The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

The Murphy Roths Large (MRL) mouse strain has "super-healing" properties that enhance recovery from injury. In mice, the DBA/2J strain intensifies many aspects of muscular dystrophy, so we evaluated the ability of the MRL strain to suppress muscular dystrophy in the Sgcg-null mouse model of limb girdle muscular dystrophy. A comparative analysis of Sgcg-null mice in the DBA/2J versus MRL strains showed greater myofiber regeneration, with reduced structural degradation of muscle in the MRL strain. Transcriptomic profiling of dystrophic muscle indicated strain-dependent expression of extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized myoscaffolds. Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix. Dystrophic myoscaffolds from the MRL background, but not the DBA/2J background, were enriched in myokines like IGF-1 and IL-6. C2C12 myoblasts seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J muscles showed the MRL background induced greater myoblast differentiation compared with dystrophic DBA/2J myoscaffolds. Thus, the MRL background imparts its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

The super-healing MRL strain promotes muscle growth in muscular dystrophy through a regenerative extracellular matrix.

Genetic background shifts the severity of muscular dystrophy. In mice, the DBA/2J strain confers a more severe muscular dystrophy phenotype, whereas the Murphy's Roth Large (MRL) strain has "super-healing" properties that reduce fibrosis. A comparative analysis of the Sgcg null model of Limb Girdle Muscular Dystrophy in the DBA/2J versus MRL strain showed the MRL background was associated with greater myofiber regeneration and reduced structural degradation of muscle. Transcriptomic profiling of dystrophic muscle in the DBA/2J and MRL strains indicated strain-dependent expression of the extracellular matrix (ECM) and TGF-ß signaling genes. To investigate the MRL ECM, cellular components were removed from dystrophic muscle sections to generate decellularized "myoscaffolds". Decellularized myoscaffolds from dystrophic mice in the protective MRL strain had significantly less deposition of collagen and matrix-bound TGF-ß1 and TGF-ß3 throughout the matrix, and dystrophic myoscaffolds from the MRL background were enriched in myokines. C2C12 myoblasts were seeded onto decellularized matrices from Sgcg-/- MRL and Sgcg-/- DBA/2J matrices. Acellular myoscaffolds from the dystrophic MRL background induced myoblast differentiation and growth compared to dystrophic myoscaffolds from the DBA/2J matrices. These studies establish that the MRL background also generates its effect through a highly regenerative ECM, which is active even in muscular dystrophy.

Small RNA Sequencing to Discover Circulating MicroRNA Biomarkers of Testicular Toxicity in Dogs.

Predictive indicators of testicular toxicity could improve drug development by allowing early in-life screening for this adverse effect before it becomes severe. We hypothesized that circulating microRNAs (miRNAs) could serve as testicular toxicity biomarkers in dogs. Herein, we describe the results of an exploratory study conducted to discover biomarkers of drug-induced testicular injury. Following a dose-selection study using the testicular toxicant ethylene glycol monomethyl ether (EGME), we chose a dose of 50 mg/kg/d EGME to avoid systemic toxicity and treated 2 groups of dogs (castrated, non-castrated) for 14 to 28 days. Castrated animals were used as negative controls to identify biomarkers specific for testicular toxicity because EGME can cause toxicity to organ systems in addition to the testis. Blood was collected daily during the dosing period, followed by recovery for 29 to 43 days with less frequent sampling. Dosing was well tolerated, resulting in mild-to-moderate degeneration in testes and epididymides. Global profiling of serum miRNAs at selected dosing and recovery time points was completed by small RNA sequencing. Bioinformatics data analysis using linear modeling demonstrated several circulating miRNAs that were differentially abundant during the dosing period compared with baseline and/or castrated control samples. Confirmatory reverse transcription quantitative polymerase chain reaction data in these animals was unable to detect sustained alterations of miRNAs in serum, except for 1 potential candidate cfa-miR-146b. Taken together, we report the results of a comprehensive exploratory study and suggest future directions for follow-up research to address the challenge of developing diagnostic biomarkers of testicular toxicity.

An Overview of Machine Learning and Big Data for Drug Toxicity Evaluation.

Drug toxicity evaluation is an essential process of drug development as it is reportedly responsible for the attrition of approximately 30% of drug candidates. The rapid increase in the number and types of large toxicology data sets together with the advances in computational methods may be used to improve many steps in drug safety evaluation. The development of in silico models to screen and understand mechanisms of drug toxicity may be particularly beneficial in the early stages of drug development where early toxicity assessment can most reduce expenses and labor time. To facilitate this, machine learning methods have been employed to evaluate drug toxicity but are often limited by small and less diverse data sets. Recent advances in machine learning methods together with the rapid increase in big toxicity data such as molecular descriptors, toxicogenomics, and high-throughput bioactivity data may help alleviate some of the current challenges. In this article, the most common machine learning methods used in toxicity assessment are reviewed together with examples of toxicity studies that have used machine learning methodology. Furthermore, a comprehensive overview of the different types of toxicity tools and data sets available to build in silico toxicity prediction models has been provided to give an overview of the current big toxicity data landscape and highlight opportunities and challenges related to them.

Rich Text Formatted EHR Narratives: A Hidden and Ignored Trove.

This study presents an approach for mining structured information from clinical narratives in Electronic Health Records (EHRs) by using Rich Text Formatted (RTF) records. RTF is adopted by many medical information management systems. There is rich structural information in these files which can be extracted and interpreted, yet such information is largely ignored. We investigate multiple types of EHR narratives in the Enterprise Data Warehouse from a multisite large healthcare chain consisting of both, an academic medical center and community hospitals. We focus on the RTF constructs related to tables and sections that are not available in plain text EHR narratives. We show how to parse these RTF constructs, analyze their prevalence and characteristics in the context of multiple types of EHR narratives. Our case study demonstrates the additional utility of the features derived from RTF constructs over plain text oriented NLP.

Cancer classification and pathway discovery using non-negative matrix factorization.

OBJECTIVES: Extracting genetic information from a full range of sequencing data is important for understanding disease. We propose a novel method to effectively explore the landscape of genetic mutations and aggregate them to predict cancer type. DESIGN: We applied non-smooth non-negative matrix factorization (nsNMF) and support vector machine (SVM) to utilize the full range of sequencing data, aiming to better aggregate genetic mutations and improve their power to predict disease type. More specifically, we introduce a novel classifier to distinguish cancer types using somatic mutations obtained from whole-exome sequencing data. Mutations were identified from multiple cancers and scored using SIFT, PP2, and CADD, and collapsed at the individual gene level. nsNMF was then applied to reduce dimensionality and obtain coefficient and basis matrices. A feature matrix was derived from the obtained matrices to train a classifier for cancer type classification with the SVM model. RESULTS: We have demonstrated that the classifier was able to distinguish four cancer types with reasonable accuracy. In five-fold cross-validations using mutation counts as features, the average prediction accuracy was 80% (SEM = 0.1%), significantly outperforming baselines and outperforming models using mutation scores as features. CONCLUSION: Using the factor matrices derived from the nsNMF, we identified multiple genes and pathways that are significantly associated with each cancer type. This study presents a generic and complete pipeline to study the associations between somatic mutations and cancers. The proposed method can be adapted to other studies for disease status classification and pathway discovery.

Distinct pathological signatures in human cellular models of myotonic dystrophy subtypes.

Myotonic dystrophy (DM) is the most common autosomal dominant muscular dystrophy and encompasses both skeletal muscle and cardiac complications. DM is nucleotide repeat expansion disorder in which type 1 (DM1) is due to a trinucleotide repeat expansion on chromosome 19 and type 2 (DM2) arises from a tetranucleotide repeat expansion on chromosome 3. Developing representative models of DM in animals has been challenging due to instability of nucleotide repeat expansions, especially for DM2, which is characterized by nucleotide repeat expansions often greater than 5,000 copies. To investigate mechanisms of human DM, we generated cellular models of DM1 and DM2. We used regulated MyoD expression to reprogram urine-derived cells into myotubes. In this myogenic cell model, we found impaired dystrophin expression, in the presence of muscleblind-like 1 (MBNL1) foci, and aberrant splicing in DM1 but not in DM2 cells. We generated induced pluripotent stem cells (iPSC) from healthy controls and DM1 and DM2 subjects, and we differentiated these into cardiomyocytes. DM1 and DM2 cells displayed an increase in RNA foci concomitant with cellular differentiation. iPSC-derived cardiomyocytes from DM1 but not DM2 had aberrant splicing of known target genes and MBNL sequestration. High-resolution imaging revealed tight association between MBNL clusters and RNA foci in DM1. Ca2+ transients differed between DM1- and DM2 iPSC-derived cardiomyocytes, and each differed from healthy control cells. RNA-sequencing from DM1- and DM2 iPSC-derived cardiomyocytes revealed distinct misregulation of gene expression, as well as differential aberrant splicing patterns. Together, these data support that DM1 and DM2, despite some shared clinical and molecular features, have distinct pathological signatures.

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia.

Essentials Loss of fibrinogen in zebrafish has been previously shown to result in adult onset hemorrhage Hemostatic defects were discovered in early fga-/- embryos but well tolerated until adulthood Afibrinogenemia and thrombocytopenia results in synthetic lethality in zebrafish. Testing human FGA variants of uncertain significance in zebrafish identified causative mutations SUMMARY: Background Mutations in the alpha chain of fibrinogen (FGA), such as deficiencies in other fibrinogen subunits, lead to rare inherited autosomal recessive hemostatic disorders. These range from asymptomatic to catastrophic life-threatening bleeds and the molecular basis of inherited fibrinogen deficiencies is only partially understood. Zinc finger nucleases have been used to produce mutations in zebrafish fga, resulting in overt adult-onset hemorrhage and reduced survival. Objectives To determine the age of onset of hemostatic defects in afibrinogenemic zebrafish and model human fibrinogen deficiencies. Methods TALEN genome editing (transcription activator-like effector nucleases) was used to generate a zebrafish fga mutant. Hemostatic defects were assessed through survival, gross anatomical and histological observation and laser-induced endothelial injury. Human FGA variants with unknown pathologies were engineered into the orthologous positions in zebrafish fga. Results Loss of Fga decreased survival and resulted in synthetic lethality when combined with thrombocytopenia. Zebrafish fga mutants exhibit a severe hemostatic defect by 3 days of life, but without visible hemorrhage. Induced thrombus formation through venous endothelial injury was completely absent in mutant embryos and larvae. This hemostatic defect was restored by microinjection of wild-type fga cDNA plasmid or purified human fibrinogen. This system was used to determine whether unknown human variants were pathological by engineering them into fga. Conclusions These studies confirm that loss of fibrinogen in zebrafish results in the absence of hemostasis from the embryonic period through adulthood. When combined with thrombocytopenia, zebrafish exhibit synthetic lethality, demonstrating that thrombocytes are necessary for survival in response to hemorrhage.

Dusp6 is a genetic modifier of growth through enhanced ERK activity.

Like other single-gene disorders, muscular dystrophy displays a range of phenotypic heterogeneity even with the same primary mutation. Identifying genetic modifiers capable of altering the course of muscular dystrophy is one approach to deciphering gene-gene interactions that can be exploited for therapy development. To this end, we used an intercross strategy in mice to map modifiers of muscular dystrophy. We interrogated genes of interest in an interval on mouse chromosome 10 associated with body mass in muscular dystrophy as skeletal muscle contributes significantly to total body mass. Using whole-genome sequencing of the two parental mouse strains combined with deep RNA sequencing, we identified the Met62Ile substitution in the dual-specificity phosphatase 6 (Dusp6) gene from the DBA/2 J (D2) mouse strain. DUSP6 is a broadly expressed dual-specificity phosphatase protein, which binds and dephosphorylates extracellular-signal-regulated kinase (ERK), leading to decreased ERK activity. We found that the Met62Ile substitution reduced the interaction between DUSP6 and ERK resulting in increased ERK phosphorylation and ERK activity. In dystrophic muscle, DUSP6 Met62Ile is strongly upregulated to counteract its reduced activity. We found that myoblasts from the D2 background were insensitive to a specific small molecule inhibitor of DUSP6, while myoblasts expressing the canonical DUSP6 displayed enhanced proliferation after exposure to DUSP6 inhibition. These data identify DUSP6 as an important regulator of ERK activity in the setting of muscle growth and muscular dystrophy.

Nfe2 is dispensable for early but required for adult thrombocyte formation and function in zebrafish.

The NFE2 transcription factor is expressed in multiple hematopoietic lineages with a well-defined role in regulating megakaryocyte biogenesis and platelet production in mammals. Mice deficient in NFE2 develop severe thrombocytopenia with lethality resulting from neonatal hemorrhage. Recent data in mammals reveal potential differences in embryonic and adult thrombopoiesis. Multiple studies in zebrafish have revealed mechanistic insights into hematopoiesis, although thrombopoiesis has been less studied. Rather than platelets, zebrafish possess thrombocytes, which are nucleated cells with similar functional properties. Using transcription activator-like effector nucleases to generate mutations in nfe2, we show that unlike mammals, zebrafish survive to adulthood in the absence of Nfe2. Despite developing severe thrombocytopenia, homozygous mutants do not display overt hemorrhage or reduced survival. Surprisingly, quantification of circulating thrombocytes in mutant 6-day-old larvae revealed no significant differences from wild-type siblings. Both wild-type and nfe2 null larvae formed thrombocyte-rich clots in response to endothelial injury. In addition, ex vivo thrombocytic colony formation was intact in nfe2 mutants, and adult kidney marrow displayed expansion of hematopoietic progenitors. These data suggest that loss of Nfe2 results in a late block in adult thrombopoiesis, with secondary expansion of precursors: features consistent with mammals. Overall, our data suggest parallels with erythropoiesis, including distinct primitive and definitive pathways of development and potential for a previously unknown Nfe2-independent pathway of embryonic thrombopoiesis. Long-term homozygous mutant survival will facilitate in-depth study of Nfe2 deficiency in vivo, and further investigation could lead to alternative methodologies for the enhancement of platelet production.

Efficient exon skipping of SGCG mutations mediated by phosphorodiamidate morpholino oligomers.

Exon skipping uses chemically modified antisense oligonucleotides to modulate RNA splicing. Therapeutically, exon skipping can bypass mutations and restore reading frame disruption by generating internally truncated, functional proteins to rescue the loss of native gene expression. Limb-girdle muscular dystrophy type 2C is caused by autosomal recessive mutations in the SGCG gene, which encodes the dystrophin-associated protein γ-sarcoglycan. The most common SGCG mutations disrupt the transcript reading frame abrogating γ-sarcoglycan protein expression. In order to treat most SGCG gene mutations, it is necessary to skip 4 exons in order to restore the SGCG transcript reading frame, creating an internally truncated protein referred to as Mini-Gamma. Using direct reprogramming of human cells with MyoD, myogenic cells were tested with 2 antisense oligonucleotide chemistries, 2'-O-methyl phosphorothioate oligonucleotides and vivo-phosphorodiamidate morpholino oligomers, to induce exon skipping. Treatment with vivo-phosphorodiamidate morpholino oligomers demonstrated efficient skipping of the targeted exons and corrected the mutant reading frame, resulting in the expression of a functional Mini-Gamma protein. Antisense-induced exon skipping of SGCG occurred in normal cells and those with multiple distinct SGCG mutations, including the most common 521ΔT mutation. These findings demonstrate a multiexon-skipping strategy applicable to the majority of limb-girdle muscular dystrophy 2C patients.

Genetic modifiers of muscular dystrophy act on sarcolemmal resealing and recovery from injury.

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor ß (TGFß) pathway, osteopontin encoded by the SPP1 gene and latent TGFß binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFß and TGFß-associated pathways. We identified that increased TGFß resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFß and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.

Experimental Modeling Supports a Role for MyBP-HL as a Novel Myofilament Component in Arrhythmia and Dilated Cardiomyopathy.

BACKGROUND: Cardiomyopathy and arrhythmias are under significant genetic influence. Here, we studied a family with dilated cardiomyopathy and associated conduction system disease in whom prior clinical cardiac gene panel testing was unrevealing. METHODS: Whole-genome sequencing and induced pluripotent stem cells were used to examine a family with dilated cardiomyopathy and atrial and ventricular arrhythmias. We also characterized a mouse model with heterozygous and homozygous deletion of Mybphl. RESULTS: Whole-genome sequencing identified a premature stop codon, R255X, in the MYBPHL gene encoding MyBP-HL (myosin-binding protein-H like), a novel member of the myosin-binding protein family. MYBPHL was found to have high atrial expression with low ventricular expression. We determined that MyBP-HL protein was myofilament associated in the atria, and truncated MyBP-HL protein failed to incorporate into the myofilament. Human cell modeling demonstrated reduced expression from the mutant MYBPHL allele. Echocardiography of Mybphl heterozygous and null mouse hearts exhibited a 36% reduction in fractional shortening and an increased diastolic ventricular chamber size. Atria weight normalized to total heart weight was significantly increased in Mybphl heterozygous and null mice. Using a reporter system, we detected robust expression of Mybphl in the atria, and in discrete puncta throughout the right ventricular wall and septum, as well. Telemetric electrocardiogram recordings in Mybphl mice revealed cardiac conduction system abnormalities with aberrant atrioventricular conduction and an increased rate of arrhythmia in heterozygous and null mice. CONCLUSIONS: The findings of reduced ventricular function and conduction system defects in Mybphl mice support that MYBPHL truncations may increase risk for human arrhythmias and cardiomyopathy.

Intermittent glucocorticoid steroid dosing enhances muscle repair without eliciting muscle atrophy.

Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions such as Duchenne muscular dystrophy, where their use is associated with prolonged ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are paradoxical because these steroids are also known to trigger muscle atrophy. Chronic steroid use usually involves once-daily dosing, although weekly dosing in children has been suggested for its reduced side effects on behavior. In this work, we tested steroid dosing in mice and found that a single pulse of glucocorticoid steroids improved sarcolemmal repair through increased expression of annexins A1 and A6, which mediate myofiber repair. This increased expression was dependent on glucocorticoid response elements upstream of annexins and was reinforced by the expression of forkhead box O1 (FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle injury and in muscular dystrophy and determined that both regimens provided comparable benefits in terms of annexin gene expression and muscle repair. However, daily dosing activated atrophic pathways, including F-box protein 32 (Fbxo32), which encodes atrogin-1. Conversely, weekly steroid treatment in mdx mice improved muscle function and histopathology and concomitantly induced the ergogenic transcription factor Krüppel-like factor 15 (Klf15) while decreasing Fbxo32. These findings suggest that intermittent, rather than daily, glucocorticoid steroid regimen promotes sarcolemmal repair and muscle recovery from injury while limiting atrophic remodeling.

The transcription factor, Nuclear factor, erythroid 2 (Nfe2), is a regulator of the oxidative stress response during Danio rerio development.

Development is a complex and well-defined process characterized by rapid cell proliferation and apoptosis. At this stage in life, a developmentally young organism is more sensitive to toxicants as compared to an adult. In response to pro-oxidant exposure, members of the Cap'n'Collar (CNC) basic leucine zipper (b-ZIP) transcription factor family (including Nfe2 and Nfe2-related factors, Nrfs) activate the expression of genes whose protein products contribute to reduced toxicity. Here, we studied the role of the CNC protein, Nfe2, in the developmental response to pro-oxidant exposure in the zebrafish (Danio rerio). Following acute waterborne exposures to diquat or tert-buytlhydroperoxide (tBOOH) at one of three developmental stages, wildtype (WT) and nfe2 knockout (KO) embryos and larvae were morphologically scored and their transcriptomes sequenced. Early in development, KO animals suffered from hypochromia that was made more severe through exposure to pro-oxidants; this phenotype in the KO may be linked to decreased expression of alas2, a gene involved in heme synthesis. WT and KO eleutheroembryos and larvae were phenotypically equally affected by exposure to pro-oxidants, where tBOOH caused more pronounced phenotypes as compared to diquat. Comparing diquat and tBOOH exposed embryos relative to the WT untreated control, a greater number of genes were up-regulated in the tBOOH condition as compared to diquat (tBOOH: 304 vs diquat: 148), including those commonly found to be differentially regulated in the vertebrate oxidative stress response (OSR) (e.g. hsp70.2, txn1, and gsr). When comparing WT and KO across all treatments and times, there were 1170 genes that were differentially expressed, of which 33 are known targets of the Nrf proteins Nrf1 and Nrf2. More specifically, in animals exposed to pro-oxidants a total of 968 genes were differentially expressed between WT and KO across developmental time, representing pathways involved in coagulation, embryonic organ development, body fluid level regulation, erythrocyte differentiation, and oxidation-reduction, amongst others. The greatest number of genes that changed in expression between WT and KO occurred in animals exposed to diquat at 2h post fertilization (hpf). Across time and treatment, there were six genes (dhx40, cfap70, dnajb9b, slc35f4, spi-c, and gpr19) that were significantly up-regulated in KO compared to WT and four genes (fhad1, cyp4v7, nlrp12, and slc16a6a) that were significantly down-regulated. None of these genes have been previously identified as targets of Nfe2 or the Nrf family. These results demonstrate that the zebrafish Nfe2 may be a regulator of both primitive erythropoiesis and the OSR during development.

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation.

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

Overexpression of Latent TGFß Binding Protein 4 in Muscle Ameliorates Muscular Dystrophy through Myostatin and TGFß.

Latent TGFß binding proteins (LTBPs) regulate the extracellular availability of latent TGFß. LTBP4 was identified as a genetic modifier of muscular dystrophy in mice and humans. An in-frame insertion polymorphism in the murine Ltbp4 gene associates with partial protection against muscular dystrophy. In humans, nonsynonymous single nucleotide polymorphisms in LTBP4 associate with prolonged ambulation in Duchenne muscular dystrophy. To better understand LTBP4 and its role in modifying muscular dystrophy, we created transgenic mice overexpressing the protective murine allele of LTBP4 specifically in mature myofibers using the human skeletal actin promoter. Overexpression of LTBP4 protein was associated with increased muscle mass and proportionally increased strength compared to age-matched controls. In order to assess the effects of LTBP4 in muscular dystrophy, LTBP4 overexpressing mice were bred to mdx mice, a model of Duchenne muscular dystrophy. In this model, increased LTBP4 led to greater muscle mass with proportionally increased strength, and decreased fibrosis. The increase in muscle mass and reduction in fibrosis were similar to what occurs when myostatin, a related TGFß family member and negative regulator of muscle mass, was deleted in mdx mice. Supporting this, we found that myostatin forms a complex with LTBP4 and that overexpression of LTBP4 led to a decrease in myostatin levels. LTBP4 also interacted with TGFß and GDF11, a protein highly related to myostatin. These data identify LTBP4 as a multi-TGFß family ligand binding protein with the capacity to modify muscle disease through overexpression.

Modifier genes and their effect on Duchenne muscular dystrophy.

PURPOSE OF REVIEW: Recently, genetic pathways that modify the clinical severity of Duchenne muscular dystrophy (DMD) have been identified. The pathways uncovered as modifiers are useful to predict prognosis and also elucidate molecular signatures that can be manipulated therapeutically. RECENT FINDINGS: Modifiers have been identified using combinations of transcriptome and genome profiling. Osteopontin, encoded by the SPP1 gene, was found using gene expression profiling. Latent TGFß binding protein 4, encoding latent TGFß binding protein 4 was initially discovered using a genome-wide screen in mice and then validated in cohorts of DMD patients. These two pathways converge in that they both regulate TGFß. A third modifier, Anxa6 that specifies annexin A6, is a calcium binding protein that has been identified using mouse models, and regulates the injury pathway and sarcolemmal resealing. SUMMARY: Genetic modifiers can serve as biomarkers for outcomes in DMD. Modifiers can alter strength and ambulation in muscular dystrophy, and these same features can be used as endpoints used in clinical trials. Moreover, because genetic modifiers can influence outcomes, these genetic markers should be considered when stratifying results in muscular dystrophy.

Targeted mutagenesis of zebrafish antithrombin III triggers disseminated intravascular coagulation and thrombosis, revealing insight into function.

Pathologic blood clotting is a leading cause of morbidity and mortality in the developed world, underlying deep vein thrombosis, myocardial infarction, and stroke. Genetic predisposition to thrombosis is still poorly understood, and we hypothesize that there are many additional risk alleles and modifying factors remaining to be discovered. Mammalian models have contributed to our understanding of thrombosis, but are low throughput and costly. We have turned to the zebrafish, a tool for high-throughput genetic analysis. Using zinc finger nucleases, we show that disruption of the zebrafish antithrombin III (at3) locus results in spontaneous venous thrombosis in larvae. Although homozygous mutants survive into early adulthood, they eventually succumb to massive intracardiac thrombosis. Characterization of null fish revealed disseminated intravascular coagulation in larvae secondary to unopposed thrombin activity and fibrinogen consumption, which could be rescued by both human and zebrafish at3 complementary DNAs. Mutation of the human AT3-reactive center loop abolished the ability to rescue, but the heparin-binding site was dispensable. These results demonstrate overall conservation of AT3 function in zebrafish, but reveal developmental variances in the ability to tolerate excessive clot formation. The accessibility of early zebrafish development will provide unique methods for dissection of the underlying mechanisms of thrombosis.

Annexin A6 modifies muscular dystrophy by mediating sarcolemmal repair.

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.