or caffeine and pathogenic or likely pathogenic variants in arrhythmogenic genes. Specific substances could therefore play an essential role as triggers of SCD in genetically predisposed young people.

Rare variants in genes encoding structural myocyte contribute to a thickened ventricular septum in sudden death population without ventricular alterations.

Unexpected cardiac deaths are a current challenge to healthcare systems. In adults, coronary artery disease and acquired cardiomyopathies are the most frequent causes of sudden cardiac death while in younger than 35 years old, the main cause is represented by non-ischemic diseases, usually inherited. Nowadays, around 10%-15% of unexpected deaths remain without a definite cause of decease after a complete autopsy, then classified as deaths potentially due to an inherited arrhythmia. Discrete abnormalities in some of the heart measures have been considered as potential predictors or risk factors for sudden cardiac death. However, role of non-benign genetic variants in these scattered heart alterations remains to be clarified, especially if variants are classified of ambiguous role. Clinicians usually only take into consideration pathogenic variants for decision-making. It is yet unclear what the role of VUS genetic variants in modifying the anatomical parameters of the heart. We hypothesize that some heart measures might be influenced by polygenic components as some variants may individually confer minor risk but may actually produce additive effects when combined with others. Our aim was to investigate whether carrying non-benign rare variants in genes related to inherited arrhythmias may contribute to scattered cardiac alterations in anatomical normal hearts. The study is composed by 761 samples collected from autopsies of SD suffered by adults from 18 to 50 years of age who occurred in Catalonia (Spain) in a 9-year period. Complete medico-legal autopsy was performed to determine the cause of death. Molecular autopsy was performed as part of our forensic protocol, including genes associated with inherited diseases.To evaluate the effect of genetic rare variants into hearts measures we performed a linear regression model and data were presented as regression. This study showed, for the first time, that rare variants, regardless of significance (pathogenic, probably pathogenic or uncertain significance), may contribute to interventricular septum width in the structurally normal heart. While the cohort is based on sudden death cases, further studies and case-control studies will be necessary to conclude that the genetic determinants of septal thickness contributes to sudden cardiac death. We conclude that non-benign rare variants contribute to modify scattered septum width in structural normal hearts, being a potential risk factor of arrhythmia in genetic harbors. These evidence support the current recommendation in forensic protocols of including histologic analysis of septum when inherited arrhythmogenic disease is suspicious cause of decease.

Out-of-Hospital Tissue Donation: Multidisciplinary Donor Circuit in a Forensic Institute.

OBJECTIVE: We aimed to implement an out-of-hospital system of generating donors that increases donation and answers the growing demand for tissue for therapeutic purposes. MATERIAL: The Catalan Health Service issued the 4/2015 instruction promoting the integration of the donation network through collaboration with the Donor Center of Catalonia (DCC). The creation of DCC facilitated the signing of an agreement between The Blood and Tissue Bank, the Department of Justice of the Generalitat de Catalunya, the Emergency Medical System, and the Hospital Clínic Barcelona for the procurement of tissues in the Institute of Legal Medicine and Forensic Sciences of Catalonia (IMLCFC), where the autopsies of all judicial deaths in the province of Barcelona are performed. METHODS: The Emergency Medical System informed the DCC of those instances that ended with the code "deceased." DCC assessed the possible donor on arrival at the IMLCFC, checked the medical history through the shared clinical record, and obtained family consent by telephone interview. If consent was obtained, then judicial authorization was sought. RESULTS: In 2016, 152 donors of corneas were obtained (9.7% of the annual amount in Catalonia), 149 in 2017 (9.4% of the annual amount), and 133 donations in 2018. At the end of 2017, we started multitissue retrieval and obtained in 2018 a total of 76 donors. CONCLUSIONS: Out-of-hospital tissue donation in a forensic institute is possible. In less than 3 years, IMLCFC has become the third largest eye tissue contributor among the Catalan tissue donation network and the first contributor in musculoskeletal tissues in 2018.

All-inside arthroscopic allograft reconstruction of the anterior talo-fibular ligament using an accesory transfibular portal.

BACKGROUND: Anatomic graft reconstruction of the anterior talo-fibular ligament is an alternative for patients who are bad candidates for standard procedures such as a Broström-Gould reconstruction (high-demand athletes, obesity, hyperlaxity or collagen disorders, capsular insufficiency or talar avulsions). The purpose of this study is to describe an all-inside arthroscopic technique for ATFL reconstruction, and the results in a series of patients with chronic ankle instability. METHODS: We reviewed patients with chronic ATFL ruptures treated with an all-inside arthroscopic allograft reconstruction of the ATFL, with a minimum 2-year follow-up. Twenty-two patients with lateral ankle instability were included. Mean follow-up was 34±2.5 months. RESULTS: The mean AOFAS score improved from 62.3±6.7 points preoperatively to 97.2±3.2 points at final follow-up. Three patients suffered complications: one case each of ankle rigidity, superficial peroneal nerve injury and fibular fracture. CONCLUSIONS: Chronic ATFL injuries are amenable to all-inside arthroscopic allograft reconstruction fixed with tenodesis screws. This procedure simplifies other reported techniques in that it facilitates identification and bone tunnel placement of the talar ATFL insertion.

Sudden Arrhythmic Death During Exercise: A Post-Mortem Genetic Analysis.

BACKGROUND: Sudden cardiac death is a natural and unexpected death that occurs within 1 h of the first symptom. Most sudden cardiac deaths occur during exercise, mostly as a result of myocardial infarction. After autopsy, some cases, especially in the young, are diagnosed as cardiomyopathies or remain without a conclusive cause of death. In both situations, genetic alterations may explain the arrhythmia. OBJECTIVE: Our aim was to identify a genetic predisposition to sudden cardiac death in a cohort of post-mortem cases of individuals who died during exercise, with a structurally normal heart, and were classified as arrhythmogenic death. METHODS: We analyzed a cohort of 52 post-mortem samples from individuals <50 years old who had a negative autopsy. Next-generation sequencing technology was used to screen genes associated with sudden cardiac death. RESULTS: Our cohort showed a male prevalence (12:1). Half of the deaths occurred in individuals 41-50 years of age. Running was the most common exercise activity during the fatal event, accounting for 46.15% of cases. Genetic analysis identified 83 rare variants in 37 samples (71.15% of all samples). Of all rare variants, 36.14% were classified as deleterious, being present in 53.84% of all cases. CONCLUSIONS: A comprehensive analysis of sudden cardiac death-related genes in individuals who died suddenly while exercising enabled the identification of potentially causative variants. However, many genetic variants remain of indeterminate significance, thus further work is needed before clinical translation. Nonetheless, comprehensive genetic analysis of individuals who died during exercise enables the detection of potentially causative variants and helps to identify at-risk relatives.

Correction: Natural and Undetermined Sudden Death: Value of Post-Mortem Genetic Investigation.

[This corrects the article DOI: 10.1371/journal.pone.0167358.].

Genetic analysis in post-mortem samples with micro-ischemic alterations.

Sudden cardiac arrest is a leading cause of death worldwide. Most cardiac arrests happen in patients who have previously suffered a myocardial infarct. The risk of sudden death after infarction may increase in people who carry a pathogenic genetic alteration in cardiac ion channels. We hypothesized that micro-ischemia could trigger lethal arrhythmogenesis, thus we sought to identify genetic alterations in cardiac ion channels in patients with micro-ischemic disease. We studied a cohort of 56 post-mortem samples. Autopsy studies identified myocardial infarction as the cause of death in each case. We used both Sanger sequencing and next-generation sequencing to screen candidate genes associated with sudden cardiac death. We identified six rare missense genetic variations in five unrelated patients. Two variants have been previously reported; one is associated with atrial fibrillation (SCN5A_p.H445D), and the other is predicted to be benign (ANK2_p.T2059M). The novel variants were predicted in silico as benign, except for one (RyR2_p.M4019T), which was classified as deleterious. Our post-mortem, micro-infarction cohort displayed a rate of nearly 10% non-common genetic variants. However, the clinical significance of most of the identified variants remains unknown due to lack of family assessment. Further analyses should be performed in large cohorts to clarify the role of ion-channel gene analysis in samples showing microscopic ischemic alterations.

Natural and Undetermined Sudden Death: Value of Post-Mortem Genetic Investigation.

BACKGROUND: Sudden unexplained death may be the first manifestation of an unknown inherited cardiac disease. Current genetic technologies may enable the unraveling of an etiology and the identification of relatives at risk. The aim of our study was to define the etiology of natural deaths, younger than 50 years of age, and to investigate whether genetic defects associated with cardiac diseases could provide a potential etiology for the unexplained cases. METHODS AND FINDINGS: Our cohort included a total of 789 consecutive cases (77.19% males) <50 years old (average 38.6±12.2 years old) who died suddenly from non-violent causes. A comprehensive autopsy was performed according to current forensic guidelines. During autopsy a cause of death was identified in most cases (81.1%), mainly due to cardiac alterations (56.87%). In unexplained cases, genetic analysis of the main genes associated with sudden cardiac death was performed using Next Generation Sequencing technology. Genetic analysis was performed in suspected inherited diseases (cardiomyopathy) and in unexplained death, with identification of potentially pathogenic variants in nearly 50% and 40% of samples, respectively. CONCLUSIONS: Cardiac disease is the most important cause of sudden death, especially after the age of 40. Close to 10% of cases may remain unexplained after a complete autopsy investigation. Molecular autopsy may provide an explanation for a significant part of these unexplained cases. Identification of genetic variations enables genetic counseling and undertaking of preventive measures in relatives at risk.

Post-mortem genetic analysis in juvenile cases of sudden cardiac death.

BACKGROUND: The reason behind a sudden death of a young individual remains unknown in up to 50% of postmortem cases. Pathogenic mutations in genes encoding heart proteins are known to cause sudden cardiac death. OBJECTIVE: The aim of our study was to ascertain whether genetic alterations could provide an explanation for sudden cardiac death in a juvenile cohort with no-conclusive cause of death after comprehensive autopsy. METHODS: Twenty-nine cases <15 years showing no-conclusive cause of death after a complete autopsy were studied. Genetic analysis of 7 main genes associated with sudden cardiac death was performed using Sanger technology in low quality DNA cases, while in good quality cases the analysis of 55 genes associated with sudden cardiac death was performed using Next Generation Sequencing technology. RESULTS: Thirty-five genetic variants were identified in 12 cases (41.37%). Ten genetic/variants in genes encoding cardiac ion channels were identified in 8 cases (27.58%). We also identified 9 cases (31.03%) carrying 25 genetic variants in genes encoding structural cardiac proteins. Nine cases carried more than one genetic variation, 5 of them combining structural and non-structural genes. CONCLUSIONS: Our study supports the inclusion of molecular autopsy in forensic routine protocols when no conclusive cause of death is identified. Around 40% of sudden cardiac death young cases carry a genetic variant that could provide an explanation for the cause of death. Because relatives could be at risk of sudden cardiac death, our data reinforce their need of clinical assessment and, if indicated, of genetic analysis.

Estudio retrospectivo de la muerte súbita cardiovascular extrahospitalaria de adultos de mediana edad en la ciudad de Barcelona / Retrospective study of out-of-hospital sudden cardiovascular death in middle-aged adults in Barcelona city

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Traumatic bilateral vertebral artery dissection.

Traumatic vertebral artery dissection is not often seen by forensic pathologists, and cases investigated are scarce in the forensic literature. We present the case of a 40-year-old woman cyclist who was struck by a car while wearing a helmet, and was neurologically near normal immediately thereafter at Emergency. She presented 48 h later with acute right hemiparesis, decreasing level of consciousness, and unsteadiness. CT revealed massive cerebellar infarction. CT angiography was normal. The patient died in coma 7 days after injury and autopsy revealed bilateral edematous cerebellar infarction and bilateral vertebral artery dissection. Rotational neck injury and mural tear in the wall of the Atlantic parts of both vertebral arteries is suggested as the possible mechanism of the arterial injury. Head and neck injuries are reported as a precipitating cause of vertebral artery injury. The possible influence of trauma may be further underestimated if longer intervals between vessel dissection and ischemia occur. The current case illustrates that "talk-and-die" syndrome may be due to occult vertebral artery dissection, possibly bilateral. In forensic cases of delayed death after mild trauma to the head and neck, the vertebral arteries should be examined for the cause of death.

A proposed dissection procedure for vertebral arteries in forensic pathology.

Vertebral artery removing constitutes a significant forensic pathology challenge. Dissection techniques during head-neck autopsy are based on anterior approach, a difficult method, which is unable to assess the transverse part of the artery. This work presents an original and simple method for dissecting vertebral arteries by a posterior approach, opening the vertebroarterial canal through the spinal canal without any special equipment. Once the spinal cord is removed, the transversarium foramens are opened by an internal cut at the pedicle and an external cut at the transverse process. This enables us to visualize vertebral arteries in its entirety. The method improves both the examination of the upper extracranial segment of the vertebral artery and the neuropathological study when arterial injury is suspected. Applying this method routinely is both feasible and useful in suspected cases of vertebral artery trauma and could contribute to assess more precisely the actual incidence of this injury.

Injury profiles of road traffic deaths.

The objective is to describe injuries of road traffic deaths in Barcelona and identify injury profiles by road user type, through a cross-sectional study including road traffic deaths for the period 1997-2004. The data source was the Institut de Medicina Legal de Catalunya. Diagnoses were coded using the International Classification of Diseases, 9th revision, Clinical Modification, and classified using the Barell Matrix. Of the 719 deaths studied, 45% were pedestrians, 32% two wheel motor vehicle users, and 23% car occupants. The injury profile of the road traffic deaths in Barcelona is internal injuries and fractures to the torso and to the head/neck. This profile is repeated in all the road user groups, although pedestrians present higher frequencies of fractures and contusions to extremities and contusions to the head/neck, and fewer internal torso injuries than car occupants or two wheel motor vehicle users.

Sensitivity to punishment and sensitivity to reward and traffic violations.

The aim of our study consists of contributing information on the relationship between the personality variables derived from Gray's model and the conduct that accompanies the infringement of the road traffic rules. Seven hundred and ninety-two adults of both sexes took part in the study (389 men and 403 women), all of whom had driving licences and drove frequently. The subjects answered "The Sensitivity to Punishment and Sensitivity to Reward Questionnaire", a scale of monotony avoidance, and two Likert scales of attitude and behaviour in connection with traffic violations. We found a high positive relationship between attitude and behaviour, with the men infringing the rules more than the women. Hypotheses regarding a relationship between traffic offences and sensibility to reward and monotony avoidance were confirmed. Those people with high scores in sensitivity to punishment and low ones in sensitivity to reward were those who drove within the law, while those with low sensitivity to punishment and high sensitivity to reward were those who broke it more. Sensitivity to reward was a stronger determinant in encouraging infringement of the rules than was sensitivity to punishment in discouraging the subjects to do so.