Gain-Of-Function Mutations in the Mechanically Activated Ion Channel PIEZO2 Cause a Subtype of Distal Arthrogryposis

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 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 GENETICS 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 | March 19, 2013 | vol. 110 | no. 12 | 4667–4672 Downloaded by guest on September 27, 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 hydrophobicity 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 found to be associated with an absence of anterior sequences: one was a three base pair deletion c.8179_8181del in cruciate ligaments and small menisci in both knees. The son also PIEZO2 (Ref. seq. NM_022068.2), resulting in an in-frame single has unstable knees compatible with similar intra-articular liga- amino acid deletion p.Glu2727del (Fig. 1E); the other was a missense variant [p.Arg341Gln] in the acyl-CoA synthetase medium- ment agenesis (Table 1). fi In the second kindred, the only affected individual (individual chain family member 4 gene, ACSM4. Sanger sequencing con rmed both the in-frame deletion and missense variant as authentic de 3), a female, was born at 7 mo to a healthy mother and diagnosed novo changes in individual 3. Interestingly, the E2727del PIEZO2 with DA5 as a newborn. Between the ages of 2–15 y, she re- variant falls within the C-terminal portion of PIEZO2, in a highly quired multiple orthopedic procedures on her hips, knees, and conserved pentapeptide motif Leu-Glu-Glu-Asp-Leu (LEEDL) feet to improve function. As an adult, she exhibited decreased embedded within a ∼550 residue domain of unknown function facial expression with deep-set eyes and ptosis (Table 1); she also (DUF3595) that is highly conserved across metazoa from human exhibited ophthalmoplegia that was most severe upon upward to hydra. The I802F variant falls within one of ∼37 predicted gaze, globally decreased muscle mass and spinal stiffness, limited transmembrane domains in PIEZO2, and this mutation is also in fi forearm rotation, long ngers with congenital contractures, and a region of the protein that is conserved in all mammals and poorly formed phalangeal and palmar creases (Fig. 1B). Fun- chicken. The occurrence of these unique de novo I802F and doscopic examination revealed bilateral macular striae (Fig. 1C). E2727del variants in PIEZO2 is consistent with autosomal dom- She also had severe restrictive lung disease (FEV1/FVC 28%/ inant inheritance and suggests pathogenic roles for both variants 26%) (Fig. 1D) and required chronic supplemental nocturnal ox- in the DA5 patients. ygen. Taken together, affected individuals in both kindreds exhibit generalized arthrogryposis with characteristic ophthalmoplegia Electrophysiologic Studies of I802F and E2727del. Piezos in mice are and ptosis, and with moderately severe to severe restrictive lung expressed in many tissues that respond to mechanical force (15). disease as a key feature of this particular DA5 subtype. Drosophila Piezo functions in nociception (16), and human PIEZO1

4668 | www.pnas.org/cgi/doi/10.1073/pnas.1221400110 Coste et al. Downloaded by guest on September 27, 2021 Table 1. Clinical features in three cases of DA5 Individual 1 (I802F) Individual 2 (I802F) Individual 3 (Δ2727E) Phenotype (35-y-old mother) (5-y-old son) (38-y-old female)

Short stature Yes Yes Yes Narrow/high palate Yes Yes Yes Reduced ability to open mouth Yes Yes Yes Decreased facial expression Yes Yes Yes Ophthalmoplegia Yes Yes Yes Deep-set eyes Yes Yes Yes Blepharophimosis No Yes No Ptosis Yes Yes Yes Hypermetropia Yes (+7 o.u.) Yes (+8/+10) Yes Duane anomaly Nd Nd Yes Abnormal retinal pigmentation No Nd Yes Macular retinal folds No Nd Yes Hunched anteverted shoulders Yes Yes Yes Restrictive lung disease Yes Likely Yes Spine stiffness Yes Yes Yes Hypermobile ﬁrst metacarpophalageal Yes Yes Yes Absent phalangeal creases Yes Yes Yes Poorly formed palmar creases Yes Yes Yes Limited wrist extension Yes Yes Yes Absent ACL (knee) Yes Probably No Camptodactyly Yes Yes Yes Clinodactyly Digits III–V Digit V Yes Club feet No Yes, left No Dimples over large joints Yes Yes No Exertional dyspnea Yes Yes Yes Constriction of urethra Yes No No FEV1/FVC 60%/53% Nd 28%/26% Increased muscle tone Yes No Yes Tendon reﬂexes Weak and absent in knees and ankles Weak Weak Normal intelligence Yes Yes Yes

Hearing loss No Nd Yes (subjective) GENETICS Musculoskeletal pain Yes, major problem Nd No Altered pain sensation Possibly as child High threshold suspected Nd

ACL: anterior cruciate ligaments; Nd: not determined; o.u.: oculus uterque.

missense mutations were recently identified in a disorder of currents were similar to wild-type currents, suggesting another erythrocyte volume homeostasis, hereditary xerocytosis (OMIM causal mechanism for this putative mutation. 194380) (17). However, the spectrum of PIEZO physiologic We next assayed the kinetics of recovery from inactivation of functions and the biological consequences of PIEZO mutations MA currents, which describe the ability of MA channels to re- remain unknown. To investigate the functional effects of the two spond to repetitive stimulation (Fig. 3). Interestingly, both I802F presumptive PIEZO2 mutations identified in DA5 patients, we and E2727del recover more quickly from inactivation than wild- generated the I802F and E2727del point mutations in full-length type, with recovery from inactivation time constants, τ, of 0.43 ± human PIEZO2 cDNA and characterized their effects on MA 0.07 and 0.51 ± 0.10 s, respectively, versus 1.08 ± 0.16 s for wild currents. Cells transfected with wild-type, I802F, or E2727del type (Fig. 3). These results suggest that both I802F and E2727del PIEZO2 cDNAs were stimulated by applying force to the cell channels spend about twofold less time in an inactivated state surface while patch clamp recording in the whole cell configu- following mechanical stimulation than wild-type channels, and ration was at –80 mV, as previously described (15, 18, 19). thus can be reactivated quicker. Large MA currents were present in cells transfected with wild- These results suggest that increased response to mechanical type, I802F, and E2727del (Fig. 2A). I802F and E2727del MA force may explain the phenotype of DA5 patients. However, it is currents have similar amplitudes compared with wild-type PIEZO2 also possible that PIEZO2 mutants are active without mechanical currents, as well as a linear current voltage relationship with a re- stimuli, and that cation influx from constitutively open ion channels versal potential close to 0 mV under the recording conditions (Fig. could contribute to cell toxicity (20), or could cause DA5-related 2 B and C). Human PIEZO2 MA currents have a time constant of phenotypes. However, cells transfected with the mutant PIEZO2 inactivation τ of 6.2 ± 0.6 ms (n = 12 cells) at –80 mV when fitted constructs do not show stimulus-independent PIEZO-like channel with a monoexponential equation, which is comparable to mouse activity (Fig. 2 and Fig. 3) nor enhanced cytotoxicity (Fig. S1). Piezo2 (∼7 ms). I802F-induced MA current inactivation kinetics Therefore, the DA5 phenotype in patients with PIEZO2 mutations are similar to wild-type; however, E2727del-induced MA current may reflect ion channel hyperresponsiveness to forces engendered inactivation kinetics are clearly slower at all potentials tested from during development, and later during postnatal life. –80 mV to +80 mV (Fig. 2 D and E). These differences in the kinetics of inactivation suggest that for a given mechanical stimu- Discussion lation that elicits currents of similar amplitude, the current charge MA ion channels, also termed stretch-activated ion channels, will be larger for E2727del than for wild-type. Interestingly, I802F are present in a variety of cell types, including somatosensory

Coste et al. PNAS | March 19, 2013 | vol. 110 | no. 12 | 4669 Downloaded by guest on September 27, 2021 transduction. Since DA5 is manifest as a congenital disorder present at birth, this implies that MA ion channels play a crucial role in the integrated development of the joints and neuromuscular and respiratory systems during embryogenesis. Further insight into the nature of this embryonic requirement for mechanotransduction (which cell types and at what embryonic time points) awaits analyses of loss-of-function mutations in PIEZO2. The identiﬁcation of mutations in PIEZO2 in a subtype of DA5 affords further insight into the potential pathogenic mechanisms in arthrogryposis. In the adult mouse, PIEZO2 RNA is expressed in a variety of tissues including dorsal root ganglion (DRG) sensory neurons and the lung (15). Therefore, this syndrome could result from altered behavior of stretch sensors like muscle spindles and Golgi tendon organs that are important for pro- prioception and muscle tone. Interestingly, mutation of ECEL1 was also recently shown to cause a DA subtype (10, 11). However, patients with mutations in ECEL1 are phenotypically distinct from the patients reported here, as the former exhibit knee extensions and micrognathia but not ophthalmoplegia or shoulder girdle contractures. Nonetheless, like PIEZO2, ECEL1 is expressed in neuronal cell types, and knockout of Ecel1 in mice reveals that

Fig. 2. E2727del PIEZO2 channels display slower inactivation kinetics compared with wild type. (A) Representative traces of MA inward currents at –80 mV in cells transfected with the indicated constructs and subjected to a series of mechanical steps in 1 μm increments. (B) Average maximal current amplitude of MA inward currents at –80 mV. The number of cells tested is shown above bars. (C) Averaged current-voltage relationships of MA currents in cells transfected with hPiezo2 (n = 7 cells), I802F (n = 8 cells), or E2727del (n = 8 cells). The insets show representative MA currents evoked at holding potentials ranging from –80 to +80 mV; inset scale bars are 1 nA and 100 ms. (D) Representative traces of MA currents elicited at –80 and +80 mV holding potentials. Traces were normalized to the peak current, and blue, red, and green dashed lines represent ﬁts of inactivation with a monoexponential equation of human PIEZO2, I802F, and E2727del currents, respectively. (E) Time-constant of inactivation (tau) of PIEZO2 (black dots, n = 7 cells), I802F (red dots, n = 8 cells), and E2727del (green dots, n = 8 cells) currents at different holding potentials. Dots and bars represent mean ± SEM. ns, not statistically different; **P < 0.01; ***P < 0.001; Mann–Whitney test.

neurons, inner ear hair cells, muscle cells, endothelial cells, and osteoblasts (21). The role of such ion channels in touch, pain, and hearing—where mechanical forces are transduced to biological signals within milliseconds—are relatively clear. Dur- ing fetal development and during the normal physiological function of many biological systems, including the neuromuscular, musculoskeletal, and respiratory systems, mechotransduction has been proposed to be mediated by a variety of molecules, in- Fig. 3. E2727del and I802F PIEZO2 channels recover faster from inactivation cluding integrins, kinases, and ion channels (22). However, the compared with wild type. (A) Typical recording traces of PIEZO2, I802F, and E2727del MA currents recorded at –80 mV. The protocol depicted on the top identify and function of MA ion channels in most of these bi- consists of two consecutive mechanical stimulation steps separated by in- ological systems remains unknown. creasing delay and aims at testing recovery from inactivation. The traces for Our results show that mutations in PIEZO2 can cause a subtype each construct represent superimposition of multiple test-control stimula- of DA5 that restricts respiratory function, as well as cause the tion pairings at different delta-t intervals. The test current traces are each generalized contractures, ptosis, and ophthalmoplegia that char- normalized to the peak of control currents. Peak of test currents are fitted acterize DA5 in general (Table 1). In particular, electrophysio- with a monoexponential equation. (B) Average of test peak current/control = = logical analysis of mutant PIEZO2 proteins suggests that these peak current for PIEZO2 (black, n 8 cells), I802F (red, n 7 cells), and fi E2727del (green, n = 7 cells) are fitted with monoexponential equation. (C) channels are more ef cient at transducing repetitive mechanical Average of time-constant (tau) of recovery from inactivation determined for signals. In addition, we found no evidence that these channels are each individual cell exemplified in A. The number of cells tested is shown constitutively active (without mechanical force), arguing that the above bars. Dots and bars represent mean ± SEM. **P < 0.01; ***P < 0.001; observed phenotype most likely reflects dysregulated mechano- Mann–Whitney test.

4670 | www.pnas.org/cgi/doi/10.1073/pnas.1221400110 Coste et al. Downloaded by guest on September 27, 2021 Ecel1 (also called DINE, or damage-induced neuronal endopep- human reference sequence [University of California Santa Cruz (UCSC) HG19 tidase) is required for the proper intramuscular axonal branching (Human Genome version 19) build] using the Burroughs-Wheeler Alignment of motor neurons in skeletal muscle during embryogenesis (23,24). tool (28) in paired-end mode followed by base quality recalibration and These results suggest that mutations in neuronally expressed targeted local realignment focused around known short insertions and deletions (indels) using the Genome Analysis Toolkit (GATK) (29, 30). Dupli- genes such as PIEZO2 and ECEL1, as well as in the already cated reads from sequencing the same DNA fragment were discarded. known DA genes that are expressed in muscle, can perturb the Single nucleotide substitutions and indels were identified for all three neuromuscular pathway that controls the development of muscle samples simultaneously using the Unified Genotyper tool from the GATK in tone during embryogenesis. multisample calling mode. Variant quality score recalibration was performed Arthrogryposis could also result from abnormal tissue modeling using the GATK to identify a set of high-confidence variants. The functional during development, such that hyperactive PIEZO2 signaling consequence of the resulting set of variants was predicted using the Variant regulates morphogenesis in ways that constrain joint extension, Effect Predictor from Ensembl (31). lung or thorax expansion, and oculomotor movement. Expression Novel variants were defined as those not found in dbSNP (Single Nucle- analyses of PIEZO2 in human or mouse embryos might shed light otide Polymorphism Database) build 135 (32). A stringent set of putative de on how this ion channel causes arthrogryposis. Interestingly, an- novo variants was defined as those found to be heterozygote in the pro- terior cruciate ligament agenesis was identified in the first kindred, band without evidence of any sequencing read supporting the alternate and similar DA5 patients have been described before (25). Per- allele in both parents and absence of the alternate allele in both dbSNP build 135 and the Exome Sequencing Project (http://evs.gs.washington.edu/EVS/). turbations in PIEZO2 function may therefore hold relevance for For individual 3 and her unaffected parents, an average of 1.15 × 1011 structural syndromes involving abnormal joint mobility. Further- bases of sequence were generated per individual, resulting in an average of more, the delineation of two different types of PIEZO2 gain-of- 43-fold coverage across the genome. A total of 4,165,074 SNVs were iden- function mutations [viz., slower inactivation and accelerated tified in the family along with 817,283 short insertions or deletions (indels). recovery from inactivation (E2727del) vs. only accelerated recovery Of these SNVs, 45,626 were novel. By comparing individual 3’s genome se- (I802F)] may prove to have genotype–phenotype implications, quence with that of her unaffected parents, 62 potential de novo SNVs were with composite gain-of-function mutations (E2727del) having discovered along with 75 potential de novo indels; as not all of these var- more severe clinical consequences. iants were validated, some may represent false positives. However, two of It has been proposed that congenital contractures could result the de novo variants were predicted to alter their respective protein from decreased fetal movement brought about by primary neural sequences: one was a three base pair deletion resulting in an in-frame single amino acid deletion in the PIEZO2 (p.E2727del); the other was a missense or muscular disorders, or from other abnormalities in utero (3, 7, fi fi variant identi ed in the acyl-CoA synthetase medium-chain family member 26). Interestingly, previously identi ed DA mutations reside in 4 gene (ACSM4, p.R341Q). Sanger sequencing confirmed that the in-frame MYH3 and MYH8 that encode sarcomeric proteins, or in TNNI2, deletion and missense variant were both de novo changes in individual TNNT3 and TPM2, respectively (2, 12–14). These proteins are 3—that is, present in the heterozygous state and absent in her parents. expressed in fast twitch fibers, and functional studies have shown Consent to perform whole genome sequencing under Clinical Laboratory that DA (DA1 and DA2) mutations in TNNI2, TNNT3, and Improvement Amendments (CLIA) conditions was obtained under protocols TPM2 produce increased contractile function in troponin- approved by the Institutional Review Board (IRB) of Partners HealthCare, replaced rabbit psoas fibers (13). This could suggest that these Inc. Explicit permission to publish the findings contained herein was fi obtained from individual 3.

speci c DA mutations cause congenital contractures on the basis GENETICS of hypercontractility and increased tension in developing fast twitch skeletal muscles, resulting in diminished muscle movement Whole Exome Sequencing. Whole exome sequencing for trio 1 was in principle performed as previously described (33). Briefly, massively parallel sequencing in utero. The possibility that PIEZO2 senses forces associated – fi of genomic DNA from an affected individual parent trio was performed at with muscle contraction, combined with the nding that mutant the Radboud University Nijmegen Medical Centre by using the SOLiD 5500xl PIEZO2 proteins have increased activity, is likewise consistent platform (Life Technologies). Enrichment of exonic sequences was achieved with the view that the integrated development of muscles and by using the human SureSelect 50 Mb set (Agilent, v2), which targets joints during embryogenesis is an active rather than a passive ∼21,000 human genes. On average, we obtained >136 million mappable process (13). For example, increased ion flux could result in in- sequencing reads (50 bp single-end) and 6.6 Gb of mappable sequence data creased neural firing during fetal muscle movement, and this in per individual after multiplex sequencing, using six exomes per flowchip. turn could inhibit overall muscle movement via a negative feed- Color space reads were mapped to the hg19 reference genome with SOLiD back loop that integrates muscle contraction and movement. LifeScope software version 2.1, which uses an iterative mapping approach. > This model could predict that as yet unidentified PIEZO2 On average, 88% of bases mapped on or near (50 bp proximity) the tar- — gets, resulting in a mean coverage of 104-fold (median of 82-fold). In total, variants might have the opposite effect that is, diminished >81% of the targeted exons were covered ≥20 times (>86% ≥10 times) PIEZO2 function may be associated with joint hypermobility, (Table S2). SNVs were subsequently called by the DiBayes algorithm using a common condition of suspected multifactorial etiology and high-stringency calling settings, and small insertions and deletions were dominant inheritance pattern. detected using the SOLiD Small Indel Tool. Mechanotransduction has been implicated in several physio- We identified a total of 44,606 variants in the proband of trio 1 (individual logical processes, including the sensing of hemodynamic forces 1). All variants were annotated using an in-house annotation pipeline built in during development and the function of various organs (22), but Nijmegen. These variants were filtered and analyzed for potential de novo not as a cause of defective muscle development and arthrogry- occurrence using an automated prioritization scheme (34) involving an au- posis. Here, we show that a mechanosensitive ion channel plays tomated check of all private, nonsynonymous variants identified in the af- a key role in the development and function of both the neuro- fected individuals with high quality (>4 variant reads, >25% variation) for fi muscular and respiratory systems. Further analyses will be re- occurrence in the respective parental bam les. Candidate de novo events (Table S3) were then verified using conventional Sanger sequencing. The quired to elucidate the frequency of PIEZO2 mutations in other only validated de novo mutation was the I802F missense variant in PIEZO2. individuals with DA and to determine whether these mutations The regional ethical review board as well as the Norwegian health affect other classical mechanotransduction-dependent processes authorities have approved whole exome sequencing as a method to find such as hearing and pain perception. de novo disease-causing mutation. The patients have given informed consent to the publishing of clinical information and pictures. Methods Whole Genome Sequencing. Whole genome sequencing was performed by the Cell Culture and Transient Transfection. Human embryonic kidney 293T Illumina Clinical Services Laboratory (Illumina, Inc.). Briefly, genomic DNA was (HEK293T) cells were grown in Dulbecco’s Modified Eagle Medium con- − − randomly fragmented and then sequenced using 100 base pair paired-end taining 4.5 mg·mL 1 glucose, 10% (vol/vol) FBS, 50 U·mL 1 penicillin, and 50 reads on an Illumina HiSeq Sequencer (27). The resulting output was con- mg·mL−1 streptomycin. Cells were plated onto poly-lysine-coated 12-mm verted to FASTQ format. The paired-end FASTQ files were aligned to the round glass coverslips placed in 24-well plates and transfected using

Coste et al. PNAS | March 19, 2013 | vol. 110 | no. 12 | 4671 Downloaded by guest on September 27, 2021 lipofectamine 2000 (Invitrogen) according to the manufacturer’s instruction. Toxicity/Viability Assay. To exclude the possibility that the PIEZO2 mutations A total of 800 ng·mL−1 of plasmid DNA was cotransfected with 300 ng·mL−1 tested are constitutively active and cause cellular toxicity on that basis, we of GFP plasmid, and GFP positive cells were recorded 12–48 h later. directly tested if human PIEZO2 mutants incurred toxicity or affected the viability of HEK cells. We used wild-type and constitutively active (F640L) Electrophysiology. Patch-clamp experiments were performed in standard versions of TRPV1, the latter as a positive control. The F640L TRPV1 mutation whole-cell recordings using an Axopatch 200B amplifier (Axon Instruments). exhibited an increased cytotoxicity ratio compared with wild-type TRPV1, whereas PIEZO2 I802F and E2727del did not (Fig. S1). HEK293T cells were Patch pipettes had a resistance of 1.5–2.5 MΩ when filled with an internal transfected with various cDNAs and seeded in a 384-well plate (TransIT-LT1, solution consisting of (in mM) 133 CsCl, 10 Hepes, 5 EGTA, 1 CaCl , 1 MgCl , 2 2 Mirus, 9,000 cells/well; 62.5 ng cDNA/well; eight wells per cDNA). Two days 4 MgATP, and 0.4 Na GTP (pH adjusted to 7.3 with CsOH). The extracellular 2 after transfection, cells were tested using the MultiTox-Fluor Multiplex Cy- solution consisted of (in mM) 130 NaCl, 3 KCl, 1 MgCl , 10 Hepes, 2.5 CaCl , 2 2 totoxicity Assay per manufacturer’s instructions (Promega; measures two 10 glucose (pH adjusted to 7.3 with NaOH). All experiments were done at protease activities correlated with number of dead and live cells). Briefly, fi room temperature. Currents were sampled at 20 kHz and ltered at 2 kHz. reagents were incubated with cells for 2–3 h at 37 °C after which fluores- Voltages were not corrected for a liquid junction potential. Leak currents cence at Ex485/Em520 (dead-cell fluorescence or cytotoxicity) and Ex405/ before mechanical stimulations were subtracted off-line from the cur- Em492 (live-cell fluorescence or viability) were measured using EnVision rent traces. Multilabel Plate Reader (Perkin-Elmer). Average cytotoxicity–viability fluorescence ratio was calculated and normalized to the average ratio of Mechanical Stimulation. Mechanical stimulation was achieved using a fire- pcDNA3.1 controls. Results of four independent experiments were analyzed polished glass pipette (tip diameter 3–4 μm) positioned at an angle of 80° to for significance using Prism 6 (Graphpad). Rat TRPV1 cloned in pcDNA5-FRT the cell being recorded. Downward movement of the probe toward the cell and the toxic TRPV1-F640L mutant (Quikchange, Agilent Technologies) were was driven by a Clampex controlled piezo-electric crystal microstage (E625 used as controls (20). LVPZT Controller/Amplifier; Physik Instrumente). The probe had a velocity of − 1 μm·ms 1 during the ramp segment of the command for forward motion, ACKNOWLEDGMENTS. We thank the patients and their families for partici- pation in this study. We also thank Stuart Cahalan for cloning contributions. and the stimulus was applied for 150 ms. To assess the mechanical sensitivity We thank Christian Gilissen and Peer Arts from the genomic disorders group of a cell, a series of mechanical steps in 1 μm increments was applied every Nijmegen for the technical support in analyzing the exomes from kindred 2. 10 s, which allowed for full recovery of mechanosensitive currents. Inward This work was supported by Helse Vest, National Institutes of Health (NIH) MA currents were recorded at a holding potential of –80 mV. For I–Vre- Grants R01HD060050, R01MH084676, and R01DE022358, and by funds from ’ lationship recordings, voltage steps were applied 0.7 s before the mechan- Brigham and Women s Hospital. N.S. was supported in part by a career de- – velopment award from the NIH/National Heart, Lung, and Blood Institute ical stimulation from a holding potential of 60 mV. The protocol used to (HL114642). A.H. was supported by a grant from the Netherlands Organiza- characterize the recovery from inactivation kinetics was applied every 15 s, tion for Health Research and Development (ZonMW 917-66-363). J.C. was and each of the two consecutive mechanical stimulus steps lasted 100 ms. supported by a Medical Research Council Career Development Fellowship.

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