The potential of microRNAs (miRNA) as non-invasive diagnostic, prognostic, and predictive biomarkers, as well as therapeutic targets, has recently been recognized. Previous studies have highlighted the importance of consistency in the methodology used, but to our knowledge, no study has described the methodology of sample preparation and storage systematically with respect to miRNAs as blood biomarkers. The aim of this study was to investigate the stability of miRNAs in blood under various relevant clinical and research conditions: different collection tubes, storage at different temperatures, physical disturbance, as well as serial freeze-thaw cycles. Blood samples were collected from 12 healthy donors into different collection tubes containing anticoagulants, including EDTA, citrate and lithium-heparin, as well as into serum collection tubes. MiRNA stability was evaluated by measuring expression changes of miR-1, miR-21 and miR-29b at different conditions: varying processing time of whole blood (up to 72 hours (h)), long-term storage (9 months at -80°C), physical disturbance (1 and 8 h), as well as in a series of freeze/thaw cycles (1 and 4 times). Different collection tubes revealed comparable concentrations of miR-1, miR-21 and miR-29b. Tubes with lithium-heparin were found unsuitable for miRNA quantification. MiRNA levels were stable for at least 24 h at room temperature in whole blood, while separated fractions did show alterations within 24 h. There were significant changes in the miR-21 and miR-29b levels after 72 h incubation of whole blood at room temperature (p<0.01 for both). Both miR-1 and miR-21 showed decreased levels after physical disturbance for 8 h in separated plasma and miR-1 in serum whole blood, while after 1 h of disturbance no changes were observed. Storage of samples at -80°C extended the miRNA stability remarkably, however, miRNA levels in long-term stored (9 months) whole blood samples were significantly changed, which is in contrast to the plasma samples, where miR-21 or miR-29b levels were found to be stable. Repetitive (n = 4) freeze-thaw cycles resulted in a significant reduction of miRNA concentration both in plasma and serum samples. This study highlights the importance of proper and systematic sample collection and preparation when measuring circulating miRNAs, e.g., in context of clinical trials. We demonstrated that the type of collection tubes, preparation, handling and storage of samples should be standardized to avoid confounding variables influencing the results.

Asthma is a common chronic disease among young adults, and several studies have reported increased mortality rates in patients with asthma. However, no study has described sudden unexpected death in a nationwide setting in patients with uncontrolled asthma. We defined uncontrolled asthma as a previous hospital admittance because of asthma (of any severity) or when asthma was considered to have influenced the death according to the death certificate. The purpose of this study is to increase the medical focus on young persons with uncontrolled asthma and thereby hopefully aid in preventing sudden unexpected deaths. We therefore aimed to describe clinical characteristics, symptoms, causes of death, and contact with the healthcare system prior to sudden unexpected death in young persons with uncontrolled asthma. Through the review of death certificates, we found 625 sudden unexpected death cases in individuals aged 1–35 years in Denmark from 2000 to 2006. Of those, 49 persons with uncontrolled asthma were identified. Previous contacts with the healthcare system were identified, and available records from general practitioners were retrieved. We identified 49 individuals who suffered from uncontrolled asthma. This corresponds to an incidence rate of 0.32 per 100,000 person-years. The cause of death in 31 cases (63%) was sudden cardiac death, and in 13 cases (27%), it was a fatal asthma attack. Symptoms (chest pain, dyspnea, seizures, general malaise, syncope, and palpitations) prior to death were reported in 41 (84%) of the cases. In 34 (69%) of the cases, antecedent symptoms (symptoms >24 hours before death) were present, and 28 (57%) patients had prodromal symptoms (symptoms <24 hours before death). The most common antecedent symptoms were dyspnea and chest pain, whereas the most common prodromal symptoms were dyspnea, general malaise, and/or fatigue. Twenty-eight patients (57%) sought medical advice from a general practitioner and/or emergency department due to these symptoms. The cause of death was predominantly sudden cardiac death followed by fatal asthma attack. We found that 41 (84%) of patients suffered from symptoms prior to death and that 28 (57%) sought medical advice from the emergency department and/or general practitioners.

The genetic basis of cardiac electrical phenotypes has in the last 25 years been the subject of intense investigation. While in the first years, such efforts were dominated by the study of familial arrhythmia syndromes, in recent years, large consortia of investigators have successfully pursued genome-wide association studies (GWAS) for the identification of single-nucleotide polymorphisms that govern inter-individual variability in electrocardiographic parameters in the general population. We here provide a review of GWAS conducted on cardiac electrical phenotypes in the last 14 years and discuss the implications of these discoveries for our understanding of the genetic basis of disease susceptibility and variability in disease severity. Furthermore, we review functional follow-up studies that have been conducted on GWAS loci associated with cardiac electrical phenotypes and highlight the challenges and opportunities offered by such studies.

Although sudden infant death syndrome (SIDS) is a leading cause of natural death during the first year of life,^1-3 the underlying mechanisms causing sudden death remain largely unknown, and prevention is still a challenge. SIDS is a diagnosis of exclusion applied to the death of an infant younger than 1 year after an extensive postmortem investigation.⁴ Causes of SIDS are considered to be complex and multifactorial.⁵ There is a triple-risk hypothesis with 3 risk factors that predispose to SIDS: (1) an exogenous stressor (eg, sleeping prone, smoke exposure), (2) a critical development period (usually age 2-4 months), and (3) an underlying vulnerability, such as genetic susceptibility.^6,7 To date, it is not possible to predict infants at risk of SIDS. Environmental factors may be risk factors in an infant who is genetically susceptible and quite innocuous to a nonsusceptible infant. A positive familial aggregation study is an important step to suggest the usefulness of clinical genetic work-up of the families. Even though positive family history of SIDS has been described,^8-14 it is unknown whether a familial aggregation exists in the general population. Previous studies of familial aggregation were performed on small and/or selected populations^8-12; therefore, a nationwide study on an unselected pediatric population with information on family history is warranted to establish a familial clustering of children who died of SIDS. More knowledge is warranted because an association between a family history of SIDS and subsequent SIDS will have public health implications to identify children at risk of SIDS, with the goal of preventing of further SIDS within families. Thus, the aim of this study was to evaluate whether siblings of children who died of SIDS have a higher risk of SIDS compared with the general pediatric population. We hypothesized that the risk of SIDS would be increased among siblings of children who died of SIDS.

Brugada syndrome (BrS) is an inherited arrhythmia with a higher disease prevalence and more lethal arrhythmic events in Asians than in Europeans. Genome-wide association studies (GWAS) have revealed its polygenic architecture mainly in European populations. The aim of this study was to identify novel BrS-associated loci and to compare allelic effects across ancestries. A GWAS was conducted in Japanese participants, involving 940 cases and 1634 controls, followed by a cross-ancestry meta-analysis of Japanese and European GWAS (total of 3760 cases and 11 635 controls). The novel loci were characterized by fine-mapping, gene expression, and splicing quantitative trait associations in the human heart. The Japanese-specific GWAS identified one novel locus near ZSCAN20 (P = 1.0 × 10⁻⁸), and the cross-ancestry meta-analysis identified 17 association signals, including six novel loci. The effect directions of the 17 lead variants were consistent (94.1%; P for sign test = 2.7 × 10⁻⁴), and their allelic effects were highly correlated across ancestries (Pearson’s R = .91; P = 2.9 × 10⁻⁷). The genetic risk score derived from the BrS GWAS of European ancestry was significantly associated with the risk of BrS in the Japanese population [odds ratio 2.12 (95% confidence interval 1.94–2.31); P = 1.2 × 10⁻⁶¹], suggesting a shared genetic architecture across ancestries. Functional characterization revealed that a lead variant in CAMK2D promotes alternative splicing, resulting in an isoform switch of calmodulin kinase II-δ, favouring a pro-inflammatory/pro-death pathway. This study demonstrates novel susceptibility loci implicating potentially novel pathogenesis underlying BrS. Despite differences in clinical expressivity and epidemiology, the polygenic architecture of BrS was substantially shared across ancestries. Japanese-specific GWAS (940 cases and 1634 controls) followed by a cross-ancestry GWAS meta-analysis using Japanese (JPN) and European (EUR) populations (2820 cases and 10001 controls) have identified six novel BrS risk loci including CAMK2D and shared polygenic architectures across JPN and EUR. BrS, Brugada syndrome; GRS, genetic risk score; GWAS, genome-wide association study; LAE, lethal arrhythmic event; QTL, quantitative trait locus; sQTL, cis-splice quantitative trait locus.