Recessive MYF5 Mutations Cause External Ophthalmoplegia, Rib, and Vertebral Anomalies
2018; Elsevier BV; Volume: 103; Issue: 1 Linguagem: Inglês
10.1016/j.ajhg.2018.05.003
ISSN1537-6605
AutoresSilvio Alessandro Di Gioia, Sherin Shaaban, Beyhan Tüysüz, Nursel Elçioğlu, Wai‐Man Chan, Caroline D. Robson, Kirsten Ecklund, Nicole M. Gilette, Azmi Hamzaoğlu, Gulsen Akay Tayfun, Elias I. Traboulsi, Elizabeth C. Engle,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoMYF5 is member of the Myc-like basic helix-loop-helix transcription factor family and, in cooperation with other myogenic regulatory factors MYOD and MYF5, is a key regulator of early stages of myogenesis. Here, we report three consanguineous families with biallelic homozygous loss-of-function mutations in MYF5 who define a clinical disorder characterized by congenital ophthalmoplegia with scoliosis and vertebral and rib anomalies. The clinical phenotype overlaps strikingly with that reported in several Myf5 knockout mouse models. Affected members of two families share a haploidentical region that contains a homozygous 10 bp frameshift mutation in exon 1 of MYF5 (c.23_32delAGTTCTCACC [p.Gln8Leufs∗86]) predicted to undergo nonsense-mediated decay. Affected members of the third family harbor a homozygous missense change in exon 1 of MYF5 (c.283C>T [p.Arg95Cys]). Using in vitro assays, we show that this missense mutation acts as a loss-of-function allele by impairing MYF5 DNA binding and nuclear localization. We performed whole-genome sequencing in one affected individual with the frameshift mutation and did not identify additional rare variants in the haploidentical region that might account for differences in severity among the families. These data support the direct role of MYF5 in rib, spine, and extraocular muscle formation in humans. MYF5 is member of the Myc-like basic helix-loop-helix transcription factor family and, in cooperation with other myogenic regulatory factors MYOD and MYF5, is a key regulator of early stages of myogenesis. Here, we report three consanguineous families with biallelic homozygous loss-of-function mutations in MYF5 who define a clinical disorder characterized by congenital ophthalmoplegia with scoliosis and vertebral and rib anomalies. The clinical phenotype overlaps strikingly with that reported in several Myf5 knockout mouse models. Affected members of two families share a haploidentical region that contains a homozygous 10 bp frameshift mutation in exon 1 of MYF5 (c.23_32delAGTTCTCACC [p.Gln8Leufs∗86]) predicted to undergo nonsense-mediated decay. Affected members of the third family harbor a homozygous missense change in exon 1 of MYF5 (c.283C>T [p.Arg95Cys]). Using in vitro assays, we show that this missense mutation acts as a loss-of-function allele by impairing MYF5 DNA binding and nuclear localization. We performed whole-genome sequencing in one affected individual with the frameshift mutation and did not identify additional rare variants in the haploidentical region that might account for differences in severity among the families. These data support the direct role of MYF5 in rib, spine, and extraocular muscle formation in humans. Myogenesis is determined by MYOD, MYF5, and MYF6 basic helix-loop-helix (bHLH) transcription factors (TFs).1Rudnicki M.A. Schnegelsberg P.N. Stead R.H. Braun T. Arnold H.H. Jaenisch R. MyoD or Myf-5 is required for the formation of skeletal muscle.Cell. 1993; 75: 1351-1359Abstract Full Text PDF PubMed Scopus (1323) Google Scholar, 2Kassar-Duchossoy L. Gayraud-Morel B. Gomès D. Rocancourt D. Buckingham M. Shinin V. Tajbakhsh S. Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice.Nature. 2004; 431: 466-471Crossref PubMed Scopus (477) Google Scholar Mouse knockout studies have revealed that any one of these factors can compensate for the other two during skeletal muscle development, and muscles of the body, neck, jaw, and face develop as long as any one of the three factors is expressed. Likely because of this redundancy, mutations in these genes have not been convincingly demonstrated to cause human disease. By contrast, extraocular muscles (EOMs) are composed of striated fibers distinct from skeletal muscle3Porter J.D. Baker R.S. Muscles of a different ‘color’: the unusual properties of the extraocular muscles may predispose or protect them in neurogenic and myogenic disease.Neurology. 1996; 46: 30-37Crossref PubMed Scopus (131) Google Scholar and lack a Myf5/Myf6-independent pathway to activate myogenesis through Myod. Thus, EOMs fail to form in mice lacking expression of both Myf5 and Myf6 and are hypoplastic with loss of either factor alone.4Sambasivan R. Gayraud-Morel B. Dumas G. Cimper C. Paisant S. Kelly R.G. Tajbakhsh S. Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates.Dev. Cell. 2009; 16: 810-821Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar In addition, expression of Myf5 and Myf6 in the hypaxial myotome promotes rib and vertebral formation in the adjacent sclerotome though a non-cell-autonomous effect mediated by PDGF and FGF signaling,5Braun T. Rudnicki M.A. Arnold H.H. Jaenisch R. Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death.Cell. 1992; 71: 369-382Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 6Yoon J.K. Olson E.N. Arnold H.H. Wold B.J. Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.Dev. Biol. 1997; 188: 349-362Crossref PubMed Scopus (50) Google Scholar and mice lacking expression of both Myf5 and Myf6 can also develop rib and vertebral anomalies and die perinatally.2Kassar-Duchossoy L. Gayraud-Morel B. Gomès D. Rocancourt D. Buckingham M. Shinin V. Tajbakhsh S. Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice.Nature. 2004; 431: 466-471Crossref PubMed Scopus (477) Google Scholar, 5Braun T. Rudnicki M.A. Arnold H.H. Jaenisch R. Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death.Cell. 1992; 71: 369-382Abstract Full Text PDF PubMed Scopus (555) Google Scholar, 6Yoon J.K. Olson E.N. Arnold H.H. Wold B.J. Different MRF4 knockout alleles differentially disrupt Myf-5 expression: cis-regulatory interactions at the MRF4/Myf-5 locus.Dev. Biol. 1997; 188: 349-362Crossref PubMed Scopus (50) Google Scholar, 7Tajbakhsh S. Rocancourt D. Buckingham M. Muscle progenitor cells failing to respond to positional cues adopt non-myogenic fates in myf-5 null mice.Nature. 1996; 384: 266-270Crossref PubMed Scopus (247) Google Scholar, 8Tallquist M.D. Weismann K.E. Hellström M. Soriano P. Early myotome specification regulates PDGFA expression and axial skeleton development.Development. 2000; 127: 5059-5070Crossref PubMed Google Scholar Errors in development of EOMs or their innervating motor neurons can result in congenital ophthalmoplegia.9Engle E.C. Human genetic disorders of axon guidance.Cold Spring Harb. Perspect. Biol. 2010; 2: a001784Crossref PubMed Scopus (149) Google Scholar, 10Nugent A.A. Kolpak A.L. Engle E.C. Human disorders of axon guidance.Curr. Opin. Neurobiol. 2012; 22: 837-843Crossref PubMed Scopus (63) Google Scholar, 11Jones K.J. North K.N. External ophthalmoplegia in neuromuscular disorders: case report and review of the literature.Neuromuscul. Disord. 1997; 7: 143-151Abstract Full Text PDF PubMed Scopus (13) Google Scholar In an effort to identify genetic causes of congenital ophthalmoplegia, we enrolled affected probands and their family members into an ongoing genetic study approved by the institutional review board of Boston Children’s Hospital. Written informed consent was obtained from participating family members or their parents, and investigations were conducted in accordance with the principles of the Declaration of Helsinki. Clinical data were ascertained through examinations and review of medical records, and DNA was extracted from blood or saliva from all participating family members for genetic analysis. Here, we report that members of three families affected with congenital ophthalmoplegia and scoliosis, with documented vertebral and rib anomalies in two, harbor homozygous recessive loss-of-function mutations in MYF5 (MIM: 159990) and define a previously undescribed human disorder we refer to as the “MYF5 syndrome” (Table 1).Table 1Clinical Features of MYF5 SyndromePedigreeALOALOCHOBXBIndividual ID12345SexFMMFFGeographic locationTurkey (ALO:II-1)Turkey (ALO:II-2)Turkey (CHO:II-2)Yemen (BX:IV-4)Yemen (BX:IV-6)Homozygous MYF5 variantc.23_32delAGTTCTCACCc.23_32delAGTTCTCACCc.23_32delAGTTCTCACCc.283C>Tc.283C>TMYF5 proteinp.Gln8Leufs∗86p.Gln8Leufs∗86p.Gln8Leufs∗86p.Arg95Cysp.Arg95CysPhenotypeStrabismusXT++, HT R+XT++HT++XT+++, HT L+XT +External ophthalmoplegiayesyesyesyesyesForced ductionsNDNDNDNDpositivePtosis++R; +L++R; +L+++no+LTorticollisyesyesyes?possiblyScoliosisyesyesyesyes?Motor developmentnormalnormaldelayednormalnormalHypotonia/weaknessnononononoCognitionnormalnormalnormalnormalnormalImagingOrbital MRINDNDEOMs hypoplastic to absentNDNDRibsdysmorphic, hypoplastic, fusion anomalies, fused sternumdysmorphic, hypoplastic, shortened, fusion anomaliesdysmorphic, hypoplastic, shortened and missing ribs, fusion anomalies, pseudarthrosisNDNDSpinecervical scoliosis and fusions, clivus malformations, basilar invagination, narrow disc spacesNDcervical and thoracic scoliosis, cervical fusions, clivus malformations, basilar invagination, narrow disc spacesNDlumbar scoliosisAbbreviations: F, female; M, male; years, years; +, mild; ++, moderate; +++, severe, ND, no data; R, right; L, left; XT, exotropia; HT, hypotropia; ?, unknown. Open table in a new tab Abbreviations: F, female; M, male; years, years; +, mild; ++, moderate; +++, severe, ND, no data; R, right; L, left; XT, exotropia; HT, hypotropia; ?, unknown. Pedigree BX is a previously published consanguineous Yemenite family12Traboulsi E.I. Lee B.A. Mousawi A. Khamis A.R. Engle E.C. Evidence of genetic heterogeneity in autosomal recessive congenital fibrosis of the extraocular muscles.Am. J. Ophthalmol. 2000; 129: 658-662Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar with two affected (IV-4 and IV-6) and six unaffected siblings born to unaffected parents, all of whom participated in the research study (Figure 1A). The affected daughters were examined at 15 months (IV-6) and 4 years (IV-4) of age,12Traboulsi E.I. Lee B.A. Mousawi A. Khamis A.R. Engle E.C. Evidence of genetic heterogeneity in autosomal recessive congenital fibrosis of the extraocular muscles.Am. J. Ophthalmol. 2000; 129: 658-662Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar and again as young adults. They had congenital bilateral nonprogressive external ophthalmoplegia with mild left-sided ptosis in IV-6 and absence of ptosis in IV-4. IV-4 developed scoliosis between age 4 and young adulthood. In photos, IV-6 was noted to have uneven shoulders suggestive of scoliosis and/or torticollis. Family history was noncontributory. On examination in primary position, IV-6 had a large bilateral exotropia and left hypotropia, while IV-4 had a small exotropia. Both had absence of elevation, marked limitation of adduction and depression, and moderate limitation of abduction. IV-6 underwent forced duction testing that revealed restriction to passive eye movement. Their cranial nerve exams were otherwise reported to be normal, as were their intellectual, social, and motor development. Further details of their general physical examinations were not available. Pedigree ALO is a consanguineous Turkish pedigree with two affected children (II-1 and II-2) and one unaffected healthy sibling (II-4) born to unaffected first-cousin parents, all of whom participated in the research study (Figure 1A). The parents also had one fetal loss of unknown gestational age and affection status (II-3). The affected children were born with nonprogressive ptosis and ophthalmoplegia and developed torticollis and scoliosis by 6 years of age. Both had normal intellectual, social, and motor development. Family history was noncontributory. On examination at ages 19 (II-1) and 9 (II-2) years, both had bilateral ptosis (R > L) and their primary eye positions were exotropic (Figures 1B and 1H). Residual eye movements were limited and no aberrant eye movements were noted. Cranial nerve testing was otherwise normal, and both had normal pupillary responses and full facial, jaw, neck, and skeletal muscle strength. Muscle tone, reflexes, coordination, sensation, and gait were also normal. No limb anomalies or dysmorphologies were noted. Family CHO is a Turkish pedigree with one affected (II-2) and one unaffected (II-1) son born to unaffected parents who originated from the same village as each other and family ALO, but were not known to be related. Family history was noncontributory. The affected son was born after an uncomplicated pregnancy and delivery. He had nonprogressive bilateral ptosis and ophthalmoplegia from birth. He was noted to have torticollis in infancy and pectus carinatum as a child, and both progressed with age. He had normal intellectual and social development and mildly delayed motor milestones. When he came to medical attention at age 16, he had bilateral ptosis (R > L) with a chin-up and rotated head position, limited eye movements, down-slanting palpebral fissures, torticollis, narrow shoulders, thoracic scoliosis, and truncal obesity (Figures 1L and 1M). No limb anomalies or other dysmorphologies were noted, and his karyotype was normal. CHO II-2 underwent magnetic resonance imaging (MRI) of the brain (which was normal) and of the orbits that revealed absence of the extraocular muscles (Figures 1N and S1E). Frontal radiographs and CT images of ALO II-1, ALO II-2, and CHO II-2 revealed a spectrum of dysmorphic ribs with hypoplasia, fusion anomalies, pseudarthrosis, missing ribs, and failure of some remaining ribs to extend anteriorly toward the sternum (Figures 1D, 1E, 1J, 1K, and 1O–1Q). The sternum of ALO II-1 was fused (Figure 1F). In addition, spine CT images of ALO II-1 and CHO II-2 revealed scoliosis, narrowed thoracic disc spaces, variable fusion anomalies of cervical and thoracic vertebral bodies and posterior elements, abnormally shaped and subluxed cervical facets, malformations of the clivus with basilar invagination and clefting of the anterior arch of C1, and abnormally shaped thoracic vertebrae. CHO II-2 also demonstrated atlantooccipital fusion (Figures 1G, 1R, and S1). BX IV-4 underwent MRI as a young adult; these images were not available for review, but were reported to reveal 25 degrees of dextroscoliosis centered at L1-L2 and mild nonspecific degenerative changes in the lower lumbar spine. No other imaging of the affected members of BX was performed. Genome-wide linkage analysis of pedigree BX, generated from Affymetrix Human Mapping 10K SNP array (ThermoFisher) data assuming recessive inheritance and a disease allele frequency of 0.001, yielded the maximum potential lod score of 2.1 at loci spanning 69 Mb and 1.4 Mb on chromosomes 12p12.1–12q21.31 and 18q12.3, respectively, and haplotype analysis revealed homozygosity across both regions (Figure 2A). Homozygosity mapping using Omni2.5-8 SNP array data from Illumina was performed for pedigrees ALO and CHO. Analysis of ALO revealed a 72 Mb region of homozygosity on chromosome 12p12.1–12q22 and a 50 Mb region on chromosome 17p13.2-17q22. Analysis of pedigree CHO revealed a 48 Mb region of homozygosity on chromosome 12q13.12–12q22, a 53 Mb region on 17p13.3–17q22 that overlapped with ALO but had an alternate haplotype, and 59 and 119 Mb regions on chromosome 3p25.3–3p14.1 and 8p21.1–8q24.3, respectively (Figure 2A). The regions of homozygosity on chromosome 12 overlapped in the three families for 42.6 Mb and, within this region, pedigrees ALO and CHO shared a 1.2 Mb region of haploidentity (chr12: 80,696,475–81,927,644) that encompasses PTPRQ (MIM: 613317), MYF5, MYF6 (MIM: 159991), LIN7A (MIM: 603380), ACSS3 (MIM: 614356), and portions of OTOGL (MIM: 614925) and PPFIA2 (MIM: 603143) (Figures 2B and 2C). To identify causal variants in the three pedigrees, whole-exome sequencing (WES) was performed on DNA from BX III-1, III-2, IV-4, and IV-6, ALO I-2, II-1, and II-2, and CHO II-2. Libraries were prepared using Agilent SureSelect Human All Exon versions 4X, 5X, and 5+UTRs+MitoX for ALO, CHO, and BX, respectively, and sequenced on a HiSeq2000. Homozygous recessive variants of moderate to high impact and allele frequencies less than 0.01 were then identified using the seqr software platform (Table S1). Evaluation of WES data from pedigree BX revealed nine rare coding variants that were homozygous in the affected daughters and heterozygous in the parents (Table S1). Of these, six were located within the pedigree’s 69 Mb region of homozygosity on chromosome 12, including two of particular interest: STAC3 (MIM: 615521) (hg19, GenBank: NM_145064.2; c.322C>G [p.Arg108Gly]) and MYF5 (hg19, GenBank: NM_005593.2; c.283C>T [p.Arg95Cys], M2), which are located outside of and within the 1.2 Mb region of shared haploidentity between ALO and CHO, respectively. A STAC3 biallelic missense (p.Trp284Ser) and truncating variants have been reported to cause a myopathic phenotype in four families distinct from BX: affected individuals had ptosis but not significant restriction of eye movements, and had facial weakness, generalized hypotonia and weakness, and susceptibility to malignant hyperthermia with or without cleft palate, scoliosis, and joint contractures.13Grzybowski M. Schänzer A. Pepler A. Heller C. Neubauer B.A. Hahn A. Novel STAC3 Mutations in the first non-Amerindian patient with Native American myopathy.Neuropediatrics. 2017; 48: 451-455Crossref PubMed Scopus (20) Google Scholar, 14Horstick E.J. Linsley J.W. Dowling J.J. Hauser M.A. McDonald K.K. Ashley-Koch A. Saint-Amant L. Satish A. Cui W.W. Zhou W. et al.Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy.Nat. Commun. 2013; 4: 1952Crossref PubMed Scopus (166) Google Scholar, 15Telegrafi A. Webb B.D. Robbins S.M. Speck-Martins C.E. FitzPatrick D. Fleming L. Redett R. Dufke A. Houge G. van Harssel J.J.T. et al.Moebius Syndrome Research ConsortiumIdentification of STAC3 variants in non-Native American families with overlapping features of Carey-Fineman-Ziter syndrome and Moebius syndrome.Am. J. Med. Genet. A. 2017; 173: 2763-2771Crossref PubMed Scopus (20) Google Scholar By contrast, mutations in MYF5 had not been reported, and the p.Arg95Cys variant had a CADD score of 34 and was predicted to be disease causing by MutationTaster, damaging by Fathmm, and probably damaging by PolyPhen. Evaluation of WES data from pedigree ALO revealed ten rare coding variants that were homozygous in the two affected children and heterozygous in the mother (Table S1). Of these, nine were located within the homozygous region on chromosome 12, of which only one, a 10 base pair deletion in MYF5 (hg19, GenBank: NM_005593.2; c.23_32delAGTTCTCACC [p.Gln8Leufs∗86], M1) was in the haploidentical region shared with pedigree CHO. None was located in the homozygous region on chromosome 17. Evaluation of WES data from the affected child in pedigree CHO revealed only six rare homozygous coding variants within the regions of homozygosity: one on chromosome 1, four on chromosome 3, and one on chromosome 12 (Table S1). The single homozygous variant on chromosome 12 fell within the 1.2 Mb region of shared haploidentity and was the identical MYF5 10 base pair deletion harbored by the affected members of ALO (Figures 2D–2F). To exclude the presence of other homozygous structural variations (SVs) or non-coding variants not reported by exome sequencing within the haploidentical region of CHO and ALO, we performed PCR-free whole-genome sequencing in individual CHO-1 (30× average coverage, New York Genome Center, NY). After careful annotation of each non-coding variant and SV, we did not identify any novel homozygous rare events (cumulative MAF < 0.01) that might be linked to the observed phenotype (Table S2). Both the MYF5 deletion and MYF5 missense variant were absent from the 1000 Genomes, GnomAD, and ExAC databases, and Sanger sequencing confirmed their appropriate segregation within each family (Figures 1A and S2; forward primer, 5′-TTACCGGAGCGACAGACTAG-3′; reverse primer, 5′-CCACACCAGGTTAAATGAGGT-3′). No other MYF5 variants or polymorphisms were present in any of the family members, and no other genes harbored rare coding variants in more than one family (Table S1). We also screened MYF5 sequence in probands from 28 recessive, 26 dominant, and 51 simplex unsolved pedigrees with congenital ophthalmoplegia and did not identify rare homozygous or compound heterozygous variants. While some probands had been diagnosed with “congenital fibrosis of the extraocular muscles” (CFEOM) similar to pedigree BX, none were known to have rib anomalies. MYF5 contains a basic domain that interacts with DNA and a helix-loop-helix domain necessary for homo- and hetero-dimerization (Figure 3A).16Braun T. Winter B. Bober E. Arnold H.H. Transcriptional activation domain of the muscle-specific gene-regulatory protein myf5.Nature. 1990; 346: 663-665Crossref PubMed Scopus (89) Google Scholar The frameshift deletion identified in families ALO and CHO is predicted to be the target of nonsense-mediated decay or, in case of escape, to generate a protein product containing only the first 7 MYF5 amino acids followed by 85 altered residues prior to a new stop codon (p.Gln8Leufs∗86), thus resulting in complete loss of MYF5 function (Figures 2F and S3). The missense mutation identified in family BX is predicted to alter an arginine at amino acid position 95, located at the end of the MYF5 basic domain. MYF5 Arg95 is highly conserved in other species (Figure 2G) as well as in 104 of the 113 human Myc-type bHLH TFs (Prosite: PS50888; Figure 3B), supporting its functional importance for TF binding activity. Moreover, missense mutations that alter this conserved arginine in the bHLH TF TWIST1 cause Saethre-Chotzen syndrome,17Kress W. Schropp C. Lieb G. Petersen B. Büsse-Ratzka M. Kunz J. Reinhart E. Schäfer W.D. Sold J. Hoppe F. et al.Saethre-Chotzen syndrome caused by TWIST 1 gene mutations: functional differentiation from Muenke coronal synostosis syndrome.Eur. J. Hum. Genet. 2006; 14: 39-48Crossref PubMed Scopus (99) Google Scholar, 18de Heer I.M. de Klein A. van den Ouweland A.M. Vermeij-Keers C. Wouters C.H. Vaandrager J.M. Hovius S.E. Hoogeboom J.M. Clinical and genetic analysis of patients with Saethre-Chotzen syndrome.Plast. Reconstr. Surg. 2005; 115 (discussion 1903–1895): 1894-1902Crossref PubMed Scopus (50) Google Scholar, 19Gripp K.W. Zackai E.H. Stolle C.A. Mutations in the human TWIST gene.Hum. Mutat. 2000; 15: 150-155Crossref PubMed Scopus (90) Google Scholar and amino acid substitutions elsewhere in the basic domain of bHLH TF proteins are associated with additional human disorders.20Malecki M.T. Jhala U.S. Antonellis A. Fields L. Doria A. Orban T. Saad M. Warram J.H. Montminy M. Krolewski A.S. Mutations in NEUROD1 are associated with the development of type 2 diabetes mellitus.Nat. Genet. 1999; 23: 323-328Crossref PubMed Scopus (510) Google Scholar, 21El Ghouzzi V. Legeai-Mallet L. Benoist-Lasselin C. Lajeunie E. Renier D. Munnich A. Bonaventure J. Mutations in the basic domain and the loop-helix II junction of TWIST abolish DNA binding in Saethre-Chotzen syndrome.FEBS Lett. 2001; 492: 112-118Crossref PubMed Scopus (53) Google Scholar, 22Marchegiani S. Davis T. Tessadori F. van Haaften G. Brancati F. Hoischen A. Huang H. Valkanas E. Pusey B. Schanze D. et al.Recurrent mutations in the basic domain of TWIST2 cause ablepharon macrostomia and Barber-Say syndromes.Am. J. Hum. Genet. 2015; 97: 99-110Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 23El Ghouzzi V. Legeai-Mallet L. Aresta S. Benoist C. Munnich A. de Gunzburg J. Bonaventure J. Saethre-Chotzen mutations cause TWIST protein degradation or impaired nuclear location.Hum. Mol. Genet. 2000; 9: 813-819Crossref PubMed Scopus (78) Google Scholar Consistent with these data, in silico modeling of the Arg95 amino acid using PyMOL and the MYOD structure24Ma P.C. Rould M.A. Weintraub H. Pabo C.O. Crystal structure of MyoD bHLH domain-DNA complex: perspectives on DNA recognition and implications for transcriptional activation.Cell. 1994; 77: 451-459Abstract Full Text PDF PubMed Scopus (399) Google Scholar as a backbone highlights the direct interaction of this arginine residue with DNA, and loss of this DNA interaction when a cysteine is substituted for the arginine (Figure 3A). To assess whether the MYF5 p.Arg95Cys amino acid substitution impairs MYF5 transcriptional function, we obtained a MYF5 expressing plasmid with a C-terminal Myc-FLAG tag (MYF5WT-FLAG, RC210156, OriGene) and mutagenized the plasmid to substitute Arg95 with a cysteine (MYF5R95C-FLAG). Mutagenesis was performed by GENEWIZ and confirmed by qualitative restriction digestion and sequencing. Both MYF5WT-FLAG and MYF5R95C-FLAG constructs were co-transfected with a previously generated myogenin-luciferase reporter25Parker M.H. Perry R.L.S. Fauteux M.C. Berkes C.A. Rudnicki M.A. MyoD synergizes with the E-protein HEB β to induce myogenic differentiation.Mol. Cell. Biol. 2006; 26: 5771-5783Crossref PubMed Scopus (51) Google Scholar in C3H10T1/2 cells (clone 8, ATCC CCL-226). Dual promoter luciferase assay (Promega) was then used to evaluate the relative activation of the luciferase compared to Renilla activation (Figure 3C). Cells transfected with MYF5WT-FLAG plasmid strongly activated the luciferase reporter while, by contrast, very minimal activation was observed in cells transfected with the same amount of MYF5R95C-FLAG plasmid, despite higher protein level of the latter as shown by western blot. These data establish that the mutant protein is deficient in promoting transcriptional activation in vitro. To function as a TF, MYF5 must first translocate to the nucleus. MYF5 has a nuclear localization signal (NLS) in its basic domain26Vandromme M. Cavadore J.C. Bonnieu A. Froeschlé A. Lamb N. Fernandez A. Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently.Proc. Natl. Acad. Sci. USA. 1995; 92: 4646-4650Crossref PubMed Scopus (55) Google Scholar, 27Wang Y.H. Chen Y.H. Lu J.H. Tsai H.J. A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor myf5 into nucleolus.DNA Cell Biol. 2005; 24: 651-660Crossref PubMed Scopus (14) Google Scholar and, in zebrafish, a 23 amino acid sequence from the basic domain of Myf5 is sufficient and necessary to translocate a GFP protein to the nucleus and the nucleolus.27Wang Y.H. Chen Y.H. Lu J.H. Tsai H.J. A 23-amino acid motif spanning the basic domain targets zebrafish myogenic regulatory factor myf5 into nucleolus.DNA Cell Biol. 2005; 24: 651-660Crossref PubMed Scopus (14) Google Scholar Remarkably, Arg95 not only interacts with DNA but also is one of the residues within this 23 amino acid bipartite NLS ([KRKASTVDRRRAATMRERRRLKK] in which the two NLS are underlined and Arg96 is italicized). To investigate whether p.Arg95Cys impairs nuclear localization as well as transcription, we first performed nuclear fractionation of transfected C3H10T1/2 cells and observed FLAG signal in the nuclear fraction of cells transfected with MYF5WT-FLAG but not MYF5R95C-FLAG (Figure 3D). Next, we examined and quantified the presence of the overexpressed proteins in the nucleus of fixed C3H10T1/2 cells (Figures 3E–3G). Both constructs showed cytoplasmic localization, but MYF5WT-FLAG localized to the nucleus more often (average ∼20% of nuclei) than MYF5R95C-FLAG (<10% of nuclei). While quantification of MYF5R95C-FLAG nuclear localization was not significantly different than that of the empty plasmid, we did observe MYF5R95C-FLAG in the nucleus of transfected cells (Figure 3F), suggesting that nuclear transport may have not been completely abolished by the mutation. Taken together, these data support the pathogenicity of p.Arg95Cys by demonstrating both impaired nuclear localization and transcriptional activity of the mutant MYF5 protein. Notably, Arg95 is located at the edge of the basic domain adjacent to helix 1 of the MYF5 protein (Figure 3B), and amino acid substitutions of resides in helix 1 of TWIST1 were reported to similarly both reduce nuclear localization and abolish DNA binding of the TWIST1 protein.21El Ghouzzi V. Legeai-Mallet L. Benoist-Lasselin C. Lajeunie E. Renier D. Munnich A. Bonaventure J. Mutations in the basic domain and the loop-helix II junction of TWIST abolish DNA binding in Saethre-Chotzen syndrome.FEBS Lett. 2001; 492: 112-118Crossref PubMed Scopus (53) Google Scholar, 23El Ghouzzi V. Legeai-Mallet L. Aresta S. Benoist C. Munnich A. de Gunzburg J. Bonaventure J. Saethre-Chotzen mutations cause TWIST protein degradation or impaired nuclear location.Hum. Mol. Genet. 2000; 9: 813-819Crossref PubMed Scopus (78) Google Scholar The pathogenicity of the human MYF5 variants harbored by these three families is supported by the remarkable phenotypic similarities of the human syndrome to that reported in a series of Myf5−/− mouse models generated using different constructs and cutting sites (Figure S4, Table S3). The reported phenotypes of Myf5−/− mice range from absent EOMs4Sambasivan R. Gayraud-Morel B. Dumas G. Cimper C. Paisant S. Kelly R.G. Tajbakhsh S. Distinct regulatory cascades govern extraocular and pharyngeal arch muscle progenitor cell fates.Dev. Cell. 2009; 16: 810-821Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 28Michalak S.M. Whitman M.C. Park J.G. Tischfield M.A. Nguyen E.H. Engle E.C. Ocular motor nerve development in the presence and absence of extraocular muscle.Invest. Ophthalmol. Vis. Sci. 2017; 58: 2388-2396PubMed Google Scholar and absent ribs5Braun T. Rudnicki M.A. Arnold H.H. Jaenisch R. Targeted inactivation of the muscle regulatory gene Myf-5 results in abnorm
Referência(s)