CLINICAL REPORT
Novel SMAD4 Mutation Causing Myhre Syndrome Viviana Caputo,1* Gianfranco Bocchinfuso,2 Marco Castori,3 Alice Traversa,4,5 Antonio Pizzuti,1 Lorenzo Stella,2 Paola Grammatico,3 and Marco Tartaglia5 1
Dipartimento di Medicina Sperimentale, Sapienza Universita` di Roma, Rome, Italy Dipartimento di Scienze e Tecnologie Chimiche, Universita` di Roma “Tor Vergata”, Rome, Italy 3 Genetica Medica, Dipartimento di Medicina Molecolare, Sapienza Universita` di Roma, Azienda Ospedaliera S. Camillo Forlanini, Rome, Italy 4 Dipartimento di Medicina Molecolare, Sapienza Universita` di Roma, Rome, Italy 5 Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanita`, Rome, Italy 2
Manuscript Received: 23 December 2013; Manuscript Accepted: 23 February 2014
Myhre syndrome (MYHRS, OMIM 139210) is an autosomal dominant disorder characterized by developmental and growth delay, athletic muscular built, variable cognitive deficits, skeletal anomalies, stiffness of joints, distinctive facial gestalt and deafness. Recently, SMAD4 (OMIM 600993) was identified by exome sequencing as the disease gene mutated in MYHRS. Previously only three missense mutations affecting Ile500 (p.Ile500Thr, p. Ile500Val, and p.Ile500Met) have been described in 22 unrelated subjects with MYHRS or a clinically related phenotype. Here we report on a 15-year-old boy with typical MYHRS and a novel heterozygous SMAD4 missense mutation affecting residue Arg496. This finding provides further information about the distinctive SMAD4 mutation spectrum in MYHRS. In silico structural analyses exploring the impact of the Arg-to-Cys change at codon 496 suggested that conformational changes promoted by replacement of Arg496 impact the stability of the SMAD heterotrimer and/or proper SMAD4 ubiquitination. Ó 2014 Wiley Periodicals, Inc.
Key words: mutation analysis; Myhre syndrome; SMAD4; structural analyses; TGF-b pathway
INTRODUCTION Myhre syndrome (MYHRS, OMIM 139210) is an autosomal dominant disorder characterized by developmental and growth delay, an athletic muscular built, stiffness of joints, distinctive facial gestalt, minor skeletal anomalies, and deafness [Myhre et al., 1981]. MYHRS is a rare condition, with less than 50 affected individuals reported. It has partial clinical overlap with GOMBO syndrome (OMIM 233270), geleophysic dysplasia (OMIM 231050 and OMIM 614185), acromicric dysplasia (OMIM 102370), WeillMarchesani (OMIM 277600 and OMIM 608328), Moore-Federman (OMIM 127200), and stiff skin syndrome (OMIM 184900). Although the molecular bases for some of these clinically related conditions are unknown, increasing evidence supports the crucial role of transforming growth factor beta (TGF-b) signaling dysregulation in their pathogenesis [Faivre et al., 2003; Dagoneau
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Caputo V, Bocchinfuso G, Castori M, Traversa A, Pizzuti A, Stella L, Grammatico P, Tartaglia M. 2014. Novel SMAD4 mutation causing Myhre syndrome. Am J Med Genet Part A 9999:1–6.
et al., 2004; Le Goff et al., 2008, 2011; Loeys et al., 2010]. By using an exome sequencing approach, our group identified SMAD4 (OMIM 600993) as the gene mutated in MYHRS [Caputo et al., 2012]. Two distinct missense changes affected an isoleucine residue at codon 500 (p.Ile500Thr and p.Ile500Val) in 8 unrelated patients. An independent study confirmed SMAD4 as the MYHRS disease gene, with heterozygous missense mutations affecting Ile500 (p.Ile500Thr, p.Ile500Val, and p.Ile500Met) in 11 unrelated subjects [Le Goff et al., 2012]. Here we report on a patient with clinical features of MYHRS carrying a novel heterozygous SMAD4 missense mutation affecting the residue Arg496.
MATERIALS AND METHODS Clinical Data and Biological Material Collection Clinical data and biological material collection and storage were attained from the participating family in accordance with the ethical standards of the institutional review board (San CamilloConflict of interest: none. Grant sponsor: Istituto Superiore di Sanita`-Ricerca Corrente 2013. Correspondence to: Viviana Caputo, Ph.D., Dipartimento di Medicina Sperimentale, Sapienza Universita` di Roma, Viale Regina Elena, 324 00161–Rome, Italy. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2014 DOI 10.1002/ajmg.a.36544
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AMERICAN JOURNAL OF MEDICAL GENETICS PART A
2 Forlanini Hospital, Rome, Italy) and after written informed consent. Genomic DNA was isolated from hair bulb cells, peripheral blood leukocytes, and buccal mucosal epithelial cells using standard protocols.
CLINICAL REPORT This patient was born at 36th weeks gestation by cesarean for maternal eclampsia. He was the first child of a 22-year-old healthy mother and her unrelated 22-year-old husband. Family history was unremarkable. Birth parameters included length 45 cm (10–25th centile), weight 2,850 g (50–75th centile), and OFC 34 cm (50–75th centile), while Apgar scores were 71/85. He had generalized joint stiffness from birth. He had no feeding, sleep, urinary, or gastrointestinal problem. Early psychomotor development was normal, as the patient walked independently at 14 months without ever crawling and said first words at 12 months. He never toe-walked. Subsequently, parents noted clumsiness and poor vocabulary. At age 4 years a child neurology consultation revealed lack of coordination of fine movements in the absence of a true developmental delay and poor articulation calling for speech therapy and psychological support for the following 5 years. Education followed standard programs and his scholastic performance was always satisfying. Nonetheless, the parents were concerned about his
habitus and requested many consultations without conclusive diagnosis. Heart, kidney and abdominal ultrasound, electromyography and electroneurography, brain and total spine magnetic resonance imaging, and screening for bone turnover had normal results, except for minimal liver enlargement in the absence of biochemical markers of liver dysfunction. Standard karyotype and FMR1 analyses were negative. Various standard radiographs were performed without a diagnostic impact. At age 15 years, measurements included height 163 cm (25th centile), weight 80 kg (95–97th centile), OFC 58 cm (97th centile), inner canthal distance 3.2 cm (50th centile), outer canthal distance 92 mm (75th centile), philtrum length 17 mm (50–75th centile), ear length 57 mm ( 1 SD). The patient appeared athletic with a muscular built, although he did not perform intense sport activity (Fig. 1a). Arm span was proportionate to his height (156 cm), while hands were short (Fig. 1b,c), with total hand length 19 cm (3rd– 25th centile), middle finger length 7 cm (3rd centile) and middle finger/total hand ratio 36.8% (T missense change (p. Arg496Cys) (Fig. S1a - see Supporting Information online). Genotyping of parental DNAs demonstrated de novo origin of the mutation and confirmed paternity. Sequencing of the relevant exon using genomic DNA obtained from hair bulb and buccal mucosal epithelial cells of the proband provided evidence for its germline origin. Arg496 is conserved in orthologs (Fig. S1b - see Supporting Information online), and a conservative missense change affecting this residue, p.Arg496His, had previously been
3 reported as somatically acquired lesions in human cancers [Lazzereschi et al., 2005; Fleming et al., 2013]. The affected amino acid is close to Ile500, previously reported to be invariantly affected in both SMAD4 mutation-positive MYHRS and clinically related traits [Asakura et al., 2012; Caputo et al., 2012; Le Goff et al., 2012; Lindor et al., 2012].
Structural Analysis Previous works reported the impact of the disease-causing amino acid changes affecting Ile500 on SMAD4 binding to other SMAD proteins and on ubiquitination, which is known to control SMAD4 degradation via proteasome [Caputo et al., 2012; Le Goff et al., 2012]. Structural analyses explored the consequences of the novel MYHRS-causing mutation on SMAD4 function, Arg496 is located in the MH2 domain (Fig. 2a), which mediates SMAD4 binding to activated receptor-regulated SMAD proteins (R-SMADs), and formation of the functionally active heterotrimer complex [Chacko et al., 2001; Wu et al., 2001; Kuang and Chen, 2004; Bourgeois et al., 2013]. The MH2 domain contains two subdomains, the three-helix bundle extension and the b-sandwich core. This domain is involved in R-SMAD binding through two different interfaces (interface 1 and interface 2) (Fig. 2a). Residue Arg496 is located within the three-helix bundle at interface 1 and is specific for SMAD4 proteins, being substituted by a neutral residue in paralogs (Fig. S1c-see Supporting Information online). To investigate the role of Arg496 in trimer stability, we identified the R-SMADs interacting residues. The sequence homology between the MH2 domain of SMAD3 and those of SMAD1, SMAD5, and SMAD9 is about 80%. In all proteins, a highly conserved motif comprising four invariant residues (i.e., Gly272, Ser275, Asn276 and Arg279, in SMAD3) was identified. Arg496 was not observed to form any specific interaction (salt bridges or strong H-bonds) with these residues, questioning its direct contribution to the complex stability. Arg496 interacts with Ile500 which is located close to the protein surface, is solvent accessible for only 6% of the residue surface, and interacts with the hydrocarbon part of the side chains of residues Arg497, Arg502, Glu526, and His528. These residues constitute a basic pocket with a characteristic structural topology and very peculiar electrostatic features. Of note, this pocket is placed close to Lys519, which is one of two ubiquitination sites in SMAD4 (Fig. 2b). Searching for available structures possessing a group of residues in a topology similar to this motif identified three structures with a root mean square displacement of their Ca and Cb atoms lower than 1.0 A with respect to the reference motif (pdb codes: 1n3g, RMSD 0.63 A; 1h8c RMSD 0.71 A; 1ghp, RMSD 0.93 A). Among them, 1h8c corresponds to the structure of an UBX domain, where the motif residues corresponding to those of SMAD4 are Lys46, Arg53, Arg54, Glu79, and Phe77 (Fig. 2c). The conformation of this domain is almost superimposable with the structure of the ubiquitin domain dimer (pdb code 1aar), which contains a basic pocket similar to that investigated here [Cook et al., 1992; Buchberger et al., 2001]. In particular, the 1aar structure residues Arg74, Arg72, Arg42, and Asp39 are located close to the interface between the two ubiquitin domains, and undergo a conformation rearrangement
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
4
during dimerization [Cook et al., 1992]. This structural similarity suggests a role of this motif in the priming interaction with ubiquitin required for ubiquitination of Lys519.
DISCUSSION
FIG. 2. Structural analyses. a: Location of Arg496 in the structure of the SMAD3/SMAD4 MH2 domain complex (pdb code 1u7f). The SMAD4 chain is represented as an orange ribbon, the heavy atoms of the Arg496 side chain are depicted as ball and stick (C in brown and N in blue). The two sites of ubiquitination in SMAD4 (Lys519 and Lys507) are reported as green sticks. The SMAD3 MH2 domains are reported as surfaces. The two chains reported in the pdb file are colored in dark (chain C) and light (chain A) gray. In SMAD3, chain A, the serine residues that are subjected to phosphorylation are colored in cyan. b: Region surrounding Arg496 in the SMAD4 MH-2 domain. Coordinates are taken from the structure of the SMAD4 and SMAD3 MH-2 domains complex (pdb code 1u7f). SMAD4 and Arg496 are reported as in (a), SMAD3 chains are shown as semi-transparent light gray (chain A) and dark gray (chain C) ribbon. Other residues are also evidenced as sticks. The other arginine residues belonging to the basic pocket discussed in the text are colored in blue (Arg497 and Arg502), Glu526 is colored in red and His528 in pink. The ubiquitination sites in SMAD4 (Lys519 and Lys507) are colored in green. c: Structure of the UBX domain (pdb code 1h8c). Residues Arg53, Arg54, Lys46, Glu79, and Phe77, matching the structural motif in SMAD4, are reported as sticks (N is colored in blue, O in red, and C in brown). For comparison, the residues constituting the structural motif in SMAD4 (Arg496, Arg497, Arg502, Glu526, and His528) are also reported with the same convention used in (b).
Myhre syndrome (MYHRS) is a rare developmental disorder characterized by cognitive deficits, facial dysmorphism, deafness, and distinctive musculoskeletal features. To date, a narrow spectrum of missense mutations at a single codon site (Ile500) of the SMAD4 gene was reported in 20 MYHRS patients [Asakura et al., 2012; Caputo et al., 2012; Le Goff et al., 2012], and in two patients with LAPS syndrome [Lindor et al., 2012], indicating that these conditions belong to the same autosomal dominant disorder. Of note, SMAD4 had previously been recognized as a tumor suppressor somatically mutated in cancer, and germline loss-offunction mutations and deletions cause disorders clinically distinct from MYHRS, predisposing individuals to gastrointestinal cancer and vascular malformations [Howe et al., 1998; Gallione et al., 2004]. The distinctive clinical impact of the SMAD4 mutations affecting Ile500 strongly suggests a specific perturbing role of these mutations on SMAD4 function. Here, we reported on a patient with clinical features fitting MYHRS harboring a novel SMAD4 non-conservative missense change affecting Arg496, a residue mutated (conservative Arg496His substitution) in cancer [Lazzereschi et al., 2005; Fleming et al., 2013]. Structural analysis of the SMAD4R496C mutant protein and clinical features of our patient support the functional equivalence to missense mutations affecting Ile500. On the other hand, the evidence that cancer-associated missense mutations affecting residues mutated in MYHRS (i.e., Arg496) or spatially close codons (Asp493, Asp494, Leu495, Cys499, Arg502, Ser504, Phe505, and Val506) (COSMIC database, http://cancer.sanger.ac.uk/cancergenome/ projects/cosmic/) highlights the dramatically diverse impact of apparently subtle perturbation of SMAD4 structure and function on intracellular signaling and gene expression, still requiring further delineation. SMAD4 encodes a member of the SMAD family of signal transduction proteins activated by transmembrane serine-threonine receptor kinases in response to TGF-b signaling, and form complexes that translocate to the nucleus to regulate transcription of targets [Massague´ et al., 2005]. The nuclear translocation of the complexes is finely regulated and requires the MH2 domain. Previous analyses reported that MYHRS-causing mutations affecting Ile500 might affect either the SMAD heterotrimer function, causing dysregulation of TGF-b-mediated transcriptional control [Le Goff et al., 2012], or proper SMAD4 ubiquitination and defective degradation [Caputo et al., 2012; Le Goff et al., 2012]. The present structural analyses directed to analyze the impact of the p.Arg496Cys substitution excluded a major contribution of this amino acid change to the complex stability. Consistent with these considerations, previous studies documented that the Arg496Ser change does not prevent heterotrimer formation, although it was found to partially impair the transcriptional activity of the complex itself [Chacko et al., 2001]. The possibility that this substitution affects complex stability by indirectly perturbing the interface structure cannot be ruled out, as Arg496 is predicted to form an
CAPUTO ET AL. intramolecular salt bridge with Asp493 that, in turn, interacts electrostatically with two arginine residues strongly conserved in R-SMADs (Arg279 and Arg287 in SMAD3) [Chacko et al., 2004]. On the other hand, a possible effect of the p.Arg496Cys change on the selectivity of SMAD4 interaction with different R-SMADs can be excluded, since the interacting residues in the heteromeric interface are strictly conserved. Finally, the structural analyses reported above provided evidence that mutations affecting Arg496 might perturb the regulatory mechanism controlling SMAD4 ubiquitination. Interestingly, ligation of ubiquitin at Lys519 affects protein function by regulating its degradation and by impairing the ability of SMAD4 to form the active heterotrimer [Izzi and Attisano, 2006; Dupont et al., 2009]. Overall, our structural data indicate that conformational changes promoted by the Arg496 replacement affect the interface structure of the SMAD heterotrimer, and/or impair proper SMAD4 ubiquitination, perturbing signal flow as a result of enhanced levels of nonubiquinated SMAD4. It should be stressed, however, that other concomitant effects of the mutation cannot be ruled out. While the MH2 domain is not directly involved in association with DNA, mutations affecting Arg497, which is adjacent to the residue mutated in the present report, perturb the ability of SMAD4 to bind DNA [Kuang and Chen, 2004]. Functional studies are required to understand the differential impact of cancer-associated and MYHRS-causing SMAD4 mutations on protein function and intracellular signaling.
ACKNOWLEDGMENTS We are indebted to the family who participated in the study. This work was supported, in part, by funding from the Istituto Superiore di Sanita`-Ricerca Corrente 2013 (to M.T.).
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SUPPORTING INFORMATION
Massague´ J, Seoane J, Wotton D. 2005. Smad transcription factors. Genes Dev 19:2783–2810.
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
Novel SMAD4 Mutation Causing Myhre Syndrome Viviana Caputo,1* Gianfranco Bocchinfuso,2 Marco Castori,3 Alice Traversa,4,5 Antonio Pizzuti,1 Lorenzo Stella,2 Paola Grammatico,3 and Marco Tartaglia5 1
Dipartimento di Medicina Sperimentale, Sapienza Universita` di Roma, Rome, Italy Dipartimento di Scienze e Tecnologie Chimiche, Universita` di Roma “Tor Vergata”, Rome, Italy 3 Genetica Medica, Dipartimento di Medicina Molecolare, Sapienza Universita` di Roma, Azienda Ospedaliera S. Camillo Forlanini, Rome, Italy 4 Dipartimento di Medicina Molecolare, Sapienza Universita` di Roma, Rome, Italy 5 Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanita`, Rome, Italy 2
Manuscript Received: 23 December 2013; Manuscript Accepted: 23 February 2014
Myhre syndrome (MYHRS, OMIM 139210) is an autosomal dominant disorder characterized by developmental and growth delay, athletic muscular built, variable cognitive deficits, skeletal anomalies, stiffness of joints, distinctive facial gestalt and deafness. Recently, SMAD4 (OMIM 600993) was identified by exome sequencing as the disease gene mutated in MYHRS. Previously only three missense mutations affecting Ile500 (p.Ile500Thr, p. Ile500Val, and p.Ile500Met) have been described in 22 unrelated subjects with MYHRS or a clinically related phenotype. Here we report on a 15-year-old boy with typical MYHRS and a novel heterozygous SMAD4 missense mutation affecting residue Arg496. This finding provides further information about the distinctive SMAD4 mutation spectrum in MYHRS. In silico structural analyses exploring the impact of the Arg-to-Cys change at codon 496 suggested that conformational changes promoted by replacement of Arg496 impact the stability of the SMAD heterotrimer and/or proper SMAD4 ubiquitination. Ó 2014 Wiley Periodicals, Inc.
Key words: mutation analysis; Myhre syndrome; SMAD4; structural analyses; TGF-b pathway
INTRODUCTION Myhre syndrome (MYHRS, OMIM 139210) is an autosomal dominant disorder characterized by developmental and growth delay, an athletic muscular built, stiffness of joints, distinctive facial gestalt, minor skeletal anomalies, and deafness [Myhre et al., 1981]. MYHRS is a rare condition, with less than 50 affected individuals reported. It has partial clinical overlap with GOMBO syndrome (OMIM 233270), geleophysic dysplasia (OMIM 231050 and OMIM 614185), acromicric dysplasia (OMIM 102370), WeillMarchesani (OMIM 277600 and OMIM 608328), Moore-Federman (OMIM 127200), and stiff skin syndrome (OMIM 184900). Although the molecular bases for some of these clinically related conditions are unknown, increasing evidence supports the crucial role of transforming growth factor beta (TGF-b) signaling dysregulation in their pathogenesis [Faivre et al., 2003; Dagoneau
Ó 2014 Wiley Periodicals, Inc.
How to Cite this Article: Caputo V, Bocchinfuso G, Castori M, Traversa A, Pizzuti A, Stella L, Grammatico P, Tartaglia M. 2014. Novel SMAD4 mutation causing Myhre syndrome. Am J Med Genet Part A 9999:1–6.
et al., 2004; Le Goff et al., 2008, 2011; Loeys et al., 2010]. By using an exome sequencing approach, our group identified SMAD4 (OMIM 600993) as the gene mutated in MYHRS [Caputo et al., 2012]. Two distinct missense changes affected an isoleucine residue at codon 500 (p.Ile500Thr and p.Ile500Val) in 8 unrelated patients. An independent study confirmed SMAD4 as the MYHRS disease gene, with heterozygous missense mutations affecting Ile500 (p.Ile500Thr, p.Ile500Val, and p.Ile500Met) in 11 unrelated subjects [Le Goff et al., 2012]. Here we report on a patient with clinical features of MYHRS carrying a novel heterozygous SMAD4 missense mutation affecting the residue Arg496.
MATERIALS AND METHODS Clinical Data and Biological Material Collection Clinical data and biological material collection and storage were attained from the participating family in accordance with the ethical standards of the institutional review board (San CamilloConflict of interest: none. Grant sponsor: Istituto Superiore di Sanita`-Ricerca Corrente 2013. Correspondence to: Viviana Caputo, Ph.D., Dipartimento di Medicina Sperimentale, Sapienza Universita` di Roma, Viale Regina Elena, 324 00161–Rome, Italy. E-mail: [email protected] Article first published online in Wiley Online Library (wileyonlinelibrary.com): 00 Month 2014 DOI 10.1002/ajmg.a.36544
1
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
2 Forlanini Hospital, Rome, Italy) and after written informed consent. Genomic DNA was isolated from hair bulb cells, peripheral blood leukocytes, and buccal mucosal epithelial cells using standard protocols.
CLINICAL REPORT This patient was born at 36th weeks gestation by cesarean for maternal eclampsia. He was the first child of a 22-year-old healthy mother and her unrelated 22-year-old husband. Family history was unremarkable. Birth parameters included length 45 cm (10–25th centile), weight 2,850 g (50–75th centile), and OFC 34 cm (50–75th centile), while Apgar scores were 71/85. He had generalized joint stiffness from birth. He had no feeding, sleep, urinary, or gastrointestinal problem. Early psychomotor development was normal, as the patient walked independently at 14 months without ever crawling and said first words at 12 months. He never toe-walked. Subsequently, parents noted clumsiness and poor vocabulary. At age 4 years a child neurology consultation revealed lack of coordination of fine movements in the absence of a true developmental delay and poor articulation calling for speech therapy and psychological support for the following 5 years. Education followed standard programs and his scholastic performance was always satisfying. Nonetheless, the parents were concerned about his
habitus and requested many consultations without conclusive diagnosis. Heart, kidney and abdominal ultrasound, electromyography and electroneurography, brain and total spine magnetic resonance imaging, and screening for bone turnover had normal results, except for minimal liver enlargement in the absence of biochemical markers of liver dysfunction. Standard karyotype and FMR1 analyses were negative. Various standard radiographs were performed without a diagnostic impact. At age 15 years, measurements included height 163 cm (25th centile), weight 80 kg (95–97th centile), OFC 58 cm (97th centile), inner canthal distance 3.2 cm (50th centile), outer canthal distance 92 mm (75th centile), philtrum length 17 mm (50–75th centile), ear length 57 mm ( 1 SD). The patient appeared athletic with a muscular built, although he did not perform intense sport activity (Fig. 1a). Arm span was proportionate to his height (156 cm), while hands were short (Fig. 1b,c), with total hand length 19 cm (3rd– 25th centile), middle finger length 7 cm (3rd centile) and middle finger/total hand ratio 36.8% (T missense change (p. Arg496Cys) (Fig. S1a - see Supporting Information online). Genotyping of parental DNAs demonstrated de novo origin of the mutation and confirmed paternity. Sequencing of the relevant exon using genomic DNA obtained from hair bulb and buccal mucosal epithelial cells of the proband provided evidence for its germline origin. Arg496 is conserved in orthologs (Fig. S1b - see Supporting Information online), and a conservative missense change affecting this residue, p.Arg496His, had previously been
3 reported as somatically acquired lesions in human cancers [Lazzereschi et al., 2005; Fleming et al., 2013]. The affected amino acid is close to Ile500, previously reported to be invariantly affected in both SMAD4 mutation-positive MYHRS and clinically related traits [Asakura et al., 2012; Caputo et al., 2012; Le Goff et al., 2012; Lindor et al., 2012].
Structural Analysis Previous works reported the impact of the disease-causing amino acid changes affecting Ile500 on SMAD4 binding to other SMAD proteins and on ubiquitination, which is known to control SMAD4 degradation via proteasome [Caputo et al., 2012; Le Goff et al., 2012]. Structural analyses explored the consequences of the novel MYHRS-causing mutation on SMAD4 function, Arg496 is located in the MH2 domain (Fig. 2a), which mediates SMAD4 binding to activated receptor-regulated SMAD proteins (R-SMADs), and formation of the functionally active heterotrimer complex [Chacko et al., 2001; Wu et al., 2001; Kuang and Chen, 2004; Bourgeois et al., 2013]. The MH2 domain contains two subdomains, the three-helix bundle extension and the b-sandwich core. This domain is involved in R-SMAD binding through two different interfaces (interface 1 and interface 2) (Fig. 2a). Residue Arg496 is located within the three-helix bundle at interface 1 and is specific for SMAD4 proteins, being substituted by a neutral residue in paralogs (Fig. S1c-see Supporting Information online). To investigate the role of Arg496 in trimer stability, we identified the R-SMADs interacting residues. The sequence homology between the MH2 domain of SMAD3 and those of SMAD1, SMAD5, and SMAD9 is about 80%. In all proteins, a highly conserved motif comprising four invariant residues (i.e., Gly272, Ser275, Asn276 and Arg279, in SMAD3) was identified. Arg496 was not observed to form any specific interaction (salt bridges or strong H-bonds) with these residues, questioning its direct contribution to the complex stability. Arg496 interacts with Ile500 which is located close to the protein surface, is solvent accessible for only 6% of the residue surface, and interacts with the hydrocarbon part of the side chains of residues Arg497, Arg502, Glu526, and His528. These residues constitute a basic pocket with a characteristic structural topology and very peculiar electrostatic features. Of note, this pocket is placed close to Lys519, which is one of two ubiquitination sites in SMAD4 (Fig. 2b). Searching for available structures possessing a group of residues in a topology similar to this motif identified three structures with a root mean square displacement of their Ca and Cb atoms lower than 1.0 A with respect to the reference motif (pdb codes: 1n3g, RMSD 0.63 A; 1h8c RMSD 0.71 A; 1ghp, RMSD 0.93 A). Among them, 1h8c corresponds to the structure of an UBX domain, where the motif residues corresponding to those of SMAD4 are Lys46, Arg53, Arg54, Glu79, and Phe77 (Fig. 2c). The conformation of this domain is almost superimposable with the structure of the ubiquitin domain dimer (pdb code 1aar), which contains a basic pocket similar to that investigated here [Cook et al., 1992; Buchberger et al., 2001]. In particular, the 1aar structure residues Arg74, Arg72, Arg42, and Asp39 are located close to the interface between the two ubiquitin domains, and undergo a conformation rearrangement
AMERICAN JOURNAL OF MEDICAL GENETICS PART A
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during dimerization [Cook et al., 1992]. This structural similarity suggests a role of this motif in the priming interaction with ubiquitin required for ubiquitination of Lys519.
DISCUSSION
FIG. 2. Structural analyses. a: Location of Arg496 in the structure of the SMAD3/SMAD4 MH2 domain complex (pdb code 1u7f). The SMAD4 chain is represented as an orange ribbon, the heavy atoms of the Arg496 side chain are depicted as ball and stick (C in brown and N in blue). The two sites of ubiquitination in SMAD4 (Lys519 and Lys507) are reported as green sticks. The SMAD3 MH2 domains are reported as surfaces. The two chains reported in the pdb file are colored in dark (chain C) and light (chain A) gray. In SMAD3, chain A, the serine residues that are subjected to phosphorylation are colored in cyan. b: Region surrounding Arg496 in the SMAD4 MH-2 domain. Coordinates are taken from the structure of the SMAD4 and SMAD3 MH-2 domains complex (pdb code 1u7f). SMAD4 and Arg496 are reported as in (a), SMAD3 chains are shown as semi-transparent light gray (chain A) and dark gray (chain C) ribbon. Other residues are also evidenced as sticks. The other arginine residues belonging to the basic pocket discussed in the text are colored in blue (Arg497 and Arg502), Glu526 is colored in red and His528 in pink. The ubiquitination sites in SMAD4 (Lys519 and Lys507) are colored in green. c: Structure of the UBX domain (pdb code 1h8c). Residues Arg53, Arg54, Lys46, Glu79, and Phe77, matching the structural motif in SMAD4, are reported as sticks (N is colored in blue, O in red, and C in brown). For comparison, the residues constituting the structural motif in SMAD4 (Arg496, Arg497, Arg502, Glu526, and His528) are also reported with the same convention used in (b).
Myhre syndrome (MYHRS) is a rare developmental disorder characterized by cognitive deficits, facial dysmorphism, deafness, and distinctive musculoskeletal features. To date, a narrow spectrum of missense mutations at a single codon site (Ile500) of the SMAD4 gene was reported in 20 MYHRS patients [Asakura et al., 2012; Caputo et al., 2012; Le Goff et al., 2012], and in two patients with LAPS syndrome [Lindor et al., 2012], indicating that these conditions belong to the same autosomal dominant disorder. Of note, SMAD4 had previously been recognized as a tumor suppressor somatically mutated in cancer, and germline loss-offunction mutations and deletions cause disorders clinically distinct from MYHRS, predisposing individuals to gastrointestinal cancer and vascular malformations [Howe et al., 1998; Gallione et al., 2004]. The distinctive clinical impact of the SMAD4 mutations affecting Ile500 strongly suggests a specific perturbing role of these mutations on SMAD4 function. Here, we reported on a patient with clinical features fitting MYHRS harboring a novel SMAD4 non-conservative missense change affecting Arg496, a residue mutated (conservative Arg496His substitution) in cancer [Lazzereschi et al., 2005; Fleming et al., 2013]. Structural analysis of the SMAD4R496C mutant protein and clinical features of our patient support the functional equivalence to missense mutations affecting Ile500. On the other hand, the evidence that cancer-associated missense mutations affecting residues mutated in MYHRS (i.e., Arg496) or spatially close codons (Asp493, Asp494, Leu495, Cys499, Arg502, Ser504, Phe505, and Val506) (COSMIC database, http://cancer.sanger.ac.uk/cancergenome/ projects/cosmic/) highlights the dramatically diverse impact of apparently subtle perturbation of SMAD4 structure and function on intracellular signaling and gene expression, still requiring further delineation. SMAD4 encodes a member of the SMAD family of signal transduction proteins activated by transmembrane serine-threonine receptor kinases in response to TGF-b signaling, and form complexes that translocate to the nucleus to regulate transcription of targets [Massague´ et al., 2005]. The nuclear translocation of the complexes is finely regulated and requires the MH2 domain. Previous analyses reported that MYHRS-causing mutations affecting Ile500 might affect either the SMAD heterotrimer function, causing dysregulation of TGF-b-mediated transcriptional control [Le Goff et al., 2012], or proper SMAD4 ubiquitination and defective degradation [Caputo et al., 2012; Le Goff et al., 2012]. The present structural analyses directed to analyze the impact of the p.Arg496Cys substitution excluded a major contribution of this amino acid change to the complex stability. Consistent with these considerations, previous studies documented that the Arg496Ser change does not prevent heterotrimer formation, although it was found to partially impair the transcriptional activity of the complex itself [Chacko et al., 2001]. The possibility that this substitution affects complex stability by indirectly perturbing the interface structure cannot be ruled out, as Arg496 is predicted to form an
CAPUTO ET AL. intramolecular salt bridge with Asp493 that, in turn, interacts electrostatically with two arginine residues strongly conserved in R-SMADs (Arg279 and Arg287 in SMAD3) [Chacko et al., 2004]. On the other hand, a possible effect of the p.Arg496Cys change on the selectivity of SMAD4 interaction with different R-SMADs can be excluded, since the interacting residues in the heteromeric interface are strictly conserved. Finally, the structural analyses reported above provided evidence that mutations affecting Arg496 might perturb the regulatory mechanism controlling SMAD4 ubiquitination. Interestingly, ligation of ubiquitin at Lys519 affects protein function by regulating its degradation and by impairing the ability of SMAD4 to form the active heterotrimer [Izzi and Attisano, 2006; Dupont et al., 2009]. Overall, our structural data indicate that conformational changes promoted by the Arg496 replacement affect the interface structure of the SMAD heterotrimer, and/or impair proper SMAD4 ubiquitination, perturbing signal flow as a result of enhanced levels of nonubiquinated SMAD4. It should be stressed, however, that other concomitant effects of the mutation cannot be ruled out. While the MH2 domain is not directly involved in association with DNA, mutations affecting Arg497, which is adjacent to the residue mutated in the present report, perturb the ability of SMAD4 to bind DNA [Kuang and Chen, 2004]. Functional studies are required to understand the differential impact of cancer-associated and MYHRS-causing SMAD4 mutations on protein function and intracellular signaling.
ACKNOWLEDGMENTS We are indebted to the family who participated in the study. This work was supported, in part, by funding from the Istituto Superiore di Sanita`-Ricerca Corrente 2013 (to M.T.).
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SUPPORTING INFORMATION
Massague´ J, Seoane J, Wotton D. 2005. Smad transcription factors. Genes Dev 19:2783–2810.
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
