Blanc SVSE 1 - Sciences de la vie, de la santé et des écosystèmes : Physiologie, métabolisme, physiopathologie, santé publique

Molecular and cellular mechanisms of Meckel/Joubert ciliopathy – FOETOCILPATH

Submission summary

Meckel syndrome (MKS) is a severe autosomal recessive ciliopathy, which is often lethal prenatally or at birth, characterized by pleiotropic phenotypes such as multicystic kidney and hepatic duct dysplasia, occipital meningo-encephalocele and posterior fossa abnormalities (including cerebellar vermis agenesis), polydactyly, and other malformations. Since the identification of the two first genes responsible for MKS in 2006, MKS1 and TMEM67/MKS3, we have undertaken the molecular characterisation of a large series of 120 fetuses with Meckel syndrome or a closely related phenotype. We described a strong phenotype-genotype correlation, depending on the mutated gene. The identification of MKS3 mutations in fetuses with vermis agenesis led us to screen this gene in another ciliopathy, characterized by vermis agenesis and a brainstem anomaly, known as Joubert syndrome (JBS). This allowed us to describe the allelism between these two disorders JBS6/MKS3 locus. Since then, we identified and characterized two additional genes involved in MKS: CEP290/MKS4 and RPGRIP1L/MKS5.
In view of the close relationships between MKS and JBS, we have undertaken a collaboration with two other groups working on the JBS2 locus (Dr Joe Gleeson, San Diego, USA and Dr. Enza-Maria Valente, Rome, Italy). Our combined efforts allowed us to identify the JBS2/MKS2 gene as TMEM216 on chromosome 11 (article in revision in Nature Genetics). MKS2 encodes a previously uncharacterized tetraspan transmembrane protein, interacting with MKS3 and MKS4 proteins and expressed is cilia. MKS2 mutated fibroblasts of fetuses show absent or bent short cilia. To date, very little is known about the multisubunit complex in which MKS proteins are involved, and what signaling pathways they coordinate. We have found that MKS2 interacts with MKS3, MKS4, RhoA and Dishevelled-1 (Dvl-1), and that MKS2 inactivation in cellular models provokes defective basal body docking, with concomitant hyperactivation of RhoA and phosphorylation of Dvl-1. These data strongly suggest that MKS2 acts in a multisubunit complex that is essential for basal docking and ciliogenesis.
We therefore think that a top priority is to identify TMEM216/MKS2 interacting partners and signaling pathways by the following strategies: 1) protein purification of the complex after MKS2 immunoprecipitation; 2) identification of MKS2 partners through a yeast two-hybrid approach; 3) analysis of ciliogenesis (number and morphology of cilia, actin cytoskeleton remodeling and basal body/centrosome positioning) and of ciliary signaling pathways (Wnt/PCP, Shh) in MKS2 mutant human fibroblasts and in shRNA knockdown MKS2 renal tubular cells; and 4) functional analysis by RNAi and complementation by wild type and mutants human cDNAs in Paramecium used as a novel cellular model system. Indeed, the study of MKS protein function in Paramecium brings an important added value owing to the sensory, signal transduction and motile properties of cilia in this cell type.
Another aim of our project is to undertake a systematic analysis of the functional effect of mutations in all MKS genes, based on our unique resource of human fetal fibroblasts available from fetuses mutated for each gene, and on the Paramecium cellular model. A transcriptomic analysis of mutant fibroblasts cell lines and of MKS depleted Paramecium will allow us to define the signaling pathways regulated by MKS proteins.
Finally, a MKS gene mutation is identified in only 50% of our cohort, showing the extreme genetic heterogeneity of this condition. We will use genetic analysis in cases with no molecular explanation to identify and characterise new genes responsible for JBS/MKS or related nephronophtisis (NPHP) and Senior-Loken syndrome (SLS).

Project coordination

Tania ATTIE-BITACH (INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION DE PARIS V) – tania.attie@inserm.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSTITUT DES MALADIES GENETIQUES INSTITUT DES MALADIES GENETIQUES - IMAGINE
FRE3144 CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR SUD
U781 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION DE PARIS V
U983 INSTITUT NATIONAL DE LA SANTE ET DE LA RECHERCHE MEDICALE - DELEGATION DE PARIS V

Help of the ANR 528,320 euros
Beginning and duration of the scientific project: - 36 Months

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