Gain-of-function mutations in the mechanically activated ion channel PIEZO2 cause a subtype of Distal Arthrogryposis Bertrand Costea,1,2, Gunnar Hougeb,c,2, Michael F. Murrayd,2, Nathan Stitzield,3, Michael Bandelle, Monica A. Giovannid, Anthony Philippakisd, Alexander Hoischenb,f, Gunnar Riemerg, Unni Steeng, Vidar Martin Steenb,c, Jayanti Mathure, James Coxh, Matthew Leboi, Heidi Rehmi, Scott T. Weissi, John N. Woodh, Richard L. Maasd, Shamil R. Sunyaevd,4, and Ardem Patapoutiana,e,4 aMolecular and Cellular Neuroscience Department, Dorris Neuroscience Center, The Scripps Research Institute, La Jolla, CA 92037; bCenter for Medical Genetics and Molecular Medicine, Haukeland University Hospital, 5021 Bergen, Norway; cDepartment of Clinical Medicine, University of Bergen, 5021 Bergen, Norway; dDivision of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, MA 02115; eGenomics Institute of the Novartis Research Foundation, San Diego, CA 92121; fDepartment of Human Genetics, Nijmegen Centre for Molecular Life Sciences and Institute for Genetic and Metabolic Disorders, Radboud University Nijmegen Medical Centre, 6525 Nijmegen, The Netherlands; gTRS National Resource Centre for Rare Disorders, Sunnaas Rehabilitation Hospital, 1450 Nesoddtangen, Norway; hMolecular Nociception Group, University College London, London WC1E 6BT, United Kingdom; and iPartners Center for Personalized Genetic Medicine, Partners HealthCare, Boston, MA 02115 Edited by Ramon Latorre, Centro Interdisciplinario de Neurociencias, Universidad de Valparaíso, Valparaíso, Chile, and approved February 6, 2013 (received for review December 11, 2012) Mechanotransduction, the pathway by which mechanical forces enzyme-like 1 (ECEL1) have been shown to be responsible for are translated to biological signals, plays important but poorly a specific, recessively inherited subtype of DA (10, 11). Mean- characterized roles in physiology. PIEZOs are recently identified, while, other types of DAs have been shown to involve mutations widely expressed, mechanically activated ion channels that are in the myosin heavy chain genes MYH3 and MYH8, in the my- hypothesized to play a role in mechanotransduction in mammals. osin-binding protein C gene, MYBPC1, and in the genes TNNI2, Here, we describe two distinct PIEZO2 mutations in patients with TNNT3 and TPM2 that encode the muscle regulatory proteins GENETICS a subtype of Distal Arthrogryposis Type 5 characterized by gener- troponin I, troponin T and beta-tropomyosin, respectively (2, 12– alized autosomal dominant contractures with limited eye move- 14). Here, we show that a subtype of DA5 that includes restric- ments, restrictive lung disease, and variable absence of cruciate tive pulmonary disease is caused by gain-of-function mutations in knee ligaments. Electrophysiological studies reveal that the two the mechanically activated (MA) cation channel PIEZO2 (15). PIEZO2 mutations affect biophysical properties related to channel inactivation: both E2727del and I802F mutations cause the PIEZO2- Results dependent, mechanically activated currents to recover faster from Clinical Assessment. To identify the genetic cause of DA5, we inactivation, while E2727del also causes a slowing of inactivation. ascertained two affected unrelated kindreds for next generation Both types of changes in kinetics result in increased channel activity DNA sequencing. In the first kindred, the index case (individual 1) in response to a given mechanical stimulus, suggesting that Distal presented with DA5 and subsequently gave birth to a son with left Arthrogryposis Type 5 can be caused by gain-of-function mutations talipes equinovarus (club foot) (individual 2; Fig. 1A). Both mother in PIEZO2. We further show that overexpression of mutated PIEZO2 and son exhibited generalized arthrogryposis, deep-set hyperme- cDNAs does not cause constitutive activity or toxicity to cells, indi- tropic eyes, ptosis and ophthalmoplegia, and restricted movements cating that the observed phenotype is likely due to a mechanotrans- of the jaw, neck and spine, thorax and lungs, and large and small duction defect. Our studies identify a type of channelopathy and link joints of the extremities (Table 1, Fig. 1 A and B). The contractures the dysfunction of mechanically activated ion channels to develop- respond to physiotherapy, but severe contractures remain despite mental malformations and joint contractures. such treatment. During childhood, the mother was operated on for hypertrophic, constricted neck muscles, and in adulthood, the congenital contractures | neuromuscular system | whole exome mother developed significant musculoskeletal pain resulting in sequencing | whole genome sequencing disability. However, muscle strength and structure were normal on clinical investigation, muscle biopsy, and computed tomography rthrogryposis, or multiple congenital contractures, is a char- (CT) scan. Both mother and son exhibited shortness of breath Aacteristic of over 300 different disorders of highly variable upon exercise, and the mother had pulmonary function tests of etiology (1–3). Such malformations can be secondary to envi- [forced expiratory capacity in 1 second (% normal value) / forced ronmental factors such as decreased intrauterine movement or result from disorders of neurological, muscle, or connective tissue development. Some of these neuromuscular and connective tissue Author contributions: B.C., G.H., M.F.M., R.L.M., S.R.S., and A. Patapoutian designed re- disorders are classified as distal arthrogryposes (DAs). Currently, search; B.C., G.H., M.F.M., N.S., M.B., M.A.G., A. Philippakis, A.H., G.R., U.S., V.M.S., J.M., M.L., H.R., and S.T.W. performed research; J.C. and J.N.W. contributed new reagents/ DAs are subdivided into 10 types, depending on the number and analytic tools; and B.C., G.H., R.L.M., S.R.S., and A. Patapoutian wrote the paper. nature of additional features (2, 4). Of these, DA Type 5 (DA5) The authors declare no conflict of interest. [online Mendelian inheritance in man (OMIM) 108145] has been This article is a PNAS Direct Submission. described in the literature for more than 70 y and can be further 1Present address: Ion Channels and Signaling Group, Centre de Recherche en Neurobiologie- divided into several subtypes based on additional phenotypic Neurophysiologie de Marseille, 13015 Marseille, France. – features (1, 2, 4 9). DA5 itself is an autosomal dominant multi- 2B.C., G.H., and M.F.M. contributed equally to this work. system disorder characterized by multiple distal contractures, 3Present address: Cardiovascular Division, Department of Medicine, Washington Univer- characteristic facies, ophthalmoplegia with ptosis, (2), and in sity School of Medicine, St. Louis, MO 63110. some cases restrictive lung disease with pulmonary hypertension 4To whom correspondence may be addressed. E-mail: [email protected] or ssunyaev@ (4, 9). Although DA5 is often dominantly inherited and several rics.bwh.harvard.edu. large families have been described (4, 5, 9), molecular insight is This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. lacking. Recently, loss-of-function mutations in endothelin converting 1073/pnas.1221400110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1221400110 PNAS Early Edition | 1of6 Downloaded by guest on October 1, 2021 Fig. 1. Dysmorphic features of DA5 individuals. (A) Infant photographs of individual 2 showing deep-set small eyes with mild ptosis, restricted shoulder ab- duction, and flexion contractures in the knees. Deep skin dimples can be seen on the shoulder (red circle) and sternum. (B) The DA of individual 2, 3, and 1 (from top to bottom). Note flexion contractures of the interphalangeal joints, increasing in severity from thumb to little finger. (C) Retinal photograph of individual 3 shows pigmented macular striae. This was not found in individual 1. (D) Flow volume loop of individual 3 showing markedly reduced total lung capacity (∼1 L) and restricted flow dynamics during both inspiration and expiration. (E) Hydrophobicity plot of human PIEZO2 showing Kyte–Doolittle hy- drophobicity analysis (19-residue window) done with ProtScale program (Expasy). Triangles indicate the position of I802F and E2727del mutations. vital volume (% normal value)] = 60%/53%, characteristic of Whole Genome and Exome Sequencing. Individuals 1 and 2 and moderately severe restrictive lung disease. More detailed exami- their respective unaffected parents gave consent for clinical whole nation of major joint movements revealed severely limited joint exome and whole genome sequencing, respectively. Exome se- extension but largely normal joint flexion (Table S1). Other quencing of individual 1 identifiedadenovoc.2404A> T findings were severe scoliosis in individual 2, and relatively short [p.Ile802Phe] missense variant in PIEZO2 (Fig. 1E)thatwas stature, both in individual 1 (length 158 cm, i.e., 10th centile; head confirmed by Sanger sequencing in both individuals 1 and 2. ’ circumference 55 cm, i.e., 50th centile; her mother’s length was In individual 3 s genome sequence, analyzed independently, 62 174 cm and her father’s length was 176 cm) and individual 2 (his potential de novo single nucleotide variants (SNVs) were dis- growth has followed the 2.5 centile, weight and head circum- covered along with 75 potential de novo indels. Two of these de ferences on the 50th centile). In the mother, recurrent knee sub- novo variants were predicted to alter the respective protein luxations were