Primary inherited disorders of muscle include both dystrophies and non-dystrophic myopathies. These conditions, characterized by muscle weakness and impaired locomotion, form a group of heredity diseases affecting both children and adults. Complexity due to genetic, phenotypic and clinical heterogeneity poses a great challenge in identifying causative genes underlying muscle disorders. Furthermore, even in instances where a defective gene has been identified, that knowledge has often not translated into development of specific and effective therapies for the relevant neuromuscular disorders. Suitable animal models are needed to better understand the pathophysiology, and to provide high throughput assay systems to identify potentially therapeutic drugs. Therefore, to identify novel genes involved in neuromuscular disorders, the applicant performed a forward genetics screen in a vertebrate animal model zebrafish (Danio rerio) and identified 13 unique mutants with defective skeletal muscle. This K01 proposal is aimed at supporting the career development of Dr. Vandana Gupta as she explores the cellular processes and molecular mechanisms associated with skeletal muscle growth and diseases, by studying a zebrafish mutant, osoi. Genetic mapping in osoi fish identified a 20 base pair deletion in a novel DEAD-box RNA helicase, a RNA binding protein, resulting in a frameshift mutation. Dr. Gupta's preliminary work firmly establishes mutation in this RNA helicase as a cause of skeletal muscle hypotrophy. The pathological findings identified smaller size myofibers with sarcomeric disorganization associated with central nuclei. The central nucleation is reminiscent of centronuclear myopathy, a form of human congenital myopathy and myotonic dystrophies, and myofiber hypotrophy in general is a central feature of congenital myopathies and several forms of dystrophies. In skeletal muscle diseases, strong efforts are currently being devoted to develop therapies aimed at increasing myofiber size. However, lack of suitable targets has been a major hindrance in development of successful treatments. DEAD-box RNA helicases are involved in all cellular processes involving RNA, from transcription, mRNA splicing, translation, RNA modification, transport, ribosome biogenesis, miRNA biosynthesis, RNA/protein complex assembly and RNA degradation. Dr. Gupta's preliminary data identifies a ribosomal defect in osoi mutant and puts forward a novel path to investigate ribosomal regulation in muscle growth and diseases. Through better understanding of the molecular pathways in skeletal muscle hypotrophy, the applicant hopes to begin development of corrective therapies for such muscle defects.
Specific Aim 1 will investigate the phenotypic and pathological changes associated with osoi loci in vivo and in vitro. Findings from this will be applied to study the genetic basis of skeletal muscle diseases in patients with similar muscle defects.
Specific Aim 2 will explore the ribosomal biogenesis defects observed in osoi fish employing biochemical approaches.
This aim will further investigate the structure-functional relationship of different conserved helicase motis in ribosomal biogenesis and skeletal muscle hypotrophy.
Specific Aim 3 will explore the molecular functions of osoi loci by identifying RNA targets bound to this RNA helicase in vivo using molecular and system biology methods. The in vivo functional significance of targets identified in this aim will be tested by knock-downs using morpholino technology in zebrafish. Dr. Gupta's long term aim as an independent investigator is to study the genetic causes of human neuromuscular disorders and their molecular basis to devise treatment strategies using model organisms. Her research position in Beggs Laboratory at Children's Hospital Boston provides an ideal opportunity for her training, with the lab's long standing interest and experience in studying genetic causes and treatments of muscle disorders, excellent mentorship, available resources and collaborative opportunities within Children's Hospital and the Harvard Medical School community. The support provided with K01 award will help her to complete her training and with provide her with a launch pad to obtain data for a successful R01 application to fund her future work.

Public Health Relevance

Primary inherited disorders of skeletal muscles constitute the largest genetic group of diseases affecting children and adults, for which no treatment is available yet. The goal of this proposal is to study the role of a novel RNA helicase in identifyin cellular processes and molecular pathways crucial for skeletal muscle growth and diseases. This knowledge gained from this work will significantly contribute towards designing treatments for muscle disorders

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Scientist Development Award - Research & Training (K01)
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Arthritis and Musculoskeletal and Skin Diseases Special Grants Review Committee (AMS)
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Boyce, Amanda T
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Children's Hospital Boston
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Winter, Josine M de; Joureau, Barbara; Lee, Eun-Jeong et al. (2016) Mutation-specific effects on thin filament length in thin filament myopathy. Ann Neurol 79:959-69
Shamseldin, Hanan E; Smith, Laura L; Kentab, Amal et al. (2016) Mutation of the mitochondrial carrier SLC25A42 causes a novel form of mitochondrial myopathy in humans. Hum Genet 135:21-30
Shamseldin, Hanan E; Bennett, Alexis H; Alfadhel, Majid et al. (2016) GOLGA2, encoding a master regulator of golgi apparatus, is mutated in a patient with a neuromuscular disorder. Hum Genet 135:245-51
Smith, Laura L; Gupta, Vandana A; Beggs, Alan H (2014) Bridging integrator 1 (Bin1) deficiency in zebrafish results in centronuclear myopathy. Hum Mol Genet 23:3566-78
Di Costanzo, Stefania; Balasubramanian, Anuradha; Pond, Heather L et al. (2014) POMK mutations disrupt muscle development leading to a spectrum of neuromuscular presentations. Hum Mol Genet 23:5781-92
Gupta, Vandana A; Beggs, Alan H (2014) Kelch proteins: emerging roles in skeletal muscle development and diseases. Skelet Muscle 4:11
Patel, Nisha; Smith, Laura L; Faqeih, Eissa et al. (2014) ZBTB42 mutation defines a novel lethal congenital contracture syndrome (LCCS6). Hum Mol Genet 23:6584-93
Yuen, Michaela; Sandaradura, Sarah A; Dowling, James J et al. (2014) Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy. J Clin Invest 124:4693-708
Balasubramanian, Anuradha; Kawahara, Genri; Gupta, Vandana A et al. (2014) Fam65b is important for formation of the HDAC6-dysferlin protein complex during myogenic cell differentiation. FASEB J 28:2955-69
Gupta, Vandana A; Ravenscroft, Gianina; Shaheen, Ranad et al. (2013) Identification of KLHL41 Mutations Implicates BTB-Kelch-Mediated Ubiquitination as an Alternate Pathway to Myofibrillar Disruption in Nemaline Myopathy. Am J Hum Genet 93:1108-17

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