Original article Mutations in JPH2-encoded junctophilin-2 associated with hypertrophic cardiomyopathy in humans Andrew P. Landstrom a,d,1 , Noah Weisleder b,1 , Karin B. Batalden a , J. Martijn Bos c , David J. Tester d , Steve R. Ommen a,c , Xander H.T. Wehrens e , William C. Claycomb f , Jae-Kyun Ko b , Moonsun Hwang b , Zui Pan b , Jianjie Ma b , Michael J. Ackerman a,c,d,g, ⁎ a Mayo Medical School, Mayo Clinic College of Medicine, Rochester, MN, USA b Department of Physiology and Biophysics, Robert Wood Johnson Medical School, Piscataway, NJ, USA c Department of Internal Medicine, Division of Cardiovascular Diseases, Mayo Clinic College of Medicine, Rochester, MN, USA d Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic College of Medicine, Rochester, MN, USA e Department of Molecular Physiology and Biophysics, and Medicine (Cardiology), Baylor College of Medicine, Houston, TX, USA f Department of Biochemistry and Molecular Biology, Louisiana State University Health Sciences Center, New Orleans, LA, USA g Department of Pediatrics, Division of Pediatric Cardiology, Mayo Clinic College of Medicine, Rochester, MN, USA Received 10 January 2007; received in revised form 9 April 2007; accepted 11 April 2007 Available online 18 April 2007 Abstract Junctophilin-2 (JPH2) is a cardiac specific member of the junctophilins, a newly characterized family of junctional membrane complex proteins important in physically approximating the plasmalemmal L-type calcium channel and the sarcoplasmic reticulum ryanodine receptor for calcium-induced calcium release. JPH2 knockout mice showed disrupted calcium transients, altered junctional membrane complex formation, cardiomyopathy, and embryonic lethality. Furthermore, JPH2 gene expression is down-regulated in murine cardiomyopathy models. To this end, we explored JPH2 as a novel candidate gene for the pathogenesis of hypertrophic cardiomyopathy (HCM) in humans. Using polymerase chain reaction, denaturing high performance liquid chromatography, and direct DNA sequencing, comprehensive open reading frame/splice site mutational analysis of JPH2 was performed on DNA obtained from 388 unrelated patients with HCM. HCM-associated JPH2 mutations were engineered and functionally characterized using immunocytochemistry, cell morphometry measurements, and live cell confocal calcium imaging. Three novel HCM-susceptibility mutations: S101R, Y141H and S165F, which localize to key functional domains, were discovered in 3/388 unrelated patients with HCM and were absent in 1000 ethnic-matched reference alleles. Functionally, each human mutation caused (i) protein reorganization of junctophilin-2, (ii) perturbations in intracellular calcium signaling, and (iii) marked cardiomyocyte hyperplasia. The molecular and functional evidence implicates defective junctophilin-2 and disrupted calcium signaling as a novel pathogenic mechanism for HCM and establishes HCM as the first human disease associated with genetic defects in JPH2. Whether susceptibility for other cardiomyopathies, such as dilated cardiomyopathy, can be conferred by mutations in JPH2 warrants investigation. © 2007 Elsevier Inc. All rights reserved. Keywords: Genetics; Calcium; Cardiomyopathy; Hypertrophy; Junctophilin; JPH2 1. Introduction Hypertrophic cardiomyopathy (HCM) is a genotypically and phenotypically heterogeneous disorder. Clinically defined as cardiac hypertrophy in the absence of extrinsic causes such as hypertension and valvular disease, HCM affects approximately 1 in 500 persons and is one of the most common causes of sudden cardiac death in young athletes [1,2]. To date, hundreds Journal of Molecular and Cellular Cardiology 42 (2007) 1026 – 1035 www.elsevier.com/locate/yjmcc Abbreviations: CICR, calcium-induced calcium release; DHPLC, denatur- ing high performance liquid chromatography; HCM, hypertrophic cardiomyo- pathy; JPH2, junctophilin-2; MORN, membrane occupation and recognition nexus; SR, sarcoplasmic reticulum. ⁎ Corresponding author. Windland Smith Rice Sudden Death Genomics Laboratory, Guggenheim 501, Mayo Clinic, Rochester, MN 55905, USA. Tel.: +1 507 284 0101; fax: +1 507 284 3757. E-mail address: ackerman.michael@mayo.edu (M.J. Ackerman). 1 APL and NW contributed equally and are co-equal first authors. 0022-2828/$ - see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.yjmcc.2007.04.006