A Premature Stop Codon in MYO18B Is Associated with Severe Nemaline Myopathy with Cardiomyopathy

Journal of Neuromuscular Diseases 2 (2015) 219–227 219 DOI 10.3233/JND-150085 IOS Press Research Report

A Premature Stop Codon in MYO18B is Associated with Severe Nemaline Myopathy with Cardiomyopathy

Edoardo Malfattia,b,1, Johann Bohm¨ c,d,e,f,1, Emmanuelle Lacene` a,b,g, Maud Beuvina,b, Guy Brochiera,b,g, Norma B. Romeroa,b,g,2,∗ and Jocelyn Laportec,d,e,f,2,∗ aSorbonne Universités, UPMC Univ Paris 06, INSERM UMRS974, CNRS FRE3617, Center for Research in Myology, GH Pitié-Salpêtrière, 47 Boulevard de l’hôpital, 75013 Paris, France bUnité de Morphologie Neuromusculaire, Institut de Myologie, Groupe Hospitalier Universitaire La Pitié-Salpêtrière; Paris, France cDepartment of Translational Medecine, IGBMC, Illkirch, France dINSERM U964, Illkirch, France eCNRS, UMR7104, Illkirch, France f Strasbourg University, Illkirch, France gCentre de référence de Pathologie Neuromusculaire Paris-Est, Institut de Myologie, GHU La Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris; Paris, France

Abstract. Background: Nemaline myopathies (NM) are rare and severe muscle diseases characterized by the presence of nemaline bodies (rods) in muscle fibers. Although ten genes have been implicated in the etiology of NM, an important number of patients remain without a molecular diagnosis. Objective: Here we describe the clinical and histopathological features of a sporadic case presenting with severe NM and cardiomyopathy. Using exome sequencing, we aimed to identify the causative gene. Results: We identified a homozygous nonsense mutation in the last exon of MYO18B, leading to a truncated protein lacking the most C-terminal part. MYO18B codes for an unconventional myosin protein and it is mainly expressed in skeletal and cardiac muscles, two tissues severely affected in the patient. We showed that the mutation does not impact on mRNA stability. Immunostaining and Western blot confirmed the absence of the full-length protein. Conclusion: We propose MYO18B as a novel gene associated with nemaline myopathy and cardiomyopathy.

Keywords: Congenital myopathy, nemaline myopathy, cardiomyopathy, MYO18B, myosin

INTRODUCTION abnormalities in skeletal muscle [1, 2]. Nearly half of the congenital myopathy patients still await molecular Congenital myopathies are rare monogenic characterization and cannot benefit from adequate disorders characterized by distinctive morphologic genetic counselling and healthcare delivery [1]. The identification of additional implicated genes is not 1,2These authors contributed equally to this work. only important for molecular diagnosis, but also ∗Correspondence to: Norma B. Romero, Institut de Myolo- suggests potential drug targets. gie, Groupe Hospitalier Universitaire La Pitie-Salp´ etriˆ ere,` 47-83 Nemaline myopathy (NM) is a congenital mus- boulevard de l’Hepital,ˆ 75013 Paris, France. Tel.: +33 1 42 16 37 74; cle disorder associating hypotonia, muscle weakness, E-mail: [email protected] and Jocelyn Laporte, IGBMC, 1 Rue Laurent Fries, 67404 Illkirch. Tel.: +33 3 88 65 and often skeletal deformities with the presence of 34 12; E-mail: [email protected]. numerous nemaline bodies (rods) in muscle fibers [3].

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The disorder has a marked clinical variability, ranging 9.4, 4.63, 4.35 staining were also performed, but are not from neonatal lethality to mild and non-progressive shown. Pictures of each section were obtained with a forms with childhood and adulthood onset [4]. Zeiss AxioCam HRc linked to a Zeiss Axioplan Bright To date, ten genes have been implicated in NM, Field Microscope and processed with the Axio Vision (ACTA1, MIM#161800; NEB, MIM#256030; TPM2, 4.4 software (Zeiss, Germany). MIM#609285; TPM3, MIM#609284; TNNT1, MIM# Resin-embedded blocks for ultrastructural analy- 605355; KBTBD13, MIM#609273; CFL2, MIM# ses were not prepared at time of the muscle biopsy. 610687; KLHL40 MIM#615340; KLHL41 MIM# We therefore fixed frozen muscle sections (40␮m) 615731, and LMOD3 MIM#616112), encoding pro- in osmium tetroxide (1%), dehydrated and embed- teins of the skeletal muscle thin filament or Kelch ded them in epoxy resin (EMBed-812, Electron domain containing proteins [5–14]. Microscopy Sciences, USA). Ultra-thin sections Although dilated and hypertrophic cardiomy- (80 nm) were stained with uranyl acetate and lead cit- opathies have been previously reported in some rate. The grids were analyzed with a Philips CM120 nemaline myopathy patients [15–17], NM is not typ- electron microscope (80 kV; Philips Electronics NV, ically associated with cardiomyopathy. The majority Eindhoven, The Netherlands) and imaged using a of the reported cases were genetically undiagnosed, Morada digital camera (Soft Imaging System, France). apart from three cases with de novo ACTA1 mutations, presenting congenital NM and hypertrophic cardiomy- Exome sequencing, variants prioritization and opathy (HCM), or dilated cardiomyopathy (DCM) mutation analysis [18–20]. With the aim to identify novel genes responsible The nemaline myopathy genes ACTA1, NEB, TPM2, for NM with cardiomyopathy, we performed exome TPM3, and TNNT1, as well as spinal muscular atro- sequencing in a severe sporadic case. We found a phy (SMA), Prader-Willi syndrome, and myotonic premature stop codon in the C-terminal part of the dystrophy-1 (DM1) were excluded by conventional unconventional myosin MYO18B, and we confirmed techniques. Exome sequencing was performed on the presence of a truncated protein on muscle biopsy. DNA samples from the index patient on a HiSeq 2000 We propose MYO18B as a novel NM gene, which (Illumina, San Diego, USA) using the Agilent 44M v2 should be considered as candidate gene for severe SureSelect Exon enrichment kit at the BGI (Shenzhen, cases, especially if a cardiomyopathy is diagnosed. China). Variant calling was performed with the SOAP software. The mean coverage of the exome was 57x, and the percentage of the exome covered at least 20x MATERIALS AND METHODS was 77.2%. Variants with a frequency <20% of the total reads for a specific position were excluded. Vari- Ethical issues, biopsy, histology, and electron ant filtering was performed by comparison with SNV microscopy databases as dbSNP,Exome VariantServer and EXAC, and polymorphisms with a minor allele frequency The parents of the index patient gave informed (MAF) of more than 0.5% were discarded. Variant pri- consent for the genetic analysis and muscle biopsy oritization was done with the VaRank program [21]. according to French legislation (Comite´ de Protection Sanger sequencing was performed as reported [22]. des Personnes Est IV DC-2012-1693). Genomic DNA The position of the mutation was numbered according was extracted from blood by standard methods. to NM 032608.5 and NP 115997.5. The index patient underwent an open muscle biopsy from vastus lateralis at 14 days of life. RT-PCR For conventional histochemical techniques, 10 ␮m thick cryostat sections were stained with modified The patient’s RNA was extracted from the muscle Gomori trichrome (mGT). Haematoxylin and eosin biopsy with TRI-Reagent (Sigma, St. Louis, USA), and (H&E), Periodic acid Schiff technique (PAS), Oil reverse transcribed using the SuperScript II Reverse red O, reduced nicotinamide adenine dinucleotide Transcriptase (Invitrogen, Carlsbad, USA) and random dehydrogenase-tetrazolium reductase (NADH-TR), hexamer primers following the manufacturer’s proto- succinic dehydrogenase (SDH), cytochrome c oxidase col. MYO18B-specific primers encompassing several (COX), and adenosine triphosphatase (ATPase) at pH exons and the mutation were used to assess mRNA E. Malfatti et al. / MYO18B Mutation in Nemaline Myopathy 221 steady state levels. NEB primers were used as internal and pulmonary hypertension. The patient was tachy- controls. cardic (170 bt/min), and a therapy with diuretics was started. The discontinuation of diuretics at one Immunolabeling and protein analysis month of age precipitated hemodynamic instability leading to the sudden development of acute lung Immunofluorescence analyses were performed on edema promptly recuperated with high doses of diuret- frozen muscle samples from the index patient and ics and digitalis treatment. Cerebral MRI showed a control biopsy without histopathological lesions. minimal signs of subependimal hemorrhage. EEG Anti-myosin 18B C-ter (Sigma, St. Louis, MO, was normal. Karyotype was normal, and spinal mus- USA), anti-myosin 18B C-ter (GTX104872, Gene- cular atrophy (SMA), Prader-Willi syndrome, and Tex, Euromedex, Paris France) and anti-myosin 18B neonatal myotonic dystrophy-1 (DM1) were excluded N-ter polyclonal (kind gift from Professor Gerolamo by molecular analysis. Metabolic work-up including Lanfranchi, University of Padua, Italy) antibodies, serum and CRL lactate assessment, mitochondrial were applied on 10-␮m-thick cryosections overnight respiratory chain complexes enzymatic assay, chro- ◦ at 4 C. The next day, the sections were incubated matography of blood and urinary aminoacids and with appropriate conjugated secondary antibodies urinary organic acid was normal. The course was pro- following routine protocols. Slides without primary gressive with degradation of the hemodynamic and antibodies were used as controls. respiratory status, and failure to thrive. The patient Western blot was performed on skeletal muscle pro- deceased at 4.5 months following a septic shock. tein extracts by routine methods using the anti-myosin Autopsy was not performed. 18B C-ter antibody (Sigma), as well as an anti-myosin heavy chain antibody (Sigma. St. Louis, USA) as load- ing control. Muscle morphology

Muscle biopsy revealed marked fiber size vari- RESULTS ability, and a contingent of atrophic muscle fibers was observed. A few of them had a single cen- Clinical findings tralized nucleus. Modified Engel-Gomori trichrome staining revealed the presence of fuchsinophilic inclu- The index patient was a girl born to healthy con- sions in around 40% of the muscle fibers (Fig. 1D). sanguineous parents of Portuguese origin. The mother The inclusions corresponded to small nemaline reported three previous miscarriages. Pregnancy was bodies (minirods), found both in subsarcolemmal areas uneventful until the discovery of a cardiomyopathy and dispersed in the cytoplasm. The minirods occu- characterized by reduced left ventricle and aortic valve pied most of the cytoplasm of marked atrophic fibers growth during a routine ultrasound at 22 weeks of (Fig. 1D; asterisk). Oxidative techniques failed to show gestation. Birth took place at term by natural vagi- unevenly stained areas or negative COX fibers. Ultra- nal delivery. The weight was 2830 grams (-2 SD), structural studies confirmed the presence of minirods the height 49 cm, and the cranial circumference presenting the typical lattice structure of Z-disc mate- 32.5 cm. Physical examination revealed severe axial rial (Fig. 1E). In conclusion, the histopathological and and peripheral hypotonia, reduced spontaneous periph- ultrastructural findings were strongly suggestive of eral movements, tendon contractures, and absence of nemaline myopathy. nursing necessitating nasogastric feeding (Fig. 1A, B, and C). The patient also presented dysmorphic features including transversal enlargement of the skull, Identification of a MYO18B-homozygous small and horizontal palpebral fissure, high-arched premature stop codon in the patient palate, low-set ears, pectum excavatum, large and lowly implanted thumbs (Fig. 1A), and clinodactyly There are currently 10 known genes implicated of both fourth and fifth fingers of the hands. Car- in nemaline myopathy. However, a large number of diac ultrasound confirmed the presence of small nemaline myopathy patients do not harbor mutations and mildly hypertrophic left cavities, compensatory in any of those genes, indicating that additional genes dilatation of right cavities, interatrial communication, remain to be discovered. Owing to the fact that the 222 E. Malfatti et al. / MYO18B Mutation in Nemaline Myopathy

Fig. 1. Clinical and histological features of the nemaline myopathy patient. A) Clinical examination of the patient revealed a floppy infant with pectus excavatum, and severely reduced active movements. B) The patient showed severe axial muscle weakness, and C) absence of antigravity reflexes, impossible neck extension or brief head control. D) Modified Engel-Gomori trichrome staining revealed the presence of marked fiber size variation, and the presence of nemaline bodies in 40-50% of fibers. The asterisk indicates a severely atrophic fiber filled by minirods. E) Electron microscopy confirmed the presence of small nemaline bodies (minirods). major nemaline myopathy genes ACTA1, NEB, TPM2, chromosome 22. The variant was confirmed by Sanger TPM3, and TNNT1 have previously been excluded by sequencing of a second DNA sample from the patient. conventional techniques, we performed exome The mutation was not listed in the human variation sequencing on DNA samples from the patient. Data databases as EVS, dbSNP or ExAC, or in our in-house analysis did not reveal any potentially pathogenic database encompassing 676 exomes. Segregation sequence aberration in any of the 10 well covered analysis revealed that both healthy parents carry the nemaline myopathy genes. We detected a homozy- mutation in the heterozygous state (Fig. 2A and 2B). gous nonsense mutation in MYO18B (c.6496G>T; The index patient did not have siblings, and DNA p.Glu2166*), residing within a large homozygous samples from the miscarriages or other relatives were region encompassing the entire MYO18B gene on not available. The mutation creates a premature stop E. Malfatti et al. / MYO18B Mutation in Nemaline Myopathy 223

1 2

1 3

3 33

Fig. 2. Identification of a homozygous nonsense mutation in MYO18B. A) Pedigree of the consanguineous family. B) Chromatopherograms showing the segregation of the MYO18B c.6496G>T mutation. Both healthy parents (1 and 2) are heterozygous carriers of the MYO18B mutation; the patient (3) is homozygous. C) RT-PCR analysis of skeletal muscle cDNA. The MYO18B amplicon encompassing the MYO18B mutation was detected at comparable levels in the patient and an age-matched control. A NEB amplicon of similar size was used as internal control. codon in the last of the 42 coding exons of MYO18B, The MYO18B premature stop codon leads to a and is predicted to remove the last 401 amino acids truncated protein out of 2567 at the protein level. In the human variation databases, MYO18B nonsense or frameshift variants In order to verify the presence of the predicted leading to a potential protein truncation are rare and truncated myosin 18B protein in the patient’s mus- never found to be homozygous. This goes along with cle, we performed immunofluorescence on muscle the idea that heterozygous carriers are healthy and sections using N-terminal and C-terminal antibodies. that homozygous mutations may be associated with Immunolabeling with an N-terminal antibody did not disease. Other rare homozygous variants detected in reveal any difference in protein localization and sig- the patient’s exome are listed in Supplemental Table 1. nal intensity between the patient and age-matched controls (Fig. 3A, and B). Myosin 18B appeared to be nuclear and cytoplasmic. In a subset of fibers, The MYO18B premature stop codon does not probably type 2 fibers, we detected a more intense involve major nonsense mRNA decay punctate cytoplasmic signal. Immunolabeling using the C-terminal myosin 18B antibody revealed absence Premature stop codons most often trigger nonsense- of staining in the patient as compared to the con- mediated mRNA decay (NMD), except if they occur in trol (Fig. 3C, and D). Western blot analysis using the the last or second but last exon [23]. In order to assess C-terminal myosin 18B antibody revealed a 285 kDa whether the MYO18B p.Glu2166* variant affects the band corresponding to myosin 18B in protein extracts mRNA steady state level, we extracted mRNA from fromanage-matchedmusclebiopsy,whilenosignalwas the patient’s muscle biopsy, reverse transcribed it into detectable in the biopsy from the index patient. Taken cDNA, and amplified a fragment encompassing the together, our results suggest the presence of a truncated MYO18B exons 40 to 42 and the mutation. No dif- myosin18BproteindeprivedofitsmostC-terminalpart. ference was detected in the signal intensity between the patient and an age-matched control (Fig. 2C), sug- DISCUSSION gesting that the overall cDNA level was comparable and that the MYO18B mRNA is not subject to major Here we propose MYO18B as novel gene associated degradation through NMD. with congenital nemaline myopathy. The index patient 224 E. Malfatti et al. / MYO18B Mutation in Nemaline Myopathy

Fig. 3. Impact of the MYO18B premature stop codon. Immunolabeling of the muscle biopsy of the index patient and an age-matched control using N-terminal and C-terminal myosin 18B antibodies. A) and B) Immunostaining with N-terminal myosin 18B antibodies showed a similar pattern of cytoplasmic staining in frozen muscle sections from the patient and the control. C) and D) Immunostaining using a C-ter myosin 18B antibody demonstrated the absence of staining in the patient’s section compared to the control, validating the presence of a truncated myosin 18B in the patient. E) Western blot using the C-terminal anti-myosin 18B antibody shows absence of signal in muscle extracts from the patient.

presented with severe hypotonia, muscle weakness and detected a homozygous nonsense mutation in MYO18B cardiomyopathy. Muscle histopathology was indica- leading to a truncated protein. The index patient was tive of nemaline myopathy, and exome sequencing part of a cohort analyzed by exome sequencing and E. Malfatti et al. / MYO18B Mutation in Nemaline Myopathy 225 encompassing 34 patients with mild nemaline myopa- revealing symptom in the patient reported by D’Amico thy, and 17 unrelated patients with severe nemaline et al. [18]. myopathy, including two patients with additional car- The cardiac disease encountered in our case was diomyopathy. The index patient was the only one rather peculiar and consisted of antenatal reduced harboring a MYO18B mutation. growth of the left ventricle and aortic valve. Soon Following lines of evidence suggest the implica- after birth, the presence of small and mildly hyper- tion of the MYO18B mutation in the development trophic left cavities, compensatory dilatation of right of the disease: we detected a homozygous non- cavities, and interatrial communication were noted sense mutation in MYO18B by exome sequencing, by ultrasound. If the muscle symptoms, as frequently immunolabeling confirmed the presence of a trun- observed in congenital nemaline myopathy, remained cated protein, and Western blot using a C-terminal stable, the cardiac phenotype progressed rapidly antibody showed the absence of the full-length pro- with the development of congestive heart failure. tein. Both healthy parents were heterozygous for The presence of a complex cardiac disorder com- the mutation, confirming autosomal recessive disease bining multiple malformations and HCM suggested inheritance. In accordance, homozygous MYO18B a cardiac myogenesis/development dysfunction. Our truncations were not listed in the public exome patient deceased more than twenty years ago and, his- datasets. MYO18B encodes an unconventional myosin tologic studies on autoptic cardiac muscle were not specifically expressed in skeletal and cardiac muscle, performed. the two main affected tissues in our patient [24]. More- In addition to the cardiac involvement, dysmorphic over, Myo18b knockout mice were embryonic lethal, features and clinodactyly may represent specific fea- and presented a cardiac dysfunction similar to the tures related to the MYO18B truncation, and these patient [25]. signs might be helpful in orientating molecular inves- Interestingly, a recent paper described two patients tigations towards MYO18B in patients with nemaline with Klippel-Feil anomaly and severe neonatal muscle myopathy. MYO18B codes for an unconventional mus- weakness harboring a homozygous MYO18B non- cle myosin heavy chain identified for the first time in sense mutation [26]. Muscle examinations detected the 2003 [24]. The protein is mainly expressed in human presence of nemaline bodies, confirming MYO18B as cardiac and skeletal muscle, and at lower level in novel gene for nemaline myopathy. Additional clinical testis. In mature human skeletal and cardiac muscle, features, in particular cervical spine fusion, were diag- it is localized in both nucleus and cytoplasm [24]. nosed during childhood for the Klippel-Feil patients, Although myosin 18B contains two overlapping C- while our patient deceased at 4.5 months of age. A terminal nuclear localization signals, we did not cardiomyopathy as in our patient was however not observe any difference in nuclear versus cytoplas- diagnosed in the Klippel-Feil patients, and this differ- mic localization between the patient’s and the control ence might be mutation-specific. Indeed, we detected biopsy. Conventional myosins are known to be local- a truncated myosin 18B protein in muscle extracts of ized at the A-bands and function as a molecular motor our patient, while the Klippel-Feil mutation was shown for muscle contraction. In contrast, myosin 18B was to involve nonsense-mediated mRNA decay (NMD), found at the Z-lines of myofibrils in murine striated resulting in total myosin 18B protein loss. These find- muscles [25]. Noteworthy, most proteins previously ings suggest a toxic effect of truncated myosin 18B found mutated in nemaline myopathy are located at in heart, but the exact pathomechanisms remain to be the Z-lines and are implicated in the F-actin pathway. uncovered. The sarcomeric localization of myosin 18B suggests a An association of rods with cardiomyopathy has possible role in functioning and/or maintenance of the been reported in less than 20 patients, all presenting contractile apparatus, and might explain the presence general skeletal muscle involvement as primary mani- of nemaline bodies encountered in the patient. festations [20]. Ten patients developed cardiac failure Several of the most recent genes implicated in nema- in adulthood, and six manifested cardiac involvement line myopathy encode proteins presumably implicated in infancy or childhood. A molecular diagnosis was in the protein degradation pathway, as KLHL40 and available only for three pediatric patients [18–20], KLHL41. Both belong to the BTB-BACK-kelch (BBK) all carrying dominant de novo mutations in ACTA1. family of proteins, some of which have been shown Two of them manifested hypertrophic cardiomyopa- to promote degradation of their substrates [12, 27]. thy (HCM) [18, 19], and one childhood-onset dilated It should be noted that myosin 18B interacts with cardiomyopathy [20]. Intriguingly, HCM was the the proteasomal subunit Sug1 and is degraded by 226 E. Malfatti et al. / MYO18B Mutation in Nemaline Myopathy the ubiquitin-proteasome pathway [28], supporting the REFERENCES hypothesis that myosin 18B may link the contractile apparatus and protein degradation, representing the [1] Romero NB, Clarke NF. Congenital myopathies. Handb Clin two main pathways implicated in nemaline myopathy Neurol. 2013;113:1321-36. [2] Nance JR, et al. Congenital myopathies: An update. Curr to date. Neurol Neurosci Rep. 2012;12(2):165-74. In conclusion, our clinical, histological, ultrastruc- [3] Malfatti E, et al. 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