Functional characterization of the OFD1 protein reveals a nuclear localization and physical interaction with subunits of a chromatin remodeling complex.

Oral-facial-digital (OFD) type I syndrome is an X-linked dominant disease (MIM311200) characterized by malformations of oral cavity, face, and digits and by cystic kidneys. We previously identified OFD1, the gene responsible for this disorder, which encodes for a centrosomal protein with an unknown function. We now report that OFD1 localizes both to the primary cilium and to the nucleus. Moreover, we demonstrate that the OFD1 protein is able to self-associate and that this interaction is mediated by its coiled-coil rich region. Interestingly, we identify an OFD1-interacting protein RuvBl1, a protein belonging to the AAA(+)-family of ATPases, which has been recently associated to cystic kidney in zebrafish and to ciliary assembly and function in Chlamydomonas reinhardtii. We also provide experimental evidence that OFD1, together with RuvBl1, is able to coimmunoprecipitate with subunits of the human TIP60 histone acetyltransferase (HAT) multisubunit complex. On the basis of these results, we hypothesize that OFD1 may be part of a multi-protein complex and could play different biological functions in the centrosome-primary cilium organelles as well as in the nuclear compartment.

Mutational spectrum of the oral-facial-digital type I syndrome: a study on a large collection of patients.

Oral-facial-digital type I (OFDI) syndrome is a male-lethal X-linked dominant developmental disorder belonging to the heterogeneous group of oral-facial-digital syndromes (OFDS). OFDI is characterized by malformations of the face, oral cavity, and digits. Central nervous system (CNS) abnormalities and cystic kidney disease can also be part of this condition. This rare genetic disorder is due to mutations in the OFD1 gene that encodes a centrosome/basal body protein necessary for primary cilium assembly and for left-right axis determination, thus ascribing OFDI to the growing number of disorders associated to ciliary dysfunction. We now report a mutation analysis study in a cohort of 100 unrelated affected individuals collected worldwide. Putative disease-causing mutations were identified in 81 patients (81%). We describe 67 different mutations, 64 of which represent novel mutations, including 36 frameshift, nine missense, 11 splice-site, and 11 nonsense mutations. Most of them concentrate in exons 3, 8, 9, 12, 13, and 16, suggesting that these exons may represent mutational hotspots. Phenotypic characterization of the patients provided a better definition of the clinical features of OFDI syndrome. Our results indicate that renal cystic disease is present in 60% of cases >18 years of age. Genotype-phenotype correlation did not reveal significant associations apart for the high-arched/cleft palate most frequently associated to missense and splice-site mutations. Our results contribute to further expand our knowledge on the molecular basis of OFDI syndrome.

Novel functional features of the Lis-H domain: role in protein dimerization, half-life and cellular localization.

The presence of a conserved protein motif usually implies common functional features. Here, we focused on the LisH (LIS1 homology) domain, which is found in multiple proteins, and have focused on three involved in human genetic diseases; LIS1, Transducin beta-like 1X (TBL1) and Oral-facial-digital type 1 (OFD1). The recently solved structure of the LisH domain in the N-terminal region of LIS1 depicted it as a novel dimerization motif. Our findings indicated that the LisH domain of both LIS1 and TBL1 is essential for in vitro oligomerization. Furthermore, our study disclosed novel in vivo features of the LisH motif. Mutations in conserved LisH amino acids significantly reduced both the protein half-life of LIS1, TBL1, and OFD1, and dramatically affected specific intracellular localizations of these proteins. LIS1 mutated in the LisH domain induced its localization to the actin filaments. TBL1 mutated in the LisH domain was not imported into the nucleus. Mutations in OFD1 modified its localization to the Golgi apparatus and in some cases also to the nucleus. In summary, the LisH domain may participate in protein dimerization, affect protein half-life, and may influence specific cellular localizations. Our results allow the prediction that mutations within the LisH motif are likely to result in pathogenic consequences in genes associated with genetic diseases.

OFD1, the gene mutated in oral-facial-digital syndrome type 1, is expressed in the metanephros and in human embryonic renal mesenchymal cells.

Oral-facial-digital syndrome type 1 (OFD1) causes polycystic kidney disease (PKD) and malformations of the mouth, face and digits. Recently, a gene on Xp22, OFD1, was reported to be mutated in a limited set of OFD1 patients. This study describes mutation analysis in six further OFD1 families. Additionally, gene expression was sought in human development. In two OFD1 kindreds affected by PKD, a frameshift mutation and a splice-site mutation were detected. In four apparently sporadic cases, three frameshift and a missense mutation were found. Using RT-PCR of RNA from first-trimester normal human embryos, both alternative splice forms of mRNA (OFD1a and OFD1b) were found to be widely expressed in organogenesis. Northern blot detected OFD1 mRNA in metanephros, brain, tongue, and limb, all organs affected in the syndrome. A polyclonal antibody directed to a C-terminal OFD1a epitope detected a 120-kD protein in the metanephros and in human renal mesenchymal cell lines. In normal human embryos, OFD1a immunolocalized to the metanephric mesenchyme, oral mucosa, nasal and cranial cartilage, and brain. Moreover, using normal human renal mesenchymal cell lines, the immunoreactive protein colocalized with gamma-tubulin, suggesting that OFD1 is associated with the centrosome. First, it is concluded that OFD1 mutations would generally be predicted to result in unstable transcripts or nonfunctional proteins. Second, OFD1 is expressed in human organogenesis; on the basis of the metanephric expression pattern, the results suggest that OFD1 plays a role in differentiation of metanephric precursor cells.