Genetic Analysis and New Gene Discovery in Nemaline Myopathy. V. A. GUPTA1, G. RAVENSCROFT2, R. SHAHEEN3, E. J. TODD2, L. C. SWANSON1, M. SHIINA4, K. OGATA4, C. HSU1, N. F. CLARKE6, B. T. DARRAS7, M. FARRAR8, A. HASHEM3, N. MANTON2, F. MUNTONI9, K. N. NORTH10, S. SANDARADURA8, I. NISHINO11, Y. K. HAYASHI11, C. A. SEWRY9, E. THOMPSON2, T. W. YU1, C. A. BROWNSTEIN1, R. ALLCOCK2, M. R. DAVIS2, C. WALLGREN-PETTERSSON12, N. MATSUMOTO5, F. S. ALKURAYA3, N. G. LAING2, A. H. BEGGS1 1) Genomics Program and Division of Genetics, The Manton Center for Orphan Disease Research, Boston Children's Hospital, Harvard Medical School, Boston, MA 02115, USA; 2) Western Australian Institute for Medical Research and the Centre for Medical Research, University of Western Australia, Nedlands, Western Australia; 3) Developmental Genetics Unit, King Faisal Specialist Hospital and Research Center, Riyad 11211, Saudi Arabia; 4) Department of Biochemistry, Yokohama City University, Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; 5) Department of Human Genetics, Yokohama City University, Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Japan; 6) Institute for Neuroscience and Muscle Research, Childrens Hospital at Westmead, Sydney, NSW2145, Australia; 7) Department of Neurology, Boston Childrens Hospital, Harvard Medical School, Boston, MA 02115, USA; 8) Department of Neurology, Sydney Childrens Hospital and School of Womens and Children Health, University of South Wales, Australia; 9) Dubowitz Neuromuscular Centre, Institute of Child Health and Great Ormond Street Hospital, London WC1N 1EH, UK; 10) Murdoch Childrens Research Institute, The Royal Childrens Hospital, Flemington Road Parkville Victoria 3052, Australia; 11) 11Department of Neuromuscular Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo 187-8502, Japan; 12) The Folkhälsan Institute of Genetics and Department of Medical Genetics, Haartman Institute, University of Helsinki, Biomedicum Helsinki, Finland.

   Nemaline myopathy (NM) is one of the most common congenital myopathies and is characterized by generalized skeletal muscle weakness and the presence of nemaline bodies in affected myofibers. Clinically, the NMs form a heterogeneous group of myopathies ranging from fetal akinesia, through the severe congenital forms with death from respiratory failure during first year of life, to a mild-childhood onset myopathy with survival into adulthood. Substantial progress has been made in understanding the genetic basis of these conditions. NMs with known genetic cause include mutations in ACTA1, NEB, TPM3, TPM2, CFL2, TNNT1 and KBTBD13 and the most recently identified, KLHL40. Mutations in these genes account for only ~ 60% of all genetically identified cases. To identify mutations in cases with unknown genetic cause, we performed next generation sequencing on 60 NM patients. Data analysis and subsequent validation by Sanger sequencing of this cohort identified novel mutations in known genes associated with NM as well as novel disease genes. We identified 26 NEB, 3 ACTA1, 1 TPM3 and 1 TNNT1 mutation in the 60 patients. WES also resulted in the identification of a new disease locus, KLHL41, in five families affected with NM. Kelch-like protein 41 (KLHL41) belongs to the family of Kelch and BTB domain containing proteins that includes two other NM loci, KLHL40 and KBTBD13. Mutations in KLHL41 showed a clear phenotype-genotype correlation. Small deletions resulted in severe phenotypes with neonatal death, whereas missense changes resulted in an impaired motor function with survival in to late childhood and/or early adulthood. Functional studies in zebrafish showed that loss of klhl41 results in highly diminished motor function and myofibrillar disorganization with thickened Z-lines and nemaline body formation, the pathological hallmark of NM. The BTB domain containing Kelch family members interact with Cul3 ubiquitin ligase to form a functional ubiquitination complex. The interaction of KLHL40, KLHL41 and KBTBD13 with E3 ubiquitin ligases implicates the involvement of Kelch proteins mediated ubiquitination pathway in disease pathology in NM. Further studies on the mechanism of Kelch protein regulated pathways will allow a clearer understanding of NM and may assist in future therapeutics developments.

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