Many monogenic disorders, including the muscular dystrophies, display phenotypic variability despite the same disease-causing mutation. To identify genetic modifiers of muscular dystrophy and its associated cardiomyopathy, we used quantitative trait locus mapping and whole genome sequencing in a mouse model. This approach uncovered a modifier locus on chromosome 11 associated with sarcolemmal membrane damage and heart mass. Whole genome and RNA sequencing identified Anxa6, encoding annexin A6, as a modifier gene. A synonymous variant in exon 11 creates a cryptic splice donor, resulting in a truncated annexin A6 protein called ANXA6N32. Live cell imaging showed that annexin A6 orchestrates a repair zone and cap at the site of membrane disruption. In contrast, ANXA6N32 dramatically disrupted the annexin A6-rich cap and the associated repair zone, permitting membrane leak. Anxa6 is a modifier of muscular dystrophy and membrane repair after injury.dystrophin | muscle | plasma membrane

The performance of classic regression-based and modern tree-based variable selection methods is associated with the size of the clinical dataset used. Classic regression-based variable selection methods seem to achieve better parsimony in clinical prediction problems in smaller datasets while modern tree-based methods perform better in larger datasets.

Cardiac conduction system (CCS) disease, which results in disrupted conduction and impaired cardiac rhythm, is common with significant morbidity and mortality. Current treatment options are limited, and rational efforts to develop cell-based and regenerative therapies require knowledge of the molecular networks that establish and maintain CCS function. Recent genome-wide association studies (GWAS) have identified numerous loci associated with adult human CCS function, including TBX5 and SCN5A. We hypothesized that TBX5, a critical developmental transcription factor, regulates transcriptional networks required for mature CCS function. We found that deletion of Tbx5 from the mature murine ventricular conduction system (VCS), including the AV bundle and bundle branches, resulted in severe VCS functional consequences, including loss of fast conduction, arrhythmias, and sudden death. Ventricular contractile function and the VCS fate map remained unchanged in VCS-specific Tbx5 knockouts. However, key mediators of fast conduction, including Na v 1.5, which is encoded by Scn5a, and connexin 40 (Cx40), demonstrated Tbx5-dependent expression in the VCS. We identified a TBX5-responsive enhancer downstream of Scn5a sufficient to drive VCS expression in vivo, dependent on canonical T-box binding sites. Our results establish a direct molecular link between Tbx5 and Scn5a and elucidate a hierarchy between human GWAS loci that affects function of the mature VCS, establishing a paradigm for understanding the molecular pathology of CCS disease. IntroductionThe cardiac conduction system (CCS) consists of a network of specialized cardiomyocytes that generate and propagate the electrical impulses that organize cardiac contraction. The CCS is composed of the slowly propagating atrial nodes, including the sinoatrial (SA) and atrioventricular (AV) nodes, and the rapidly propagating ventricular conduction system (VCS), including the AV (His) bundle and right and left bundle branches. The VCS is uniquely adapted for fast conduction in order to rapidly transmit the electrical impulse governing ventricular contraction from the AV node to the ventricular apex. Disorders of the VCS are common, carry significant morbidity, and are poorly understood from a molecular perspective.The transcriptional networks required to maintain function of the adult CCS are undefined. Our current understanding of the molecular mediators of CCS function stems largely from heritable monogenic disorders and mouse models that have identified a limited number of genes essential for maintaining cardiac rhythm, most of which encode ion channels and their interacting partners (reviewed in ref. 1). Similar approaches have also begun to uncover the transcriptional networks required for CCS development (reviewed in ref.2). Recent genome-wide association studies (GWAS) have identified loci implicated in ECG interval variation (3-8), providing candidate genes with potentially important roles in CCS function in the general population. Specifically, numerous loci near genes e...

Abstract-Heart failure (HF) is associated with reduced cardiac Na ϩ channel (SCN5A) current. We hypothesized that abnormal transcriptional regulation of this ion channel during HF could help explain the reduced current. Using human hearts explanted at the transplantation, we have identified 3 human C-terminal SCN5A mRNA splicing variants predicted to result in truncated, nonfunctional channels. As compared with normal hearts, the explanted ventricles showed an upregulation of 2 of the variants and a downregulation of the full-length mRNA transcript such that the E28A transcript represented only 48.5% (PϽ0.01) of the total SCN5A mRNA. This correlated with a 62.8% (PϽ0.01) reduction in Na ϩ channel protein. Lymphoblasts and skeletal muscle expressing SCN5A also showed identical C-terminal splicing variants. Variants showed reduced membrane protein and no functional current. Transfection of truncation variants into a cell line stably transfected with the full-length Na ϩ channel resulted in dose-dependent reductions in channel mRNA and current. Introduction of a premature truncation in the C-terminal region in a single allele of the mouse SCN5A resulted in embryonic lethality. Embryonic stem cell-derived cardiomyocytes expressing the construct showed reductions in Na ϩ channel-dependent electrophysiological parameters, suggesting that the presence of truncated Na ϩ channel mRNA at levels seen in HF is likely to be physiologically significant. In summary, chronic HF was associated with an increase in 2 truncated SCN5A variants and a decrease in the native mRNA. These splice variations may help explain a loss of Na Key Words: sodium channels Ⅲ transcriptional regulation Ⅲ mRNA splice variations Ⅲ heart failure Ⅲ arrhythmia H uman heart failure (HF) is associated with decreased cardiac voltage-gated sodium channel current. 1,2 Genetically mediated decreases in Na ϩ current have been implicated in the risk for sudden death, [3][4][5] and Na ϩ channel changes may contribute to the increased risk of sudden death in HF. 6,7 Because transcriptional alterations in other ion channels have been noted to contribute to current changes in HF, 8,9 we investigated Na ϩ channel protein and mRNA abundance in hearts explanted during cardiac transplantation to determine whether there were changes that might explain the reduced Na ϩ current previously reported in this tissue. Materials and Methods Detection of Human SCN5A 3UTR Variants by Rapid Amplification of cDNA Ends PCRTotal human RNA from normal fetal and adult whole hearts was purchased from Clontech (Mountain View, Calif). The RNA ligasemediated rapid amplification of cDNA ends (RACE) method was used to characterize the 3Ј ends of the human SCN5A mRNA using the GeneRacer kit (Invitrogen, Carlsbad, Calif). Primary and nested PCR reactions were performed with primers HE26F (on exon 26) and HE27F (on exon 27) specific to the human SCN5A gene and the GeneRacer 3Ј primer for amplifying the 3Ј-end fragment. The nested PCR products were cloned into pCR4-TOPO vector (Invitrogen) and se...

Background Hypertrophic cardiomyopathy and dilated cardiomyopathy arise from mutations in genes encoding sarcomere proteins including MYH7, MYBPC3, and TTN. Genetic diagnosis of cardiomyopathy relies on complete sequencing of the gene coding regions, and most pathogenic variation is rare. The 1000 Genomes project is an ongoing consortium designed to deliver whole genome sequence information from an ethnically diverse population and therefore is a rich source to determine both common and rare genetic variants. Methods and Results We queried the 1000 Genomes database of 1,092 individuals for exonic variants within three sarcomere genes MHY7, MYBPC3, and TTN. We focused our analysis on protein-altering variation, including nonsynonymous single nucleotide polymorphisms, insertion/deletion polymorphisms, or splice site altering variants. We identified known and predicted pathogenic variation in MYBPC3 and MYH7 at a higher frequency than what would be expected based on the known prevalence of cardiomyopathy. We also found substantial variation, including protein-disrupting sequences, in TTN. Conclusions Cardiomyopathy is a genetically heterogeneous disorder caused by mutations in multiple genes. The frequency of predicted pathogenic protein altering variation in cardiomyopathy genes suggests that many of these variants may be insufficient to cause disease on their own but may modify phenotype in a genetically susceptible host. This is suggested by the high prevalence of TTN insertion/deletions in the 1000 Genomes cohort. Given the possibility of additional genetic variants that modify the phenotype of a primary driver mutation, broad-based genetic testing should be employed.