Autosomal recessive intellectual disability (ARID) is vastly heterogeneous. Truncating mutations of TRAPPC9 were reported in 8 ARID families. Autosomal recessive primary microcephaly (MCPH) represents another subgroup of ARID, itself very heterogeneous, where the size of the brain is very small since birth. MCPH1 plays a role at the centrosome via a BRCT1 domain, and in DNA Damage Repair (DDR) via BRCT2 and BRCT3, and it is not clear which of these two mechanisms causes MCPH in man.

Primary microcephaly (PM) is characterized by a small head since birth and is vastly heterogeneous both genetically and phenotypically. While most cases are monogenic, genetic interactions between Aspm and Wdr62 have recently been described in a mouse model of PM. Here, we used two complementary, holistic in vivo approaches: high throughput DNA sequencing of multiple PM genes in human patients with PM, and genome-edited zebrafish modeling for the digenic inheritance of PM. Exomes of patients with PM showed a significant burden of variants in 75 PM genes, that persisted after removing monogenic causes of PM (e.g., biallelic pathogenic variants in CEP152). This observation was replicated in an independent cohort of patients with PM, where a PM gene panel showed in addition that the burden was carried by six centrosomal genes. Allelic frequencies were consistent with digenic inheritance. In zebrafish, non-centrosomal gene casc5 -/- produced a severe PM phenotype, that was not modified by centrosomal genes aspm or wdr62 invalidation. A digenic, quadriallelic PM phenotype was produced by aspm and wdr62. Our observations provide strong evidence for digenic inheritance of human PM, involving centrosomal genes. Absence of genetic interaction between casc5 and aspm or wdr62 further delineates centrosomal and non-centrosomal pathways in PM.

Bi-allelic variants affecting one of the four genes encoding the AP4 subunits are responsible for the "AP4 deficiency syndrome." Core features include hypotonia that progresses to hypertonia and spastic paraplegia, intellectual disability, postnatal microcephaly, epilepsy, and neuroimaging features. Namely, AP4M1 (SPG50) is involved in autosomal recessive spastic paraplegia 50 (MIM#612936). We report on three patients with core features from three unrelated consanguineous families originating from the Middle East. Exome sequencing identified the same homozygous nonsense variant: NM_004722.4(AP4M1):c.1012C>T p.Arg338* (rs146262009). So far, four patients from three other families carrying this homozygous variant have been reported worldwide. We describe their phenotype and compare it to the phenotype of patients with other variants in AP4M1. We construct a shared single-nucleotide polymorphism (SNP) haplotype around AP4M1 in four families and suggest a probable founder effect of Arg338* AP4M1 variant with a common ancestor most likely of Turkish origin.

Rhophilin-2 or p76(RBE), a protein whose expression is induced by the cyclic AMP pathway in thyrocytes, contains several protein-protein interaction domains including HR-1, Bro1 and PDZ domains, and is a partner of RhoB in its GTP-bound form (Eur J Biochem, 269(24): 6241-9, 2002). We here define its subcellular localization and dissect the significance of its domains. By subcellular fractionation and colocalization experiments, rhophilin-2 is recruited to subcellular organelles by activated RhoB-GTP. As for its yeast homologue, Npi3/Bro1p, the Bro1 domain of rhophilin-2 is necessary to its recruitment to the vesicular structures, which are not labeled for EEA1 nor Lamp1, but well with the late endosome marker CD63.

A tight control of the machineries regulating membrane bending and actin dynamics is very important for the generation of membrane protrusions, which are crucial for cell migration and invasion. Protein/protein and protein/phosphoinositides complexes assemble and disassemble to coordinate these mechanisms, the scaffold properties of the involved proteins playing a prominent role in this organization. The PI 5-phosphatase SHIP2 is a critical enzyme modulating PI(3,4,5)P3, PI(4,5)P2 and PI(3,4)P2 content in the cell. The scaffold properties of SHIP2 contribute to the specific targeting or retention of the protein in particular subcellular domains. Here, we identified IRSp53 as a new binding interactor of SHIP2 proline-rich domain. Both proteins are costained in HEK293T cells protrusions, upon transfection. We showed that the SH3-binding polyproline motif recognized by IRSp53 in SHIP2 is different from the regions targeted by other PRR binding partners i.e., CIN85, ITSN or even Mena a common interactor of both SHIP2 and IRSp53. We presented evidence that IRSp53 phosphorylation on S366 did not influence its interaction with SHIP2 and that Mena is not necessary for the association of SHIP2 with IRSp53 in MDA-MB-231 cells. The absence of Mena in MDA-MB-231 cells decreased the intracellular content in F-actin and modified the subcellular localization of SHIP2 and IRSp53 by increasing their relative content at the plasma membrane. Together our data suggest that SHIP2, through interaction with the cell protrusion regulators IRSp53 and Mena, participate to the formation of multi-protein complexes. This ensures the appropriate modulations of PIs which is important for regulation of membrane dynamics.

Congenital hydrocephalus is characterized by ventriculomegaly, defined as a dilatation of cerebral ventricles, and thought to be due to impaired cerebrospinal fluid (CSF) homeostasis. Primary congenital hydrocephalus is a subset of cases with prenatal onset and absence of another primary cause, e.g., brain hemorrhage. Published series report a Mendelian cause in only a minority of cases. In this study, we analyzed exome data of PCH patients in search of novel causal genes and addressed the possibility of an underlying oligogenic mode of inheritance for PCH.

Hundreds of double homeobox (DUX) genes map within 3.3-kb repeated elements dispersed in the human genome and encode DNA-binding proteins. Among these, we identified DUX4, a potent transcription factor that causes facioscapulohumeral muscular dystrophy (FSHD). In the present study, we performed yeast two-hybrid screens and protein co-purifications with HaloTag-DUX fusions or GST-DUX4 pull-down to identify protein partners of DUX4, DUX4c (which is identical to DUX4 except for the end of the carboxyl terminal domain) and DUX1 (which is limited to the double homeodomain). Unexpectedly, we identified and validated (by co-immunoprecipitation, GST pull-down, co-immunofluorescence and in situ Proximal Ligation Assay) the interaction of DUX4, DUX4c and DUX1 with type III intermediate filament protein desmin in the cytoplasm and at the nuclear periphery. Desmin filaments link adjacent sarcomere at the Z-discs, connect them to sarcolemma proteins and interact with mitochondria. These intermediate filament also contact the nuclear lamina and contribute to positioning of the nuclei. Another Z-disc protein, LMCD1 that contains a LIM domain was also validated as a DUX4 partner. The functionality of DUX4 or DUX4c interactions with cytoplasmic proteins is underscored by the cytoplasmic detection of DUX4/DUX4c upon myoblast fusion. In addition, we identified and validated (by co-immunoprecipitation, co-immunofluorescence and in situ Proximal Ligation Assay) as DUX4/4c partners several RNA-binding proteins such as C1QBP, SRSF9, RBM3, FUS/TLS and SFPQ that are involved in mRNA splicing and translation. FUS and SFPQ are nuclear proteins, however their cytoplasmic translocation was reported in neuronal cells where they associated with ribonucleoparticles (RNPs). Several other validated or identified DUX4/DUX4c partners are also contained in mRNP granules, and the co-localizations with cytoplasmic DAPI-positive spots is in keeping with such an association. Large muscle RNPs were recently shown to exit the nucleus via a novel mechanism of nuclear envelope budding. Following DUX4 or DUX4c overexpression in muscle cell cultures, we observed their association with similar nuclear buds. In conclusion, our study demonstrated unexpected interactions of DUX4/4c with cytoplasmic proteins playing major roles during muscle differentiation. Further investigations are on-going to evaluate whether these interactions play roles during muscle regeneration as previously suggested for DUX4c.

The Super-Conserved Receptors Expressed in the Brain (SREBs) form a subfamily of orphan G protein-coupled receptors, highly conserved in evolution and characterized by a predominant expression in the brain. The signaling pathways activated by these receptors (if any) are presently unclear. Given the strong conservation of their intracellular loops, we used a BioID2 proximity-labeling assay to identify protein partners of SREBs that would interact with these conserved domains. Using streptavidin pull-down followed by mass spectrometry analysis, we identified the amino acid transporter SLC3A2, the AKAP protein LRBA, and the 4.1 protein EPB41L2 as potential interactors of these GPCRs. Using co-immunoprecipitation experiments, we confirmed the physical association of these proteins with the receptors. We then studied the functional relevance of the interaction between EPB41L2 and SREB1. Immunofluorescence microscopy revealed that SREB1 and EPB41L2 co-localize at the plasma membrane and that SREB1 is enriched in the β-catenin-positive cell membranes. siRNA knockdown experiments revealed that EPB41L2 promotes the localization of SREB1 at the plasma membrane and increases the solubilization of SREB1 when using detergents, suggesting a modification of its membrane microenvironment. Altogether, these data suggest that EPB41L2 could regulate the subcellular compartmentalization of SREBs and, as proposed for other GPCRs, could affect their stability or activation.

We describe a new syndrome of young onset diabetes, short stature and microcephaly with intellectual disability in a large consanguineous family with three affected children. Linkage analysis and whole exome sequencing were used to identify the causal nonsense mutation, which changed an arginine codon into a stop at position 127 of the tRNA methyltransferase homolog gene TRMT10A (also called RG9MTD2). TRMT10A mRNA and protein were absent in lymphoblasts from the affected siblings. TRMT10A is ubiquitously expressed but enriched in brain and pancreatic islets, consistent with the tissues affected in this syndrome. In situ hybridization studies showed that TRMT10A is expressed in human embryonic and fetal brain. TRMT10A is the mammalian ortholog of S. cerevisiae TRM10, previously shown to catalyze the methylation of guanine 9 (m(1)G9) in several tRNAs. Consistent with this putative function, in silico topology prediction indicated that TRMT10A has predominant nuclear localization, which we experimentally confirmed by immunofluorescence and confocal microscopy. TRMT10A localizes to the nucleolus of β- and non-β-cells, where tRNA modifications occur. TRMT10A silencing induces rat and human β-cell apoptosis. Taken together, we propose that TRMT10A deficiency negatively affects β-cell mass and the pool of neurons in the developing brain. This is the first study describing the impact of TRMT10A deficiency in mammals, highlighting a role in the pathogenesis of microcephaly and early onset diabetes. In light of the recent report that the type 2 diabetes candidate gene CDKAL1 is a tRNA methylthiotransferase, the findings in this family suggest broader relevance of tRNA methyltransferases in the pathogenesis of type 2 diabetes.

Primary microcephaly (PM) is defined as a significant reduction in occipitofrontal circumference (OFC) of prenatal onset. Clinical and genetic heterogeneity of PM represents a diagnostic challenge.