Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies. Ciliopathies are a continuum of genetically highly heterogeneous disorders with varying severity and organ involvement and are all caused by genes involved in ciliary function or biogenesis. Among the most severe ciliopathies are the short-rib polydactyly (SRP) group of lethal skeletal dysplasia (SRPI [MIM: 613091], SRPII [MIM: 263520], SRPIII [MIM: 613091], and SRPIV [MIM: 269860]), hydrolethalus syndrome (HLS [MIM: 236680]), and Meckel syndrome (MKS [MIM: 249000]). All three represent the extreme phenotype of viable ciliopathies, namely Jeune asphyxiating thoracic dystrophy (MIM: 208500) and Ellis-van Creveld syndrome (MIM: 225500),1Huber 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 acrocallosal syndrome (MIM: 200990),2Putoux A. Thomas S. Coene K.L.M. Davis E.E. Alanay Y. Ogur G. Uz E. Buzas D. Gomes C. Patrier S. et al.KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes.Nat. Genet. 2011; 43: 601-606Crossref PubMed Scopus (172) Google Scholar and Joubert syndrome (MIM: 213300),3Baala L. Romano S. Khaddour R. Saunier S. Smith U.M. Audollent S. Ozilou C. Faivre L. Laurent N. Foliguet B. et al.The Meckel-Gruber syndrome gene, MKS3, is mutated in Joubert syndrome.Am. J. Hum. Genet. 2007; 80: 186-194Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 4Delous M. Baala L. Salomon R. Laclef C. Vierkotten J. Tory K. Golzio C. Lacoste T. Besse L. Ozilou C. et al.The ciliary gene RPGRIP1L is mutated in cerebello-oculo-renal syndrome (Joubert syndrome type B) and Meckel syndrome.Nat. Genet. 2007; 39: 875-881Crossref PubMed Scopus (366) Google Scholar, 5Valente E.M. Logan C.V. Mougou-Zerelli S. Lee J.H. Silhavy J.L. Brancati F. Iannicelli M. Travaglini L. Romani S. Illi B. et al.Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes.Nat. Genet. 2010; 42: 619-625Crossref PubMed Scopus (215) Google Scholar respectively. Here, we examined a consanguineous Lebanese family (family 1; Figure 1A) with two 15 gestational week (gw)-old fetuses presenting with severe hydrocephaly, polydactyly of the hands and feet, a cleft palate, and skeletal abnormalities (Figure 1B, Table 1, and supplemental case reports). We considered this phenotype to be similar to that of HLS and therefore sequenced HYLS1 (MIM: 610693) and KIF7 (MIM: 611254) but found no mutation. Because HLS is now known as a ciliopathy, we then combined a targeted capture strategy for candidate ciliary genes with next-generation sequencing, as described previously, by using DNA from fetus II:3.6Thomas S. Legendre M. Saunier S. Bessières B. Alby C. Bonnière M. Toutain A. Loeuillet L. Szymanska K. Jossic F. et al.TCTN3 mutations cause Mohr-Majewski syndrome.Am. J. Hum. Genet. 2012; 91: 372-378Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 7Failler M. Gee H.Y. Krug P. Joo K. Halbritter J. Belkacem L. Filhol E. Porath J.D. Braun D.A. Schueler M. et al.Mutations of CEP83 cause infantile nephronophthisis and intellectual disability.Am. J. Hum. Genet. 2014; 94: 905-914Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar In brief, ciliary exome-targeted sequencing and bioinformatics filtering were conducted with a Custom SureSelect Capture Kit (Agilent Technologies) targeting 4.5 Mb of 20,168 exons (1,221 ciliary candidate genes). Agilent SureSelect libraries were prepared from 3 μg of genomic DNA sheared with a Covaris S2 Ultrasonicator according to manufacturer’s instructions. The Ovation Ultralow System (NuGEN Technologies) was used to prepare HiSeq 2500 pre-capture barcoded libraries. The ciliome capture by hybridization was performed on a pool of 10–16 barcoded pre-capture libraries. Sequencing performed on a HiSeq 2500 (Illumina) was done on pools of barcoded ciliome libraries (16 ciliome libraries per lane of HiSeq FlowCell). Paired-end reads (100 100-bp reads) were generated and mapped on a human genome reference (NCBI Genome browser build 37) with the Burrows-Wheeler Aligner (Illumina). Downstream processing was carried out with the Genome Analysis Toolkit, SAMtools, and Picard Tools according to documented best practices from the Broad Institute. All variants were annotated with a software system developed by the Paris Descartes University bioinformatics platform. Informed consent was obtained for all participating families, and the study was approved by the ethical committee of Paris Ile de France II. Genomic DNA was extracted from frozen tissue or amniocyte cultured cells for fetal subjects and from peripheral-blood samples for parents. Finally, a total of 6,263 variants were identified, and after we filtered data by removing known SNPs and synonymous coding sequence variations and by using a recessive model of inheritance, a unique nonsense homozygous mutation remained (Table S1) in KIAA0586 (MIM: 610178). KIAA0586, the ortholog of chicken KIAA0586, was considered an excellent candidate gene given that its disruption in animal models causes defects, including polydactyly and abnormal dorsoventral patterning of the neural tube, attributed to abnormal hedgehog signaling.8Davey M.G. Paton I.R. Yin Y. Schmidt M. Bangs F.K. Morrice D.R. Smith T.G. Buxton P. Stamataki D. Tanaka M. et al.The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling.Genes Dev. 2006; 20: 1365-1377Crossref PubMed Scopus (102) Google Scholar, 9Yin Y. Bangs F. Paton I.R. Prescott A. James J. Davey M.G. Whitley P. Genikhovich G. Technau U. Burt D.W. Tickle C. The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation.Development. 2009; 136: 655-664Crossref PubMed Scopus (104) Google Scholar, 10Bangs F. Antonio N. Thongnuek P. Welten M. Davey M.G. Briscoe J. Tickle C. Generation of mice with functional inactivation of talpid3, a gene first identified in chicken.Development. 2011; 138: 3261-3272Crossref PubMed Scopus (59) Google Scholar, 11Ben J. Elworthy S. Ng A.S.M. van Eeden F. Ingham P.W. Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates.Development. 2011; 138: 4969-4978Crossref PubMed Scopus (52) Google Scholar The c.230C>G (p.Ser77∗) variant (GenBank: NM_001244189.1) segregated with the expected patterns of autosomal-recessive inheritance in all available family members and is absent from dbSNP, the NHLBI Exome Sequencing Project (ESP) Exome Variant Server (EVS), the Exome Aggregation Consortium (ExAC) Browser, and 300 Lebanese control chromosomes. This homozygous nonsense variation is located in KIAA0586 exon 2, and mRNAs produced are predicted to be targeted for nonsense-mediated decay (NMD). To test this, we extracted mRNA from the tissue of an affected individual (subject II:3, family 1) and age-matched control individuals and subsequently performed reverse transcription. Compared to control mRNA, mRNA extracted from the affected subject, showed a total absence of transcript containing KIAA0586 exon 2. Importantly, GAPDH amplification was similar in both samples (Figure 1C). We next tested KIAA0586 expression in different tissues at different human developmental stages and found those specific transcripts containing KIAA0586 exon 2 were expressed as early as 6 gw (Carnegie stage 16) in humans. They appeared ubiquitously expressed during fetal development and persisted postnatally in all human adult tissues tested (Figure S1).Table 1Clinical Data of Affected Individuals in the Four Families StudiedFamily (ID)SubjectAgeSexOriginPDCKBDPCPMTSBrain AnomaliesSkeletal AnomaliesOther AnomaliesVariationExonInheritance1 (HE)II:215 gwNDLebanon+NDND+major hydrocephalyno claviclediaphragmatic herniano DNANDNDII:315 gwfemaleLebanon+−ND+major hydrocephaly, occipital defectflat and wide iliac wingsfetal hydropsc.230C>G (p.Ser77∗)2homozygous2 (CI)II:229 gwmaleRomania+−−++VH, polymicrogyria, absent olfactory bulb, ventriculomegalyshort ribstongue hamartomas, multiple frenulaec.1815G>A (splice)14homozygousII:339 gw (died 1 hr after birth)NDRomania+−NDND+VH, abnormal gyration, mega cisterna magnashort ribs, short limbsNDc.1815G>A (splice)14homozygous3 (FA)II:1spontaneous fetal death (<10 weeks)NDHungaryNDNDNDNDNDanencephalyNDNDno DNANDNDII:2died at 13 monthsfemaleHungary+−−−+occipital meningocele, VH, hypoplasia of the hemispheres and corpus callosum, abnormal basal ganglia, pontocerebellar hypoplasiashort ribs, short limbs, PD of hands and feetdysplastic and low set ears, depressed nasal bridge, short neck, multiple frenulaec.1815G>A (splice)14homozygous4 (ME)II:4died at 1 day of lifeNDKosovo+−−++described as identical to his siblingdescribed as identical to his siblingdescribed as identical to his siblingno DNANDNDII:526 gwmaleKosovo+−−++Occipital meningocele (key hole), hypoplastic brain stem, VH, CC and septal agenesis, temporal polymicrogyriaShort ribs, short limbs (−6 to −8 SD), superior limb incurvation, bilateral postaxial PD of hands and feetRetinal dysplasia with retinal coloboma, brachyphalangism, facial dysmorphism, micropenis, frenulae nodulesc.1815G>A (splice)14homozygousThe KIAA0586 reference sequence used was GenBank: NM_001244189.1. Chromosome analysis and clinicopathological examination were performed for all affected subjects. Abbreviations are as follows: BDP, bile duct proliferation of liver; CC, corpus callosum; CK, cystic kidneys; CP, cleft palate; MTS, molar tooth sign; PD, polydactyly; VH, vermis hypoplasia, ND, no data. Open table in a new tab The KIAA0586 reference sequence used was GenBank: NM_001244189.1. Chromosome analysis and clinicopathological examination were performed for all affected subjects. Abbreviations are as follows: BDP, bile duct proliferation of liver; CC, corpus callosum; CK, cystic kidneys; CP, cleft palate; MTS, molar tooth sign; PD, polydactyly; VH, vermis hypoplasia, ND, no data. We performed additional next-generation sequencing of ciliary genes for 150 subjects presenting with lethal ciliopathies with various combinations of brain and skeletal abnormalities. This screen led us to identify a c.1815G>A (p.=) homozygous silent variant (GenBank: NM_001244189.1) in three subjects from three unrelated families (II:2 in family 2, II:2 in family 3, and II:5 in family 4) originating from Romania (family 2), Hungary (family 3), and Kosovo (family 4). The variant segregates with the expected patterns of autosomal-recessive inheritance in all available family members. All three subjects had a SRP syndrome with similar cerebral anomalies, preaxial polydactyly of the feet and postaxial polydactyly of the hands, and long-bone shortening, including short ribs. Upon neuropathological examination, all three affected subjects displayed vermis agenesis and brainstem anomalies evocative of a molar tooth sign (Figure 2A, Table 1, and supplemental case reports). The variation involves the last base of KIAA0586 exon 14, is absent from dbSNP, the EVS, and the ExAC Browser, and is predicted in silico to abolish the intron 14 donor splice site (MaxEntScan, splice site prediction by Neural Network [NNSPLICE], and Human Splicing Finder). To confirm this hypothesis, we performed RT-PCR and subsequent cDNA sequencing on total mRNA extracted from blood samples from subjects II:2 and II:3 from family 2 and from control individuals. A unique transcript lacking KIAA0586 exon 14 was observed in both affected subjects (Figure 2B) and is predicted to cause a shift in the reading frame with a premature stop codon. Given the Eastern European origin of the three families, a founder effect was highly suspected. The distance to the common ancestor was estimated by a likelihood-based method.12Genin E. Tullio-Pelet A. Begeot F. Lyonnet S. Abel L. Estimating the age of rare disease mutations: the example of Triple-A syndrome.J. Med. Genet. 2004; 41: 445-449Crossref PubMed Scopus (100) Google Scholar We selected the polymorphic markers encompassing KIAA0586 and found a similar haplotype in those three families (Figure 2C). The allele frequencies of the microsatellites used were found on the CEPH genotype database, and the mutation rate was chosen as 10−3. Because the genetic distances available for closely linked markers are generally not very accurate, we computed the rates of recombination between markers by using both the overall genetic length of the haplotype and the physical distances between markers.13Picard C. Fieschi C. Altare F. Al-Jumaah S. Al-Hajjar S. Feinberg J. Dupuis S. Soudais C. Al-Mohsen I.Z. Génin E. et al.Inherited interleukin-12 deficiency: IL12B genotype and clinical phenotype of 13 patients from six kindreds.Am. J. Hum. Genet. 2002; 70: 336-348Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar The closest markers at which affected individuals do not share any alleles were determined on both sides of KIAA0586. The distance to the common ancestor was estimated to be approximately 480 years n = 16 generations ago (ninf [the lower bound of the 95% confidence interval (CI)] = 5; nsup [the upper bound of the 95% CI] = 63; nend [the total number of iterations performed] = 232; likelihood = −3.776324). In animal and cell models, TALPID3 has been involved in very early stages of ciliogenesis, before the formation and docking of a primary ciliary vesicle at the distal appendage.14Kobayashi T. Kim S. Lin Y.-C. Inoue T. Dynlacht B.D. The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly.J. Cell Biol. 2014; 204: 215-229Crossref PubMed Scopus (78) Google Scholar TALPID3 interacts with CP110 and co-localizes to the distal ends of centrioles. CP110 and its protein-interaction network (including CEP97, CEP290, and KIF24) have been found to modulate cilium assembly (reviewed in Tsang et al.15Tsang W.Y. Dynlacht B.D. CP110 and its network of partners coordinately regulate cilia assembly.Cilia. 2013; 2: 9PubMed Google Scholar). In addition, TALPID3 localizes near the distal appendages,14Kobayashi T. Kim S. Lin Y.-C. Inoue T. Dynlacht B.D. The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly.J. Cell Biol. 2014; 204: 215-229Crossref PubMed Scopus (78) Google Scholar which localize to the site of centriole-to-membrane docking and are required for the centriole to be converted to a basal body and thus for ciliogenesis.16Tanos B.E. Yang H.-J. Soni R. Wang W.-J. Macaluso F.P. Asara J.M. Tsou M.-F.B. Centriole distal appendages promote membrane docking, leading to cilia initiation.Genes Dev. 2013; 27: 163-168Crossref PubMed Scopus (229) Google Scholar In order to examine the effect of KIAA0586 loss on cilia assembly in humans, we induced ciliogenesis by using serum-starvation-mediated cell-cycle arrest in confluent fibroblasts from both affected (family 2, subject II:2) and control subjects and visualized cilia by co-immunostaining with antibodies to ARL13B (a cilia marker), glutamylated tubulin (GT335) or acetylated α-tubulin, and pericentrin (Figure 3A and data not shown). In control fibroblasts with wild-type KIAA0586, cilia were evident in 60% of the total stained cells by 48 hr of serum starvation, and nearly all were co-stained with all markers. In mutant fibroblasts, only 20% of cells presented with cilia (Figures 3A and 3B). This result indicates that KIAA0586 is necessary for cilia biogenesis in humans, in accordance with its function in animal and cell models. We then checked centriolar morphogenesis and targeting of crucial proteins and found no differences in the staining patterns of CEP164 (distal appendages of mother centrioles) and ODF2 (subdistal appendages), indicating that assembly of these proteins to centrioles is not affected by KIAA0586 loss (Figures 3C and 3D). Finally, as suggested by TALPID3 siRNA depletion studies in which TALPID3 is required for centriolar satellite dispersal preceding the formation of mature ciliary vesicles,14Kobayashi T. Kim S. Lin Y.-C. Inoue T. Dynlacht B.D. The CP110-interacting proteins Talpid3 and Cep290 play overlapping and distinct roles in cilia assembly.J. Cell Biol. 2014; 204: 215-229Crossref PubMed Scopus (78) Google Scholar we compared the distribution of the CP110-interacting protein CEP290 in affected cells to the distribution in control cells and found an abnormal extended pattern of this satellite protein in asynchronously growing conditions (Figure 3E). We next tested the transduction of the SHH pathway in mutant fibroblasts by assaying the expression of PTCH1 and GLI1, two transcriptional targets of SHH signaling, after pathway activation by the addition of smoothened agonist. We found that both PTCH1 and GLI1 were less induced in mutant fibroblasts than in controls (Figure 4A). We also analyzed GLI3 processing by western blot and found that KIAA0586 mutant fibroblasts exhibited increased amounts of full-length, unprocessed GLI3 (GLI3-FL) and an abnormal GLI3-FL/GLI3-R ratio (Figure 4B and Figure S3). Thus, KIAA0586 mutant cells exhibit abnormal SHH responsiveness, suggesting that at least some of the defects in affected individuals with KIAA0586 variations might be secondary to abnormal SHH signaling, as observed in animal models.8Davey M.G. Paton I.R. Yin Y. Schmidt M. Bangs F.K. Morrice D.R. Smith T.G. Buxton P. Stamataki D. Tanaka M. et al.The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling.Genes Dev. 2006; 20: 1365-1377Crossref PubMed Scopus (102) Google Scholar, 9Yin Y. Bangs F. Paton I.R. Prescott A. James J. Davey M.G. Whitley P. Genikhovich G. Technau U. Burt D.W. Tickle C. The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation.Development. 2009; 136: 655-664Crossref PubMed Scopus (104) Google Scholar, 10Bangs F. Antonio N. Thongnuek P. Welten M. Davey M.G. Briscoe J. Tickle C. Generation of mice with functional inactivation of talpid3, a gene first identified in chicken.Development. 2011; 138: 3261-3272Crossref PubMed Scopus (59) Google Scholar, 11Ben J. Elworthy S. Ng A.S.M. van Eeden F. Ingham P.W. Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates.Development. 2011; 138: 4969-4978Crossref PubMed Scopus (52) Google Scholar, 17Davey M.G. McTeir L. Barrie A.M. Freem L.J. Stephen L.A. Loss of cilia causes embryonic lung hypoplasia, liver fibrosis, and cholestasis in the talpid3 ciliopathy mutant.Organogenesis. 2014; 10: 177-185Crossref PubMed Scopus (16) Google Scholar In this study, using a targeted high-throughput sequencing strategy for candidate ciliary genes, we identified homozygous nonsense and splicing KIAA0586 mutations as responsible for lethal ciliopathies. This candidate-gene strategy previously succeeded in identifying ciliopathy genes, including TCTN3 (MIM: 613847), which is associated with Mohr-Majewski syndrome (OFD4 [MIM: 258860]).6Thomas S. Legendre M. Saunier S. Bessières B. Alby C. Bonnière M. Toutain A. Loeuillet L. Szymanska K. Jossic F. et al.TCTN3 mutations cause Mohr-Majewski syndrome.Am. J. Hum. Genet. 2012; 91: 372-378Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar The phenotype of KIAA0586-affected subjects ranges from a hydrolethalus phenotype to SRP syndrome in four unrelated families. In particular, cerebral phenotypes range from anencephaly or large occipital meningocele to vermian agenesis associated with brainstem anomalies; such anomalies are classically suggestive of ciliopathies. Importantly, the only living SRP-affected individual reported in this study also presented with a molar tooth sign on brain MRI, consistent with the conclusions of neuropathological examination on fetal subjects. All affected subjects display skeletal anomalies, including a consistent polydactyly, short ribs, and micromelia with round femoral ends in families 2– 4. In addition, most of the affected subjects have a large median cleft palate. These features are reminiscent of the spectrum of SRPII and SRPIV.1Huber 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, 18El Hokayem J. Huber C. Couvé A. Aziza J. Baujat G. Bouvier R. Cavalcanti D.P. Collins F.A. Cordier M.-P. Delezoide A.-L. et al.NEK1 and DYNC2H1 are both involved in short rib polydactyly Majewski type but not in Beemer Langer cases.J. Med. Genet. 2012; 49: 227-233Crossref PubMed Scopus (46) Google Scholar, 19Schmidts M. Clinical genetics and pathobiology of ciliary chondrodysplasias.J. Pediatr. Genet. 2014; 3: 46-94PubMed Google Scholar Thus, fetuses and individuals reported in this study all display lethal phenotypes consistent with a hedgehog-signaling defect and similar to the phenotype described in KIAA0586 animal models.8Davey M.G. Paton I.R. Yin Y. Schmidt M. Bangs F.K. Morrice D.R. Smith T.G. Buxton P. Stamataki D. Tanaka M. et al.The chicken talpid3 gene encodes a novel protein essential for Hedgehog signaling.Genes Dev. 2006; 20: 1365-1377Crossref PubMed Scopus (102) Google Scholar, 9Yin Y. Bangs F. Paton I.R. Prescott A. James J. Davey M.G. Whitley P. Genikhovich G. Technau U. Burt D.W. Tickle C. The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation.Development. 2009; 136: 655-664Crossref PubMed Scopus (104) Google Scholar, 10Bangs F. Antonio N. Thongnuek P. Welten M. Davey M.G. Briscoe J. Tickle C. Generation of mice with functional inactivation of talpid3, a gene first identified in chicken.Development. 2011; 138: 3261-3272Crossref PubMed Scopus (59) Google Scholar, 11Ben J. Elworthy S. Ng A.S.M. van Eeden F. Ingham P.W. Targeted mutation of the talpid3 gene in zebrafish reveals its conserved requirement for ciliogenesis and Hedgehog signalling across the vertebrates.Development. 2011; 138: 4969-4978Crossref PubMed Scopus (52) Google Scholar, 17Davey M.G. McTeir L. Barrie A.M. Freem L.J. Stephen L.A. Loss of cilia causes embryonic lung hypoplasia, liver fibrosis, and cholestasis in the talpid3 ciliopathy mutant.Organogenesis. 2014; 10: 177-185Crossref PubMed Scopus (16) Google Scholar In particular, the KIAA0586 chicken with a frameshift mutation in exon 7 (corresponding to human exon 9) closely models the human SRP ciliopathy phenotype, including a small rib cage and polydactyly, as well as lung anomalies.17Davey M.G. McTeir L. Barrie A.M. Freem L.J. Stephen L.A. Loss of cilia causes embryonic lung hypoplasia, liver fibrosis, and cholestasis in the talpid3 ciliopathy mutant.Organogenesis. 2014; 10: 177-185Crossref PubMed Scopus (16) Google Scholar Interestingly, the c.1815G>A variation identified in families 2– 4 leads to the deletion of human exon 14, corresponding to mouse exon 12 (Figure S2), which has been shown to be essential for the function of the protein.9Yin Y. Bangs F. Paton I.R. Prescott A. James J. Davey M.G. Whitley P. Genikhovich G. Technau U. Burt D.W. Tickle C. The Talpid3 gene (KIAA0586) encodes a centrosomal protein that is essential for primary cilia formation.Development. 2009; 136: 655-664Crossref PubMed Scopus (104) Google Scholar, 10Bangs F. Antonio N. Thongnuek P. Welten M. Davey M.G. Briscoe J. Tickle C. Generation of mice with functional inactivation of talpid3, a gene first identified in chicken.Development. 2011; 138: 3261-3272Crossref PubMed Scopus (59) Google Scholar Indeed, Talpid3−/− mice in which exons 11 and 12 are constitutively deleted show abnormal Shh signaling and embryonic lethality as early as embryonic day 10.5. The c.230C>G (p.Ser77∗) nonsense variation of family 1 leads to NMD of exon-2-containing transcripts shown to be expressed early in human embryos and throughout fetal development. Finally, ciliogenesis was severely impaired in KIAA0586 mutant cells, and the SHH signaling pathway was abnormal, characteristic of other severe human ciliopathies, such as Morh-Majewski or hydrolethalus syndromes, associated with TCTN3 and KIF7 variations,2Putoux A. Thomas S. Coene K.L.M. Davis E.E. Alanay Y. Ogur G. Uz E. Buzas D. Gomes C. Patrier S. et al.KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes.Nat. Genet. 2011; 43: 601-606Crossref PubMed Scopus (172) Google Scholar, 6Thomas S. Legendre M. Saunier S. Bessières B. Alby C. Bonnière M. Toutain A. Loeuillet L. Szymanska K. Jossic F. et al.TCTN3 mutations cause Mohr-Majewski syndrome.Am. J. Hum. Genet. 2012; 91: 372-378Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar respectively, or Meckel syndrome. Overall, these results highlight the conserved function of KIAA0586 among species and its involvement in human ciliopathies. In view of the wide phenotypic spectrum of ciliopathy genes, the lethal phenotype reported here might represent the severe end of the phenotypic spectrum associated with KIAA0586 variations. We thank the families for their participation. We also thank Leila Hakkakian, Judite De Oliveira, and Nadège Gigot for technical assistance and Prof. Andrew Green for referral of affected subjects, and we are grateful to the French Society of Fetal Pathology for participating in the study. This work was supported by grants from the Agence Nationale de la Recherche (ANR; 2010 fetal ciliopathies grant BLAN-1122-01 and 2013 cilia, axonal guidance, and corpus callosum grant ANR-13-BSV1-0027 to T.A.B.). C.A. is funded by the Fondation pour la Recherche Médicale and K.P. by grants from ANR. The Institut Imagine is supported by an ANR grant (ANR-A0-IAHU-01). Download .pdf (4.14 MB) Help with pdf files Document S1. Case reports, Figures S1–S3, and Tables S1 and S2 The URLs for data presented herein are as follows:Broad Institute GATK Best Practices, https://www.broadinstitute.org/gatk/guide/best-practicesClustalW2, http://www.ebi.ac.uk/Tools/msa/clustalw2/Human Splicing Finder, http://www.umd.be/HSF/MaxEntScan, http://genes.mit.edu/burgelab/maxent/Xmaxentscan_scoreseq_acc.htmlMultAlin, http://multalin.toulouse.inra.fr/multalin/OMIM, http://www.omim.org/PolyPhen-2, http://genetics.bwh.harvard.edu/pph2/Primer3Plus, http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/SIFT, http://sift.bii.a-star.edu.sg/Splice Site Prediction by Neural Network (NNSPLICE), http://www.fruitfly.org/seq_tools/splice.htmlUCSC Genome Browser, http://genome.ucsc.edu Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly SyndromeAlby et al.The American Journal of Human GeneticsAugust 06, 2015In Brief(The American Journal of Human Genetics 97, 311–318; August 6, 2015) Full-Text PDF Open Archive