Short-Rib Polydactyly and Jeune Syndromes Are Caused by Mutations in WDR60

Short-rib polydactyly syndromes (SRPS I–V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis. Short-rib polydactyly syndromes (SRPS I–V) are a group of lethal congenital disorders characterized by shortening of the ribs and long bones, polydactyly, and a range of extraskeletal phenotypes. A number of other disorders in this grouping, including Jeune and Ellis-van Creveld syndromes, have an overlapping but generally milder phenotype. Collectively, these short-rib dysplasias (with or without polydactyly) share a common underlying defect in primary cilium function and form a subset of the ciliopathy disease spectrum. By using whole-exome capture and massive parallel sequencing of DNA from an affected Australian individual with SRPS type III, we detected two novel heterozygous mutations in WDR60, a relatively uncharacterized gene. These mutations segregated appropriately in the unaffected parents and another affected family member, confirming compound heterozygosity, and both were predicted to have a damaging effect on the protein. Analysis of an additional 54 skeletal ciliopathy exomes identified compound heterozygous mutations in WDR60 in a Spanish individual with Jeune syndrome of relatively mild presentation. Of note, these two families share one novel WDR60 missense mutation, although haplotype analysis suggested no shared ancestry. We further show that WDR60 localizes at the base of the primary cilium in wild-type human chondrocytes, and analysis of fibroblasts from affected individuals revealed a defect in ciliogenesis and aberrant accumulation of the GLI2 transcription factor at the centrosome or basal body in the absence of an obvious axoneme. These findings show that WDR60 mutations can cause skeletal ciliopathies and suggest a role for WDR60 in ciliogenesis. Ciliopathies form an expanding class of heterogeneous congenital diseases mechanistically linked by an underlying dysfunction of the primary cilium (reviewed in Waters and Beales,1Waters A.M. Beales P.L. Ciliopathies: an expanding disease spectrum.Pediatr. Nephrol. 2011; 26: 1039-1056Crossref PubMed Scopus (467) Google Scholar Hildebrandt et al.,2Hildebrandt F. Benzing T. Katsanis N. Ciliopathies.N. Engl. J. 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Garshasbi M. Tzschach A. et al.Cranioectodermal dysplasia, Sensenbrenner syndrome, is a ciliopathy caused by mutations in the IFT122 gene.Am. J. Hum. Genet. 2010; 86: 949-956Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 7Huber C. Cormier-Daire V. Ciliary disorder of the skeleton.Am. J. Med. Genet. C. Semin. Med. Genet. 2012; 160C: 165-174Crossref PubMed Scopus (168) Google Scholar The severe SRPS class is further divided into five subtypes, namely type I (Saldino-Noonan syndrome MIM 263530), type II (Majewski syndrome MIM 263520), type III (Verma-Naumoff syndrome MIM 263510), type IV (Beemer-Langer syndrome MIM 269860), and the recently recognized type V (MIM 614091).8Mill P. Lockhart P.J. Fitzpatrick E. Mountford H.S. Hall E.A. Reijns M.A. Keighren M. Bahlo M. Bromhead C.J. Budd P. et al.Human and mouse mutations in WDR35 cause short-rib polydactyly syndromes due to abnormal ciliogenesis.Am. J. Hum. Genet. 2011; 88: 508-515Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar Among the skeletal ciliopathies, the SRPS subtypes are the most severe and are incompatible with postnatal life. Skeletal ciliopathies may also manifest extraskeletal phenotypes including polycystic kidney disease, retinal degeneration, and cardiac, liver, and brain anomalies. To date many mutations causing skeletal ciliopathies affect genes encoding components of the intraflagellar transport (IFT) machinery, a motor-driven trafficking process responsible for transporting proteins required for cilia assembly and function along the axoneme.9Hao L. Scholey J.M. Intraflagellar transport at a glance.J. 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Genet. 2003; Full Text Full Text PDF PubMed Scopus (154) Google Scholar in massive parallel sequencing the to mutations for disorders in small families to With many ciliopathy at the molecular and proteins in the this approach is to the of genes mutated in we the of capture and massive parallel sequencing to mutations in WDR60, a relatively uncharacterized in two families with SRPS and Jeune syndrome, The family is a Australian family of but also with parents and two affected individuals with SRPS type III and in with short long on at at severe shortening of long with short and The at but at of the and in revealed polydactyly of both of some and mild of renal and with The affected individual from this family in an at but short ribs and short on at The at this further revealed defect in the and evidence of to of the of WDR60 and the on structure and WDR60 for the Australian SRPS family individual and the Spanish Jeune syndrome family Sequencing of DNA show of of the WDR60 mutated in the from the in family and using in and the predicted to in of for are as Sequencing of the the and with or the sequencing is in the on of human WDR60 the protein and the of the affected by the mutations in this The by the missense present in both families is conserved to C. structure and WDR60 for the Australian SRPS family individual and the Spanish Jeune syndrome family Sequencing of DNA show of of the WDR60 mutated in the from the in family and using in and the predicted to in of for are as Sequencing of the the and with or the sequencing is in the on of human WDR60 the protein and the of the affected by the mutations in this The by the missense present in both families is conserved to C. With of and whole-exome sequencing on DNA from individual in Sequencing were with the in of for capture by the and and for and with the DNA and parallel sequencing with cell the and to the Analysis for base were to the of the human the H. N. A of for 2010; PubMed Scopus Google Scholar were by H. B. A. T. J. N. G. G. R. and 2009; PubMed Scopus Google Scholar and for and base and were with the Analysis A. M. E. A. K. A. K. D. S. M. M.A. The Analysis a for DNA sequencing 2010; PubMed Scopus Google Scholar, M.A. E. R. K.V. C. A.A. G. M.A. M. et for and using DNA sequencing Genet. 2011; 43: PubMed Scopus Google Scholar and by K. M. H. of genetic variants from sequencing 2010; PubMed Scopus Google Scholar analysis of with and We and of for for for heterozygous we variants that and were to of damaging by using both and with a in D. A. of human from 2010; PubMed Scopus Google Scholar small with the G. G. 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S. the of variants on protein function using the 2009; PubMed Scopus Google Scholar predicted the to and sequencing of these mutations in the parents and other affected individual in this family compound heterozygous mutations were also in were both were predicted to and by and and sequencing that appropriately in the family and mutations in WDR60 were the most cause of disease in the Australian of this we the sequencing of WDR60 from A of and were of which of these were in and individual a compound for mutations in WDR60, that the of compound heterozygous WDR60 mutations in the human is to further of these we sequencing from an additional 54 individuals with skeletal ciliopathies, Jeune were with of the and in the this the these and as the Australian with as M. Frank V. Eisenberger T. Al Turki S. Bizet A.A. D. Rix S. C. N. M. et mutations in the intraflagellar transport gene IFT140 in skeletal ciliopathies with kidney 2013; PubMed Scopus Google Scholar were by exomes from the variants were for in and the Sequencing by a a base variants of an and variants and were genes with variants with in exomes the were and genes were for in the cilia as a of for of and identified to these one of the 54 individuals in to two damaging heterozygous mutations in of these is the novel missense by the Australian family The other is a novel in the conserved and predicted to affect of This by and sequencing of from a cell from individual and This that by aberrant in a to of the protein WDR60 mutations were by sequencing to appropriately in this family also compound heterozygous variants in (MIM and a in (MIM With to a of for an in the absence of a of in this the predicted to affect a and is to or to and although polycystic kidney disease and show no of the skeletal that Jeune J. M. H. K. K. J.H. et is an and function during PubMed Scopus Google Scholar the variants in affected conserved and as to predicted one predicted to and by these the other predicted to a by and although predicted sequencing mutations in this of variants in cause in (MIM with the Jeune syndrome phenotype in this The affected individual is the of Spanish at detected short at at and at with a polydactyly on the by and a small defect of to and at and on the of the He has no evidence of or developmental to date to the Australian SRPS this a relatively mild both families in this share one missense and recent we this a The for the variants were to the the analysis with the which identifies haplotype by the for with the are as The WDR60 gene for the and also with the which identifies short in sequencing shared were identified by a common effect in these two families sequencing analysis for the identified WDR60 mutations in additional Jeune syndrome with DNA for whole-exome sequencing further of the of WDR60, we have the of WDR60 in these WDR60 is a relatively uncharacterized conserved protein predicted to and two These have been in and in are in proteins involved in IFT and cilia L. Scholey J.M. Intraflagellar transport at a glance.J. Cell Sci. 2009; 122: 889-892Crossref PubMed Scopus (54) Google Scholar The in family and the in family are both predicted to to of the protein the but to the and the missense in both families is predicted to to an the of the of the wild-type by analysis suggests that this is to some of all mutations are to in or to the or of protein. is the in for G. N. Gleeson J.G. human disease in the 2011; Full Text Full Text PDF PubMed Scopus Google Scholar mutations in the gene can to skeletal ciliopathies for mutations in both Jeune syndrome and SRPS type N. Goulet M. Geneviève D. Sznajer Y. Martinovic J. Smithson S. Huber C. Baujat G. Flori E. Tecco L. et al.DYNC2H1 mutations cause asphyxiating thoracic dystrophy and short rib-polydactyly syndrome, type III.Am. J. Hum. Genet. 2009; 84: 706-711Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar we have Jeune syndrome individuals with including of M. Arts H.H. Bongers Oud M.M. D. L. J. et sequencing identifies mutations as a common cause of asphyxiating thoracic dystrophy without major polydactyly, renal or retinal Med. Genet. 2013; PubMed Scopus Google Scholar with is a in individuals in the families in this families share a common missense mutation, and the other in both is predicted to a These alleles may in or a protein, although of a protein with some function is that in may from WDR60 but may the effect of genetic modifying a that has also been for G. N. Gleeson J.G. human disease in the 2011; Full Text Full Text PDF PubMed Scopus Google Scholar or WDR60 is relatively two recent of cilia in mouse H. J. analysis of primary Biol. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar and in mouse E. T. of cilium and centrosome components and to human 2012; PubMed Scopus Google Scholar identified this protein, that may have a function in the of ciliogenesis to both primary and of cilia in C. identified the WDR60 and this protein in the a conserved function for WDR60 at the E.A. C. A. J. G. et of the a Biol. Full Text Full Text PDF PubMed Scopus Google Scholar of one of these of WDR60 at the cilium in H. J. analysis of primary Biol. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar We have by using a that WDR60 localizes in a at the base of the primary cilium in human We primary fibroblasts from the individuals with WDR60 mutations to the of in to cilia by to for were fibroblasts from postnatal for or for These are for individuals at and fibroblasts at The postnatal to the fibroblasts from both as an additional postnatal we a of that the of fibroblasts to form cilia is on the of The of in a although in the of also in the and and the of with a no obvious in to to that cilia were by the detected by to the in these we further in of the with the small This of with the these suggest a defect in ciliogenesis in these individuals and WDR60 to this skeletal ciliopathies have been linked to mutations in IFT we of IFT proteins by analysis in the IFT-A protein and the protein are at the which the basal body and and along the and the small of the of both of these proteins and in that to a primary both and at the centrosome or basal body by These are with findings in from mouse that cilia of in anterograde including and that IFT which are required for axonemal can to the basal body cilium assembly is Jr., K.F. Anderson K.V. The gene the of 2012; Full Text Full Text PDF PubMed Scopus Google Scholar in has been to a A. G. J.E. C. C. C. The intraflagellar transport is a protein regulating in Cell Sci. 2007; PubMed Scopus Google Scholar we to of a protein. this we which is a of hedgehog signaling and in and of the cilium in to of the hedgehog J. M. trafficking of in the primary cilium to in the Sci. 2009; PubMed Scopus (252) Google Scholar basal in GLI2 localizes to the of the cilium with further at this of with the hedgehog Jr., K.F. Ashe A. He M. Satir P. Moran J. Beier D. Wicking C. Anderson K.V. The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking.J. Cell Biol. 2012; 197: 789-800Crossref PubMed Scopus (154) Google Scholar that a cilium the wild-type of GLI2 in we a accumulation of GLI2 to the of been with GLI2 is to the these the that in WDR60 the mother centriole to the membrane and basal body but to an axoneme. a by analysis may present in these fibroblasts from for cilia by from the absence of the Jr., K.F. Ashe A. He M. Satir P. Moran J. Beier D. Wicking C. Anderson K.V. The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking.J. Cell Biol. 2012; 197: 789-800Crossref PubMed Scopus (154) Google Scholar We as an of the along with which in the cilium in the absence of localizes along the in both and and in with no obvious with at the basal body and is no evidence of cilia with at at this of these suggest that IFT GLI2 can to the basal body in the absence of an obvious axonemal at a in ciliogenesis by a This is with GLI2 is present in the is Jr., K.F. Ashe A. He M. Satir P. Moran J. Beier D. Wicking C. Anderson K.V. The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking.J. Cell Biol. 2012; 197: 789-800Crossref PubMed Scopus (154) Google Scholar the that some are to form cilia with and trafficking suggests that a of a function may required in a cell at a to ciliogenesis. by using whole-exome in one we novel mutations in WDR60 in an Australian family with SRPS type III and in a Spanish family with Jeune both families share one disease haplotype analysis suggests no shared ancestry. The caused by mutations in the gene may due to of the on protein function with other ciliopathy cases, may also in the or or other in affected By using from affected we have a defect in to in axonemal extension. the to IFT in the skeletal ciliopathies to the function of WDR60 and role in regulating this trafficking The to the families for and in the We also to de for family for from family for the on individual for the cell and for and for at the for for with the of the Australian is the of a and an Australian and by an and also from the is a and from the and from the is by the and from for and the is by an This sequencing by the which is by A of can at with and and The for are as Sequencing in