title: Dystrophinopathy carrier determination and detection of protein deficiencies in muscular dystrophy using lentiviral MyoD-forced myogenesis.
abstract: The objective of this study is to expand the applications of MyoD-forced myogenesis for research and diagnosis of human muscle disorders using a lentiviral vector (LVhMyoD) for efficient trans-differentiation of patient primary cells. LVhMyoD transduced cells readily formed striated, multinucleate myotubes expressing a wide range of genes associated with muscular dystrophy (dystrophin, dysferlin, sarcoglycans, caveolin-3) and congenital myopathy (nebulin, actin, desmin, tropomyosin, troponin). We demonstrate that MyoD gene-modified fibroblasts reproduce protein deficiencies associated with different forms of muscular dystrophy, and confirm that LVhMyoD gene-modified chorionic villus can be used successfully to determine the dystrophin status of the developing fetus, augmenting prenatal diagnosis of dystrophinopathy patients. Using muscle-specific cDNA derived from LVhMyoD gene-modified patient cells, we identified a female carrier bearing a large dystrophin deletion and a previously unidentified non-coding splice-site mutation within dystrophin in a Becker muscular dystrophy patient. This study highlights the significant potential of lentiviral MyoD-forced myogenesis for study of a wide range of human muscle disorders; a field constrained by the limited availability of human tissue. LVhMyoD gene-modified patient cells provide a renewable source of mutant protein and muscle-specific mRNA, facilitating accelerated mutation screening of large genes, molecular analyses of splicing abnormalities and study of disease-causing mutations.
authors.affiliation: Neurogenetics Research Unit and Institute for Neuromuscular Research, The Children's Hospital at Westmead, Locked Bag 4001, Sydney, NSW 2145, Australia. SandraC3@chw.edu.au
authors.forename: Sandra T
authors.initials: ST
authors.lastname: Cooper
authors.forename: Eddy
authors.initials: E
authors.lastname: Kizana
authors.forename: Jonathon D
authors.initials: JD
authors.lastname: Yates
authors.forename: Harriet P
authors.initials: HP
authors.lastname: Lo
authors.forename: Nan
authors.initials: N
authors.lastname: Yang
authors.forename: Zhan He
authors.initials: ZH
authors.lastname: Wu
authors.forename: Ian E
authors.initials: IE
authors.lastname: Alexander
authors.forename: Kathryn N
authors.initials: KN
authors.lastname: North
mesh_terms: D000185:Actinin / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D002824:Chorionic Villi / Q000473:pathology; D004252:DNA Mutational Analysis; D016189:Dystrophin / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000473:pathology; D006801:Humans; D016086:Lentivirus / Q000502:physiology*; D008297:Male; D024510:Muscle Development / Q000502:physiology; D009136:Muscular Dystrophies / Q000473:pathology*; D009154:Mutation; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D011488:Protein Deficiency / Q000378:metabolism*; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods
chemical_list: D016189:Dystrophin; D017570:MyoD Protein; D012333:RNA, Messenger; D000185:Actinin