A Restricted Spectrum of Mutations in the SMAD4 Tumor-Suppressor Gene Underlies Myhre Syndrome

Abstract

Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor β and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth. Myhre syndrome is a developmental disorder characterized by reduced growth, generalized muscular hypertrophy, facial dysmorphism, deafness, cognitive deficits, joint stiffness, and skeletal anomalies. Here, by performing exome sequencing of a single affected individual and coupling the results to a hypothesis-driven filtering strategy, we establish that heterozygous mutations in SMAD4, which encodes for a transducer mediating transforming growth factor β and bone morphogenetic protein signaling branches, underlie this rare Mendelian trait. Two recurrent de novo SMAD4 mutations were identified in eight unrelated subjects. Both mutations were missense changes altering Ile500 within the evolutionary conserved MAD homology 2 domain, a well known mutational hot spot in malignancies. Structural analyses suggest that the substituted residues are likely to perturb the binding properties of the mutant protein to signaling partners. Although SMAD4 has been established as a tumor suppressor gene somatically mutated in pancreatic, gastrointestinal, and skin cancers, and germline loss-of-function lesions and deletions of this gene have been documented to cause disorders that predispose individuals to gastrointestinal cancer and vascular dysplasias, the present report identifies a previously unrecognized class of mutations in the gene with profound impact on development and growth. Myhre syndrome (MIM 139210) is the eponym for the disorder originally described 30 years ago in two unrelated males exhibiting distinctive dysmorphic features, short stature, generalized muscular hypertrophy, cryptorchidism, limitation of joints, deafness, and intellectual disability.1Myhre S.A. Ruvalcaba R.H.A. Graham C.B. A new growth deficiency syndrome.Clin. Genet. 1981; 20: 1-5Crossref PubMed Scopus (64) Google Scholar Myhre syndrome is a rare condition with less than 30 affected individuals, mostly males, reported to date.2Burglen L. Héron D. Moerman A. Dieux-Coeslier A. Bourguignon J.P. Bachy A. Carel J.C. Cormier-Daire V. Manouvrier S. Verloes A. Myhre syndrome: New reports, review, and differential diagnosis.J. Med. Genet. 2003; 40: 546-551Crossref PubMed Scopus (36) Google Scholar, 3Lopez-Cardona M.G. Garcia-Cruz D. Garcia-Ortiz J.E. Davalos N.O. Feria-Velasco A. Rodriguez-Rojas L.X. Garcia-Cruz M.O. Figuera-Villanueva L.E. Stephens A. Larios-Arceo F. Sanchez-Corona J. Second female case of Myhre syndrome.Clin. Dysmorphol. 2004; 13: 91-94Crossref PubMed Scopus (21) Google Scholar, 4van Steensel M.A.M. Vreeburg M. Steijlen P.M. de Die-Smulders C. Myhre syndrome in a female with previously undescribed symptoms: Further delineation of the phenotype.Am. J. Med. Genet. A. 2005; 139A: 127-130Crossref PubMed Scopus (18) Google Scholar, 5Rulli I. Ferrero G.B. Belligni E. Delmonaco A.G. Defilippi C. Silengo M. Myhre's syndrome in a girl with normal intelligence.Am. J. Med. Genet. A. 2005; 134A: 100-102Crossref PubMed Scopus (15) Google Scholar, 6Becerra-Solano L.E. Díaz-Rodriguez M. Nastasi-Catanese J.A. Toscano-Flores J.J. Bañuelos-Robles O. Figuera L.E. Matute E. de Lourdes Ramírez-Dueñas M. The fifth female patient with Myhre syndrome: Further delineation.Clin. Dysmorphol. 2008; 17: 113-117Crossref PubMed Scopus (13) Google Scholar, 7Bachmann-Gagescu R. Hisama F.M. Yuen A.L. Myhre syndrome with ataxia and cerebellar Atrophy.Clin. Dysmorphol. 2011; 20: 156-159Crossref PubMed Scopus (4) Google Scholar, 8McGowan R. Gulati R. McHenry P. Cooke A. Butler S. Keng W.T. Murday V. Whiteford M. Dikkers F.G. Sikkema-Raddatz B. et al.Clinical features and respiratory complications in Myhre syndrome.Eur. J. Med. Genet. 2011; 54: e553-e559PubMed Google Scholar Based on the available records, cardinal features of Myhre syndrome include reduced growth, generalized muscular hypertrophy, variable cognitive deficits, a recognizable pattern of facial features (i.e., narrow palpebral fissures, midface hypoplasia, a small mouth with a thin upper lip, and prognathism), and skeletal anomalies (i.e., thick skull bones, platispondyly, large vertebral pedicles, broad ribs, hypoplastic iliac wings, and brachydactyly), joint stiffness, and deafness. Additional recurrent features include a cleft lip and/or palate, congenital heart defects, stiff skin, cryptorchidism, hypertension, and ocular defects. Myhre syndrome has clinical overlap with growth retardation, ocular abnormalities, microcephaly, brachydactyly, and oligophrenia (GOMBO) syndrome (MIM 233270); laryngotracheal stenosis, progressive, with short stature and arthropathy syndrome (LAPS) syndrome (MIM 603391); geleophysic dysplasia (GPHYSD1 [MIM 231050] and GPHYSD2 [MIM 614185]); acromicric dysplasia (ACMICD [MIM 102370]); Weill-Marchesani syndrome (WMS1 [MIM 277600] and WMS2 [MIM 608328]); and Moore-Federman syndrome (MIM 127200); genetic relatedness among these disorders has been suggested.9Bottani A. Verloes A. Myhre-GOMBO syndrome: Possible lumping of two “old” new syndromes.Am. J. Med. Genet. 1995; 59: 523-524Crossref PubMed Scopus (9) Google Scholar, 10Figuera L.E. Geleophysic dysplasia vs. Myhre syndrome.Am. J. Med. Genet. 1996; 65: 361-362Crossref PubMed Google Scholar, 11Lindor N.M. LAPS syndrome and Myhre syndrome: Two disorders or one?.Am. J. Med. Genet. A. 2009; 149A: 798-799Crossref PubMed Scopus (8) Google Scholar Although the molecular basis for some of these clinically related conditions is unknown, increasing evidence supports the relevant role of transforming growth factor β (TGFβ) signaling dysregulation in the pathogenesis of GPHYSD, ACMICD, and WMS.12Le Goff C. Morice-Picard F. Dagoneau N. Wang L.W. Perrot C. Crow Y.J. Bauer F. Flori E. Prost-Squarcioni C. Krakow D. et al.ADAMTSL2 mutations in geleophysic dysplasia demonstrate a role for ADAMTS-like proteins in TGF-beta bioavailability regulation.Nat. Genet. 2008; 40: 1119-1123Crossref PubMed Scopus (169) Google Scholar, 13Le Goff C. Mahaut C. Wang L.W. Allali S. Abhyankar A. Jensen S. Zylberberg L. Collod-Beroud G. Bonnet D. Alanay Y. et al.Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias.Am. J. Hum. Genet. 2011; 89: 7-14Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar Here, an exome sequencing-based approach was used to identify similar to mothers against decapentaplegic (SMAD) family member 4 (SMAD4 [MIM 600993]), previously known as mothers against decapentaplegic homolog 4 (Drosophila) (MADH4), which encodes a protein with a crucial role in TGFβ/bone morphogenetic protein (BMP) signal transduction, as the gene mutated in Myhre syndrome. Eight subjects with clinical features fitting Myhre syndrome were included in the study (Table 1 and Figure 1A ). Clinical data and biological material collection and storage were attained from the participating families in accordance with the ethical standards of the institutional review boards (Ospedale “Bambino Gesù”, Rome; Università di Torino, Turin; Arcispedale Santa Maria Nuova, Reggio Emilia; Ospedale Microcitemico, Cagliari; Università “Federico II”, Naples) and after written informed consent was secured. Genomic DNA was isolated from skin fibroblasts, hair bulb cells, peripheral blood leukocytes, saliva, and/or buccal mucosal epithelial cells with standard protocols. In all subjects, karyotype and subtelomeric fluorescence in situ hybridization (FISH) analyses provided normal results. High-resolution array- comparative genomic hybridization (CGH) or SNP array analyses were performed on three out of the eight subjects and did not disclose any disease-causing genomic rearrangement.Table 1Clinical Features of the Affected Subjects Included in the StudyFeaturesSubjectsOBG_01TO_01TO_02TO_03RE_01NA_01NA_02CA_01Nucleotide substitution1498A>G1499T>C1498A>G1498A>G1499T>C1498A>G1498A>G1499T>CPredicted amino acid changeIle500ValIle500ThrIle500ValIle500ValIle500ThrIle500ValIle500ValIle500ThrPaternal age at birth (years)3534383631332836Length at birth (centile)n.a.3rd<3rd3rd<3rdn.a.n.a.<3rdWeight at birth (centile)<3rd3rd<3rd3rd<3rd<3rdn.a.<3rdHead circumference at birth (centile)n.a.3rd<3rdn.a.<3rdn.a.n.a.n.a.Age at examination (years)42134.52613143413GendermalemalefemalefemalemalemalefemalemaleHeight (centile)<3rd<3rd<3rd<3rd<3rd<3rd<3rd<3rdObesity+−−++++−Macrocephaly−+−−+−−−Facial FeaturesNarrow palpebral fissures++++++++Midfacial hypoplasia++++++++Short philtrum++++++++Narrow mouth and/or thin upper lip+++++−−+Cleft lip and/or palate−+aUnilateral cleft lip.+bSubmucous cleft palate.+−−−−Prognathism++++++++Ocular AnomaliesRefractive errors+cAstigmatism.+−+−+−−Cataract−−−−−n.a.−+Hearing loss+dSensorineural deafness.+eMixed hearing loss.+fConductive.+eMixed hearing loss.+fConductive.+eMixed hearing loss.+eMixed hearing loss.+dSensorineural deafness.Cognitive impairmentmildmoderate+gDelayed psychomotor and language development.−hPsychological and behavioral problems.+gDelayed psychomotor and language development.+++Autistic behavior−−−−−+−+Muscular hypertrophy++++++++Joint stiffness++++++++Skeletal AnomaliesThick calvarium++++++++Hypoplastic iliac wings++++++++Platispondyly++++−−++Large vertebral pedicles+−n.a.+++++Fused vertebrae−−n.a.−−−−−Broad Ribs−+−++−−+Brachycamptodactyly++++++++Congenital heart defects+iRight ventricular hypertrophy.−+jVentricular and atrial septal defects, patent ductus arteriosus.+kAortic valve stenosis.+lAortic coarctation.−+kAortic valve stenosis.+mPatent ductus arteriosus.Cryptorchidism−+−+−Hypertension+−−−−−−+Skin AnomaliesStiff skin++−++++−Thick skin−+−+++++Other features−+nBenign mesencephalic glioma, fibrous dysplasia of the maxilla.+oHirschprung disease diagnosed at birth.−+pPericarditis with cardiac tamponade.−+qSecondary amennorrea.−n.a. is used as an abbreviation for data not available. Subjects TO_3 and NA_01 were previously reported by Rulli et al.5Rulli I. Ferrero G.B. Belligni E. Delmonaco A.G. Defilippi C. Silengo M. Myhre's syndrome in a girl with normal intelligence.Am. J. Med. Genet. A. 2005; 134A: 100-102Crossref PubMed Scopus (15) Google Scholar and Titomanlio et al.45Titomanlio L. Marzano M.G. Rossi E. D'Armiento M. De Brasi D. Vega G.R. Andreucci M.V. Orsini A.V. Santoro L. Sebastio G. Case of Myhre syndrome with autism and peculiar skin histological findings.Am. J. Med. Genet. 2001; 103: 163-165Crossref PubMed Scopus (30) Google Scholara Unilateral cleft lip.b Submucous cleft palate.c Astigmatism.d Sensorineural deafness.e Mixed hearing loss.f Conductive.g Delayed psychomotor and language development.h Psychological and behavioral problems.i Right ventricular hypertrophy.j Ventricular and atrial septal defects, patent ductus arteriosus.k Aortic valve stenosis.l Aortic coarctation.m Patent ductus arteriosus.n Benign mesencephalic glioma, fibrous dysplasia of the maxilla.o Hirschprung disease diagnosed at birth.p Pericarditis with cardiac tamponade.q Secondary amennorrea. Open table in a new tab n.a. is used as an abbreviation for data not available. Subjects TO_3 and NA_01 were previously reported by Rulli et al.5Rulli I. Ferrero G.B. Belligni E. Delmonaco A.G. Defilippi C. Silengo M. Myhre's syndrome in a girl with normal intelligence.Am. J. Med. Genet. A. 2005; 134A: 100-102Crossref PubMed Scopus (15) Google Scholar and Titomanlio et al.45Titomanlio L. Marzano M.G. Rossi E. D'Armiento M. De Brasi D. Vega G.R. Andreucci M.V. Orsini A.V. Santoro L. Sebastio G. Case of Myhre syndrome with autism and peculiar skin histological findings.Am. J. Med. Genet. 2001; 103: 163-165Crossref PubMed Scopus (30) Google Scholar Targeted enrichment and parallel sequencing experiments were performed on genomic DNA extracted from circulating leukocytes of a single affected subject (OBG_01). After fragment size selection, GS FLX Titanium adaptors (Roche) were ligated to the sheared genomic fragments to construct a shotgun sequencing library that was hybridized with a Sequence Capture Human Exome 2.1M Array v.2 (NimbleGen). Captured fragments were then amplified through emPCR and sequenced with Titanium reagents in a two-lane gasket PicoTiterPlate device (70X75) with a GS FLX instrument (Roche 454). Image analysis and base calling were performed with the GS FLX pipeline software (version 2.5.3). Approximately 1.2 Gb of genomic sequence from two GS FLX runs was obtained. The average read length was 380 bp, and fragment sizes ranged from 40 to 1,100 bp. Sequencing data were initially processed with the GSMapper software package v.2.5.3 (Roche), and high-quality sequencing reads were aligned to the human genome reference sequence (USCS hg19, GRCh37). Ninety-four percent of sequence reads mapped to the human reference genome. Targeted sequence coverage was 98%, and there was an average sequence depth of 10×. Sequence alignment and functional annotation of the identified data were with A of for genomic PubMed Scopus Google Scholar or extracted from reference with was performed with a of with the GSMapper in the of a gene of the against the changes mapped within and were and exome and aligned to the human genome the of and S. L. A. P. A and for missense PubMed Scopus Google Scholar and P. S. the of on protein the 2009; PubMed Scopus Google Scholar analyses a on protein and/or in of Based on the evidence that all affected subjects were to we for a de novo as the likely the trait. was by age in the of the reported as well as in subjects of the present (Table In Myhre the differential Based on the of TGFβ signaling to the Goff C. Morice-Picard F. Dagoneau N. Wang L.W. Perrot C. Crow Y.J. Bauer F. Flori E. Prost-Squarcioni C. Krakow D. et al.ADAMTSL2 mutations in geleophysic dysplasia demonstrate a role for ADAMTS-like proteins in TGF-beta bioavailability regulation.Nat. Genet. 2008; 40: 1119-1123Crossref PubMed Scopus (169) Google Scholar, 13Le Goff C. Mahaut C. Wang L.W. Allali S. Abhyankar A. Jensen S. Zylberberg L. Collod-Beroud G. Bonnet D. Alanay Y. et al.Mutations in the TGFβ binding-protein-like domain 5 of FBN1 are responsible for acromicric and geleophysic dysplasias.Am. J. Hum. Genet. 2011; 89: 7-14Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar we performed a filtering to gene to this signaling data analyses were performed with the and analysis of large gene 2009; PubMed Scopus Google Scholar an biological and at biological from large gene used the functional annotation table to annotation for gene and were related to TGFβ signaling of and approach the of three (MIM SMAD4 and (MIM were a F. A. gene at genomic 2011; PubMed Scopus Google Scholar that gene on analysis of the of from the clinical of Myhre syndrome short stature, joint stiffness, muscular hypertrophy, brachydactyly, deafness, and as biological and a for the of the and the for the gene the of available of and of a of [MIM [MIM [MIM [MIM [MIM [MIM [MIM SMAD4, and [MIM as the crucial role of SMAD4 in and relevant in and developmental L. The 2001; 2 PubMed Google Scholar we this gene as the sequencing of the relevant for the in the affected and sequencing of reference evidence for de novo The was documented in the skin as well as hair bulb and buccal mucosal epithelial against the of a available with the that this SMAD4 de novo was the the out to from of the two to SMAD4 as the gene mutated in Myhre syndrome. the of SMAD4 in Myhre the sequence of the gene was for mutations in DNA from circulating leukocytes from an unrelated affected subjects by sequencing with an and the Sequencing to and are in analysis identified heterozygous SMAD4 mutations in all the subjects, the the a amino acid at the was identified in the three individuals In all family DNA the de novo of and and/or epithelial cells were available for three of the subjects, and sequencing documented the the germline of these with these the and were not in than individuals by analysis and The Ile500 in the MAD homology 2 domain, a at the of the protein that is conserved among SMAD4 and and and known to the mutational hot spot for and germline lesions in and disorders C. developmental disorder to all in the Genet. 2003; PubMed Scopus Google Scholar The domain with a of and to and is for J. J. D. 2005; PubMed Scopus Google Scholar which is for signal through this signaling through of and that and which in with the then the to of A. S. in TGF-beta signal 2001; PubMed Google Scholar In the of the SMAD4 domain, Ile500 is to the protein is for of the is by and with the of the of residues and the of these residues are conserved in SMAD4 and and a binding for B. of a Full Text Full Text PDF PubMed Scopus Google Scholar The of this and a likely for and to SMAD4 binding to signaling at this as the by the identified The characterized by SMAD4 are from with (MIM or (MIM and evidence that these are SMAD4 and two The is characterized by three two of which of the SMAD4 A. G. S. De M. Structural basis of protein in TGF-beta 2004; Full Text Full Text PDF PubMed Scopus Google Scholar to the of the and A. G. S. De M. Structural basis of protein in TGF-beta 2004; Full Text Full Text PDF PubMed Scopus Google Scholar Ile500 is within of the two in and the residues in the and with residues of the Although a of missense mutations conserved residues of this which were documented or to SMAD4 and has been identified as and/or germline lesions in and of in and Human and Figure missense Ile500 has previously been reported in human the of the identified mutations on SMAD4 we performed that residues and which are to Ile500 and are in in a in the available SMAD4 in the of a is at the to an of the to from the is of Ile500 by or in SMAD4 is to with of the residues by a and/or is to perturb the of which of the that the Ile500 is for with signaling partners. the and have been to the of the B. J.J. de The and protein 2001; PubMed Scopus Google Scholar is that the identified mutations relevant of the mutant is of SMAD4 is not by is by L. L. of signaling in Full Text PDF PubMed Scopus Google Scholar which at and has been to the of the protein to a with in S. A. M. M. L. M. G. S. L. et a for 2009; Full Text Full Text PDF PubMed Scopus Google Scholar the basis of these that the by the Myhre amino acid SMAD4 and perturb signal by increasing the of clinical was for all of the subjects heterozygous for a SMAD4 (Table these subjects with a features of the short stature a recognizable facial generalized muscular hypertrophy, hearing short distinctive skeletal and joint were in all subjects with birth with Delayed psychomotor and/or language development and variable intellectual was documented in all subject and behavioral A of congenital heart and skin anomalies in the cleft lip and/or palate, and were less was documented in two subjects. of the eight subjects any vascular or of skin, pancreatic, or gastrointestinal malignancies. Benign mesencephalic and fibrous dysplasia of the were reported in individual (Table The TGFβ signaling a array of biological and in a J. signaling in growth and 103: Full Text Full Text PDF PubMed Scopus Google Scholar, V. J. The A. of Scholar, J. The transforming growth PubMed Scopus Google Scholar The TGFβ of than 30 related proteins to the two and S. of a developmental analyses of transforming growth family and signal PubMed Google Scholar TGFβ family a role in G. S.A. M.V. M. J. growth is for Genet. 1995; PubMed Scopus Google Scholar, I. R. have developmental that are with PubMed Google Scholar the of the family and developmental and skeletal morphogenetic proteins in Genet. 1996; PubMed Scopus Google Scholar, A. for are and have in and cardiac 1996; PubMed Google Scholar, N. Y. L. R. of in a by a gene related to 1996; PubMed Google Scholar, S. G. in heterozygous and Genet. PubMed Scopus Google Scholar the signaling SMAD4 a is as a to have J. J. D. 2005; PubMed Scopus Google Scholar Based on this crucial dysregulation of SMAD4 is to have which well with the cardinal features of Myhre syndrome that from of developmental by and skeletal muscular and and mutations in SMAD4 are known to cause syndrome [MIM and [MIM S. J.C. P. S. et al.Mutations in the gene in PubMed Scopus Google Scholar, E. S. G. E. A syndrome of and with mutations in 2004; Full Text Full Text PDF PubMed Scopus Google Scholar The is an disorder diagnosed within the of and characterized by of the gastrointestinal and an of gastrointestinal C. PubMed Scopus Google Scholar In the the disease is with vascular dysplasia of of the skin, and and of the and gastrointestinal which by the A clinical and J. Hum. Genet. 2009; 17: PubMed Scopus Google Scholar SMAD4 mutations are the gene and include and as well as and gene deletions and Figure C. J. B. C. C. J. J. et of SMAD4 mutations in and syndrome.Am. J. Med. Genet. A. PubMed Scopus Google Scholar a loss-of-function of with of SMAD4 or to lesions or gene deletions in of the gastrointestinal and skin In the and of Myhre mutations the that these genetic lesions have and on SMAD4 Although the of these is to the molecular in of growth and development by this previously unrecognized class of SMAD4 analysis that are likely to with the of the protein to signaling by SMAD4 binding and/or missense SMAD4 mutations were identified in all affected subjects with Myhre syndrome included in the supports the that Myhre syndrome is a at that SMAD4 for the of The of mutations to the this the basis of the clinical of SMAD4 that unrecognized of gene underlie clinically related LAPS Moore-Federman and and in sequence and parallel sequencing new for of the molecular of Mendelian J.C. Exome The spot Genet. PubMed Scopus Google Scholar, C. A. J.A. Mendelian disease exome 2011; PubMed Scopus Google Scholar the of affected gene filtering on the of of features and are to the of to the sequencing of single individuals with rare are to the families in the the the subjects, and and di for the at the di for was by from and to and of to and with and The for data are as of in Mendelian in