Ptchd1 deficiency induces excitatory synaptic and cognitive dysfunctions in mouse.

Synapse development and neuronal activity represent fundamental processes for the establishment of cognitive function. Structural organization as well as signalling pathways from receptor stimulation to gene expression regulation are mediated by synaptic activity and misregulated in neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability (ID). Deleterious mutations in the PTCHD1 (Patched domain containing 1) gene have been described in male patients with X-linked ID and/or ASD. The structure of PTCHD1 protein is similar to the Patched (PTCH1) receptor; however, the cellular mechanisms and pathways associated with PTCHD1 in the developing brain are poorly determined. Here we show that PTCHD1 displays a C-terminal PDZ-binding motif that binds to the postsynaptic proteins PSD95 and SAP102. We also report that PTCHD1 is unable to rescue the canonical sonic hedgehog (SHH) pathway in cells depleted of PTCH1, suggesting that both proteins are involved in distinct cellular signalling pathways. We find that Ptchd1 deficiency in male mice (Ptchd1-/y) induces global changes in synaptic gene expression, affects the expression of the immediate-early expression genes Egr1 and Npas4 and finally impairs excitatory synaptic structure and neuronal excitatory activity in the hippocampus, leading to cognitive dysfunction, motor disabilities and hyperactivity. Thus our results support that PTCHD1 deficiency induces a neurodevelopmental disorder causing excitatory synaptic dysfunction.

Autism spectrum disorder recurrence, resulting of germline mosaicism for a CHD2 gene missense variant.

Germline mosaicism for a novel missense variant p.Thr645Met located in the SNF2-related ATP dependent helicase domain of CHD2 in 2 affected siblings with autism spectrum disorder.

X-exome sequencing of 405 unresolved families identifies seven novel intellectual disability genes.

X-linked intellectual disability (XLID) is a clinically and genetically heterogeneous disorder. During the past two decades in excess of 100 X-chromosome ID genes have been identified. Yet, a large number of families mapping to the X-chromosome remained unresolved suggesting that more XLID genes or loci are yet to be identified. Here, we have investigated 405 unresolved families with XLID. We employed massively parallel sequencing of all X-chromosome exons in the index males. The majority of these males were previously tested negative for copy number variations and for mutations in a subset of known XLID genes by Sanger sequencing. In total, 745 X-chromosomal genes were screened. After stringent filtering, a total of 1297 non-recurrent exonic variants remained for prioritization. Co-segregation analysis of potential clinically relevant changes revealed that 80 families (20%) carried pathogenic variants in established XLID genes. In 19 families, we detected likely causative protein truncating and missense variants in 7 novel and validated XLID genes (CLCN4, CNKSR2, FRMPD4, KLHL15, LAS1L, RLIM and USP27X) and potentially deleterious variants in 2 novel candidate XLID genes (CDK16 and TAF1). We show that the CLCN4 and CNKSR2 variants impair protein functions as indicated by electrophysiological studies and altered differentiation of cultured primary neurons from Clcn4(-/-) mice or after mRNA knock-down. The newly identified and candidate XLID proteins belong to pathways and networks with established roles in cognitive function and intellectual disability in particular. We suggest that systematic sequencing of all X-chromosomal genes in a cohort of patients with genetic evidence for X-chromosome locus involvement may resolve up to 58% of Fragile X-negative cases.

Emerging major synaptic signaling pathways involved in intellectual disability.

Genetic causes of intellectual disability (ID) include mutations in proteins with various functions. However, many of these proteins are enriched in synapses and recent investigations point out their crucial role in the subtle regulation of synaptic activity and dendritic spine morphogenesis. Moreover, in addition to genetic data, functional and animal model studies are providing compelling evidence that supports the emerging unifying synapse-based theory for cognitive deficit. In this review, we highlight ID-related gene products involved in synaptic morphogenesis and function, with a particular focus on the emergent signaling pathways involved in synaptic plasticity whose disruption results in cognitive deficit.

A new gene involved in X-linked mental retardation identified by analysis of an X;2 balanced translocation.

X-linked forms of mental retardation (MR) affect approximately 1 in 600 males and are likely to be highly heterogeneous. They can be categorized into syndromic (MRXS) and nonspecific (MRX) forms. In MRX forms, affected patients have no distinctive clinical or biochemical features. At least five MRX genes have been identified by positional cloning, but each accounts for only 0.5%-1.0% of MRX cases. Here we show that the gene TM4SF2 at Xp11.4 is inactivated by the X breakpoint of an X;2 balanced translocation in a patient with MR. Further investigation led to identification of TM4SF2 mutations in 2 of 33 other MRX families. RNA in situ hybridization showed that TM4SF2 is highly expressed in the central nervous system, including the cerebral cortex and hippocampus. TM4SF2 encodes a member of the tetraspanin family of proteins, which are known to contribute in molecular complexes including beta-1 integrins. We speculate that through this interaction, TM4SF2 might have a role in the control of neurite outgrowth.

Identification of intellectual disability genes showing circadian clock-dependent expression in the mouse hippocampus.

Sleep is strongly implicated in learning, especially in the reprocessing of recently acquired memory. Children with intellectual disability (ID) tend to have sleep-wake disturbances, which may contribute to the pathophysiology of the disease. Given that sleep is partly controlled by the circadian clock, we decided to study the rhythmic expression of genes in the hippocampus, a brain structure which plays a key role in memory in humans and rodents. By investigating the hippocampal transcriptome of adult mice, we identified 663 circadian clock controlled (CCC) genes, which we divided into four categories based on their temporal pattern of expression. In addition to the standard core clock genes, enrichment analysis identified several transcription factors among these hippocampal CCC genes, and our findings suggest that genes from one cluster regulate the expression of those in another. Interestingly, these hippocampal CCC genes were highly enriched in sleep/wakefulness-related genes. We show here that several genes in the glucocorticoid signaling pathway, which is involved in memory, show a CCC pattern of expression. However, ID genes were not enriched among these CCC genes, suggesting that sleep or learning and memory disturbances observed in patients with ID are probably not related to the circadian clock in the hippocampus.

Loss of oligophrenin1 leads to uncontrolled Rho activation and increased thrombus formation in mice.

BACKGROUND: Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets and induce filopodia and lamellipodia formation and actin polymerization to strongly increase the platelet surface upon activation. Moreover, they are important for platelet secretion, integrin activation and arterial thrombus formation. OBJECTIVES: Rho GTPases are regulated by GTPase-activating proteins (GAPs) that stimulate their GTPase activity to terminate Rho signaling. The regulation of Rho GTPase activity in platelets is not well defined. Recently, we identified oligophrenin1 (OPHN1), a RhoGAP in platelets that exhibits strong GTPase-stimulating activity towards RhoA, Cdc42 and Rac1. RESULTS: In the present study we show for the first time, that deficiency of OPHN1 led to abnormal Rho activation and increased platelet cytoskeletal reorganization, including cell adhesion and lamellipodia formation on fibrinogen. Furthermore, platelets from ophn1(-/-) mice showed enhanced susceptibility to platelet activation with alterations in actin distribution and early release of granules. Platelet activation was enhanced following GPVI and PAR4 stimulation. This translated into elevated platelet thrombus formation and promoted arterial thrombosis under low shear conditions with altered hemostasis, as detected by tail bleeding time. CONCLUSIONS: The results of the present study identified OPHN1 as an important regulator of platelet cytoskeletal reorganization and demonstrate that abnormal regulation of Rho proteins leads to increased platelet adhesion and thrombus formation under low shear conditions in vitro and in vivo, suggesting a prothrombotic phenotype of mice critical for acute thrombotic occlusions.

Mapping of the X-breakpoint involved in a balanced X;12 translocation in a female with mild mental retardation.

Balanced chromosomal abnormalities such as translocations and inversions have been identified in many genetic diseases. Cloning of the breakpoints involved in these abnormalities has led to the identification of the disease-related genes. Recent reports suggest the presence of a mental retardation locus at Xq11-12. We have identified a female patient with a balanced translocation t (X;12) (q11;q15) associated with mild mental retardation. We identified a yeast artificial chromosome spanning the X-chromosome breakpoint by using fluorescent in situ hybridization techniques. A cosmid library of this YAC has been constructed and the search for candidate genes is in progress.

Gene for nonspecific X-linked mental retardation (MRX 47) is located in Xq22.3-q24.

We describe a large family with nonspecific X-linked mental retardation (MRX 47). An X-linked recessive transmission is suggested by the inheritance from the mothers in two generations of a moderate to severe form of mental retardation in six males, without any specific clinical findings. Two point linkage analysis demonstrated significant linkage between the disorder and two markers in Xq23 (Zmax = 3.75, theta = 0). Multipoint linkage analyses confirmed the significant linkage with a maximum lod score (Z = 3.96, theta = 0) at DXS1059. Recombination events observed with the flanking markers DXS1105 and DXS8067 delineate a 17 cM interval. This interval overlaps with several loci of XLMR disorders previously localized in Xq23-q24, which are reviewed herein.

X-linked neurodegenerative syndrome with congenital ataxia, late-onset progressive myoclonic encephalopathy and selective macular degeneration, linked to Xp22.33-pter.

Linkage analysis was performed in a previously described family segregating for an X-linked progressive neurological disorder [Bertini et al., 1992]. In three generations, the disease was inherited from the mothers in seven affected males (Fig. 1). Five had severe congenital hypotonia and died during the first year of life. Two other boys (maternal cousins) were found to have severe congenital ataxia, late-onset progressive myoclonic encephalopathy, and selective macular degeneration; brain CT-scan showed moderate cerebellar vermis hypoplasia. Linkage analysis was carried out in 12 informative relatives using 35 microsatellite markers (Généthon) evenly distributed on the X chromosome. A multipoint analysis showed a significant linkage (Z > 2) between the disease and three markers in the Xp22.33 region: DYS403 (Z = 2.37, theta = 0) which maps in the pseudoautosomal region, DXS7099 (Z = 2.45, theta = 0), and DXS7100 (Z = 2.48, theta = 0). Further linkage analysis with more telomeric markers will refine the location of this severe X-linked encephalopathy.

Oligophrenin 1 mutations frequently cause X-linked mental retardation with cerebellar hypoplasia.

BACKGROUND: Mutations of oligophrenin 1, one of the first genes identified in nonspecific X-linked mental retardation (MRX), have been described in patients with moderate to severe cognitive impairment and predominant cerebellar hypoplasia, in the vermis. OBJECTIVE: To further delineate the phenotypic and mutational spectrum of the syndrome, by screening oligophrenin 1 in two cohorts of male patients with mental retardation (MR) with or without known posterior fossa anomalies. METHODS: Clinical examination, cognitive testing, MRI studies, and mutational analysis (denaturing gradient gel electrophoresis and direct sequencing) on blood lymphocytes were performed in 213 unrelated affected individuals: 196 patients classified as MRX and 17 patients with MR and previously detected cerebellar anomalies. RESULTS: Four novel oligophrenin 1 mutations were identified. In the MRX group, two nonsense mutations were detected. In the MR group, two mutations were found: a deletion of exons 16 to 17 and a splice site mutation. All patients shared characteristic clinical, radiologic, and distinctive features with a degree of intrafamilial variability in motor and cognitive deficits. CONCLUSIONS: Oligophrenin 1 mutations were found in 12% (2/17) of individuals with mental retardatin and known cerebellar anomalies and in 1% (2/196) of the X-linked mental retardation group.

Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway.

Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. p190 inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by p190. Local regulation of p190 could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of p190 by integrin and Src, both implicated in neural plasticity.

A gene for dominant nonspecific X-linked mental retardation is located in Xq28.

A large family (MRX48) with a nonspecific X-linked mental retardation condition is described. An X-linked semidominant inheritance is suggested by the segregation in three generations of a moderate to severe mental retardation in seven males and by a milder intellectual impairment in two females, without any specific clinical, radiological, or biological feature. Two-point linkage analysis demonstrated significant linkage between the disorder and several markers in Xq28 (maximum LOD score [Zmax] = 2.71 at recombination fraction [theta] = 0); multipoint linkage analyses confirmed the significant linkage with a Zmax of 3.3 at theta = 0, at DXS1684. A recombination event observed with the flanking marker DXS8011 delineates a locus between this marker and the telomere. The approximate length of this locus is 8-9 cM, corresponding to 5.5-6 Mb. In an attempt to explain the variable intellectual impairment in females, we examined X-chromosome inactivation in all females of the family. Inactivation patterns in lymphocytes were random or moderately skewed, and no correlation between the phenotypic status and a specific inactivation pattern was observed. The interval of assignment noted in this family overlaps with five MRX loci previously reported in Xq28.

Determination of the gene structure of human oligophrenin-1 and identification of three novel polymorphisms by screening of DNA from 164 patients with non-specific X-linked mental retardation.

We have recently shown that mutations in oligophrenin-1 (OPHN1) are responsible for non-specific X-linked mental retardation (MRX). The structure of the gene encoding the OPHN1 protein was determined by isolation of genomic DNA clones from the human cosmid library. Genomic fragments containing exons were sequenced, and the sequences of the exons and flanking introns were defined. Knowledge of the genomic structure of the OPHN1 gene, which spans at least 500 kb and consists of 25 exons, will facilitate the search for additional mutations in OPHN1. OPHN1 was screened for mutations in 164 subjects with non-specific mental retardation. Three nucleotide substitutions were identified, one of which was a silent mutation in the codon threonine 301 at position 903 (G-->C). The other substitutions were located in exon 2, a G-->A substitution at position 133 (A45T), and in exon 10, a C-->T substitution at position 902 (T301M), but these are common polymorphisms rather than disease-causing mutations.

Inherited microdeletion in Xp21.3-22.1 involved in non-specific mental retardation.

X-linked mental retardation (XLMR) is a genetically and clinically heterogeneous common disorder. A cumulative frequency of about 1/600 male births was estimated by different authors, including the fragile X syndrome, which affects 1/4000 males. Given this very high cumulative frequency, identification of genes and molecular mechanisms involved in other XLMRs, represents a challenging task of considerable medical importance. In this report we describe clinical and molecular investigations in the family of a mentally retarded boy for whom a microdeletion in Xp21.3-22.1 was detected within the frame of a previously reported systematic search for deletion using STS-PCR screening. Thorough clinical investigation of the sibling showed that two affected brothers exhibit a moderate non-specific mental retardation without any additional neurological impairment, statural growth deficiency or characteristic dysmorphy. Molecular analysis revealed that the microdeletion observed in this family is an inherited defect which cosegregates with mental retardation as an X-linked recessive condition, since both non-deleted boys and transmitting mother are normal. These results and the inherited microdeletion detected within the same region associated with non-specific MR, reported by Raeymaekers et al., suggest that Xp21.3 MR locus is prone to deletions. Therefore, search for microdeletions in the eight families assigned by linkage analysis to this region might allow a better definition of the critical region and an identification of the gene involved in this X-linked mental retardation.

Doublecortin interacts with mu subunits of clathrin adaptor complexes in the developing nervous system.

Doublecortin is a microtubule-associated protein required for normal corticogenesis in the developing brain. We carried out a yeast two-hybrid screen to identify interacting proteins. One of the isolated clones encodes the mu1 subunit of the adaptor complex AP-1 involved in clathrin-dependent protein sorting. We found that Doublecortin also interacts in yeast with mu2 from the AP-2 complex. Mutagenesis and pull-down experiments showed that these interactions were mediated through a tyrosine-based sorting signal (YLPL) in the C-terminal part of Doublecortin. The functional relevance of these interactions was suggested by the coimmunoprecipitation of Doublecortin with AP-1 and AP-2 from mouse brain extracts. This interaction was further supported by RNA in situ hybridization and immunofluorescence studies. Taken together these data indicate that a certain proportion of Doublecortin interacts with AP-1 and/or AP-2 in vivo and are consistent with a potential involvement of Doublecortin in protein sorting or vesicular trafficking.

Identification by STS PCR screening of a microdeletion in Xp21.3-22.1 associated with non-specific mental retardation.

X-linked non-specific mental retardation (MRX) is a heterogeneous condition in which mental retardation (MR) appears to be the only consistent manifestation. The genetic and phenotypic heterogeneity exclude any possibility of pooling families and, therefore, of fine-mapping the related disease genes. In order to identify genomic critical regions involved in the MRX condition assigned to Xp21.3-22.1 region, we have implemented the PCR screening of non fragile X MR patients for the presence of deletions in this region. The amplification by PCR of 12 markers located between POLA and DXS704 using genomic DNA from 192 MR males led to the identification, in a 9 year old mentally retarded boy, of a microdeletion which extends from DXS1202 to DXS1065. None of the known genes, POLA, MAGE genes cluster, DAX1, GK and DMD, that map in the Xp21.3-22.1 region is affected by this deletion. This approach, which could easily be applied to several other MRX loci, allowed not only a confirmation of the presence of a potential locus in Xp21.3-22.1 involved in non-specific mental retardation, but also a better definition of the genomic critical region corresponding to this locus.

A novel CNS gene required for neuronal migration and involved in X-linked subcortical laminar heterotopia and lissencephaly syndrome.

X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex. It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment. We report the characterization of a novel CNS gene encoding a 40 kDa predicted protein that we named Doublecortin and the identification of mutations in four unrelated X-SCLH/LIS cases. The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part. This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors. The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion.