De novo missense variants in phosphatidylinositol kinase PIP5KIγ underlie a neurodevelopmental syndrome associated with altered phosphoinositide signaling.

Phosphoinositides (PIs) are membrane phospholipids produced through the local activity of PI kinases and phosphatases that selectively add or remove phosphate groups from the inositol head group. PIs control membrane composition and play key roles in many cellular processes including actin dynamics, endosomal trafficking, autophagy, and nuclear functions. Mutations in phosphatidylinositol 4,5 bisphosphate [PI(4,5)P2] phosphatases cause a broad spectrum of neurodevelopmental disorders such as Lowe and Joubert syndromes and congenital muscular dystrophy with cataracts and intellectual disability, which are thus associated with increased levels of PI(4,5)P2. Here, we describe a neurodevelopmental disorder associated with an increase in the production of PI(4,5)P2 and with PI-signaling dysfunction. We identified three de novo heterozygous missense variants in PIP5K1C, which encodes an isoform of the phosphatidylinositol 4-phosphate 5-kinase (PIP5KIγ), in nine unrelated children exhibiting intellectual disability, developmental delay, acquired microcephaly, seizures, visual abnormalities, and dysmorphic features. We provide evidence that the PIP5K1C variants result in an increase of the endosomal PI(4,5)P2 pool, giving rise to ectopic recruitment of filamentous actin at early endosomes (EEs) that in turn causes dysfunction in EE trafficking. In addition, we generated an in vivo zebrafish model that recapitulates the disorder we describe with developmental defects affecting the forebrain, including the eyes, as well as craniofacial abnormalities, further demonstrating the pathogenic effect of the PIP5K1C variants.

Differential alternative splicing analysis links variation in ZRSR2 to a novel type of oral-facial-digital syndrome.

PURPOSE: Oral-facial-digital (OFD) syndromes are genetically heterogeneous developmental disorders, caused by pathogenic variants in genes involved in primary cilia formation and function. We identified a previously undescribed type of OFD with brain anomalies, ranging from alobar holoprosencephaly to pituitary anomalies, in 6 unrelated families. METHODS: Exome sequencing of affected probands was supplemented with alternative splicing analysis in patient and control lymphoblastoid and fibroblast cell lines, and primary cilia structure analysis in patient fibroblasts. RESULTS: In 1 family with 2 affected males, we identified a germline variant in the last exon of ZRSR2, NM_005089.4:c.1211_1212del NP_005080.1:p.(Gly404GlufsTer23), whereas 7 affected males from 5 unrelated families were hemizygous for the ZRSR2 variant NM_005089.4:c.1207_1208del NP_005080.1:p.(Arg403GlyfsTer24), either occurring de novo or inherited in an X-linked recessive pattern. ZRSR2, located on chromosome Xp22.2, encodes a splicing factor of the minor spliceosome complex, which recognizes minor introns, representing 0.35% of human introns. Patient samples showed significant enrichment of minor intron retention. Among differentially spliced targets are ciliopathy-related genes, such as TMEM107 and CIBAR1. Primary fibroblasts containing the NM_005089.4:c.1207_1208del ZRSR2 variant had abnormally elongated cilia, confirming an association between defective U12-type intron splicing, OFD and abnormal primary cilia formation. CONCLUSION: We introduce a novel type of OFD associated with elongated cilia and differential splicing of minor intron-containing genes due to germline variation in ZRSR2.

OFD1: One gene, several disorders.

The OFD1 protein is necessary for the formation of primary cilia and left-right asymmetry establishment but additional functions have also been ascribed to this multitask protein. When mutated, this protein results in a variety of phenotypes ranging from multiorgan involvement, such as OFD type I (OFDI) and Joubert syndromes (JBS10), and Primary ciliary dyskinesia (PCD), to the engagement of single tissues such as in the case of retinitis pigmentosa (RP23). The inheritance pattern of these condition differs from X-linked dominant male-lethal (OFDI) to X-linked recessive (JBS10, PCD, and RP23). Distinctive biological peculiarities of the protein, which can contribute to explain the extreme clinical variability and the genetic mechanisms underlying the different disorders are discussed. The extensive spectrum of clinical manifestations observed in OFD1-mutated patients represents a paradigmatic example of the complexity of genetic diseases. The elucidation of the mechanisms underlying this complexity will expand our comprehension of inherited disorders and will improve the clinical management of patients.

Biallelic variants in CENPF causing a phenotype distinct from Strømme syndrome.

Biallelic loss-of-function (LoF) variants in CENPF gene are responsible for Strømme syndrome, a condition presenting with intestinal atresia, anterior ocular chamber anomalies, and microcephaly. Through an international collaboration, four individuals (three males and one female) carrying CENPF biallelic variants, including two missense variants in homozygous state and four LoF variants, were identified by exome sequencing. All individuals had variable degree of developmental delay/intellectual disability and microcephaly (ranging from -2.9 SDS to -5.6 SDS) and a recognizable pattern of dysmorphic facial features including inverted-V shaped interrupted eyebrows, epicanthal fold, depressed nasal bridge, and pointed chin. Although one of the cases had duodenal atresia, all four individuals did not have the combination of internal organ malformations of Strømme syndrome (intestinal atresia and anterior eye segment abnormalities). Immunofluorescence analysis on skin fibroblasts on one of the four cases with the antibody for ARL13B that decorates primary cilia revealed shorter primary cilia that are consistent with a ciliary defect. This case-series of individuals with biallelic CENPF variants suggests the spectrum of clinical manifestations of the disorder that may be related to CENPF variants is broad and can include phenotypes lacking the cardinal features of Strømme syndrome.

The deubiquitinating enzyme Usp14 controls ciliogenesis and Hedgehog signaling.

Primary cilia are hair-like organelles that play crucial roles in vertebrate development, organogenesis and when dysfunctional result in pleiotropic human genetic disorders called ciliopathies, characterized by overlapping phenotypes, such as renal and hepatic cysts, skeletal defects, retinal degeneration and central nervous system malformations. Primary cilia act as communication hubs to transfer extracellular signals into intracellular responses and are essential for Hedgehog (Hh) signal transduction in mammals. Despite the renewed interest in this ancient organelle of growing biomedical importance, the molecular mechanisms that trigger cilia formation, extension and ciliary signal transduction are still not fully understood. Here we provide, for the first time, evidence that the deubiquitinase ubiquitin-specific protease-14 (Usp14), a major regulator of the ubiquitin proteasome system (UPS), controls ciliogenesis, cilia elongation and Hh signal transduction. Moreover, we show that pharmacological inhibition of Usp14 positively affects Hh signal transduction in a model of autosomal dominant polycystic kidney disease. These findings provide new insight into the spectrum of action of UPS in cilia biology and may provide novel opportunities for therapeutic intervention in human conditions associated with ciliary dysfunction.

Mutations in COX7B cause microphthalmia with linear skin lesions, an unconventional mitochondrial disease.

Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possible candidates, we analyzed the X-linked COX7B and found deleterious de novo mutations in two simplex cases and a nonsense mutation, which segregates with the disease, in a familial case. COX7B encodes a poorly characterized structural subunit of cytochrome c oxidase (COX), the MRC complex IV. We demonstrated that COX7B is indispensable for COX assembly, COX activity, and mitochondrial respiration. Downregulation of the COX7B ortholog (cox7B) in medaka (Oryzias latipes) resulted in microcephaly and microphthalmia that recapitulated the MLS phenotype and demonstrated an essential function of complex IV activity in vertebrate CNS development. Our results indicate an evolutionary conserved role of the MRC complexes III and IV for the proper development of the CNS in vertebrates and uncover a group of mitochondrial diseases hallmarked by a developmental phenotype.

Ofd1 is required in limb bud patterning and endochondral bone development.

Oral-facial-digital type I (OFDI) syndrome is an X-linked male lethal developmental disorder. It is ascribed to ciliary dysfunction and characterized by malformation of the face, oral cavity, and digits. Conditional inactivation using different Cre lines allowed us to study the role of the Ofd1 transcript in limb development. Immunofluorescence and ultrastructural studies showed that Ofd1 is necessary for correct ciliogenesis in the limb bud but not for cilia outgrowth, in contrast to what was previously shown for the embryonic node. Mutants with mesenchymal Ofd1 inactivation display severe polydactyly with loss of antero-posterior (A/P) digit patterning and shortened long bones. Loss of digit identity was found to be associated with a progressive loss of Shh signaling and an impaired processing of Gli3, whereas defects in limb outgrowth were due to defective Ihh signaling and to mineralization defects during endochondral bone formation. Our data demonstrate that Ofd1 plays a role in regulating digit number and identity during limb and skeletal patterning increasing insight on the functional role of primary cilia during development.

Targeting the MITF/APAF-1 axis as salvage therapy for MAPK inhibitors in resistant melanoma.

Melanoma is a deadly form of cancer characterized by remarkable therapy resistance. Analyzing the transcriptome of MAPK inhibitor sensitive- and resistant-melanoma, we discovered that APAF-1 is negatively regulated by MITF in resistant tumors. This study identifies the MITF/APAF-1 axis as a molecular driver of MAPK inhibitor resistance. A drug-repositioning screen identified quinacrine and methylbenzethonium as potent activators of apoptosis in a context that mimics drug resistance mediated by APAF-1 inactivation. The compounds showed anti-tumor activity in in vitro and in vivo models, linked to suppression of MITF function. Both drugs profoundly sensitize melanoma cells to MAPK inhibitors, regulating key signaling networks in melanoma, including the MITF/APAF-1 axis. Significant activity of the two compounds in inhibiting specific epigenetic modulators of MITF/APAF-1 expression, such as histone deacetylases, was observed. In summary, we demonstrate that targeting the MITF/APAF-1 axis may overcome resistance and could be exploited as a potential therapeutic approach to treat resistant melanoma.

The centrosomal/basal body protein OFD1 is required for microtubule organization and cell cycle progression.

Oral-Facial-Digital type I (OFD1) is a rare inherited form of renal cystic disease associated with ciliary dysfunction. This disorder is due to mutations in the OFD1 gene that encodes a protein localized to centrosomes and basal bodies in different cell types. Immunofluorescence analysis demonstrated that OFD1 displays a dynamic distribution during cell cycle. High-content microscopy analysis of Ofd1-depleted fibroblasts revealed impaired cell cycle progression. Immunofluorescence analysis and cell proliferation assays also indicated the presence of a variety of defects such as centrosome accumulation, nuclear abnormalities and aneuploidy. In addition, Ofd1-depleted cells displayed an abnormal microtubule network that may underlie these defects. All together our results suggest that OFD1 contributes to the function of the microtubule organizing center (MTOC) in the cell, controlling cell cycle progression both in vitro and in vivo.

miR-181a/b downregulation exerts a protective action on mitochondrial disease models.

Mitochondrial diseases (MDs) are a heterogeneous group of devastating and often fatal disorders due to defective oxidative phosphorylation. Despite the recent advances in mitochondrial medicine, effective therapies are still not available for these conditions. Here, we demonstrate that the microRNAs miR-181a and miR-181b (miR-181a/b) regulate key genes involved in mitochondrial biogenesis and function and that downregulation of these miRNAs enhances mitochondrial turnover in the retina through the coordinated activation of mitochondrial biogenesis and mitophagy. We thus tested the effect of miR-181a/b inactivation in different animal models of MDs, such as microphthalmia with linear skin lesions and Leber's hereditary optic neuropathy. We found that miR-181a/b downregulation strongly protects retinal neurons from cell death and significantly ameliorates the disease phenotype in all tested models. Altogether, our results demonstrate that miR-181a/b regulate mitochondrial homeostasis and that these miRNAs may be effective gene-independent therapeutic targets for MDs characterized by neuronal degeneration.

The centrosomal OFD1 protein interacts with the translation machinery and regulates the synthesis of specific targets.

Protein synthesis is traditionally associated with specific cytoplasmic compartments. We now show that OFD1, a centrosomal/basal body protein, interacts with components of the Preinitiation complex of translation (PIC) and of the eukaryotic Initiation Factor (eIF)4F complex and modulates the translation of specific mRNA targets in the kidney. We demonstrate that OFD1 cooperates with the mRNA binding protein Bicc1 to functionally control the protein synthesis machinery at the centrosome where also the PIC and eIF4F components were shown to localize in mammalian cells. Interestingly, Ofd1 and Bicc1 are both involved in renal cystogenesis and selected targets were shown to accumulate in two models of inherited renal cystic disease. Our results suggest a possible role for the centrosome as a specialized station to modulate translation for specific functions of the nearby ciliary structures and may provide functional clues for the understanding of renal cystic disease.

Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides.

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G>A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

Synthetic long non-coding RNAs [SINEUPs] rescue defective gene expression in vivo.

Non-coding RNAs provide additional regulatory layers to gene expression as well as the potential to being exploited as therapeutic tools. Non-coding RNA-based therapeutic approaches have been attempted in dominant diseases, however their use for treatment of genetic diseases caused by insufficient gene dosage is currently more challenging. SINEUPs are long antisense non-coding RNAs that up-regulate translation in mammalian cells in a gene-specific manner, although, so far evidence of SINEUP efficacy has only been demonstrated in in vitro systems. We now show that synthetic SINEUPs effectively and specifically increase protein levels of a gene of interest in vivo. We demonstrated that SINEUPs rescue haploinsufficient gene dosage in a medakafish model of a human disorder leading to amelioration of the disease phenotype. Our results demonstrate that SINEUPs act through mechanisms conserved among vertebrates and that SINEUP technology can be successfully applied in vivo as a new research and therapeutic tool for gene-specific up-regulation of endogenous functional proteins.

Ofd1 controls dorso-ventral patterning and axoneme elongation during embryonic brain development.

Oral-facial-digital type I syndrome (OFDI) is a human X-linked dominant-male-lethal developmental disorder caused by mutations in the OFD1 gene. Similar to other inherited disorders associated to ciliary dysfunction OFD type I patients display neurological abnormalities. We characterized the neuronal phenotype that results from Ofd1 inactivation in early phases of mouse embryonic development and at post-natal stages. We determined that Ofd1 plays a crucial role in forebrain development, and in particular, in the control of dorso-ventral patterning and early corticogenesis. We observed abnormal activation of Sonic hedgehog (Shh), a major pathway modulating brain development. Ultrastructural studies demonstrated that early Ofd1 inactivation results in the absence of ciliary axonemes despite the presence of mature basal bodies that are correctly orientated and docked. Ofd1 inducible-mediated inactivation at birth does not affect ciliogenesis in the cortex, suggesting a developmental stage-dependent role for a basal body protein in ciliogenesis. Moreover, we showed defects in cytoskeletal organization and apical-basal polarity in Ofd1 mutant embryos, most likely due to lack of ciliary axonemes. Thus, the present study identifies Ofd1 as a developmental disease gene that is critical for forebrain development and ciliogenesis in embryonic life, and indicates that Ofd1 functions after docking and before elaboration of the axoneme in vivo.

Apoptosis in retinal degeneration involves cross-talk between apoptosis-inducing factor (AIF) and caspase-12 and is blocked by calpain inhibitors.

Molecular mechanisms underlying apoptosis in retinitis pigmentosa, as in other neurodegenerative diseases, are still elusive, and this fact hampers the development of a cure for this blinding disease. We show that two apoptotic pathways, one from the mitochondrion and one from the endoplasmic reticulum, are coactivated during the degenerative process in an animal model of retinitis pigmentosa, the rd1 mouse. We found that both AIF and caspase-12 translocate to the nucleus of dying photoreceptors in vivo and in an in vitro cellular model. Translocation of both apoptotic factors depends on changes in intracellular calcium homeostasis and on calpain activity. Knockdown experiments defined that AIF plays the major role in this apoptotic event, whereas caspase-12 has a reinforcing effect. This study provides a link between two executor caspase-independent apoptotic pathways involving mitochondrion and endoplasmic reticulum in a degenerating neuron.