A heterozygous variant in the human cardiac miR-133 gene, MIR133A2, alters miRNA duplex processing and strand abundance

BACKGROUND MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Sequential cleavage of miRNA precursors results in a ~22 nucleotide duplex of which one strand, the mature miRNA, is typically loaded into the RNA-induced silencing complex (RISC) while the passenger strand is degraded. Very little is known about how genetic variation might affect miRNA biogenesis and function. RESULTS We re-sequenced the MIR1-1, MIR1-2, MIR133A1, MIR133A2, and MIR133B genes, that encode the cardiac-enriched miRNAs, miR-1 and miR-133, in 120 individuals with familial atrial fibrillation and identified 10 variants, including a novel 79T > C MIR133A2 substitution. This variant lies within the duplex at the 3' end of the mature strand, miR-133a-3p, and is predicted to prevent base-pairing and weaken thermostability at this site, favoring incorporation of the passenger strand, miR-133a-5p, into RISC. Genomic DNA fragments containing miR-133a-2 precursor sequences with 79T and 79C alleles were transfected into HeLa cells. On Northern blotting the 79T allele showed strong expression of miR-133a-3p with weak expression of miR-133a-5p. In contrast, the 79C allele had no effect on miR-133a-3p but there was a significant increase (mean 3.6-fold) in miR-133a-5p levels. Deep sequencing of small RNA libraries prepared from normal human and murine atria confirmed that nearly all the mature miR-133a was comprised of miR-133a-3p and that levels of miR-133a-5p were very low. A number of isomiRs with variations at 5' and 3' ends were identified for both miR-133a-3p and miR-133a-5p, with 2 predominant miR-133a-3p isomiRs and one predominant miR-133a-5p isomiR. Bioinformatics analyses indicate that the major miR-133a-3p and 5p isomiRs have numerous predicted target mRNAs, only a few of which are in common. CONCLUSIONS Multiple miR-133a isomiRs with potential different mRNA target profiles are present in the atrium in humans and mice. We identified a human 79T > C MIR133A2 variant that alters miRNA processing and results in accumulation of the miR-133a-5p strand that is usually degraded

Percutaneous balloon mitral valvotomy with the Inoue single-balloon catheter: Commissural morphology as a determinant of outcome

AbstractObjectives. The aim of this study was to determine the importance to outcome and the predictability of commissural splitting in patients undergoing percutaneous mitral valvotomy with the Inoue single-balloon catheter.Background. Echocardiographic scoring systems devised to predict mitral valvotomy outcome are based on assessment of leaflet and subvalvular morphology, but the specific importance of commissural morphology has not been examined.Methods. In 30 consecutive patients, commissural splitting was predicted on the basis of the two-dimensional echocardiographic commissural morphology: the extent of fusion, fibrosis or calcification of each commissure. Valve morphology also was evaluated according to a previously described echocardiographic scoring system.Results. Splitting of one or both commissures occurred in 24 patients (80%) and was associated with a significantly greater mean increase in valve area (85%) than if neither commissure was split (13%). A good outcome from valvotomy (defined as valve area >1.5 cm2and increase in valve area >25%) was achieved in 96% of those in whom one or both commissures split, but in none of the patients in whom neither commissure split Whether or not splitting of at least one commissure would occur was predicted accurately in 28 (93%) of the 30 patients. Consequently, the prediction that one or both commissures would split was associated with a good outcome in 23 (89%) of 26 patients, whereas the prediction that neither commissure would split was not associated with a good outcome in any patient. There was no significant difference in the increase in mitral valve area between those with a mitral echocardiographic score <8 and those with a score ≥8. New or worsening mitral regurgitation occurred in nine patients, most commonly as a jet directed through a split commissure.Conclusions. Commissural splitting is the dominant mechanism by which mitral valve area is increased with the Inoue balloon technique, and it can be predicted by echocardiographic assessment of commissural morphology. Commissural morphology is a better predictor of outcome than is the mitral echocardiographic score

Comparison of automated candidate gene prediction systems using genes implicated in type 2 diabetes by genome-wide association studies

BackgroundAutomated candidate gene prediction systems allow geneticists to hone in on disease genes more rapidly by identifying the most probable candidate genes linked to the disease phenotypes under investigation. Here we assessed the ability of eight different candidate gene prediction systems to predict disease genes in intervals previously associated with type 2 diabetes by benchmarking their performance against genes implicated by recent genome-wide association studies.ResultsUsing a search space of 9556 genes, all but one of the systems pruned the genome in favour of genes associated with moderate to highly significant SNPs. Of the 11 genes associated with highly significant SNPs identified by the genome-wide association studies, eight were flagged as likely candidates by at least one of the prediction systems. A list of candidates produced by a previous consensus approach did not match any of the genes implicated by 706 moderate to highly significant SNPs flagged by the genome-wide association studies. We prioritized genes associated with medium significance SNPs.ConclusionThe study appraises the relative success of several candidate gene prediction systems against independent genetic data. Even when confronted with challengingly large intervals, the candidate gene prediction systems can successfully select likely disease genes. Furthermore, they can be used to filter statistically less-well-supported genetic data to select more likely candidates. We suggest consensus approaches fail because they penalize novel predictions made from independent underlying databases. To realize their full potential further work needs to be done on prioritization and annotation of genes.<br /

Analysis of genome-wide association study data using the protein knowledge base

Article 9

Gentrepid V2.0: a web server for candidate disease gene prediction

Contains fulltext : 124935.pdf (publisher's version ) (Open Access)BACKGROUND: Candidate disease gene prediction is a rapidly developing area of bioinformatics research with the potential to deliver great benefits to human health. As experimental studies detecting associations between genetic intervals and disease proliferate, better bioinformatic techniques that can expand and exploit the data are required. DESCRIPTION: Gentrepid is a web resource which predicts and prioritizes candidate disease genes for both Mendelian and complex diseases. The system can take input from linkage analysis of single genetic intervals or multiple marker loci from genome-wide association studies. The underlying database of the Gentrepid tool sources data from numerous gene and protein resources, taking advantage of the wealth of biological information available. Using known disease gene information from OMIM, the system predicts and prioritizes disease gene candidates that participate in the same protein pathways or share similar protein domains. Alternatively, using an ab initio approach, the system can detect enrichment of these protein annotations without prior knowledge of the phenotype. CONCLUSIONS: The system aims to integrate the wealth of protein information currently available with known and novel phenotype/genotype information to acquire knowledge of biological mechanisms underpinning disease. We have updated the system to facilitate analysis of GWAS data and the study of complex diseases. Application of the system to GWAS data on hypertension using the ICBP data is provided as an example. An interesting prediction is a ZIP transporter additional to the one found by the ICBP analysis. The webserver URL is https://www.gentrepid.org/

Complexity of murine cardiomyocyte miRNA biogenesis, sequence variant expression and function

microRNAs (miRNAs) are critical to heart development and disease. Emerging research indicates that regulated precursor processing can give rise to an unexpected diversity of miRNA variants. We subjected small RNA from murine HL-1 cardiomyocyte cells to next generation sequencing to investigate the relevance of such diversity to cardiac biology. ∼40 million tags were mapped to known miRNA hairpin sequences as deposited in miRBase version 16, calling 403 generic miRNAs as appreciably expressed. Hairpin arm bias broadly agreed with miRBase annotation, although 44 miR* were unexpectedly abundant (>20% of tags); conversely, 33 -5p/-3p annotated hairpins were asymmetrically expressed. Overall, variability was infrequent at the 5' start but common at the 3' end of miRNAs (5.2% and 52.3% of tags, respectively). Nevertheless, 105 miRNAs showed marked 5' isomiR expression (>20% of tags). Among these was miR-133a, a miRNA with important cardiac functions, and we demonstrated differential mRNA targeting by two of its prevalent 5' isomiRs. Analyses of miRNA termini and base-pairing patterns around Drosha and Dicer cleavage regions confirmed the known bias towards uridine at the 5' most position of miRNAs, as well as supporting the thermodynamic asymmetry rule for miRNA strand selection and a role for local structural distortions in fine tuning miRNA processing. We further recorded appreciable expression of 5 novel miR*, 38 extreme variants and 8 antisense miRNAs. Analysis of genome-mapped tags revealed 147 novel candidate miRNAs. In summary, we revealed pronounced sequence diversity among cardiomyocyte miRNAs, knowledge of which will underpin future research into the mechanisms involved in miRNA biogenesis and, importantly, cardiac function, disease and therapy.This work was supported by by the Victor Chang Cardiac Research Institute and grants 573726, 573731 and 514904 from the National Health & Medical Research Council awarded to TP

Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations

Background: Variants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in large affected families. Mutations of TBX5 cause Holt–Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development. Design: In order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes. Results: Two cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M). Conclusions: In addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts

Analysis of protein sequence and interaction data for candidate disease gene prediction

Linkage analysis is a successful procedure to associate diseases with specific genomic regions. These regions are often large, containing hundreds of genes, which make experimental methods employed to identify the disease gene arduous and expensive. We present two methods to prioritize candidates for further experimental study: Common Pathway Scanning (CPS) and Common Module Profiling (CMP). CPS is based on the assumption that common phenotypes are associated with dysfunction in proteins that participate in the same complex or pathway. CPS applies network data derived from protein&ndash;protein interaction (PPI) and pathway databases to identify relationships between genes. CMP identifies likely candidates using a domain-dependent sequence similarity approach, based on the hypothesis that disruption of genes of similar function will lead to the same phenotype. Both algorithms use two forms of input data: known disease genes or multiple disease loci. When using known disease genes as input, our combined methods have a sensitivity of 0.52 and a specificity of 0.97 and reduce the candidate list by 13-fold. Using multiple loci, our methods successfully identify disease genes for all benchmark diseases with a sensitivity of 0.84 and a specificity of 0.63. Our combined approach prioritizes good candidates and will accelerate the disease gene discovery process

Combined mutation screening of NKX2-5, GATA4, and TBX5 in congenital heart disease: multiple heterozygosity and novel mutations

Background: Variants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in large affected families. Mutations of TBX5 cause Holt–Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development. Design: In order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes. Results: Two cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M). Conclusions: In addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts

Risks of Ventricular Arrhythmia and Heart Failure in Carriers of RBM20 Variants

BACKGROUND: Variants in RBM20 are reported in 2% to 6% of familial cases of dilated cardiomyopathy and may be associated with fatal ventricular arrhythmia and rapid heart failure progression. We sought to determine the risk of adverse events in RBM20 variant carriers and the impact of sex on outcomes. METHODS: Consecutive probands and relatives carrying RBM20 variants were retrospectively recruited from 12 cardiomyopathy units. The primary end point was a composite of malignant ventricular arrhythmia (MVA) and end-stage heart failure (ESHF). MVA and ESHF end points were also analyzed separately and males and females compared. Left ventricular ejection fraction (LVEF) contemporary to MVA was examined. RBM20 variant carriers with left ventricular systolic dysfunction (RBM20LVSD) were compared with variant-elusive patients with idiopathic left ventricular systolic dysfunction. RESULTS: Longitudinal follow-up data were available for 143 RBM20 variant carriers (71 male; median age, 35.5 years); 7 of 143 had an MVA event at baseline. Thirty of 136 without baseline MVA (22.0%) reached the primary end point, and 16 of 136 (11.8%) had new MVA with no significant difference between males and females (log-rank P=0.07 and P=0.98, respectively). Twenty of 143 (14.0%) developed ESHF (17 males and 3 females; log-rank P35%. At 5 years, 15 of 67 (22.4%) RBM20LVSD versus 7 of 197 (3.6%) patients with idiopathic left ventricular systolic dysfunction had reached the primary end point (log-rank P<0.001). RBM20 variant carriage conferred a 6.0-fold increase in risk of the primary end point. CONCLUSIONS: RBM20 variants are associated with a high risk of MVA and ESHF compared with idiopathic left ventricular systolic dysfunction. The risk of MVA in male and female RBM20 variant carriers is similar, but male sex is strongly associated with ESHF