Mutations in Multidomain Protein MEGF8 Identify a Carpenter Syndrome Subtype Associated with Defective Lateralization

Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed. Carpenter syndrome is an autosomal-recessive multiple-congenital-malformation disorder characterized by multisuture craniosynostosis and polysyndactyly of the hands and feet; many other clinical features occur, and the most frequent include obesity, umbilical hernia, cryptorchidism, and congenital heart disease. Mutations of RAB23, encoding a small GTPase that regulates vesicular transport, are present in the majority of cases. Here, we describe a disorder caused by mutations in multiple epidermal-growth-factor-like-domains 8 (MEGF8), which exhibits substantial clinical overlap with Carpenter syndrome but is frequently associated with abnormal left-right patterning. We describe five affected individuals with similar dysmorphic facies, and three of them had either complete situs inversus, dextrocardia, or transposition of the great arteries; similar cardiac abnormalities were previously identified in a mouse mutant for the orthologous Megf8. The mutant alleles comprise one nonsense, three missense, and two splice-site mutations; we demonstrate in zebrafish that, in contrast to the wild-type protein, the proteins containing all three missense alterations provide only weak rescue of an early gastrulation phenotype induced by Megf8 knockdown. We conclude that mutations in MEGF8 cause a Carpenter syndrome subtype frequently associated with defective left-right patterning, probably through perturbation of signaling by hedgehog and nodal family members. We did not observe any subject with biallelic loss-of function mutations, suggesting that some residual MEGF8 function might be necessary for survival and might influence the phenotypes observed. The multiple-congenital-anomalies disorder Carpenter syndrome (acrocephalopolysyndactyly type II [MIM 201000]) was first described by George Carpenter in 1901 in two siblings with craniosynostosis and polysyndactyly;1Carpenter G. Two sisters showing malformations of the skull and other congenital abnormalities.Rep. Soc. Study Dis. Child. Lond. 1901; 1: 110-118Google Scholar in 1909 he added a case report of a third affected sibling in the family.2Carpenter G. Acrocephaly, with other congenital malformations.Proc. R. Soc. Med. 1909; 2: 45-53PubMed Google Scholar Over 70 further cases have been described in the clinical literature, and a large number of additional phenotypic features have been noted and include high birth weight and obesity in later life, congenital heart disease, umbilical hernia, cryptorchidism in males, and genu valgum.3Temtamy S.A. Carpenter’s syndrome: Acrocephalopolysyndactyly. An autosomal recessive syndrome.J. Pediatr. 1966; 69: 111-120Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 4Cohen D.M. Green J.G. Miller J. Gorlin R.J. Reed J.A. Acrocephalopolysyndactyly type II—Carpenter syndrome: Clinical spectrum and an attempt at unification with Goodman and Summit syndromes.Am. J. Med. Genet. 1987; 28: 311-324Crossref PubMed Scopus (43) Google Scholar, 5Gorlin R.J. Cohen Jr., M.M. Hennekam R.C.M. Carpenter syndrome (acrocephalopolysyndactyly).in: Syndromes of the Head and Neck. 4th ed. Oxford University Press, New York2001: 666-668Google Scholar Although often arising sporadically, consanguinity is frequent, and autosomal-recessive mutations are presumed to underlie most cases. In 2007 the majority of individuals with Carpenter syndrome were shown by Jenkins et al. to have biallelic mutations in RAB23 (MIM 606144), encoding a member of the family of small guanosine triphosphatases (GTPases) that regulate intracellular trafficking of membrane-associated proteins.6Jenkins D. Seelow D. Jehee F.S. Perlyn C.A. Alonso L.G. Bueno D.F. Donnai D. Josifova D. Mathijssen I.M.J. Morton J.E.V. et al.RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity.Am. J. Hum. Genet. 2007; 80: 1162-1170Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar All together, Jenkins et al.6Jenkins D. Seelow D. Jehee F.S. Perlyn C.A. Alonso L.G. Bueno D.F. Donnai D. Josifova D. Mathijssen I.M.J. Morton J.E.V. et al.RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity.Am. J. Hum. Genet. 2007; 80: 1162-1170Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar and two subsequent studies7Alessandri J.L. Dagoneau N. Laville J.M. Baruteau J. Hébert J.C. Cormier-Daire V. RAB23 mutation in a large family from Comoros Islands with Carpenter syndrome.Am. J. Med. Genet. A. 2010; 152A: 982-986Crossref PubMed Scopus (41) Google Scholar, 8Jenkins D. Baynam G. De Catte L. Elcioglu N. Gabbett M.T. Hudgins L. Hurst J.A. Jehee F.S. Oley C. Wilkie A.O.M. Carpenter syndrome: Extended RAB23 mutation spectrum and analysis of nonsense-mediated mRNA decay.Hum. Mutat. 2011; 32: E2069-E2078Crossref PubMed Scopus (31) Google Scholar have reported RAB23 mutations in a total of 24 families and 31 cases. Affected individuals in 13 families have been homozygous for the identical c.434T>A (p.Leu145∗) mutation, indicating a major founder effect in subjects of north European origin. However, an additional ten distinct mutant alleles have been described, consistent with complete loss of RAB23 function. The role of RAB23 as a negative regulator of hedgehog (HH) signaling was originally defined by the analysis of mutant phenotypes affecting the orthologous murine Rab23 (open brain mutant);9Eggenschwiler J.T. Espinoza E. Anderson K.V. Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway.Nature. 2001; 412: 194-198Crossref PubMed Scopus (310) Google Scholar homozygous mutants are more severely affected than humans in that they die in mid gestation because of neural-tube defects.9Eggenschwiler J.T. Espinoza E. Anderson K.V. Rab23 is an essential negative regulator of the mouse Sonic hedgehog signalling pathway.Nature. 2001; 412: 194-198Crossref PubMed Scopus (310) Google Scholar, 10Günther T. Struwe M. Aguzzi A. Schughart K. Open brain, a new mouse mutant with severe neural tube defects, shows altered gene expression patterns in the developing spinal cord.Development. 1994; 120: 3119-3130PubMed Google Scholar, 11Eggenschwiler J.T. Anderson K.V. Dorsal and lateral fates in the mouse neural tube require the cell-autonomous activity of the open brain gene.Dev. Biol. 2000; 227: 648-660Crossref PubMed Scopus (63) Google Scholar Abrogation of RAB23 activity in human retinal cells prevents formation of the primary cilium,12Yoshimura S. Egerer J. Fuchs E. Haas A.K. Barr F.A. Functional dissection of Rab GTPases involved in primary cilium formation.J. Cell Biol. 2007; 178: 363-369Crossref PubMed Scopus (277) Google Scholar suggesting a likely mechanism linking to HH signaling,13Lim Y.S. Chua C.E. Tang B.L. Rabs and other small GTPases in ciliary transport.Biol. Cell. 2011; 103: 209-221Crossref PubMed Scopus (56) Google Scholar but the biochemical details are poorly understood. One study of canine cells proposed that Rab23 facilitates ciliary recycling of Smoothened (Smo), a HH-signaling mediator,14Boehlke C. Bashkurov M. Buescher A. Krick T. John A.K. Nitschke R. Walz G. Kuehn E.W. Differential role of Rab proteins in ciliary trafficking: Rab23 regulates smoothened levels.J. Cell Sci. 2010; 123: 1460-1467Crossref PubMed Scopus (83) Google Scholar and genetic evidence in mice places the Rab23 mutation downstream of Smo.15Eggenschwiler J.T. Bulgakov O.V. Qin J. Li T. Anderson K.V. Mouse Rab23 regulates hedgehog signaling from smoothened to Gli proteins.Dev. Biol. 2006; 290: 1-12Crossref PubMed Scopus (115) Google Scholar Compatible with the latter observation, transcription of Gli1, a HH target, requires a physical association between RAB23 and the homolog of Drosophila suppressor of fused (SUFU), another HH-signaling component.16Chi S. Xie G. Liu H. Chen K. Zhang X. Li C. Xie J. Rab23 negatively regulates Gli1 transcriptional factor in a Su(Fu)-dependent manner.Cell. Signal. 2012; 24: 1222-1228Crossref PubMed Scopus (32) Google Scholar Notwithstanding these gaps in knowledge, the origins of the cardinal physical features of Carpenter syndrome are better understood. In the limbs, reduced proteolytic processing of the transcription factor GLI3 to its shorter repressor form is the likely cause of polydactyly;15Eggenschwiler J.T. Bulgakov O.V. Qin J. Li T. Anderson K.V. Mouse Rab23 regulates hedgehog signaling from smoothened to Gli proteins.Dev. Biol. 2006; 290: 1-12Crossref PubMed Scopus (115) Google Scholar, 17Litingtung Y. Dahn R.D. Li Y. Fallon J.F. Chiang C. Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity.Nature. 2002; 418: 979-983Crossref PubMed Scopus (472) Google Scholar in the skull, a similar mechanism might operate because Gli3-loss-of-function mouse mutants exhibit fusions of the lambdoid and interfrontal sutures.18Rice D.P.C. Connor E.C. Veltmaat J.M. Lana-Elola E. Veistinen L. Tanimoto Y. Bellusci S. Rice R. Gli3Xt-J/Xt-J mice exhibit lambdoid suture craniosynostosis which results from altered osteoprogenitor proliferation and differentiation.Hum. Mol. Genet. 2010; 19: 3457-3467Crossref PubMed Scopus (47) Google Scholar, 19Veistinen L. Takatalo M. Tanimoto Y. Kesper D.A. Vortkamp A. Rice D.P.C. Loss-of-function of Gli3 in mice causes abnormal frontal bone morphology and premature synostosis of the interfrontal suture.Front Physiol. 2012; 3: 121Crossref PubMed Scopus (23) Google Scholar The abnormalities probably reflect disturbance of HH signaling, principally mediated by sonic hedgehog (SHH) in the limbs17Litingtung Y. Dahn R.D. Li Y. Fallon J.F. Chiang C. Shh and Gli3 are dispensable for limb skeleton formation but regulate digit number and identity.Nature. 2002; 418: 979-983Crossref PubMed Scopus (472) Google Scholar and by Indian hedgehog (IHH) in the cranial sutures.20Lenton K. James A.W. Manu A. Brugmann S.A. Birker D. Nelson E.R. Leucht P. Helms J.A. Longaker M.T. Indian hedgehog positively regulates calvarial ossification and modulates bone morphogenetic protein signaling.Genesis. 2011; 49: 784-796Crossref PubMed Scopus (80) Google Scholar, 21Klopocki E. Lohan S. Brancati F. Koll R. Brehm A. Seemann P. Dathe K. Stricker S. Hecht J. 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Genet. 2011; 19: PubMed Scopus (23) Google Scholar individuals had features congenital heart disease, and of RAB23 mutations, but craniosynostosis was to the or and other features birth umbilical hernia, and in were for was a case of from was with Carpenter The study was by and the for for was from the Although subject the cardinal features craniosynostosis with and for Carpenter he had dextrocardia, which not been described in association with RAB23 mutations and of RAB23 was and that he was for a an of GLI3 by and analysis was genetic causes of Carpenter the of the we that had a autosomal-recessive for of with Mutations in and and with high and multiple type hernia, at at of at of at of at of and high and of great at of at of at of and associated with situs of with with and complete with of with of brain at of at of and with at of of subject was for of the and and of and of at of at of and severe at of at of of are feet; and of subject was for of the Open in a new The are feet; and We in the and by the and identified of the and containing that were homozygous in the and that were likely to a recessive mutation the of the of J. K. M. R. R. S. et of in individuals with autosomal recessive J. Hum. Genet. 2006; Full Text Full Text PDF PubMed Scopus Google Scholar, F. F. M. G. C. Seelow D. N. G. et to recessive in individuals from Genet. PubMed Scopus Google Scholar We C. A.W. E.W. D.A. et the cause of a Genet. 2010; PubMed Scopus Google Scholar by an All to from a from of the The was an with We of that, with C. M. and of to the human Biol. PubMed Scopus Google Scholar to the and of in an of were with the H. A. T. J. N. G. G. R. and PubMed Scopus Google Scholar We identified in the which we by present either in or five other from subjects with clinical not to previously shown to be homozygous in subject and in or or that for of a homozygous missense multiple epidermal-growth-factor-like-domains 8 [MIM which was by We to be a as the mutation because an homozygous missense mutation in the orthologous mouse Megf8 was previously described in a mutant with similar phenotypic abnormalities of in the and and of all limbs, to of the R. L. J. L. Mouse of with spectrum of cardiac PubMed Scopus Google Scholar, D. R. T. C. Y. M. et the gene Megf8 with mutation Sci. PubMed Scopus Google Scholar further genetic evidence that MEGF8 mutations be of human we of the of MEGF8 in additional in is two major by the or of a that not the The number the shorter which and a protein and We two of to from individuals who were because of a diagnosis of Carpenter syndrome but who had a RAB23 a H. A. R. The association of Carpenter syndrome and situs case J. Med. Sci. 2011; Scopus Google Scholar with Carpenter syndrome and complete situs and from individuals with of congenital heart of but craniosynostosis or limb for clinical the and that we to all We found mutations in the indicating that MEGF8 mutations are not a cause of However, we did mutations in of and in the Carpenter syndrome supporting that the MEGF8 mutation was in the The found are in and the mutations identified are in In subject we found for a missense and a mutation in subject of H. A. R. The association of Carpenter syndrome and situs case J. Med. Sci. 2011; Scopus Google Scholar was for a missense and in subject was for two splice-site mutations and of the mutations identified are in either or in the in we them from for in European by E. and N. for and in and for and in and of the splice-site mutations present in subject with the of by that the for the of had from to not that the for the of was altered from to indicating that the mutation the of but probably not we were to demonstrate that the two mutations in subject were present alleles or that they affected because of mRNA from the or from other family were for Although the evidence that MEGF8 mutations are in family is for we have the clinical details of the two affected cases because they subjects in many The clinical features of subject are in of subject were H. A. R. The association of Carpenter syndrome and situs case J. Med. Sci. 2011; Scopus Google Scholar and of subjects and sibling of subject from whom was are in the clinical features of all Mutations in with Carpenter of not from the but analysis in subject that the mutation was present in two from subject and the were not origins shown are and of the two mutations was not from subject and the were not origins shown are and of the two mutations was not not from the but analysis in subject that the mutation was present in two from subject and the were not origins shown are and of the two mutations was not Open in a new The of the mutations in the gene and protein are shown in The three missense to three of subject is in a subject in an and subject in an MEGF8 is in for between the human and Drosophila be the of T. P. to the protein 2012; PubMed Scopus Google Scholar of the in which mutations were found be identified in many and be for a protein that modulates in and mutations the obesity of the mouse by of Biol. 2011; PubMed Scopus Google Scholar the of the missense we and the mutations of family subject to a an and is between two only at the in the Li M. of human factor and its Biol. 2001; PubMed Scopus Google Scholar Also of is that the mouse Megf8 mutation at one of these D. R. T. C. Y. M. et the gene Megf8 with mutation Sci. PubMed Scopus Google Scholar subject at a of a the of a at is and in the human protein with the of a and with or and the X. Zhang D. M. of the of human Biol. PubMed Scopus Google Scholar, Li X. C.A. M. and in the of the of Biol. PubMed Scopus Google Scholar Mutations at one of these in have been reported in M. and of an in Sci. Full Text Full Text PDF PubMed Scopus Google Scholar subject in an is and are in The of the J. R. R. of three of the Mol. Biol. PubMed Scopus Google Scholar shows that is to the of at we the evidence for to be for the and subjects and of which were present in a homozygous but for the subject present in with a further the of the we and the to in G. P. C.E. P. C. E. of human proteins for Mol. Biol. PubMed Scopus Google Scholar and the by However, we found that the protein exhibits a of as for other J. V. activity of the of the protein PubMed Scopus Google Scholar the to induced by the mutation an of we the of expression of the zebrafish by two D. R. T. C. Y. M. et the gene Megf8 with mutation Sci. PubMed Scopus Google Scholar were in with In to the previously described defects, we at abnormal gastrulation characterized by a and with expression of and of the We similar of the of the identified MEGF8 missense protein we for genetic rescue of these of MEGF8 which with the of with of defects, only of with wild-type MEGF8 were indicating a rescue and that the human gene for the zebrafish We of the missense the human by and the of of any of the three missense mutant of MEGF8 did not in an rescue the reduced of to that caused by the that the mutant proteins might some residual function. the and evidence together, we conclude that biallelic mutations of MEGF8 in a phenotype Carpenter syndrome caused by RAB23 of the clinical features and that the spectrum of limb associated with mutations in the two is similar and and of the of subject and of described in Carpenter S.A. Carpenter’s syndrome: Acrocephalopolysyndactyly. An autosomal recessive syndrome.J. Pediatr. 1966; 69: 111-120Abstract Full Text PDF PubMed Scopus (63) Google Scholar, 4Cohen D.M. Green J.G. Miller J. Gorlin R.J. Reed J.A. Acrocephalopolysyndactyly type II—Carpenter syndrome: Clinical spectrum and an attempt at unification with Goodman and Summit syndromes.Am. J. Med. Genet. 1987; 28: 311-324Crossref PubMed Scopus (43) Google Scholar, D. Seelow D. Jehee F.S. Perlyn C.A. Alonso L.G. Bueno D.F. Donnai D. Josifova D. Mathijssen I.M.J. Morton J.E.V. et al.RAB23 mutations in Carpenter syndrome imply an unexpected role for hedgehog signaling in cranial-suture development and obesity.Am. J. Hum. Genet. 2007; 80: 1162-1170Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar with MEGF8 mutations additional features the RAB23 mutant and these include a high birth weight and obesity and the of cryptorchidism or in However, are in many other clinical to be severe in the case of MEGF8 mutations and only the is frequently by associated with a dysmorphic a and and of were present in at three subjects and from transposition of the great subject to subject to complete situs subject and were with and a of the the clinical was The of cardiac associated with the previously described mouse R. L. J. L. Mouse of with spectrum of cardiac PubMed Scopus Google Scholar, D. R. T. C. Y. M. et the gene Megf8 with mutation Sci. PubMed Scopus Google Scholar but is with RAB23 mutations, two cases of of defective have been J.L. Dagoneau N. Laville J.M. Baruteau J. Hébert J.C. Cormier-Daire V. RAB23 mutation in a large family from Comoros Islands with Carpenter syndrome.Am. J. Med. Genet. A. 2010; 152A: 982-986Crossref PubMed Scopus (41) Google Scholar, 8Jenkins D. Baynam G. De Catte L. Elcioglu N. Gabbett M.T. Hudgins L. Hurst J.A. Jehee F.S. Oley C. Wilkie A.O.M. Carpenter syndrome: Extended RAB23 mutation spectrum and analysis of nonsense-mediated mRNA decay.Hum. Mutat. 2011; 32: E2069-E2078Crossref PubMed Scopus (31) Google Scholar However, we the that MEGF8 mutations cause cardiac malformations to in the of additional features and abnormalities more of MEGF8 mutations; umbilical and genu two features of the of MEGF8 these the many features of the mutant phenotype that RAB23 and MEGF8 might be in role and An by is that MEGF8 a for mutation than RAB23 mutations in RAB23 to in MEGF8 by One might be the of a major founder effect for MEGF8 mutations that mutant was identified more than is that the Carpenter syndrome phenotype associated with MEGF8 mutations only in a of residual protein activity affected had two alleles complete loss of with the that for the might be in other recessive S. S. E. S. N. C. S. R. J. C. et mutations in and a Mutat. PubMed Scopus Google Scholar in the of residual activity might cause clinical in the of which did not a consistent between individuals with of the origins of which was first identified in in a for proteins containing M. D. T. N. N. of proteins with multiple by PubMed Scopus Google Scholar is its The of multiple with and a the might in However, an to an did not observe expression and described intracellular and D. R. T. C. Y. M. et the gene Megf8 with mutation Sci. PubMed Scopus Google Scholar In the was found to be and left-right of were to have D. R. T. C. Y. M. et the gene Megf8 with mutation Sci. PubMed Scopus Google Scholar Although mouse homozygous for the expression of at the that the of were the that the expression of in the lateral was a in of the from the by an D. R. T. C. Y. M. et the gene Megf8 with mutation Sci. PubMed Scopus Google Scholar to Megf8 might be is by mouse which to be the of Megf8 and similar was shown to the of by trafficking to either the or to the and mutations the obesity of the mouse by of Biol. 2011; PubMed Scopus Google Scholar an function for MEGF8 in intracellular trafficking for the of the and the phenotypic in the of MEGF8 and RAB23 We are to the families for in We John and for with for genetic and the at the at the for for John and for with and and and University of for analysis of was by the for Oxford and from the of and the European to the of and to in Clinical to to and to and and the for to and The in are of the and are not of the of with and and The for are as in by