DNA-pools targeted-sequencing as a robust cost-effective method to detect rare variants: Application to dilated cardiomyopathy genetic diagnosis.

Dilated cardiomyopathy (DCM) is a heart disease characterized by left ventricular dilatation and systolic dysfunction. In 30% of cases, pathogenic variants, essentially private to each patient, are identified in at least one of almost 50 reported genes. Thus, while costly, exons capture-based Next Generation Sequencing (NGS) of a targeted gene panel appears as the best strategy to genetically diagnose DCM. Here, we report a NGS strategy applied to pools of 8 DNAs from DCM patients and validate its robustness for rare variants detection at 4-fold reduced cost. Our pipeline uses Freebayes to detect variants with the expected 1/16 allele frequency. From the whole set of detected rare variants in 96 pools we set the variants quality parameters optimizing true positives calling. When compared to simplex DNA sequencing in a shared subset of 50 DNAs, 96% of SNVs/InsDel were accurately identified in pools. Extended to the 384 DNAs included in the study, we detected 100 variants (ACMG class 4 and 5), mostly in well-known morbid gene causing DCM such as TTN, MYH7, FLNC, and TNNT2. To conclude, we report an original pool-sequencing NGS method accurately detecting rare variants. This innovative approach is cost-effective for genetic diagnostic in rare diseases.

Integrative Analyses of Circulating Proteins and Metabolites Reveal Sex Differences in the Associations with Cardiac Function among DCM Patients.

Dilated cardiomyopathy (DCM) is characterized by reduced left ventricular ejection fraction (LVEF) and left or biventricular dilatation. We evaluated sex-specific associations of circulating proteins and metabolites with structural and functional heart parameters in DCM. Plasma samples (297 men, 71 women) were analyzed for proteins using Olink assays (targeted analysis) or LC-MS/MS (untargeted analysis), and for metabolites using LC MS/MS (Biocrates AbsoluteIDQ p180 Kit). Associations of proteins (n = 571) or metabolites (n = 163) with LVEF, measured left ventricular end diastolic diameter (LVEDDmeasured), and the dilation percentage of LVEDD from the norm (LVEDDacc. to HENRY) were examined in combined and sex-specific regression models. To disclose protein-metabolite relations, correlation analyses were performed. Associations between proteins, metabolites and LVEF were restricted to men, while associations with LVEDD were absent in both sexes. Significant metabolites were validated in a second independent DCM cohort (93 men). Integrative analyses demonstrated close relations between altered proteins and metabolites involved in lipid metabolism, inflammation, and endothelial dysfunction with declining LVEF, with kynurenine as the most prominent finding. In DCM, the loss of cardiac function was reflected by circulating proteins and metabolites with sex-specific differences. Our integrative approach demonstrated that concurrently assessing specific proteins and metabolites might help us to gain insights into the alterations associated with DCM.

Deep phenotyping of two preclinical mouse models and a cohort of RBM20 mutation carriers reveals no sex-dependent disease severity in RBM20 cardiomyopathy.

RBM20 cardiomyopathy is an arrhythmogenic form of dilated cardiomyopathy caused by mutations in the splicing factor RBM20. A recent study found a more severe phenotype in male patients with RBM20 cardiomyopathy patients than in female patients. Here, we aim to determine sex differences in an animal model of RBM20 cardiomyopathy and investigate potential underlying mechanisms. In addition, we aim to determine sex and gender differences in clinical parameters in a novel RBM20 cardiomyopathy patient cohort. We characterized an Rbm20 knockout (KO) mouse model, and show that splicing of key RBM20 targets, cardiac function, and arrhythmia susceptibility do not differ between sexes. Next, we performed deep phenotyping of these mice, and show that male and female Rbm20-KO mice possess transcriptomic and phosphoproteomic differences. Hypothesizing that these differences may influence the heart's ability to compensate for stress, we exposed Rbm20-KO mice to acute catecholaminergic stimulation and again found no functional differences. We also replicate the lack of functional differences in a mouse model with the Rbm20-R636Q mutation. Lastly, we present a patient cohort of 33 RBM20 cardiomyopathy patients and show that these patients do not possess sex and gender differences in disease severity. Current mouse models of RBM20 cardiomyopathy show more pronounced changes in gene expression and phosphorylation of cardiac proteins in male mice, but no sex differences in cardiac morphology and function. Moreover, other than reported before, male RBM20 cardiomyopathy patients do not present with worse cardiac function in a patient cohort from Germany and the Netherlands.NEW & NOTEWORTHY Optimal management of the cardiac disease is increasingly personalized, partly because of differences in outcomes between sexes. RBM20 cardiomyopathy has been described to be more severe in male patients, and this carries the risk that male patients are more scrutinized in the clinic than female patients. Our findings do not support this observation and suggest that treatment should not differ between male and female RBM20 cardiomyopathy patients, but instead should focus on the underlying disease mechanism.

Doxorubicin induces cardiotoxicity in a pluripotent stem cell model of aggressive B cell lymphoma cancer patients.

Cancer therapies with anthracyclines have been shown to induce cardiovascular complications. The aims of this study were to establish an in vitro induced pluripotent stem cell model (iPSC) of anthracycline-induced cardiotoxicity (ACT) from patients with an aggressive form of B-cell lymphoma and to examine whether doxorubicin (DOX)-treated ACT-iPSC cardiomyocytes (CM) can recapitulate the clinical features exhibited by patients, and thus help uncover a DOX-dependent pathomechanism. ACT-iPSC CM generated from individuals with CD20+ B-cell lymphoma who had received high doses of DOX and suffered cardiac dysfunction were studied and compared to control-iPSC CM from cancer survivors without cardiac symptoms. In cellular studies, ACT-iPSC CM were persistently more susceptible to DOX toxicity including augmented disorganized myofilament structure, changed mitochondrial shape, and increased apoptotic events. Consistently, ACT-iPSC CM and cardiac fibroblasts isolated from fibrotic human ACT myocardium exhibited higher DOX-dependent reactive oxygen species. In functional studies, Ca2+ transient amplitude of ACT-iPSC CM was reduced compared to control cells, and diastolic sarcoplasmic reticulum Ca2+ leak was DOX-dependently increased. This could be explained by overactive CaMKIIδ in ACT CM. Together with DOX-dependent augmented proarrhythmic cellular triggers and prolonged action potentials in ACT CM, this suggests a cellular link to arrhythmogenic events and contractile dysfunction especially found in ACT engineered human myocardium. CamKIIδ inhibition prevented proarrhythmic triggers in ACT. In contrast, control CM upregulated SERCA2a expression in a DOX-dependent manner, possibly to avoid heart failure conditions. In conclusion, we developed the first human patient-specific stem cell model of DOX-induced cardiac dysfunction from patients with B-cell lymphoma. Our results suggest that DOX-induced stress resulted in arrhythmogenic events associated with contractile dysfunction and finally in heart failure after persistent stress activation in ACT patients.

Reduction of A-to-I RNA editing in the failing human heart regulates formation of circular RNAs.

Alterations of RNA editing that affect the secondary structure of RNAs can cause human diseases. We therefore studied RNA editing in failing human hearts. Transcriptome sequencing showed that adenosine-to-inosine (A-to-I) RNA editing was responsible for 80% of the editing events in the myocardium. Failing human hearts were characterized by reduced RNA editing. This was primarily attributable to Alu elements in introns of protein-coding genes. In the failing left ventricle, 166 circRNAs were upregulated and 7 circRNAs were downregulated compared to non-failing controls. Most of the upregulated circRNAs were associated with reduced RNA editing in the host gene. ADAR2, which binds to RNA regions that are edited from A-to-I, was decreased in failing human hearts. In vitro, reduction of ADAR2 increased circRNA levels suggesting a causal effect of reduced ADAR2 levels on increased circRNAs in the failing human heart. To gain mechanistic insight, one of the identified upregulated circRNAs with a high reduction of editing in heart failure, AKAP13, was further characterized. ADAR2 reduced the formation of double-stranded structures in AKAP13 pre-mRNA, thereby reducing the stability of Alu elements and the circularization of the resulting circRNA. Overexpression of circAKAP13 impaired the sarcomere regularity of human induced pluripotent stem cell-derived cardiomyocytes. These data show that ADAR2 mediates A-to-I RNA editing in the human heart. A-to-I RNA editing represses the formation of dsRNA structures of Alu elements favoring canonical linear mRNA splicing and inhibiting the formation of circRNAs. The findings are relevant to diseases with reduced RNA editing and increased circRNA levels and provide insights into the human-specific regulation of circRNA formation.

Genome-wide association analysis in dilated cardiomyopathy reveals two new players in systolic heart failure on chromosomes 3p25.1 and 22q11.23.

AIMS: Our objective was to better understand the genetic bases of dilated cardiomyopathy (DCM), a leading cause of systolic heart failure. METHODS AND RESULTS: We conducted the largest genome-wide association study performed so far in DCM, with 2719 cases and 4440 controls in the discovery population. We identified and replicated two new DCM-associated loci on chromosome 3p25.1 [lead single-nucleotide polymorphism (SNP) rs62232870, P = 8.7 × 10-11 and 7.7 × 10-4 in the discovery and replication steps, respectively] and chromosome 22q11.23 (lead SNP rs7284877, P = 3.3 × 10-8 and 1.4 × 10-3 in the discovery and replication steps, respectively), while confirming two previously identified DCM loci on chromosomes 10 and 1, BAG3 and HSPB7. A genetic risk score constructed from the number of risk alleles at these four DCM loci revealed a 3-fold increased risk of DCM for individuals with 8 risk alleles compared to individuals with 5 risk alleles (median of the referral population). In silico annotation and functional 4C-sequencing analyses on iPSC-derived cardiomyocytes identify SLC6A6 as the most likely DCM gene at the 3p25.1 locus. This gene encodes a taurine transporter whose involvement in myocardial dysfunction and DCM is supported by numerous observations in humans and animals. At the 22q11.23 locus, in silico and data mining annotations, and to a lesser extent functional analysis, strongly suggest SMARCB1 as the candidate culprit gene. CONCLUSION: This study provides a better understanding of the genetic architecture of DCM and sheds light on novel biological pathways underlying heart failure.

Genotype Complements the Phenotype: Identification of the Pathogenicity of an LMNA Splice Variant by Nanopore Long-Read Sequencing in a Large DCM Family.

Dilated cardiomyopathy (DCM) is a common cause of heart failure (HF) and is of familial origin in 20−40% of cases. Genetic testing by next-generation sequencing (NGS) has yielded a definite diagnosis in many cases; however, some remain elusive. In this study, we used a combination of NGS, human-induced pluripotent-stem-cell-derived cardiomyocytes (iPSC-CMs) and nanopore long-read sequencing to identify the causal variant in a multi-generational pedigree of DCM. A four-generation family with familial DCM was investigated. Next-generation sequencing (NGS) was performed on 22 family members. Skin biopsies from two affected family members were used to generate iPSCs, which were then differentiated into iPSC-CMs. Short-read RNA sequencing was used for the evaluation of the target gene expression, and long-read RNA nanopore sequencing was used to evaluate the relevance of the splice variants. The pedigree suggested a highly penetrant, autosomal dominant mode of inheritance. The phenotype of the family was suggestive of laminopathy, but previous genetic testing using both Sanger and panel sequencing only yielded conflicting evidence for LMNA p.R644C (rs142000963), which was not fully segregated. By re-sequencing four additional affected family members, further non-coding LMNA variants could be detected: rs149339264, rs199686967, rs201379016, and rs794728589. To explore the roles of these variants, iPSC-CMs were generated. RNA sequencing showed the LMNA expression levels to be significantly lower in the iPSC-CMs of the LMNA variant carriers. We demonstrated a dysregulated sarcomeric structure and altered calcium homeostasis in the iPSC-CMs of the LMNA variant carriers. Using targeted nanopore long-read sequencing, we revealed the biological significance of the variant c.356+1G>A, which generates a novel 5' splice site in exon 1 of the cardiac isomer of LMNA, causing a nonsense mRNA product with almost complete RNA decay and haploinsufficiency. Using novel molecular analysis and nanopore technology, we demonstrated the pathogenesis of the rs794728589 (c.356+1G>A) splice variant in LMNA. This study highlights the importance of precise diagnostics in the clinical management and workup of cardiomyopathies.

microRNA neural networks improve diagnosis of acute coronary syndrome (ACS).

BACKGROUND: Cardiac troponins are the preferred biomarkers of acute myocardial infarction. Despite superior sensitivity, serial testing of Troponins to identify patients suffering acute coronary syndromes is still required in many cases to overcome limited specificity. Moreover, unstable angina pectoris relies on reported symptoms in the troponin-negative group. In this study, we investigated genome-wide miRNA levels in a prospective cohort of patients with clinically suspected ACS and determined their diagnostic value by applying an in silico neural network. METHODS: PAXgene blood and serum samples were drawn and hsTnT was measured in patients at initial presentation to our Chest-Pain Unit. After clinical and diagnostic workup, patients were adjudicated by senior cardiologists in duty to their final diagnosis: STEMI, NSTEMI, unstable angina pectoris and non-ACS patients. ACS patients and a cohort of healthy controls underwent deep transcriptome sequencing. Machine learning was implemented to construct diagnostic miRNA classifiers. RESULTS: We developed a neural network model which incorporates 34 validated ACS miRNAs, showing excellent classification results. By further developing additional machine learning models and selecting the best miRNAs, we achieved an accuracy of 0.96 (95% CI 0.96-0.97), sensitivity of 0.95, specificity of 0.96 and AUC of 0.99. The one-point hsTnT value reached an accuracy of 0.89, sensitivity of 0.82, specificity of 0.96, and AUC of 0.96. CONCLUSIONS: Here we show the concept of neural network based biomarkers for ACS. This approach also opens the possibility to include multi-modal data points to further increase precision and perform classification of other ACS differential diagnoses.

Dual Functional States of R406W-Desmin Assembly Complexes Cause Cardiomyopathy With Severe Intercalated Disc Derangement in Humans and in Knock-In Mice.

BACKGROUND: Mutations in the human desmin gene cause myopathies and cardiomyopathies. This study aimed to elucidate molecular mechanisms initiated by the heterozygous R406W-desmin mutation in the development of a severe and early-onset cardiac phenotype. METHODS: We report an adolescent patient who underwent cardiac transplantation as a result of restrictive cardiomyopathy caused by a heterozygous R406W-desmin mutation. Sections of the explanted heart were analyzed with antibodies specific to 406W-desmin and to intercalated disc proteins. Effects of the R406W mutation on the molecular properties of desmin were addressed by cell transfection and in vitro assembly experiments. To prove the genuine deleterious effect of the mutation on heart tissue, we further generated and analyzed R405W-desmin knock-in mice harboring the orthologous form of the human R406W-desmin. RESULTS: Microscopic analysis of the explanted heart revealed desmin aggregates and the absence of desmin filaments at intercalated discs. Structural changes within intercalated discs were revealed by the abnormal organization of desmoplakin, plectin, N-cadherin, and connexin-43. Next-generation sequencing confirmed the DES variant c.1216C>T (p.R406W) as the sole disease-causing mutation. Cell transfection studies disclosed a dual behavior of R406W-desmin with both its integration into the endogenous intermediate filament system and segregation into protein aggregates. In vitro, R406W-desmin formed unusually thick filaments that organized into complex filament aggregates and fibrillar sheets. In contrast, assembly of equimolar mixtures of mutant and wild-type desmin generated chimeric filaments of seemingly normal morphology but with occasional prominent irregularities. Heterozygous and homozygous R405W-desmin knock-in mice develop both a myopathy and a cardiomyopathy. In particular, the main histopathologic results from the patient are recapitulated in the hearts from R405W-desmin knock-in mice of both genotypes. Moreover, whereas heterozygous knock-in mice have a normal life span, homozygous animals die at 3 months of age because of a smooth muscle-related gastrointestinal phenotype. CONCLUSIONS: We demonstrate that R406W-desmin provokes its severe cardiotoxic potential by a novel pathomechanism, where the concurrent dual functional states of mutant desmin assembly complexes underlie the uncoupling of desmin filaments from intercalated discs and their structural disorganization.

Development of interleukin-17-producing γδ T cells is restricted to a functional embryonic wave.

γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.

Association between travel time to delivery unit and unplanned out-of-hospital birth, infant morbidity and mortality: A population-based cohort study.

INTRODUCTION: Over the last decade, a number of delivery units have been closed in Sweden, justified by both economic incentives and patient safety issues. However, concentrating births to larger delivery units naturally increases travel time for some parturient women, which may lead to unintended negative consequences. We aimed to investigate the association between travel time to delivery unit and unplanned out-of-hospital birth, and subsequent infant morbidity and mortality. MATERIAL AND METHODS: We performed a population-based cohort study including 365 604 women in the Swedish Pregnancy Register, giving birth between 2014 and 2017. Modified Poisson regression was used to investigate the association between travel time from home address to actual delivery unit, based on geographic information system analysis, and risk of an unplanned out-of-hospital birth. Analyses were stratified by parity and urban/rural residence. Lastly, the associations between an unplanned out-of-hospital birth and severe infant morbidity, stillbirth, peripartum, perinatal and neonatal mortality were investigated. RESULTS: Of those with an unplanned out-of-hospital birth (n = 2159), 65% had a travel time up to 30 minutes. A travel time between 31 and 60 minutes was associated with a doubled risk of unplanned out-of-hospital birth (adjusted risk ratio [RR] 1.96, 95% confidence interval [CI] 1.74-2.22) and women with a travel time of more than 1 hour had an adjusted RR of 3.19 (95% CI 2.64-3.86), compared with those with a travel time of <30 minutes. No difference in results was seen when stratified for parity and urban/rural residence. No association was found between unplanned out-of-hospital birth and severe infant morbidity. Significant associations were found in crude analyses for stillbirth (RR 1.85, 95% CI 1.09-3.13), peripartum (RR 1.93, 95% CI 1.18-3.16), perinatal (RR 2.03, 95% CI 1.28-3.23) and neonatal mortality (RR 3.08, 95% CI 1.27-7.46), although neonatal mortality was very rare (2.3/1000 out-of-hospital births). Similar effect estimates were found in the adjusted analyses, though no longer significant. CONCLUSIONS: Although the majority of unplanned out-of-hospital births occurred in the group of women with a travel time of 0-30 minutes, increasing travel time to a delivery unit is associated with unplanned out-of-hospital birth, which may increase the risk of mortality.

Identification of SCN5a p.C335R Variant in a Large Family with Dilated Cardiomyopathy and Conduction Disease.

INTRODUCTION: Familial dilated cardiomyopathy (DCM) is clinically variable and has been associated with mutations in more than 50 genes. Rapid improvements in DNA sequencing have led to the identification of diverse rare variants with unknown significance (VUS), which underlines the importance of functional analyses. In this study, by investigating human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs), we evaluated the pathogenicity of the p.C335R sodium voltage-gated channel alpha subunit 5 (SCN5a) variant in a large family with familial DCM and conduction disease. METHODS: A four-generation family with autosomal dominant familial DCM was investigated. Next-generation sequencing (NGS) was performed in all 16 family members. Clinical deep phenotyping, including endomyocardial biopsy, was performed. Skin biopsies from two patients and one healthy family member were used to generate human-induced pluripotent stem cells (iPSCs), which were then differentiated into cardiomyocytes. Patch-clamp analysis with Xenopus oocytes and iPSC-CMs were performed. RESULTS: A SCN5a variant (c.1003T>C; p.C335R) could be detected in all family members with DCM or conduction disease. A novel truncating TTN variant (p.Ser24998LysfsTer28) could also be identified in two family members with DCM. Family members with the SCN5a variant (p.C335R) showed significantly longer PQ and QRS intervals and lower left ventricular ejection fractions (LV-EF). All four patients who received CRT-D were non-responders. Electrophysiological analysis with Xenopus oocytes showed a loss of function in SCN5a p.C335R. Na+ channel currents were also reduced in iPSC-CMs from DCM patients. Furthermore, iPSC-CM with compound heterozygosity (SCN5a p.C335R and TTNtv) showed significant dysregulation of sarcomere structures, which may be contributed to the severity of the disease and earlier onset of DCM. CONCLUSION: The SCN5a p.C335R variant is causing a loss of function of peak INa in patients with DCM and cardiac conduction disease. The co-existence of genetic variants in channels and structural genes (e.g., SCN5a p.C335R and TTNtv) increases the severity of the DCM phenotype.

Energy Metabolites as Biomarkers in Ischemic and Dilated Cardiomyopathy.

With more than 25 million people affected, heart failure (HF) is a global threat. As energy production pathways are known to play a pivotal role in HF, we sought here to identify key metabolic changes in ischemic- and non-ischemic HF by using a multi-OMICS approach. Serum metabolites and mRNAseq and epigenetic DNA methylation profiles were analyzed from blood and left ventricular heart biopsy specimens of the same individuals. In total we collected serum from n = 82 patients with Dilated Cardiomyopathy (DCM) and n = 51 controls in the screening stage. We identified several metabolites involved in glycolysis and citric acid cycle to be elevated up to 5.7-fold in DCM (p = 1.7 × 10-6). Interestingly, cardiac mRNA and epigenetic changes of genes encoding rate-limiting enzymes of these pathways could also be found and validated in our second stage of metabolite assessment in n = 52 DCM, n = 39 ischemic HF and n = 57 controls. In conclusion, we identified a new set of metabolomic biomarkers for HF. We were able to identify underlying biological cascades that potentially represent suitable intervention targets.

Opiate Intoxication Caused by Epidural Infusion of Morphine: A Case Report of a Near Fatal Medication Error.

INTRODUCTION: Epidural infusion of local anesthetics with opioids is widely used for pain control during the perioperative-and peripartum-periods. Selection of the opioid, appropriate dosing, and follow-up by the acute pain service are critical in providing safe postoperative epidural analgesia. CASE REPORT SUMMARY: A 71-year-old man was scheduled for a parastomal hernia repair with midline laparotomy. The parastomal hernia was a complication from a previously performed colectomy for ulcerative colitis. Preoperatively, the patient received a lower thoracic epidural catheter. The epidural infusate (0.2% ropivacaine with 0.5 µg/mL sufentanil) was prepared and double-checked by holding area nurses. The fact that the right prescription medication label partially covered a morphine label went unnoticed. The intraoperative phase was characterized by stable parameters. Postoperatively, it was not possible to demonstrate an epidural nerve block. No pain was reported, and the patient could be transferred to the ward. The patient developed coma and delayed respiratory depression after discharge to the surgical ward, requiring intensive care unit admission and naloxone administration. Analysis of the syringe content revealed the presence of morphine (1 mg/mL). DISCUSSION: Color-coded prefilled syringes combined with the use of an epidural specific syringe connector to prevent cross-connections should become standard practice. In addition, delayed respiratory depression should be considered after epidural administration of morphine.

Identification and regulation of the long non-coding RNA Heat2 in heart failure.

AIMS: Circulating immune cells have a significant impact on progression and outcome of heart failure. Long non-coding RNAs (lncRNAs) comprise novel epigenetic regulators which control cardiovascular diseases and inflammatory disorders. We aimed to identify lncRNAs regulated in circulating immune cells of the blood of heart failure patients. METHODS AND RESULTS: Next-generation sequencing revealed 110 potentially non-coding RNA transcripts differentially expressed in peripheral blood mononuclear cells of heart failure patients with reduced ejection fraction. The up-regulated lncRNA Heat2 was further functionally characterized. Heat2 expression was detected in whole blood, PBMNCs, eosinophil and basophil granulocytes. Heat2 regulates cell division, invasion, transmigration and immune cell adhesion on endothelial cells. CONCLUSION: Heat2 is an immune cell enriched lncRNA that is elevated in the blood of heart failure patients and controls cellular functions.

ANK2 functionally interacts with KCNH2 aggravating long QT syndrome in a double mutation carrier.

Pathogenic long QT mutations often comprise high phenotypic variability and particularly variants in ANK2 (long QT syndrome 4) frequently lack QT prolongation. We sought to elucidate the genetic and functional background underlying the clinical diversity in a 3-generation family with different cardiac arrhythmias. Next-generation sequencing-based screening of patients with QT prolongation identified the index patient of the family carrying an ANK2-E1813K variant and a previously uncharacterized KCNH2-H562R mutation in a double heterozygous conformation. The patient presented with a severe clinical phenotype including a markedly prolonged QTc interval (544 ms), recurrent syncope due to Torsade de Pointes tachycardias, survived cardiopulmonary resuscitation, progressive cardiac conduction defect, and atrial fibrillation. Evaluation of other family members identified a sister and a niece solely carrying the ANK2-E1813K variant, who showed age-related conduction disease. An asymptomatic second sister solely carried the KCNH2-H562R mutation. Voltage-clamp recordings in Xenopus oocytes revealed that KCNH2-H562R subunits were non-functional but did not exert dominant-negative effects on wild-type subunits. Expression of KCNH2-H562R in HEK293 cells showed a trafficking deficiency. Co-expression of the C-terminal regulatory domain of ANK2 in Xenopus oocytes revealed that ANK2-E1813K diminished currents mediated by the combination of wild-type and H562R KCNH2 subunits. Our data suggest that ANK2 functionally interacts with KCNH2 leading to a stronger current suppression and marked aggravation of long QT syndrome in the patient carrying variants in both proteins.

A high-resolution map of the human small non-coding transcriptome.

Motivation: Although the amount of small non-coding RNA-sequencing data is continuously increasing, it is still unclear to which extent small RNAs are represented in the human genome. Results: In this study we analyzed 303 billion sequencing reads from nearly 25 000 datasets to answer this question. We determined that 0.8% of the human genome are reliably covered by 874 123 regions with an average length of 31 nt. On the basis of these regions, we found that among the known small non-coding RNA classes, microRNAs were the most prevalent. In subsequent steps, we characterized variations of miRNAs and performed a staged validation of 11 877 candidate miRNAs. Of these, many were actually expressed and significantly dysregulated in lung cancer. Selected candidates were finally validated by northern blots. Although isolated miRNAs could still be present in the human genome, our presented set likely contains the largest fraction of human miRNAs. Contact: c.backes@mx.uni-saarland.de or andreas.keller@ccb.uni-saarland.de. Supplementary information: Supplementary data are available at Bioinformatics online.

γδ T cells enhance autoimmunity by restraining regulatory T cell responses via an interleukin-23-dependent mechanism.

Mice that lack interleukin-23 (IL-23) are resistant to T cell-mediated autoimmunity. Although IL-23 is a maturation factor for T helper 17 (Th17) cells, a subset of γδ T cells expresses the IL-23 receptor (IL-23R) constitutively. Using IL-23R reporter mice, we showed that γδ T cells were the first cells to respond to IL-23 during experimental autoimmune encephalomyelitis (EAE). Although γδ T cells produced Th17 cell-associated cytokines in response to IL-23, their major function was to prevent the development of regulatory T (Treg) cell responses. IL-23-activated γδ T cells rendered αß effector T cells refractory to the suppressive activity of Treg cells and also prevented the conversion of conventional T cells into Foxp3(+) Treg cells in vivo. Thus, IL-23, which by itself has no direct effect on Treg cells, is able to disarm Treg cell responses and promote antigen-specific effector T cell responses via activating γδ T cells.

Identification and Functional Characterization of Hypoxia-Induced Endoplasmic Reticulum Stress Regulating lncRNA (HypERlnc) in Pericytes.

RATIONALE: Pericytes are essential for vessel maturation and endothelial barrier function. Long noncoding RNAs regulate many cellular functions, but their role in pericyte biology remains unexplored. OBJECTIVE: Here, we investigate the effect of hypoxia-induced endoplasmic reticulum stress regulating long noncoding RNAs (HypERlnc, also known as ENSG00000262454) on pericyte function in vitro and its regulation in human heart failure and idiopathic pulmonary arterial hypertension. METHODS AND RESULTS: RNA sequencing in human primary pericytes identified hypoxia-regulated long noncoding RNAs, including HypERlnc. Silencing of HypERlnc decreased cell viability and proliferation and resulted in pericyte dedifferentiation, which went along with increased endothelial permeability in cocultures consisting of human primary pericyte and human coronary microvascular endothelial cells. Consistently, Cas9-based transcriptional activation of HypERlnc was associated with increased expression of pericyte marker genes. Moreover, HypERlnc knockdown reduced endothelial-pericyte recruitment in Matrigel assays (P<0.05). Mechanistically, transcription factor reporter arrays demonstrated that endoplasmic reticulum stress-related transcription factors were prominently activated by HypERlnc knockdown, which was confirmed via immunoblotting for the endoplasmic reticulum stress markers IRE1α (P<0.001), ATF6 (P<0.01), and soluble BiP (P<0.001). Kyoto encyclopedia of genes and gene ontology pathway analyses of RNA sequencing experiments after HypERlnc knockdown indicate a role in cardiovascular disease states. Indeed, HypERlnc expression was significantly reduced in human cardiac tissue from patients with heart failure (P<0.05; n=19) compared with controls. In addition, HypERlnc expression significantly correlated with pericyte markers in human lungs derived from patients diagnosed with idiopathic pulmonary arterial hypertension and from donor lungs (n=14). CONCLUSIONS: Here, we show that HypERlnc regulates human pericyte function and the endoplasmic reticulum stress response. In addition, RNA sequencing analyses in conjunction with reduced expression of HypERlnc in heart failure and correlation with pericyte markers in idiopathic pulmonary arterial hypertension indicate a role of HypERlnc in human cardiopulmonary disease.

Web-based NGS data analysis using miRMaster: a large-scale meta-analysis of human miRNAs.

The analysis of small RNA NGS data together with the discovery of new small RNAs is among the foremost challenges in life science. For the analysis of raw high-throughput sequencing data we implemented the fast, accurate and comprehensive web-based tool miRMaster. Our toolbox provides a wide range of modules for quantification of miRNAs and other non-coding RNAs, discovering new miRNAs, isomiRs, mutations, exogenous RNAs and motifs. Use-cases comprising hundreds of samples are processed in less than 5 h with an accuracy of 99.4%. An integrative analysis of small RNAs from 1836 data sets (20 billion reads) indicated that context-specific miRNAs (e.g. miRNAs present only in one or few different tissues / cell types) still remain to be discovered while broadly expressed miRNAs appear to be largely known. In total, our analysis of known and novel miRNAs indicated nearly 22 000 candidates of precursors with one or two mature forms. Based on these, we designed a custom microarray comprising 11 872 potential mature miRNAs to assess the quality of our prediction. MiRMaster is a convenient-to-use tool for the comprehensive and fast analysis of miRNA NGS data. In addition, our predicted miRNA candidates provided as custom array will allow researchers to perform in depth validation of candidates interesting to them.