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Joubert syndrome (JBTS), a rare genetic disorder resulted from primary cilium defects or basal-body dysfunction, is characterized by agenesis of cerebellar vermis and abnormal brain stem. Both genotypes and phenotypes of JBTS are highly heterogeneous. The identification of pathogenic gene variation is essential for making a definite diagnosis on JBTS. Here, we found that hypoplasia of cerebellar vermis occurred in three male members in a Chinese family. Then, we performed whole exome sequencing to identify a novel missense mutation c.599T > C (p. L200P) in the OFD1 gene which is the candidate gene of X-linked JBTS (JBST10). The following analysis showed that the variant was absent in the 1000 Genomes, ExAC and the 200 female controls; the position 200 Leucine residue was highly conserved across species; the missense variant was predicted to be deleterious using PolyPhen-2, PROVEAN, SIFT and Mutation Taster. The OFD1 expression was heavily lower in the proband and an induced male fetus compared with a healthy male with a wild-type OFD1 gene. The in vitro expression analysis of transiently transfecting c.599T or c.599C plasmids into HEK-293T cells confirmed that the missense mutation caused OFD1 reduction at the protein level. And further the mutated OFD1 decreased the level of Gli1 protein, a read-out of Sonic hedgehog (SHH) signaling essential for development of central neural system. A known pathogenic variant c.515T > C (p. L172P) showed the similar results. All of these observations suggested that the missense mutation causes the loss function of OFD1, resulting in SHH signaling impairs and brain development abnormality. In addition, the three patients have Dandy-Walker malformation, macrogyria and tetralogy of Fallot, respectively, the latter two of which are firstly found in JBTS10 patients. In conclusion, our findings expand the context of genotype and phenotype in the JBTS10 patients.

Zic genes are strongly expressed in the cerebellum. This feature leads to their initial identification and their name "zic," as the abbreviation of "zinc finger protein of the cerebellum." Zic gene function in cerebellar development has been investigated mainly in mice. However, association of heterozygous loss of ZIC1 and ZIC4 with Dandy-Walker malformation, a structural birth defect of the human cerebellum, highlights the clinical relevance of these studies. Two proposed mechanisms for Zic-mediated cerebellar developmental control have been documented: regulation of neuronal progenitor proliferation-differentiation and the patterning of the cerebellar primordium. Clinical studies have also revealed that ZIC1 gain of function mutations contribute to coronal craniosynostosis, a rare skull malformation. The molecular pathways contributing to these phenotypes are not fully explored; however, embryonic interactions with sonic hedgehog signaling, retinoic acid signaling, and TGFβ signaling have been described during mouse cerebellar development. Further, Zic1/2 target a multitude of genes associated with cerebellar granule cell maturation during postnatal mouse cerebellar development.

Mutations affecting the integrity and function of cilia have been identified in various genes over the last decade accounting for a group of diseases called ciliopathies. Ciliopathies display a broad spectrum of phenotypes ranging from mild manifestations to lethal combinations of multiple severe symptoms and most of them share cystic kidneys as a common feature. Our starting point was a consanguineous pedigree with three affected fetuses showing an early embryonic phenotype with enlarged cystic kidneys, liver and pancreas and developmental heart disease. By genome-wide linkage analysis, we mapped the disease locus to chromosome 17q11 and identified a homozygous nonsense mutation in NEK8/NPHP9 that encodes a kinase involved in ciliary dynamics and cell cycle progression. Missense mutations in NEK8/NPHP9 have been identified in juvenile cystic kidney jck mice and in patients suffering from nephronophthisis (NPH), an autosomal-recessive cystic kidney disease. This work confirmed a complete loss of NEK8 expression in the affected fetuses due to nonsense-mediated decay. In cultured fibroblasts derived from these fetuses, the expression of prominent polycystic kidney disease genes (PKD1 and PKD2) was decreased, whereas the oncogene c-MYC was upregulated, providing potential explanations for the observed renal phenotype. We furthermore linked NEK8 with NPHP3, another NPH protein known to cause a very similar phenotype in case of null mutations. Both proteins interact and activate the Hippo effector TAZ. Taken together, our study demonstrates that NEK8 is essential for organ development and that the complete loss of NEK8 perturbs multiple signalling pathways resulting in a severe early embryonic phenotype.

Heterozygous deletions encompassing the ZIC1;ZIC4 locus have been identified in a subset of individuals with the common cerebellar birth defect Dandy-Walker malformation (DWM). Deletion of Zic1 and Zic4 in mice produces both cerebellar size and foliation defects similar to human DWM, confirming a requirement for these genes in cerebellar development and providing a model to delineate the developmental basis of this clinically important congenital malformation. Here, we show that reduced cerebellar size in Zic1 and Zic4 mutants results from decreased postnatal granule cell progenitor proliferation. Through genetic and molecular analyses, we show that Zic1 and Zic4 have Shh-dependent function promoting proliferation of granule cell progenitors. Expression of the Shh-downstream genes Ptch1, Gli1 and Mycn was downregulated in Zic1/4 mutants, although Shh production and Purkinje cell gene expression were normal. Reduction of Shh dose on the Zic1(+/-);Zic4(+/-) background also resulted in cerebellar size reductions and gene expression changes comparable with those observed in Zic1(-/-);Zic4(-/-) mice. Zic1 and Zic4 are additionally required to pattern anterior vermis foliation. Zic mutant folial patterning abnormalities correlated with disrupted cerebellar anlage gene expression and Purkinje cell topography during late embryonic stages; however, this phenotype was Shh independent. In Zic1(+/-);Zic4(+/-);Shh(+/-), we observed normal cerebellar anlage patterning and foliation. Furthermore, cerebellar patterning was normal in both Gli2-cko and Smo-cko mutant mice, where all Shh function was removed from the developing cerebellum. Thus, our data demonstrate that Zic1 and Zic4 have both Shh-dependent and -independent roles during cerebellar development and that multiple developmental disruptions underlie Zic1/4-related DWM.

To characterize the possible relationship between the expression of the HGF/HGF-R system with transcription factor STAT3, responsible for morphogenetic response of HGF stimulation, and the embryonic development alterations, we investigated, by immunohistochemistry, the expression of HGF, c-met and STAT3 in 9 placentas from malformed fetuses and 9 control placentas from non-malformed fetuses. The major and distinct patterns of expression characterizing the placentas from malformed fetuses were a higher percentage mean of stromal cells stained for HGF, c-met and STAT3 antibodies (60%, 66% and 54%, respectively on fibroblast cells and 44%, 57% and 42%, respectively on myofibroblast cells) and a lower percentage mean of cytotrophoblast cells stained for the same antibodies (2%, 2% and 1%, respectively), than in control placentas. In fact, in this latter group, the stromal fibroblast cells were stained in a percentage mean of 27%, 22% and 7%, respectively; the stromal myofibroblast cells in a percentage mean of 5%, 6% and 2%, respectively and the cytotrophoblast cells in a percentage mean of 25%, 34% and 18%, respectively. The expression of each antibody on stromal cells in both groups suggests an alternative role of the HGF/HGF-R system activating the via STAT3 transdution and operating on placental tissues, overall in organogenesis alteration conditions. This immunohistochemical approach could be used in the diagnostic practice of pathologists on chorionic villi biopsy when genetic alterations are absent and ultrasound aspects are doubtful for malformations.