We present a human miRNA tissue atlas by determining the abundance of 1997 miRNAs in 61 tissue biopsies of different organs from two individuals collected post-mortem. One thousand three hundred ...sixty-four miRNAs were discovered in at least one tissue, 143 were present in each tissue. To define the distribution of miRNAs, we utilized a tissue specificity index (TSI). The majority of miRNAs (82.9%) fell in a middle TSI range i.e. were neither specific for single tissues (TSI > 0.85) nor housekeeping miRNAs (TSI < 0.5). Nonetheless, we observed many different miRNAs and miRNA families that were predominantly expressed in certain tissues. Clustering of miRNA abundances revealed that tissues like several areas of the brain clustered together. Considering -3p and -5p mature forms we observed miR-150 with different tissue specificity. Analysis of additional lung and prostate biopsies indicated that inter-organism variability was significantly lower than inter-organ variability. Tissue-specific differences between the miRNA patterns appeared not to be significantly altered by storage as shown for heart and lung tissue. MiRNAs TSI values of human tissues were significantly (P = 10(-8)) correlated with those of rats; miRNAs that were highly abundant in certain human tissues were likewise abundant in according rat tissues. We implemented a web-based repository enabling scientists to access and browse the data (https://ccb-web.cs.uni-saarland.de/tissueatlas).
Information on miRNA targeting genes is growing rapidly. For high-throughput experiments, but also for targeted analyses of few genes or miRNAs, easy analysis with concise representation of results ...facilitates the work of life scientists. We developed miRTargetLink, a tool for automating respective analysis procedures that are frequently applied. Input of the web-based solution is either a single gene or single miRNA, but also sets of genes or miRNAs, can be entered. Validated and predicted targets are extracted from databases and an interaction network is presented. Users can select whether predicted targets, experimentally validated targets with strong or weak evidence, or combinations of those are considered. Central genes or miRNAs are highlighted and users can navigate through the network interactively. To discover the most relevant biochemical processes influenced by the target network, gene set analysis and miRNA set analysis are integrated. As a showcase for miRTargetLink, we analyze targets of five cardiac miRNAs. miRTargetLink is freely available without restrictions at www.ccb.uni-saarland.de/mirtargetlink.
Alternative splicing generates differing RNA isoforms that govern phenotypic complexity of eukaryotes. Its malfunction underlies many diseases, including cancer and cardiovascular diseases. ...Comparative analysis of RNA isoforms at the genome-wide scale has been difficult. Here, we establish an experimental and computational pipeline that performs de novo transcript annotation and accurately quantifies transcript isoforms from cDNA sequences with a full-length isoform detection accuracy of 97.6%. We generate a searchable, quantitative human transcriptome annotation with 31,025 known and 5,740 novel transcript isoforms ( http://steinmetzlab.embl.de/iBrowser/ ). By analyzing the isoforms in the presence of RNA Binding Motif Protein 20 (RBM20) mutations associated with aggressive dilated cardiomyopathy (DCM), we identify 121 differentially expressed transcript isoforms in 107 cardiac genes. Our approach enables quantitative dissection of complex transcript architecture instead of mere identification of inclusion or exclusion of individual exons, as exemplified by the discovery of IMMT isoforms mis-spliced by RBM20 mutations. Thereby we achieve a path to direct differential expression testing independent of an existing annotation of transcript isoforms, providing more immediate biological interpretation and higher resolution transcriptome comparisons.
In this study, we aimed to clinically and genetically characterize LVNC patients and investigate the prevalence of variants in known and novel LVNC disease genes.
Left ventricular non-compaction ...cardiomyopathy (LVNC) is an increasingly recognized cause of heart failure, arrhythmia, thromboembolism, and sudden cardiac death. We sought here to dissect its genetic causes, phenotypic presentation and outcome.
In our registry with follow-up of in the median 61 months, we analysed 95 LVNC patients (68 unrelated index patients and 27 affected relatives; definite familial LVNC = 23.5%) by cardiac phenotyping, molecular biomarkers and exome sequencing. Cardiovascular events were significantly more frequent in LVNC patients compared with an age-matched group of patients with non-ischaemic dilated cardiomyopathy (hazard ratio = 2.481, P = 0.002). Stringent genetic classification according to ACMG guidelines revealed that TTN, LMNA, and MYBPC3 are the most prevalent disease genes (13 patients are carrying a pathogenic truncating TTN variant, odds ratio = 40.7, Confidence interval = 21.6-76.6, P < 0.0001, percent spliced in 76-100%). We also identified novel candidate genes for LVNC. For RBM20, we were able to perform detailed familial, molecular and functional studies. We show that the novel variant p.R634L in the RS domain of RBM20 co-segregates with LVNC, leading to titin mis-splicing as revealed by RNA sequencing of heart tissue in mutation carriers, protein analysis, and functional splice-reporter assays.
Our data demonstrate that the clinical course of symptomatic LVNC can be severe. The identified pathogenic variants and distribution of disease genes-a titin-related pathomechanism is found in every fourth patient-should be considered in genetic counselling of patients. Pathogenic variants in the nuclear proteins Lamin A/C and RBM20 were associated with worse outcome.
Abstract
Aims
Inflammation is a key driver of atherosclerosis and myocardial infarction (MI), and beyond proteins and microRNAs (miRs), long noncoding RNAs (lncRNAs) have been implicated in ...inflammation control. To obtain further information on the possible role of lncRNAs in the context of atherosclerosis, we obtained comprehensive transcriptome maps of circulating immune cells (peripheral blood mononuclear cells, PBMCs) of early onset MI patients. One lncRNA significantly suppressed in post-MI patients was further investigated in a murine knockout model.
Methods and results
Individual RNA-sequencing (RNA-seq) was conducted on PBMCs from 28 post-MI patients with a history of MI at age ≤50 years and stable disease ≥3 months before study participation, and from 31 healthy individuals without manifest cardiovascular disease or family history of MI as controls. RNA-seq revealed deregulated protein-coding transcripts and lncRNAs in post-MI PBMCs, among which nuclear enriched abundant transcript (NEAT1) was the most highly expressed lncRNA, and the only one significantly suppressed in patients. Multivariate statistical analysis of validation cohorts of 106 post-MI patients and 85 controls indicated that the PBMC NEAT1 levels were influenced (P = 0.001) by post-MI status independent of statin intake, left ventricular ejection fraction, low-density lipoprotein or high-density lipoprotein cholesterol, or age. We investigated NEAT1−/− mice as a model of NEAT1 deficiency to evaluate if NEAT1 depletion may directly and causally alter immune regulation. RNA-seq of NEAT1−/− splenocytes identified disturbed expression and regulation of chemokines/receptors, innate immunity genes, tumour necrosis factor (TNF) and caspases, and increased production of reactive oxygen species (ROS) under baseline conditions. NEAT1−/− spleen displayed anomalous Treg and TH cell differentiation. NEAT1−/− bone marrow-derived macrophages (BMDMs) displayed altered transcriptomes with disturbed chemokine/chemokine receptor expression, increased baseline phagocytosis (P < 0.0001), and attenuated proliferation (P = 0.0013). NEAT1−/− BMDMs responded to LPS with increased (P < 0.0001) ROS production and disturbed phagocytic activity (P = 0.0318). Monocyte-macrophage differentiation was deregulated in NEAT1−/− bone marrow and blood. NEAT1−/− mice displayed aortic wall CD68+ cell infiltration, and there was evidence of myocardial inflammation which could lead to severe and potentially life-threatening structural damage in some of these animals.
Conclusion
The study indicates distinctive alterations of lncRNA expression in post-MI patient PBMCs. Regarding the monocyte-enriched NEAT1 suppressed in post-MI patients, the data from NEAT1−/− mice identify NEAT1 as a novel lncRNA-type immunoregulator affecting monocyte-macrophage functions and T cell differentiation. NEAT1 is part of a molecular circuit also involving several chemokines and interleukins persistently deregulated post-MI. Individual profiling of this circuit may contribute to identify high-risk patients likely to benefit from immunomodulatory therapies. It also appears reasonable to look for new therapeutic targets within this circuit.
Aims
Routine genetic testing in Dilated Cardiomyopathy (DCM) has recently become reality using Next-Generation Sequencing. Several studies have explored the relationship between genotypes and ...clinical phenotypes to support risk estimation and therapeutic decisions, however, most studies are small or restricted to a few genes. This study provides to our knowledge the first systematic meta-analysis on genotype-phenotype associations in DCM.
Methods and results
We retrieved PubMed/Medline literature on genotype–phenotype associations in patients with DCM and mutations in
LMNA
,
PLN
,
RBM20, MYBPC3, MYH7, TNNT2
and
TNNI3
. We summarized and extensively reviewed all studies that passed selection criteria and performed a meta-analysis on key phenotypic parameters. Together, 48 studies with 8097 patients were included. Furthermore, we reviewed recent studies investigating genotype-phenotype associations in DCM patients with
TTN
mutations. The average frequency of mutations in the investigated genes was between 1 and 5 %. The mean age of DCM onset was the beginning of the fifth decade for all genes. Heart transplantation (HTx) rate was highest in
LMNA
mutation carriers (27 %), while
RBM20
mutation carriers were transplanted at a markedly younger age (mean 28.5 years). While 73 % of DCM patients with
LMNA
mutations showed cardiac conduction diseases, low voltage was the reported ECG hallmark in
PLN
mutation carriers. The frequency of ventricular arrhythmia in DCM patients with
LMNA
(50 %) and
PLN
(43 %) mutations was significantly higher. The penetrance of DCM phenotype in subjects with
TTN
truncating variants increased with age and reached 100 % by age of 70.
Conclusion
A pooled analysis of available genotype-phenotype data shows a higher prevalence of sudden cardiac death (SCD), cardiac transplantation, or ventricular arrhythmias in
LMNA
and
PLN
mutation carriers compared to sarcomeric gene mutations. This study will further support the clinical interpretation of genetic findings.
Biochemical DNA modification resembles a crucial regulatory layer among genetic information, environmental factors, and the transcriptome. To identify epigenetic susceptibility regions and novel ...biomarkers linked to myocardial dysfunction and heart failure, we performed the first multi-omics study in myocardial tissue and blood of patients with dilated cardiomyopathy and controls.
Infinium human methylation 450 was used for high-density epigenome-wide mapping of DNA methylation in left-ventricular biopsies and whole peripheral blood of living probands. RNA deep sequencing was performed on the same samples in parallel. Whole-genome sequencing of all patients allowed exclusion of promiscuous genotype-induced methylation calls.
In the screening stage, we detected 59 epigenetic loci that are significantly associated with dilated cardiomyopathy (false discovery corrected
≤0.05), with 3 of them reaching epigenome-wide significance at
≤5×10
. Twenty-seven (46%) of these loci could be replicated in independent cohorts, underlining the role of epigenetic regulation of key cardiac transcription regulators. Using a staged multi-omics study design, we link a subset of 517 epigenetic loci with dilated cardiomyopathy and cardiac gene expression. Furthermore, we identified distinct epigenetic methylation patterns that are conserved across tissues, rendering these CpGs novel epigenetic biomarkers for heart failure.
The present study provides to our knowledge the first epigenome-wide association study in living patients with heart failure using a multi-omics approach.
Aging is a key risk factor for chronic diseases of the elderly. MicroRNAs regulate post-transcriptional gene silencing through base-pair binding on their target mRNAs. We identified nonlinear changes ...in age-related microRNAs by analyzing whole blood from 1334 healthy individuals. We observed a larger influence of the age as compared to the sex and provide evidence for a shift to the 5' mature form of miRNAs in healthy aging. The addition of 3059 diseased patients uncovered pan-disease and disease-specific alterations in aging profiles. Disease biomarker sets for all diseases were different between young and old patients. Computational deconvolution of whole-blood miRNAs into blood cell types suggests that cell intrinsic gene expression changes may impart greater significance than cell abundance changes to the whole blood miRNA profile. Altogether, these data provide a foundation for understanding the relationship between healthy aging and disease, and for the development of age-specific disease biomarkers.
Abstract Background Inherited arrhythmias were originally considered isolated electrical defects. There is growing evidence that ion channel dysfunction also contributes to myocardial disorders, but ...genetic overlap has not been reported for sinus node dysfunction (SND) and noncompaction cardiomyopathy (NCCM). Objectives The study sought to investigate a familial electromechanical disorder characterized by SND and NCCM, and to identify the underlying genetic basis. Methods The index family and a cohort of unrelated probands with sinus bradycardia were examined by electrocardiography, Holter recording, exercise stress test, echocardiography, and/or cardiac magnetic resonance imaging. Targeted next-generation and direct sequencing were used for candidate gene analysis and mutation scanning. Ion channels were expressed in HEK293 cells and studied using patch-clamp recordings. Results SND and biventricular NCCM were diagnosed in multiple members of a German family. Segregation analysis suggested autosomal-dominant inheritance of the combined phenotype. When looking for potentially disease-causing gene variants with cosegregation, a novel hyperpolarization-activated cyclic nucleotide channel 4 ( HCN4 )-G482R mutation and a common cysteine and glycine-rich protein 3 ( CSRP3 )-W4R variant were identified. HCN4 -G482R is located in the highly conserved channel pore domain. Mutant subunits were nonfunctional and exerted dominant-negative effects on wild-type current. CSRP3 -W4R has previously been linked to dilated and hypertrophic cardiomyopathy, but was also found in healthy subjects. Moreover, different truncation (695X) and missense (P883R) HCN4 mutations segregated with a similar combined phenotype in an additional, unrelated family and a single unrelated proband respectively, which both lacked CSRP3 -W4R. Conclusions The symptom complex of SND and NCCM is associated with heritable HCN4 defects. The NCCM phenotype may be aggravated by a common CSRP3 variant in one of the families.
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