Publications by authors named "Louis M Kunkel"

109 Publications

hnRNP L is essential for myogenic differentiation and modulates myotonic dystrophy pathologies.

Muscle Nerve 2021 Mar 2. Epub 2021 Mar 2.

Department of Medicine, Molecular Cardiology Research Institute, Boston, MA.

Introduction/aims: RNA binding proteins (RBPs) play an important role in skeletal muscle development and disease by regulating RNA splicing. In myotonic dystrophy type 1 (DM1), the RBP MBNL1 (Muscleblind-like) is sequestered by toxic CUG repeats, leading to mis-splicing of MBNL1 targets. Mounting evidence from the literature has implicated other factors in the pathogenesis of DM1. Here we sought to evaluate the functional role of hnRNP L in normal and DM1 muscle cells. We sought to test if modulation of hnRNP L expression affected DM1 splicing targets and myogenic outcomes.

Methods: Co-immunoprecipitation assays using hnRNPL and MBNL1 expression constructs and expression profiling in normal and DM1 muscle cell lines were performed. Zebrafish morpholinos targeting hnrnpl and hnrnpl2 were injected into one-cell zebrafish for developmental and muscle analysis. Ascochlorin administration to DM1 myoblasts was performed and expression of the CUG repeats, DM1 splicing biomarkers, and hnRNP L expression levels were evaluated.

Results: Using DM1 patient myoblast cell lines we observed the formation of abnormal hnRNP L nuclear foci within and outside the expanded CUG repeats, further suggesting a role for this factor in DM1 pathology. We showed that the antiviral and antitumorigenic isoprenoid compound ascochlorin increased MBNL1 and hnRNP L expression levels. Drug treatment of DM1 muscle cells with ascochlorin partially rescued mis-splicing of established early biomarkers of DM1 and improved the defective myotube formation displayed by DM1 muscle cells.

Discussion: Together, these studies reveal that hnRNP L modulated DM1 pathologies, and is a potential therapeutic target. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/mus.27216DOI Listing
March 2021

POLRMT mutations impair mitochondrial transcription causing neurological disease.

Nat Commun 2021 02 18;12(1):1135. Epub 2021 Feb 18.

Wellcome Centre for Mitochondrial Research, Translational and Clinical Research Institute, The Medical School, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.

While >300 disease-causing variants have been identified in the mitochondrial DNA (mtDNA) polymerase γ, no mitochondrial phenotypes have been associated with POLRMT, the RNA polymerase responsible for transcription of the mitochondrial genome. Here, we characterise the clinical and molecular nature of POLRMT variants in eight individuals from seven unrelated families. Patients present with global developmental delay, hypotonia, short stature, and speech/intellectual disability in childhood; one subject displayed an indolent progressive external ophthalmoplegia phenotype. Massive parallel sequencing of all subjects identifies recessive and dominant variants in the POLRMT gene. Patient fibroblasts have a defect in mitochondrial mRNA synthesis, but no mtDNA deletions or copy number abnormalities. The in vitro characterisation of the recombinant POLRMT mutants reveals variable, but deleterious effects on mitochondrial transcription. Together, our in vivo and in vitro functional studies of POLRMT variants establish defective mitochondrial transcription as an important disease mechanism.
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http://dx.doi.org/10.1038/s41467-021-21279-0DOI Listing
February 2021

PDE10A Inhibition Reduces the Manifestation of Pathology in DMD Zebrafish and Represses the Genetic Modifier PITPNA.

Mol Ther 2020 Nov 20. Epub 2020 Nov 20.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA; The Stem Cell Program, Boston Children's Hospital, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; The Manton Center for Orphan Disease Research at Boston Children's Hospital, Boston, MA 02115, USA. Electronic address:

Duchenne muscular dystrophy (DMD) is a severe genetic disorder caused by mutations in the DMD gene. Absence of dystrophin protein leads to progressive degradation of skeletal and cardiac function and leads to premature death. Over the years, zebrafish have been increasingly used for studying DMD and are a powerful tool for drug discovery and therapeutic development. In our study, a birefringence screening assay led to identification of phosphodiesterase 10A (PDE10A) inhibitors that reduced the manifestation of dystrophic muscle phenotype in dystrophin-deficient sapje-like zebrafish larvae. PDE10A has been validated as a therapeutic target by pde10a morpholino-mediated reduction in muscle pathology and improvement in locomotion, muscle, and vascular function as well as long-term survival in sapje-like larvae. PDE10A inhibition in zebrafish and DMD patient-derived myoblasts were also associated with reduction of PITPNA expression that has been previously identified as a protective genetic modifier in two exceptional dystrophin-deficient golden retriever muscular dystrophy (GRMD) dogs that escaped the dystrophic phenotype. The combination of a phenotypic assay and relevant functional assessments in the sapje-like zebrafish enhances the potential for the prospective discovery of DMD therapeutics. Indeed, our results suggest a new application for a PDE10A inhibitor as a potential DMD therapeutic to be investigated in a mouse model of DMD.
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http://dx.doi.org/10.1016/j.ymthe.2020.11.021DOI Listing
November 2020

Effect of serotonin modulation on dystrophin-deficient zebrafish.

Biol Open 2020 08 28;9(8). Epub 2020 Aug 28.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA, USA

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease caused by mutation of the gene. Pharmacological therapies that function independently of dystrophin and complement strategies aimed at dystrophin restoration could significantly improve patient outcomes. Previous observations have suggested that serotonin pathway modulation ameliorates dystrophic pathology, and re-application of serotonin modulators already used clinically would potentially hasten availability to DMD patients. In our study, we used dystrophin-deficient and zebrafish models of DMD for rapid and easy screening of several classes of serotonin pathway modulators as potential therapeutics. None of the candidate drugs tested significantly decreased the percentage of zebrafish exhibiting the dystrophic muscle phenotype in the short-term birefringence assay or lengthened the lifespan in the long-term survival assay. Although we did not identify an effective drug, we believe our data is of value to the DMD research community for future studies, and there is evidence that suggests serotonin modulation may still be a viable treatment strategy with further investigation. Given the widespread clinical use of selective serotonin reuptake inhibitors, tricyclic antidepressants and reversible inhibitors of monoamine oxidase, their reapplication to DMD is an attractive strategy in the field's pursuit to identify pharmacological therapies to complement dystrophin restoration strategies.
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http://dx.doi.org/10.1242/bio.053363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7473644PMC
August 2020

Applying genome-wide CRISPR-Cas9 screens for therapeutic discovery in facioscapulohumeral muscular dystrophy.

Sci Transl Med 2020 03;12(536)

Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA.

The emergence of CRISPR-Cas9 gene-editing technologies and genome-wide CRISPR-Cas9 libraries enables efficient unbiased genetic screening that can accelerate the process of therapeutic discovery for genetic disorders. Here, we demonstrate the utility of a genome-wide CRISPR-Cas9 loss-of-function library to identify therapeutic targets for facioscapulohumeral muscular dystrophy (FSHD), a genetically complex type of muscular dystrophy for which there is currently no treatment. In FSHD, both genetic and epigenetic changes lead to misexpression of , the FSHD causal gene that encodes the highly cytotoxic DUX4 protein. We performed a genome-wide CRISPR-Cas9 screen to identify genes whose loss-of-function conferred survival when DUX4 was expressed in muscle cells. Genes emerging from our screen illuminated a pathogenic link to the cellular hypoxia response, which was revealed to be the main driver of DUX4-induced cell death. Application of hypoxia signaling inhibitors resulted in increased DUX4 protein turnover and subsequent reduction of the cellular hypoxia response and cell death. In addition, these compounds proved successful in reducing FSHD disease biomarkers in patient myogenic lines, as well as improving structural and functional properties in two zebrafish models of FSHD. Our genome-wide perturbation of pathways affecting DUX4 expression has provided insight into key drivers of DUX4-induced pathogenesis and has identified existing compounds with potential therapeutic benefit for FSHD. Our experimental approach presents an accelerated paradigm toward mechanistic understanding and therapeutic discovery of a complex genetic disease, which may be translatable to other diseases with well-established phenotypic selection assays.
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http://dx.doi.org/10.1126/scitranslmed.aay0271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7304480PMC
March 2020

To dystrophin and beyond: an interview with Louis Kunkel.

Authors:
Louis M Kunkel

Dis Model Mech 2019 12 12;13(2). Epub 2019 Dec 12.

Louis Kunkel has devoted his career to understanding the causes, mechanisms and treatment of muscular dystrophies. Dr Kunkel is the past Director of the Genomics Program at Boston Children's Hospital and Professor of Genetics and Pediatrics at Harvard Medical School. In this interview, he talks about his discovery of dystrophin, including patients in preclinical research, and bearded irises.
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http://dx.doi.org/10.1242/dmm.043018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918775PMC
December 2019

The SINE Compound KPT-350 Blocks Dystrophic Pathologies in DMD Zebrafish and Mice.

Mol Ther 2020 01 3;28(1):189-201. Epub 2019 Sep 3.

Department of Pediatrics, Division of Neurology, University of Alabama at Birmingham and Children's of Alabama, Birmingham, AL 35294, USA; UAB Center for Exercise Medicine (UCEM), Birmingham, AL 35294, USA; Department of Genetics at the University of Alabama at Birmingham, Birmingham, AL 35294, USA; Civitan International Research Center at the University of Alabama at Birmingham, Birmingham, AL 35294, USA. Electronic address:

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disease that is caused by the loss of functional dystrophin protein in cardiac and skeletal muscles. DMD patient muscles become weakened, leading to eventual myofiber breakdown and replacement with fibrotic and adipose tissues. Inflammation drives the pathogenic processes through releasing inflammatory cytokines and other factors that promote skeletal muscle degeneration and contributing to the loss of motor function. Selective inhibitors of nuclear export (SINEs) are a class of compounds that function by inhibiting the nuclear export protein exportin 1 (XPO1). The XPO1 protein is an important regulator of key inflammatory and neurological factors that drive inflammation and neurotoxicity in various neurological and neuromuscular diseases. Here, we demonstrate that SINE compound KPT-350 can ameliorate dystrophic-associated pathologies in the muscles of DMD models of zebrafish and mice. Thus, SINE compounds are a promising novel strategy for blocking dystrophic symptoms and could be used in combinatorial treatments for DMD.
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http://dx.doi.org/10.1016/j.ymthe.2019.08.016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6952030PMC
January 2020

Concordance between gene expression in peripheral whole blood and colonic tissue in children with inflammatory bowel disease.

PLoS One 2019 16;14(10):e0222952. Epub 2019 Oct 16.

Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, United States of America.

Background: Presenting features of inflammatory bowel disease (IBD) are non-specific. We hypothesized that mRNA profiles could (1) identify genes and pathways involved in disease pathogenesis; (2) identify a molecular signature that differentiates IBD from other conditions; (3) provide insight into systemic and colon-specific dysregulation through study of the concordance of the gene expression.

Methods: Children (8-18 years) were prospectively recruited at the time of diagnostic colonoscopy for possible IBD. We used transcriptome-wide mRNA profiling to study gene expression in colon biopsies and paired whole blood samples. Using blood mRNA measurements, we fit a regression model for disease state prediction that was validated in an independent test set of adult subjects (GSE3365).

Results: Ninety-eight children were recruited [39 Crohn's disease, 18 ulcerative colitis, 2 IBDU, 39 non-IBD]. There were 1,118 significantly differentially (IBD vs non-IBD) expressed genes in colon tissue, and 880 in blood. The direction of relative change in expression was concordant for 106/112 genes differentially expressed in both tissue types. The regression model from the blood mRNA measurements distinguished IBD vs non-IBD disease status in the independent test set with 80% accuracy using only 6 genes. The overlap of 5 immune and metabolic pathways in the two tissue types was significant (p<0.001).

Conclusions: Blood and colon tissue from patients with IBD share a common transcriptional profile dominated by immune and metabolic pathways. Our results suggest that peripheral blood expression levels of as few as 6 genes (IL7R, UBB, TXNIP, S100A8, ALAS2, and SLC2A3) may distinguish patients with IBD from non-IBD.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0222952PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6795427PMC
March 2020

Discovery of Novel Therapeutics for Muscular Dystrophies using Zebrafish Phenotypic Screens.

J Neuromuscul Dis 2019 ;6(3):271-287

Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

The recent availability and development of mutant and transgenic zebrafish strains that model human muscular dystrophies has created new research opportunities for therapeutic development. Not only do these models mimic many pathological aspects of human dystrophies, but their small size, large clutch sizes, rapid ex utero development, body transparency, and genetic tractability enable research approaches that would be inconceivable with mammalian model systems. Here we discuss the use of zebrafish models of muscular dystrophy to rapidly screen hundreds to thousands of bioactive compounds in order to identify novel therapeutic candidates that modulate pathologic phenotypes. We review the justification and rationale behind this unbiased approach, including how zebrafish screens have identified FDA-approved drugs that are candidates for treating Duchenne and limb girdle muscular dystrophies. Not only can these drugs be re-purposed for treating dystrophies in a fraction of the time and cost of new drug development, but their identification has revealed novel, unexpected directions for future therapy development. Phenotype-driven zebrafish drug screens are an important compliment to the more established mammalian, target-based approaches for rapidly developing and validating therapeutics for muscular dystrophies.
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http://dx.doi.org/10.3233/JND-190389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6961982PMC
February 2020

Identification of a pathogenic mutation in ATP2A1 via in silico analysis of exome data for cryptic aberrant splice sites.

Mol Genet Genomic Med 2019 03 28;7(3):e552. Epub 2019 Jan 28.

Division of Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine, Gainesville, Florida.

Background: Pathogenic mutations causing aberrant splicing are often difficult to detect. Standard variant analysis of next-generation sequence (NGS) data focuses on canonical splice sites. Noncanonical splice sites are more difficult to ascertain.

Methods: We developed a bioinformatics pipeline that screens existing NGS data for potentially aberrant novel essential splice sites (PANESS) and performed a pilot study on a family with a myotonic disorder. Further analyses were performed via qRT-PCR, immunoblotting, and immunohistochemistry. RNAi knockdown studies were performed in Drosophila to model the gene deficiency.

Results: The PANESS pipeline identified a homozygous ATP2A1 variant (NC_000016.9:g.28905928G>A; NM_004320.4:c.1287G>A:p.(Glu429=)) that was predicted to cause the omission of exon 11. Aberrant splicing of ATP2A1 was confirmed via qRT-PCR, and abnormal expression of the protein product sarcoplasmic/endoplasmic reticulum Ca ATPase 1 (SERCA1) was demonstrated in quadriceps femoris tissue from the proband. Ubiquitous knockdown of SERCA led to lethality in Drosophila, as did knockdown targeting differentiating or fusing myoblasts.

Conclusions: This study confirms the potential of novel in silico algorithms to detect cryptic mutations in existing NGS data; expands the phenotypic spectrum of ATP2A1 mutations beyond classic Brody myopathy; and suggests that genetic testing of ATP2A1 should be considered in patients with clinical myotonia.
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http://dx.doi.org/10.1002/mgg3.552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418371PMC
March 2019

Impact of PYROXD1 deficiency on cellular respiration and correlations with genetic analyses of limb-girdle muscular dystrophy in Saudi Arabia and Sudan.

Physiol Genomics 2018 11 31;50(11):929-939. Epub 2018 Aug 31.

Division of Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine , Gainesville, Florida.

Next-generation sequencing is commonly used to screen for pathogenic mutations in families with Mendelian disorders, but due to the pace of discoveries, gaps have widened for some diseases between genetic and pathophysiological knowledge. We recruited and analyzed 16 families with limb-girdle muscular dystrophy (LGMD) of Arab descent from Saudi Arabia and Sudan who did not have confirmed genetic diagnoses. The analysis included both traditional and next-generation sequencing approaches. Cellular and metabolic studies were performed on Pyroxd1 siRNA C2C12 myoblasts and controls. Pathogenic mutations were identified in eight of the 16 families. One Sudanese family of Arab descent residing in Saudi Arabia harbored a homozygous c.464A>G, p.Asn155Ser mutation in PYROXD1, a gene recently reported in association with myofibrillar myopathy and whose protein product reduces thiol residues. Pyroxd1 deficiency in murine C2C12 myoblasts yielded evidence for impairments of cellular proliferation, migration, and differentiation, while CG10721 (Pyroxd1 fly homolog) knockdown in Drosophila yielded a lethal phenotype. Further investigations indicated that Pyroxd1 does not localize to mitochondria, yet Pyroxd1 deficiency is associated with decreased cellular respiration. This study identified pathogenic mutations in half of the LGMD families from the cohort, including one in PYROXD1. Developmental impairments were demonstrated in vitro for Pyroxd1 deficiency and in vivo for CG10721 deficiency, with reduced metabolic activity in vitro for Pyroxd1 deficiency.
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http://dx.doi.org/10.1152/physiolgenomics.00036.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293114PMC
November 2018

A limb-girdle muscular dystrophy 2I model of muscular dystrophy identifies corrective drug compounds for dystroglycanopathies.

JCI Insight 2018 09 20;3(18). Epub 2018 Sep 20.

Department of Pediatrics, Division of Neurology at the University of Alabama at Birmingham and Children's of Alabama, Birmingham, Alabama, USA.

Zebrafish are a powerful tool for studying muscle function owing to their high numbers of offspring, low maintenance costs, evolutionarily conserved muscle functions, and the ability to rapidly take up small molecular compounds during early larval stages. Fukutin-related protein (FKRP) is a putative protein glycosyltransferase that functions in the Golgi apparatus to modify sugar chain molecules of newly translated proteins. Patients with mutations in the FKRP gene can have a wide spectrum of clinical symptoms with varying muscle, eye, and brain pathologies depending on the location of the mutation in the FKRP protein. Patients with a common L276I FKRP mutation have mild adult-onset muscle degeneration known as limb-girdle muscular dystrophy 2I (LGMD2I), whereas patients with more C-terminal pathogenic mutations develop the severe Walker-Warburg syndrome (WWS)/muscle-eye-brain (MEB) disease. We generated fkrp-mutant zebrafish that phenocopy WWS/MEB pathologies including severe muscle breakdowns, head malformations, and early lethality. We have also generated a milder LGMD2I-model zebrafish via overexpression of a heat shock-inducible human FKRP (L276I) transgene that shows milder muscle pathology. Screening of an FDA-approved drug compound library in the LGMD2I zebrafish revealed a strong propensity towards steroids, antibacterials, and calcium regulators in ameliorating FKRP-dependent pathologies. Together, these studies demonstrate the utility of the zebrafish to both study human-specific FKRP mutations and perform compound library screenings for corrective drug compounds to treat muscular dystrophies.
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http://dx.doi.org/10.1172/jci.insight.120493DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237228PMC
September 2018

An open source microcontroller based flume for evaluating swimming performance of larval, juvenile, and adult zebrafish.

PLoS One 2018 26;13(6):e0199712. Epub 2018 Jun 26.

Division of Genetics and Genomics, The Manton Center for Orphan Disease Research, Boston Children's Hospital and Harvard Medical School, Boston, MA, United States of America.

Zebrafish are a preferred vertebrate model for delineating genotype-phenotype relationships. One of the most studied features of zebrafish is their exceptional swimming ability. By 7 days postfertilization (dpf), zebrafish spend over two-thirds of their time engaged in spontaneous swimming activity and several months later they are capable of attaining some of the fastest swimming velocities relative to body length ever recorded in the laboratory. However, laboratory-assembled flumes capable of achieving the slow flow velocities characteristics of larvae as well as the relatively fast maximal velocities of adults have not been described in sufficient detail to allow easy replication. Here we describe an easily assembled, open-source zebrafish-scaled flume for assessing swimming performance. The flume uses two independent spherical-impeller pumps modulated by a microcontroller to achieve flow velocities ranging from 1 to 70 cm s-1. The microcontroller also monitors water temperature and flow velocity and sends these data to a personal computer for real-time display and storage. Incremental protocols for assessing maximal swimming speed (Umax) were developed, stored in custom software, and then uploaded to the microcontroller in order to assess performance of larval (14, 21, 28 dpf), juvenile (35, 42 dpf), and adult (8, 22 month) zebrafish. The flume had sufficient range and sensitivity to detect developmental changes in Umax of larvae and juveniles, an 18-24% faster Umax of adult males vs. females, and a 14-20% age-related reduction in Umax for the oldest zebrafish. Detailed information is provided to assemble and operate this low-cost, versatile, and reliable tool for assessing zebrafish swimming performance.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0199712PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6019105PMC
December 2018

Repression of phosphatidylinositol transfer protein α ameliorates the pathology of Duchenne muscular dystrophy.

Proc Natl Acad Sci U S A 2017 06 22;114(23):6080-6085. Epub 2017 May 22.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115;

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by X-linked inherited mutations in the () gene. Absence of dystrophin protein from the sarcolemma causes severe muscle degeneration, fibrosis, and inflammation, ultimately leading to cardiorespiratory failure and premature death. Although there are several promising strategies under investigation to restore dystrophin protein expression, there is currently no cure for DMD, and identification of genetic modifiers as potential targets represents an alternative therapeutic strategy. In a Brazilian golden retriever muscular dystrophy (GRMD) dog colony, two related dogs demonstrated strikingly mild dystrophic phenotypes compared with those typically observed in severely affected GRMD dogs despite lacking dystrophin. Microarray analysis of these "escaper" dogs revealed reduced expression of phosphatidylinositol transfer protein-α () in escaper versus severely affected GRMD dogs. Based on these findings, we decided to pursue investigation of modulation of expression on dystrophic pathology in GRMD dogs, dystrophin-deficient sapje zebrafish, and human DMD myogenic cells. In GRMD dogs, decreased expression of was associated with increased phosphorylated Akt (pAkt) expression and decreased PTEN levels. knockdown by injection of morpholino oligonucleotides in sapje zebrafish also increased pAkt, rescued the abnormal muscle phenotype, and improved long-term sapje mutant survival. In DMD myotubes, knockdown by lentiviral shRNA increased pAkt and increased myoblast fusion index. Overall, our findings suggest as a disease modifier that accords benefits to the abnormal signaling, morphology, and function of dystrophic skeletal muscle, and may be a target for DMD and related neuromuscular diseases.
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http://dx.doi.org/10.1073/pnas.1703556114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5468635PMC
June 2017

Muscle dysfunction in a zebrafish model of Duchenne muscular dystrophy.

Physiol Genomics 2016 11 7;48(11):850-860. Epub 2016 Oct 7.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, Massachusetts.

zebrafish lack the protein dystrophin and are the smallest vertebrate model of Duchenne muscular dystrophy (DMD). Their small size makes them ideal for large-scale drug discovery screens. However, the extent that mimic the muscle dysfunction of higher vertebrate models of DMD is unclear. We used an optical birefringence assay to differentiate affected dystrophic larvae from their unaffected siblings and then studied trunk muscle contractility at 4-7 days postfertilization. Preparation cross-sectional area (CSA) was similar for affected and unaffected larvae, yet tetanic forces of affected preparations were only 30-60% of normal. ANCOVA indicated that the linear relationship observed between tetanic force and CSA for unaffected preparations was absent in the affected population. Consequently, the average force/CSA of affected larvae was depressed 30-70%. Disproportionate reductions in twitch vs. tetanic force, and a slowing of twitch tension development and relaxation, indicated that the myofibrillar disorganization evident in the birefringence assay could not explain the entire force loss. Single eccentric contractions, in which activated preparations were lengthened 5-10%, resulted in tetanic force deficits in both groups of larvae. However, deficits of affected preparations were three- to fivefold greater at all strains and ages, even after accounting for any recovery. Based on these functional assessments, we conclude that the mutant zebrafish is a phenotypically severe model of DMD. The severe contractile deficits of larvae represent novel physiological endpoints for therapeutic drug screening.
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http://dx.doi.org/10.1152/physiolgenomics.00088.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6223571PMC
November 2016

The sensitivity of exome sequencing in identifying pathogenic mutations for LGMD in the United States.

J Hum Genet 2017 Feb 6;62(2):243-252. Epub 2016 Oct 6.

Division of Pediatric Neurology, Department of Pediatrics, University of Florida College of Medicine, Gainesville, FL, USA.

The current study characterizes a cohort of limb-girdle muscular dystrophy (LGMD) in the United States using whole-exome sequencing. Fifty-five families affected by LGMD were recruited using an institutionally approved protocol. Exome sequencing was performed on probands and selected parental samples. Pathogenic mutations and cosegregation patterns were confirmed by Sanger sequencing. Twenty-two families (40%) had novel and previously reported pathogenic mutations, primarily in LGMD genes, and also in genes for Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital myopathy, myofibrillar myopathy, inclusion body myopathy and Pompe disease. One family was diagnosed via clinical testing. Dominant mutations were identified in COL6A1, COL6A3, FLNC, LMNA, RYR1, SMCHD1 and VCP, recessive mutations in ANO5, CAPN3, GAA, LAMA2, SGCA and SGCG, and X-linked mutations in DMD. A previously reported variant in DMD was confirmed to be benign. Exome sequencing is a powerful diagnostic tool for LGMD. Despite careful phenotypic screening, pathogenic mutations were found in other muscle disease genes, largely accounting for the increased sensitivity of exome sequencing. Our experience suggests that broad sequencing panels are useful for these analyses because of the phenotypic overlap of many neuromuscular conditions. The confirmation of a benign DMD variant illustrates the potential of exome sequencing to help determine pathogenicity.
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http://dx.doi.org/10.1038/jhg.2016.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5266644PMC
February 2017

CD82 Is a Marker for Prospective Isolation of Human Muscle Satellite Cells and Is Linked to Muscular Dystrophies.

Cell Stem Cell 2016 12 15;19(6):800-807. Epub 2016 Sep 15.

Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA 02115, USA. Electronic address:

Cell-surface markers for prospective isolation of stem cells from human skeletal muscle have been difficult to identify. Such markers would be powerful tools for studying satellite cell function during homeostasis and in pathogenesis of diseases such as muscular dystrophies. In this study, we show that the tetraspanin KAI/CD82 is an excellent marker for prospectively isolating stem cells from human fetal and adult skeletal muscle. Human CD82 muscle cells robustly engraft into a mouse model of muscular dystrophy. shRNA knockdown of CD82 in myogenic cells reduces myoblast proliferation, suggesting it is functionally involved in muscle homeostasis. CD82 physically interacts with alpha7beta1 integrin (α7β1-ITG) and with α-sarcoglycan, a member of the Dystrophin-Associated Glycoprotein Complex (DAPC), both of which have been linked to muscular dystrophies. Consistently, CD82 expression is decreased in Duchenne muscular dystrophy patients. Together, these findings suggest that CD82 function may be important for muscle stem cell function in muscular disorders.
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http://dx.doi.org/10.1016/j.stem.2016.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5135584PMC
December 2016

Reflections on Henry Kunkel outside the laboratory.

Clin Immunol 2016 11 18;172:21-22. Epub 2016 Jul 18.

Genetics and Pediatrics Harvard Medical School, Boston Children's Hospital, USA. Electronic address:

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http://dx.doi.org/10.1016/j.clim.2016.07.005DOI Listing
November 2016

Homozygous nonsense mutation in SGCA is a common cause of limb-girdle muscular dystrophy in Assiut, Egypt.

Muscle Nerve 2016 10 24;54(4):690-5. Epub 2016 Aug 24.

Division of Pediatric Neurology, University of Florida College of Medicine, PO Box 100296, Gainesville, Florida, USA, 32610.

Introduction: The genetic causes of limb-girdle muscular dystrophy (LGMD) have been studied in numerous countries, but such investigations have been limited in Egypt.

Methods: A cohort of 30 families with suspected LGMD from Assiut, Egypt, was studied using immunohistochemistry, homozygosity mapping, Sanger sequencing, and whole exome sequencing.

Results: Six families were confirmed to have pathogenic mutations, 4 in SGCA and 2 in DMD. Of these, 3 families harbored a single nonsense mutation in SGCA, suggesting that this may be a common mutation in Assiut, Egypt, originating from a founder effect.

Conclusions: The Assiut region in Egypt appears to share at least several of the common LGMD genes found in other parts of the world. It is notable that 4 of the 6 mutations were ascertained by means of whole exome sequencing, even though it was the last approach adopted. This illustrates the power of this technique for identifying causative mutations for muscular dystrophies. Muscle Nerve 54: 690-695, 2016.
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http://dx.doi.org/10.1002/mus.25094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5453499PMC
October 2016

Pharmacological therapeutics targeting the secondary defects and downstream pathology of Duchenne muscular dystrophy.

Expert Opin Orphan Drugs 2016 18;4(11):1179-1194. Epub 2016 Oct 18.

Boston Children's Hospital, Division of Genetics and Genomics, Boston, MA 02115.

Introduction: Since the identification of the dystrophin gene in 1986, a cure for Duchenne muscular dystrophy (DMD) has yet to be discovered. Presently, there are a number of genetic-based therapies in development aimed at restoration and/or repair of the primary defect. However, growing understanding of the pathophysiological consequences of dystrophin absence has revealed several promising downstream targets for the development of therapeutics.

Areas Covered: In this review, we discuss various strategies for DMD therapy targeting downstream consequences of dystrophin absence including loss of muscle mass, inflammation, fibrosis, calcium overload, oxidative stress, and ischemia. The rationale of each approach and the efficacy of drugs in preclinical and clinical studies are discussed.

Expert Opinion: For the last 30 years, effective DMD drug therapy has been limited to corticosteroids, which are associated with a number of negative side effects. Our knowledge of the consequences of dystrophin absence that contribute to DMD pathology has revealed several potential therapeutic targets. Some of these approaches may have potential to improve or slow disease progression independently or in combination with genetic-based approaches. The applicability of these pharmacological therapies to DMD patients irrespective of their genetic mutation, as well as the potential benefits even for advanced stage patients warrants their continued investigation.
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http://dx.doi.org/10.1080/21678707.2016.1240613DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5487007PMC
October 2016

A missense mutation in TFRC, encoding transferrin receptor 1, causes combined immunodeficiency.

Nat Genet 2016 Jan 7;48(1):74-8. Epub 2015 Dec 7.

Division of Immunology, Boston Children's Hospital, Boston, Massachusetts, USA.

Patients with a combined immunodeficiency characterized by normal numbers but impaired function of T and B cells had a homozygous p.Tyr20His substitution in transferrin receptor 1 (TfR1), encoded by TFRC. The substitution disrupts the TfR1 internalization motif, resulting in defective receptor endocytosis and markedly increased TfR1 expression on the cell surface. Iron citrate rescued the lymphocyte defects, and expression of wild-type but not mutant TfR1 rescued impaired transferrin uptake in patient-derived fibroblasts. Tfrc(Y20H/Y20H) mice recapitulated the immunological defects of patients. Despite the critical role of TfR1 in erythrocyte development and function, patients had only mild anemia and only slightly increased TfR1 expression in erythroid precursors. We show that STEAP3, a metalloreductase expressed in erythroblasts, associates with TfR1 and partially rescues transferrin uptake in patient-derived fibroblasts, suggesting that STEAP3 may provide an accessory TfR1 endocytosis signal that spares patients from severe anemia. These findings demonstrate the importance of TfR1 in adaptive immunity.
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http://dx.doi.org/10.1038/ng.3465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696875PMC
January 2016

Jagged 1 Rescues the Duchenne Muscular Dystrophy Phenotype.

Cell 2015 Nov 12;163(5):1204-1213. Epub 2015 Nov 12.

Human Genome and Stem Cell Center, Biosciences Institute, University of São Paulo, São Paulo 05508-090, Brazil. Electronic address:

Duchenne muscular dystrophy (DMD), caused by mutations at the dystrophin gene, is the most common form of muscular dystrophy. There is no cure for DMD and current therapeutic approaches to restore dystrophin expression are only partially effective. The absence of dystrophin in muscle results in dysregulation of signaling pathways, which could be targets for disease therapy and drug discovery. Previously, we identified two exceptional Golden Retriever muscular dystrophy (GRMD) dogs that are mildly affected, have functional muscle, and normal lifespan despite the complete absence of dystrophin. Now, our data on linkage, whole-genome sequencing, and transcriptome analyses of these dogs compared to severely affected GRMD and control animals reveals that increased expression of Jagged1 gene, a known regulator of the Notch signaling pathway, is a hallmark of the mild phenotype. Functional analyses demonstrate that Jagged1 overexpression ameliorates the dystrophic phenotype, suggesting that Jagged1 may represent a target for DMD therapy in a dystrophin-independent manner. PAPERCLIP.
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http://dx.doi.org/10.1016/j.cell.2015.10.049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4668935PMC
November 2015

The Pathogenesis and Therapy of Muscular Dystrophies.

Annu Rev Genomics Hum Genet 2015 4;16:281-308. Epub 2015 Jun 4.

Medical Research Council Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, OX1 3PT Oxford, United Kingdom; email: ,

Current molecular genomic approaches to human genetic disorders have led to an explosion in the identification of the genes and their encoded proteins responsible for these disorders. The identification of the gene altered by mutations in Duchenne and Becker muscular dystrophy was one of the earliest examples of this paradigm. The nearly 30 years of research partly outlined here exemplifies the road that similar current gene discovery protocols will be expected to travel, albeit much more rapidly owing to improved diagnosis of genetic disorders and an understanding of the spectrum of mutations thought to cause them. The identification of the protein dystrophin has led to a new understanding of the muscle cell membrane and the proteins involved in membrane stability, as well as new candidate genes for additional forms of muscular dystrophy. Animal models identified with naturally occurring mutations and developed by genetic manipulation have furthered the understanding of disease progression and underlying pathology. The biochemistry and molecular analysis of patient samples have led to the different dystrophin-dependent and -independent therapies that are currently close to or in human clinical trials. The lessons learned from decades of research on dystrophin have benefited the field of human genetics.
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http://dx.doi.org/10.1146/annurev-genom-090314-025003DOI Listing
May 2016

Muscular dystrophy in a family of Labrador Retrievers with no muscle dystrophin and a mild phenotype.

Neuromuscul Disord 2015 May 3;25(5):363-70. Epub 2015 Mar 3.

Department of Pathology, School of Medicine, University of California San Diego, La Jolla, CA 92093. Electronic address:

Animal models of dystrophin deficient muscular dystrophy, most notably canine X-linked muscular dystrophy, play an important role in developing new therapies for human Duchenne muscular dystrophy. Although the canine disease is a model of the human disease, the variable severity of clinical presentations in the canine may be problematic for pre-clinical trials, but also informative. Here we describe a family of Labrador Retrievers with three generations of male dogs having markedly increased serum creatine kinase activity, absence of membrane dystrophin, but with undetectable clinical signs of muscle weakness. Clinically normal young male Labrador Retriever puppies were evaluated prior to surgical neuter by screening laboratory blood work, including serum creatine kinase activity. Serum creatine kinase activities were markedly increased in the absence of clinical signs of muscle weakness. Evaluation of muscle biopsies confirmed a dystrophic phenotype with both degeneration and regeneration. Further evaluations by immunofluorescence and western blot analysis confirmed the absence of muscle dystrophin. Although dystrophin was not identified in the muscles, we did not find any detectable deletions or duplications in the dystrophin gene. Sequencing is now ongoing to search for point mutations. Our findings in this family of Labrador Retriever dogs lend support to the hypothesis that, in exceptional situations, muscle with no dystrophin may be functional. Unlocking the secrets that protect these dogs from a severe clinical myopathy is a great challenge which may have important implications for future treatment of human muscular dystrophies.
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http://dx.doi.org/10.1016/j.nmd.2015.02.012DOI Listing
May 2015

Emerging preclinical animal models for FSHD.

Trends Mol Med 2015 May 20;21(5):295-306. Epub 2015 Mar 20.

Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Children's Hospital, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; The Wellstone Program, Departments of Neurology and Cell and Developmental Biology, University of Massachusetts Medical School (UMMS), Worcester, MA 01655, USA.

Facioscapulohumeral dystrophy (FSHD) is a unique and complex genetic disease that is not entirely solved. Recent advances in the field have led to a consensus genetic premise for the disorder, enabling researchers to now pursue the design of preclinical models. In this review we explore all available FSHD models (DUX4-dependent and -independent) for their utility in therapeutic discovery and potential to yield novel disease insights. Owing to the complex nature of FSHD, there is currently no single model that accurately recapitulates the genetic and pathophysiological spectrum of the disorder. Existing models emphasize only specific aspects of the disease, highlighting the need for more collaborative research and novel paradigms to advance the translational research space of FSHD.
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http://dx.doi.org/10.1016/j.molmed.2015.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424175PMC
May 2015

Skeletal Muscle MicroRNAs: Their Diagnostic and Therapeutic Potential in Human Muscle Diseases.

J Neuromuscul Dis 2015;2(1):1-11

Division of Genetics and Genomics at Boston Children's Hospital, Boston, MA, USA; Department of Pediatrics and Genetics at Harvard Medical School, Boston, MA, USA; The Stem Cell Program at Boston Children's Hospital, Boston, MA, USA; The Manton Center for Orphan Disease Research at Boston Children's Hospital, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA.

MicroRNAs (miRNAs) are small 21-24 nucleotide RNAs that are capable of regulating multiple signaling pathways across multiple tissues. MicroRNAs are dynamically regulated and change in expression levels during periods of early development, tissue regeneration, cancer, and various other disease states. Recently, microRNAs have been isolated from whole serum and muscle biopsies to identify unique diagnostic signatures for specific neuromuscular disease states. Functional studies of microRNAs in cell lines and animal models of neuromuscular diseases have elucidated their importance in contributing to neuromuscular disease progression and pathologies. The ability of microRNAs to alter the expression of an entire signaling pathway opens up their unique ability to be used as potential therapeutic entry points for the treatment of disease. Here, we will review the recent findings of key microRNAs and their dysregulation in various neuromuscular diseases. Additionally, we will highlight the current strategies being used to regulate the expression of key microRNAs as they have become important players in the clinical treatment of some of the neuromuscular diseases.
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http://dx.doi.org/10.3233/JND-140058DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4988517PMC
January 2015

Dystrophin is a tumor suppressor in human cancers with myogenic programs.

Nat Genet 2014 Jun 4;46(6):601-6. Epub 2014 May 4.

Department of Pathology, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts, USA.

Many common human mesenchymal tumors, including gastrointestinal stromal tumor (GIST), rhabdomyosarcoma (RMS) and leiomyosarcoma (LMS), feature myogenic differentiation. Here we report that intragenic deletion of the dystrophin-encoding and muscular dystrophy-associated DMD gene is a frequent mechanism by which myogenic tumors progress to high-grade, lethal sarcomas. Dystrophin is expressed in the non-neoplastic and benign counterparts of GIST, RMS and LMS tumors, and DMD deletions inactivate larger dystrophin isoforms, including 427-kDa dystrophin, while preserving the expression of an essential 71-kDa isoform. Dystrophin inhibits myogenic sarcoma cell migration, invasion, anchorage independence and invadopodia formation, and dystrophin inactivation was found in 96%, 100% and 62% of metastatic GIST, embryonal RMS and LMS samples, respectively. These findings validate dystrophin as a tumor suppressor and likely anti-metastatic factor, suggesting that therapies in development for muscular dystrophies may also have relevance in the treatment of cancer.
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http://dx.doi.org/10.1038/ng.2974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4225780PMC
June 2014

MicroRNA-486-dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy-associated symptoms.

J Clin Invest 2014 Jun 1;124(6):2651-67. Epub 2014 May 1.

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle-specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.
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http://dx.doi.org/10.1172/JCI73579DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4038577PMC
June 2014

Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.

FASEB J 2014 Jul 31;28(7):2955-69. Epub 2014 Mar 31.

Program in Genomics, Division of Genetics and Harvard Medical School, Boston, Massachusetts, USA

Previously, we identified family with sequence similarity 65, member B (Fam65b), as a protein transiently up-regulated during differentiation and fusion of human myogenic cells. Silencing of Fam65b expression results in severe reduction of myogenin expression and consequent lack of myoblast fusion. The molecular function of Fam65b and whether misregulation of its expression could be causative of muscle diseases are unknown. Protein pulldowns were used to identify Fam65b-interacting proteins in differentiating human muscle cells and regenerating muscle tissue. In vitro, human muscle cells were treated with histone-deacetylase (HDAC) inhibitors, and expression of Fam65b and interacting proteins was studied. Nontreated cells were used as controls. In vivo, expression of Fam65b was down-regulated in developing zebrafish to determine the effects on muscle development. Fam65b binds to HDAC6 and dysferlin, the protein mutated in limb girdle muscular dystrophy 2B. The tricomplex Fam65b-HDAC6-dysferlin is transient, and Fam65b expression is necessary for the complex to form. Treatment of myogenic cells with pan-HDAC or HDAC6-specific inhibitors alters Fam65b expression, while dysferlin expression does not change. Inhibition of Fam65b expression in developing zebrafish results in abnormal muscle, with low birefringence, tears at the myosepta, and increased embryo lethality. Fam65b is an essential component of the HDAC6-dysferlin complex. Down-regulation of Fam65b in developing muscle causes changes consistent with muscle disease.-Balasubramanian, A., Kawahara, G., Gupta, V. A., Rozkalne, A., Beauvais, A., Kunkel, L. M., Gussoni, E. Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation.
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http://dx.doi.org/10.1096/fj.13-246470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4062822PMC
July 2014