Nadia Elkhartoufi

Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes

Joubert syndrome (JBTS), related disorders (JSRDs) and Meckel syndrome (MKS) are ciliopathies. We now report that MKS2 and CORS2 (JBTS2) loci are allelic and caused by mutations in TMEM216, which encodes an uncharacterized tetraspan transmembrane protein. Individuals with CORS2 frequently had nephronophthisis and polydactyly, and two affected individuals conformed to the oro-facio-digital type VI phenotype, whereas skeletal dysplasia was common in fetuses affected by MKS. A single G218T mutation (R73L in the protein) was identified in all cases of Ashkenazi Jewish descent (n = 10). TMEM216 localized to the base of primary cilia, and loss of TMEM216 in mutant fibroblasts or after knockdown caused defective ciliogenesis and centrosomal docking, with concomitant hyperactivation of RhoA and Dishevelled. TMEM216 formed a complex with Meckelin, which is encoded by a gene also mutated in JSRDs and MKS. Disruption of tmem216 expression in zebrafish caused gastrulation defects similar to those in other ciliary morphants. These data implicate a new family of proteins in the ciliopathies and further support allelism between ciliopathy disorders.

KIF7 mutations cause fetal hydrolethalus and acrocallosal syndromes

KIF7, the human ortholog of Drosophila Costal2, is a key component of the Hedgehog signaling pathway. Here we report mutations in KIF7 in individuals with hydrolethalus and acrocallosal syndromes, two multiple malformation disorders with overlapping features that include polydactyly, brain abnormalities and cleft palate. Consistent with a role of KIF7 in Hedgehog signaling, we show deregulation of most GLI transcription factor targets and impaired GLI3 processing in tissues from individuals with KIF7 mutations. KIF7 is also a likely contributor of alleles across the ciliopathy spectrum, as sequencing of a diverse cohort identified several missense mutations detrimental to protein function. In addition, in vivo genetic interaction studies indicated that knockdown of KIF7 could exacerbate the phenotype induced by knockdown of other ciliopathy transcripts. Our data show the role of KIF7 in human primary cilia, especially in the Hedgehog pathway through the regulation of GLI targets, and expand the clinical spectrum of ciliopathies.

A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the « molar tooth sign ». JS is genetically heterogeneous, involving 20 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia, and polydactyly, combined exome sequencing and mapping identified a homozygous splice‐site mutation in PDE6D, encoding a prenyl‐binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild‐type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or mental retardation, obesity, congenital retinal dystrophy, and micropenis syndromes, as novel prenyl‐dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP‐bound ARL3, which acts as a cargo‐release factor for PDE6D‐bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl‐binding‐dependent trafficking in ciliopathies.

CC2D2A mutations in Meckel and Joubert syndromes indicate a genotype-phenotype correlation.

Meckel‐Gruber syndrome (MKS) is a lethal fetal disorder characterized by diffuse renal cystic dysplasia, polydactyly, a brain malformation that is usually occipital encephalocele, and/or vermian agenesis, with intrahepatic biliary duct proliferation. Joubert syndrome (JBS) is a viable neurological disorder with a characteristic “molar tooth sign” (MTS) on axial images reflecting cerebellar vermian hypoplasia/dysplasia. Both conditions are classified as ciliopathies with an autosomal recessive mode of inheritance. Allelism of MKS and JBS has been reported for TMEM67/MKS3, CEP290/MKS4, and RPGRIP1L/MKS5. Recently, one homozygous splice mutation with a founder effect was reported in the CC2D2A gene in Finnish fetuses with MKS, defining the 6th locus for MKS. Shortly thereafter, CC2D2A mutations were also reported in JBS. The analysis of the CC2D2A gene in our series of MKS fetuses, identified 14 novel truncating mutations in 11 cases. These results confirm the involvement of CC2D2A in MKS and reveal a major contribution of CC2D2A to the disease. We also identified three missense CC2D2A mutations in two JBS cases. Therefore, and in accordance with the data reported regarding RPGRIP1L, our results indicate phenotype–genotype correlations, as missense and presumably hypomorphic mutations lead to JBS while all null alleles lead to MKS. Hum Mutat 30:1–9, 2009.

TMEM231, mutated in orofaciodigital and Meckel syndromes, organizes the ciliary transition zone

TMEM231, a functional component of the MKS complex at the ciliary transition zone, is mutated in orofaciodigital syndrome type 3 and Meckel syndrome.

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.

Phenotypic spectrum and prevalence of INPP5E mutations in Joubert Syndrome and related disorders

Joubert syndrome and related disorders (JSRD) are clinically and genetically heterogeneous ciliopathies sharing a peculiar midbrain–hindbrain malformation known as the ‘molar tooth sign’. To date, 19 causative genes have been identified, all coding for proteins of the primary cilium. There is clinical and genetic overlap with other ciliopathies, in particular with Meckel syndrome (MKS), that is allelic to JSRD at nine distinct loci. We previously identified the INPP5E gene as causative of JSRD in seven families linked to the JBTS1 locus, yet the phenotypic spectrum and prevalence of INPP5E mutations in JSRD and MKS remain largely unknown. To address this issue, we performed INPP5E mutation analysis in 483 probands, including 408 JSRD patients representative of all clinical subgroups and 75 MKS fetuses. We identified 12 different mutations in 17 probands from 11 JSRD families, with an overall 2.7% mutation frequency among JSRD. The most common clinical presentation among mutated families (7/11, 64%) was Joubert syndrome with ocular involvement (either progressive retinopathy and/or colobomas), while the remaining cases had pure JS. Kidney, liver and skeletal involvement were not observed. None of the MKS fetuses carried INPP5E mutations, indicating that the two ciliopathies are not allelic at this locus.

Novel KIF7 mutations extend the phenotypic spectrum of acrocallosal syndrome

Background Acrocallosal syndrome (ACLS) is a rare recessive disorder characterised by corpus callosum agenesis or hypoplasia, craniofacial dysmorphism, duplication of the hallux, postaxial polydactyly, and severe mental retardation. Recently, we identified mutations in KIF7, a key component of the Sonic hedgehog pathway, as being responsible for this syndrome. Methods We sequenced KIF7 in five suspected ACLS cases, one fetus and four patients, based on facial dysmorphism and brain anomalies. Results Seven mutations were identified at the KIF7 locus in these five cases, six of which are novel. We describe the first four compound heterozygous cases. In all patients, the diagnosis was suspected based on the craniofacial features, despite the absence of corpus callosum anomaly in one and of polydactyly in another. Hallux duplication was absent in 4/5 cases. Conclusions These results show that ACLS has a variable expressivity and can be diagnosed even in the absence of the two major features, namely polydactyly or agenesis or hypoplasia of the corpus callosum. Facial dysmorphism with hypertelorism and prominent forehead in all the cases, as well as vermis dysgenesis with brainstem anomalies (molar tooth sign), strongly indicated the diagnosis. KIF7 should be tested in less typical patients in whom craniofacial features are suggestive of ACLS.

Identification of a novel ARL13B variant in a Joubert syndrome-affected patient with retinal impairment and obesity

Joubert syndrome (JS) is a genetically heterogeneous autosomal recessive ciliopathy with 22 genes implicated to date, including a small, ciliary GTPase, ARL13B. ARL13B is required for cilia formation in vertebrates. JS patients display multiple symptoms characterized by ataxia due to the cerebellar vermis hypoplasia, and that can also include ocular abnormalities, renal cysts, liver fibrosis or polydactyly. These symptoms are shared with other ciliopathies, some of which display additional phenotypes, such as obesity. Here we identified a novel homozygous missense variant in ARL13B/JBTS8 in a JS patient who displayed retinal defects and obesity. We demonstrate the variant disrupts ARL13B function, as its expression did not rescue the mutant phenotype either in Arl13bscorpion zebrafish or in Arl13bhennin mouse embryonic fibroblasts, while the wild-type ARL13B did. Finally, we show that ARL13B is localized within the primary cilia of neonatal mouse hypothalamic neurons consistent with the known link between hypothalamic ciliary function and obesity. Thus our data identify a novel ARL13B variant that causes JS and retinopathy and suggest an extension of the phenotypic spectrum of ARL13B mutations to obesity.

High‐throughput sequencing of a 4.1 Mb linkage interval reveals FLVCR2 deletions and mutations in lethal cerebral vasculopathy

Rare lethal disease gene identification remains a challenging issue, but it is amenable to new techniques in high‐throughput sequencing (HTS). Cerebral proliferative glomeruloid vasculopathy (PGV), or Fowler syndrome, is a severe autosomal recessive disorder of brain angiogenesis, resulting in abnormally thickened and aberrant perforating vessels leading to hydranencephaly. In three multiplex consanguineous families, genome‐wide SNP analysis identified a locus of 14 Mb on chromosome 14. In addition, 280 consecutive SNPs were identical in two Turkish families unknown to be related, suggesting a founder mutation reducing the interval to 4.1 Mb. To identify the causative gene, we then specifically enriched for this region with sequence capture and performed HTS in a proband of seven families. Due to technical constraints related to the disease, the average coverage was only 7×. Nonetheless, iterative bioinformatic analyses of the sequence data identified mutations and a large deletion in the FLVCR2 gene, encoding a 12 transmembrane domain‐containing putative transporter. A striking absence of alpha‐smooth muscle actin immunostaining in abnormal vessels in fetal PGV brains, suggests a deficit in pericytes, cells essential for capillary stabilization and remodeling during brain angiogenesis. This is the first lethal disease‐causing gene to be identified by comprehensive HTS of an entire linkage interval. Hum Mutat 31:1–8, 2010.