REPORT SPEG Interacts with Myotubularin, and Its Deficiency Causes Centronuclear Myopathy with Dilated Cardiomyopathy Pankaj B. Agrawal, 1,2,3, * Christopher R. Pierson, 4 Mugdha Joshi, 1,3 Xiaoli Liu, 5 Gianina Ravenscroft, 6 Behzad Moghadaszadeh, 1,3 Tiffany Talabere, 7 Marissa Viola, 1 Lindsay C. Swanson, 1,3 Go ¨knur Halilo glu, 8 Beril Talim, 9 Kyle S. Yau, 6 Richard J.N. Allcock, 10 Nigel G. Laing, 6 Mark A. Perrella, 5,11 and Alan H. Beggs 1,3, * Centronuclear myopathies (CNMs) are characterized by muscle weakness and increased numbers of central nuclei within myofibers. X-linked myotubular myopathy, the most common severe form of CNM, is caused by mutations in MTM1, encoding myotubularin (MTM1), a lipid phosphatase. To increase our understanding of MTM1 function, we conducted a yeast two-hybrid screen to identify MTM1-interacting proteins. Striated muscle preferentially expressed protein kinase (SPEG), the product of SPEG complex locus (SPEG), was identified as an MTM1-interacting protein, confirmed by immunoprecipitation and immunofluorescence studies. SPEG knockout has been previously associated with severe dilated cardiomyopathy in a mouse model. Using whole-exome sequencing, we identified three unrelated CNM-affected probands, including two with documented dilated cardiomyopathy, carrying homozygous or compound-heterozygous SPEG mutations. SPEG was markedly reduced or absent in two individuals whose muscle was available for immunofluorescence and immunoblot studies. Examination of muscle samples from Speg-knockout mice revealed an increased frequency of central nuclei, as seen in human subjects. SPEG localizes in a double line, flanking desmin over the Z lines, and is apparently in alignment with the terminal cisternae of the sarcoplasmic reticulum. Examination of human and murine MTM1-deficient muscles revealed similar abnormalities in staining patterns for both desmin and SPEG. Our results suggest that mutations in SPEG, encoding SPEG, cause a CNM phenotype as a result of its interaction with MTM1. SPEG is present in cardiac muscle, where it plays a critical role; therefore, individuals with SPEG mutations additionally present with dilated cardiomyopathy. Congenital myopathies manifest early in life with varying degrees of skeletal-muscle dysfunction and hypotonia and are subclassified on the basis of histopathological find- ings. 1 Centronuclear myopathies (CNMs) are a common subtype characterized by a large number of myofibers with central nuclei in the absence of other diagnostic fea- tures. 1–3 Mutations in MTM1 (MIM 300415; encoding myotubularin [MTM1]), DNM2 (MIM 602378; encoding dynamin 2), BIN1 (MIM 601248; encoding bridging integrator 1), RYR1 (MIM 180901; encoding ryanodine re- ceptor 1), and TTN (MIM 188840; encoding titin) are associated with 60%–80% of CNM cases; the rest have an unknown genetic basis. 4–8 Alterations in proteins encoded by these genes, except for TTN, are postulated to affect the assembly or function of triads, the specialized membrane structures responsible for excitation-contraction (E-C) coupling. 6,8–12 X-linked centronuclear myopathy (MIM 310400), a severe and the most common type of CNM, is characterized by congenital onset in boys and is associated with MTM1 mutations. MTM1 encodes MTM1, a lipid phosphatase predominantly located at the junctional sarcoplasmic reticulum (SR) of the muscle triads. This pro- tein has a critical function in SR remodeling and promo- tion of SR membrane curvature and is hence essential for calcium homeostasis and E-C coupling. 9,11,13 To better understand the molecular mechanisms of MTM1 function, we used a yeast two-hybrid (Y2H) screen, performed by Hybrigenics Services (Paris), to identify MTM1-interacting proteins in a human adult and fetal skeletal muscle library. Using full-length MTM1 as the bait, we detected striated muscle preferentially expressed protein kinase (SPEG) as an MTM1-interacting partner by isolating six different overlapping clones encoding por- tions of SPEG (Figure 1A). Aligning the sequences revealed that MTM1 interacts with a region encompassing SPEG amino acids 2,530–2,674, which includes one entire Ig- like domain and ends just proximal to the second fibro- nectin type III domain. To identify MTM1 regions that interact with SPEG, we tested a peptide encompassing res- idues 2,530–2,674 of human SPEG for interaction with 1 Division of Genetics and Genomics, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA; 2 Division of Newborn Medicine, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA; 3 Manton Center for Orphan Disease Research, Boston Children’s Hospital and Harvard Medical School, Boston, MA 02115, USA; 4 Department of Pathology and Laboratory Medicine, Nationwide Children’s Hospital and the Ohio State University College of Medicine, Columbus, OH 43205, USA; 5 Department of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Boston MA 02115, USA; 6 Harry Perkins Institute of Medical Research and the Centre for Medical Research, University of Western Australia, Nedlands, WA 6009, Australia; 7 Research Institute, Nationwide Children’s Hospital and the Ohio State University College of Medicine, Columbus, OH 43205, USA; 8 Neurology Unit, Department of Pediatrics, Hacettepe University Children’s Hospital, Ankara 06100, Turkey; 9 Pathology Unit, Department of Pediatrics, Hacettepe University Children’s Hospital, Ankara 06100, Turkey; 10 Lotterywest State Biomedical Facility Genomics and School of Pathology and Laboratory Medicine, University of Western Australia, Perth, WA 6009, Australia; 11 Department of Newborn Medicine, Brigham and Women’s Hospital, Boston MA 02115, USA *Correspondence: pagrawal@enders.tch.harvard.edu (P.B.A.), beggs@enders.tch.harvard.edu (A.H.B.) http://dx.doi.org/10.1016/j.ajhg.2014.07.004. Ó2014 by The American Society of Human Genetics. All rights reserved. 218 The American Journal of Human Genetics 95, 218–226, August 7, 2014