Elma El Khouri

J Ghoumid

MD Damien Haye

MD Fabienne Giuliano

L Drevillon

A Briand-Suleau

Pierre De

PhD La A Grange

V Nau

T Gaillon

T Bienvenu

PhD Elma El Khouri

Jamal Ghoumid Md-Phd

Audrey Briand-Suleau Phd

Valérie Nau Bsc

Thierry Gaillon Bsc

Thierry Bienvenu Pharmd-Phd

PhD Hélène Jacquemin-Sablon

Michel Goossens Md-Phd

Serge Amselem Md-Phd

Irina Giurgea Md-Phd

Irina Giurgea
email: irina.giurgea@inserm.fr

Wnt/β-catenin pathway and cell adhesion deregulation in CSDE1-related intellectual disability and autism spectrum disorders

published or not. The documents may come

Wnt/β-catenin pathway and cell adhesion deregulation in CSDE1-related intellectual disability and autism spectrum disorders

INTRODUCTION

Intellectual disability (ID) is a neurodevelopmental condition characterized by significantly impaired intellectual functioning affecting the ability to learn, reasoning and problem solving and by deficits in adaptive behavior, hindering day-to-day social and practical skills [START_REF] Aspromonte | Characterization of intellectual disability and autism comorbidity through gene panel sequencing[END_REF]. ID is usually associated with other co-occurring neurodevelopmental disorders and a large majority of patients with ID also present with features of autism spectrum disorders (ASD). The co-occurrence of these two disease conditions is accounted for by their common molecular and genetic origin as several genes have been shown to be involved in both ID and ASD [START_REF] Aspromonte | Characterization of intellectual disability and autism comorbidity through gene panel sequencing[END_REF][START_REF] Srivastava | Intellectual disability and autism spectrum disorders: causal genes and molecular mechanisms[END_REF]. Many of the identified mutations are responsible for dysfunctions of specific key signaling pathways, such as the TGF-β/BMP (Transforming growth factor-beta/bone morphogenetic protein), the Wnt/β-catenin, the SHH (Sonic Hedgehog), and the retinoic acid signaling pathways (reviewed in (3)). For instance, ID-ASD-causing mutations have been reported in genes encoding proteins involved in several steps of the Wnt/βcatenin pathway, like the upstream ligands WNT1 and WNT2 (4-7), the downstream transcription factor TCF7L2 [START_REF] De Rubeis | Synaptic, transcriptional and chromatin genes disrupted in autism[END_REF][START_REF] Iossifov | The contribution of de novo coding mutations to autism spectrum disorder[END_REF] or the β-catenin (CTNNB1), the core element of this pathway [START_REF] Dubruc | A new intellectual disability syndrome caused by CTNNB1 haploinsufficiency[END_REF][START_REF] Kuechler | De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum[END_REF][START_REF] O'roak | Sporadic autism exomes reveal a highly interconnected protein network of de novo mutations[END_REF][START_REF] Krumm | A de novo convergence of autism genetics and molecular neuroscience[END_REF]. Other major players involved in the onset of ID-ASD are RNA-metabolism regulators and RNA-binding proteins (RBP). This is for instance the case for the FMR1 gene responsible for the Fragile X syndrome (FXS), the most common heritable form of ID-ASD [START_REF] Myrick | Independent role for presynaptic FMRP revealed by an FMR1 missense mutation associated with intellectual disability and seizures[END_REF], but also several other genes involved in ID-ASD (e.g. UPF3A and B, SMG6, EIF4A3, RNPS1, PQBP1, and VCX-A) [START_REF] Addington | A novel frameshift mutation in UPF3B identified in brothers affected with childhood onset schizophrenia and autism spectrum disorders[END_REF][START_REF] Bardoni | Intellectual disabilities, neuronal posttranscriptional RNA metabolism, and RNA-binding proteins: three actors for a complex scenario[END_REF][START_REF] Jolly | The UPF3B gene, implicated in intellectual disability, autism, ADHD and childhood onset schizophrenia regulates neural progenitor cell behaviour and neuronal outgrowth[END_REF][START_REF] Laumonnier | Mutations of the UPF3B gene, which encodes a protein widely expressed in neurons, are associated with nonspecific mental retardation with or without autism[END_REF][START_REF] Nguyen | Contribution of copy number variants involving nonsense-mediated mRNA decay pathway genes to neuro-developmental disorders[END_REF]. However, despite these advances in identifying ID-ASD-causing mutations, the molecular diagnosis of these conditions is challenging owing to the high clinical and genetic heterogeneity. Furthermore, whole genome sequencing or whole exome sequencing approaches are constrained by the identification of huge amount of variants in each patient and by the large number of patients that have to be studied considering the ID-ASD prevalence (1 to 3% of the general population) [START_REF] Perou | Mental health surveillance among children--United States, 2005-2011[END_REF][START_REF] Maulik | Prevalence of intellectual disability: a meta-analysis of population-based studies[END_REF][START_REF] Bourke | Population-Based Prevalence of Intellectual Disability and Autism Spectrum Disorders in Western Australia: A Comparison With Previous Estimates[END_REF]. It is therefore crucial to conduct molecular studies relying on a precise phenotype that could be associated with mutations in a given gene or set of genes.

Here, we describe the case of a patient followed from birth to the age of 21, presenting with severe ID and features resembling the Pitt-Hopkins syndrome (PTHS, MIM #610954) a neurodevelopmental disorder characterized by recognizable facial features and behavioral abnormalities, among which some overlap with ID-ASD. The patient carries no pathogenic variation in TCF4 (Transcription factor 4, MIM#602272), the gene involved in PTHS [START_REF] Amiel | Mutations in TCF4, encoding a class I basic helix-loop-helix transcription factor, are responsible for Pitt-Hopkins syndrome, a severe epileptic encephalopathy associated with autonomic dysfunction[END_REF][START_REF] Zweier | Haploinsufficiency of TCF4 causes syndromal mental retardation with intermittent hyperventilation (Pitt-Hopkins syndrome)[END_REF], a result that led us to perform an in trio exome sequencing in the patient and his healthy parents. This analysis identified a de novo heterozygous nonsense variation in CSDE1 (Cold Shock Domain-containing E1 -also known as UNR: Upstream of NRAS, MIM #191510) in the patient. CSDE1, a ubiquitously expressed RBP with high affinity for mRNAs containing A/G-rich binding motifs [START_REF] Grosset | A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex[END_REF][START_REF] Ray | Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction[END_REF], binds to regulatory regions of its targets transcripts, i.e. the 5'UTR and 3'UTR (untranslated regions) or the IRESs (Internal Ribosomal Entry Sites) to control their translation [START_REF] Ray | Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction[END_REF][START_REF] Mitchell | Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras[END_REF][START_REF] Tinton | Regulation of the cellcycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2alpha[END_REF][START_REF] Schepens | A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis[END_REF] and abundance [START_REF] Grosset | A mechanism for translationally coupled mRNA turnover: interaction between the poly(A) tail and a c-fos RNA coding determinant via a protein complex[END_REF][START_REF] Wurth | UNR/CSDE1 Drives a Post-transcriptional Program to Promote Melanoma Invasion and Metastasis[END_REF][START_REF] Lee | A posttranscriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells[END_REF]. CSDE1 is involved in a multitude of biological processes, namely cell cycle and migration, apoptosis as well as embryonic development, neurogenesis and neuronal differentiation [START_REF] Mitchell | Protein factor requirements of the Apaf-1 internal ribosome entry segment: roles of polypyrimidine tract binding protein and upstream of N-ras[END_REF][START_REF] Tinton | Regulation of the cellcycle-dependent internal ribosome entry site of the PITSLRE protein kinase: roles of Unr (upstream of N-ras) protein and phosphorylated translation initiation factor eIF-2alpha[END_REF][START_REF] Schepens | A role for hnRNP C1/C2 and Unr in internal initiation of translation during mitosis[END_REF][START_REF] Wurth | UNR/CSDE1 Drives a Post-transcriptional Program to Promote Melanoma Invasion and Metastasis[END_REF][START_REF] Lee | A posttranscriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells[END_REF][START_REF] Elatmani | The RNA-binding protein Unr prevents mouse embryonic stem cells differentiation toward the primitive endoderm lineage[END_REF]. The diverse CSDE1 targets and their associated biological processes could account for its involvement in the occurrence of multiple disease conditions (reviewed in [START_REF] Guo | The role of CSDE1 in translational reprogramming and human diseases[END_REF]) as diverse as cancers, Diamond-Blackfan anemia or ASD. CSDE1 is one of the genes mentioned in ASD large scale studies [START_REF] Sanders | De novo mutations revealed by whole-exome sequencing are strongly associated with autism[END_REF][START_REF] Xia | Common genetic variants on 1p13.2 associate with risk of autism[END_REF], and has recently been reported in patients with neurodevelopmental disabilities [START_REF] Guo | Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission[END_REF]. However, the CSDE1-related pathways involved in the pathophysiology of ID-ASD have not been explored in patient-derived cells so far.

The identification of a nonsense CDSE1 mutation in our patient, whose disease phenotype has been accurately described over two decades, prompted us to perform diverse functional ex-vivo studies with the aim to decipher the CSDE1-dependent biological pathways involved in the pathophysiology of ID-ASD.

MATERIALS AND METHODS

The affected patient and the healthy individuals -Informed consents were obtained from patient's parents and from the healthy individuals participating in the study for genetic tests, skin biopsies, data sharing and publication of the patient's photographs according to French legislation and the principles of the Declaration of Helsinki.

Whole exome sequencing -Genomic DNA was extracted from a peripheral blood sample and captured with the SureSelect HumanAll Exon V2 kit (Agilent, Santa Clara, CA). Whole-exome sequencing was performed (Integragen SA, Evry France) on a HiSeq 2000 (Illumina, San Diego, CA) according to the manufacturer's recommendations. Alignment was carried out as previously described [START_REF] Thomas | TTC12 Loss-of-Function Mutations Cause Primary Ciliary Dyskinesia and Unveil Distinct Dynein Assembly Mechanisms in Motile Cilia Versus Flagella[END_REF].

Sanger sequencing and semi-quantitative PCR -for the TCF4 and CSDE1 genes were performed as previously reported [START_REF] Whalen | Novel comprehensive diagnostic strategy in Pitt-Hopkins syndrome: clinical score and further delineation of the TCF4 mutational spectrum[END_REF].

Fibroblasts' culture -Skin biopsies were performed in the patient at the age of 14 and in five healthy donors aged between 10 and 33 years (three adults and two children -males and females). Fibroblast cultures were handled as previously described [START_REF] Drévillon | KBPcytoskeleton interactions underlie developmental anomalies in Goldberg-Shprintzen syndrome[END_REF] and used for RNA-seq analysis and for functional studies.

RNA extraction and quantitative RT-PCR assay -Total RNA from fibroblasts was isolated using RNAeasy Mini Kit (Qiagen, Germany) and 1 µg of RNA was subjected to reverse transcription using Reverse Transcriptor kit (Roche, Switzerland) according to the manufacturers' instructions. cDNA was amplified using the Mesa Blue qPCR MasterMix Plus (Eurogentec, Belgium) in the Light Cycler LC480 (Roche). Primers are provided in Supplementary Information.

RNA-seq -Total RNA was captured using TruSeq Stranded Total RNA with Ribo-Zero Gold (Illumina, San Diego, CA) sequencing was performed (Integragen SA, Evry France) on a HiSeq4000 (Illumina, San Diego, CA) according to the manufacturer's recommendations. RNA-Seq data analysis was performed by GenoSplice (www.genosplice.com). Sequencing, data quality, reads repartition, and insert size estimation were performed using FastQC, Picard-Tools, Samtools and rseqc. Reads were mapped using STARv2.4.0 (40) on the hg19 Human genome assembly. Gene expression regulation study was performed as previously described [START_REF] Noli | Discordant Growth of Monozygotic Twins Starts at the Blastocyst Stage: A Case Study[END_REF]. Further information is provided in Supplementary Information. Pathway and process enrichment analysis was performed by Metascape [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF] as described in Supplementary Information. Data from three healthy individuals were compared to those obtained from the ID-ASD patient.

Nonsense-mediated mRNA decay assessment -Fibroblasts were treated with 30 µg/mL cycloheximide (Sigma Aldrich, MO, USA) or DMSO for 4 h. Total RNA was extracted and subjected to reverse transcription. PCR amplification was carried out using the Q5 (New England Biolabs, MA, USA) PCR products were purified using ExoSap (Thermo Fisher Scientific, MA, USA) before being sequenced.

Western blot analysis -Protein expression analyses in fibroblasts were performed using a standard Western blot protocol and specific antibodies as described in Supplementary Information. Proteins were detected with Amersham ECL Select Western Blotting Detection Reagent (GE healthcare, Ill, USA) according to the manufacturer's recommendations and BioRad ChemiDoc Imaging Systems (BioRad, CA, USA) was used for detection. The ImageJ software was used for signal quantification.

Luciferase assay -Fibroblasts were transiently transfected with 2 μg of the β-catenin-responsive firefly luciferase reporter plasmid TOPFlash (Millipore, Ma, USA) or the negative control FOPFlash (Millipore) by a double transfection using Lipofectamine LTX (Thermo Fisher Scientific) and Fugene (Promega, Wis, USA) at equal amounts according to the manufacturer's instructions. 24 hours after transfection, cells were treated with 20 mM LiCl for 6 hours then lysed with Passive Lysis Buffer (Promega). Samples were incubated for 30 min at 4°C and then centrifuged for 1 min at 13 000 g at 4°C. Supernatants were collected and firefly luciferase activity was measured according to the manufacturer's instructions. The firefly luciferase activity was normalized against protein amounts and relative TOPFlash/FOPFlash activity was reported.

Adhesion assay -Fibroblasts were deprived of serum for 8 hours and then detached using 10 mM EDTA in DMEM for 10 min, washed twice with DMEM (Thermo Fisher Scientific) and resuspended in 2 mL of DMEM with 0.1% BSA. 100 μL of cell suspension was added by well of a 96-well plate precoated for 12 hours with 40 μg/mL Collagen I (Corning, NY, USA) in PBS. The plate was incubated 20 min at 37°C to allow cell adhesion. Cells were then washed and cultured for 4 hours in DMEM supplemented with 10% FBS to enable recovery. Cells were then counted using MTT cell proliferation assay kit (ATCC, VA, USA).

Statistical analyses -Statistical analyses and graph representation were performed using GraphPad Prism 5.0 software (GraphPad Software Inc.). Unpaired two-tailed Student's t tests were used for comparisons of means of two groups. The means of data provided by sets of independent experiments carried out on samples from the patient were compared to the means of data provided by sets of independent experiments carried out on samples from three healthy individuals. Data are presented as means ± SEM and individual data points are represented when scatter plots are used.

RESULTS

Case history and patient's phenotype

The medical history of the patient (individual II.1 in Figure 1A & 1B) from birth to the age of 21 is the following: the patient was born to non-consanguineous healthy parents; increased fetal nuchal skinfold thickness was observed during the 2nd trimester of pregnancy. Birth was uneventful and birth measurements were normal. He presents no malformation or Hirschsprung disease. He was first referred for genetic counseling at the age of 5 years for hypotonia and developmental delay. The diagnosis of Pitt-Hopkins syndrome was suspected at the age of 10 based on facial features, smiling appearance, ataxic gait, and repetitive behavior, associated with severe ID and absence of speech.

Motor milestones were delayed as he walked at the age of 8. He also experiences generalized tonicclonic seizures with a frequency of one seizure every 2 months. Facial features comprise long thin eyebrows, long eyelashes, narrow palpebral fissures, epicanthic folds, divergent squint, a short nose with broad nasal bridge, macrostomia with thick lips and small spaced teeth. He also presents pectus excavatum, genu flexum, amyotrophy, flat feet, a brachydactyly of the third toe and mild camptodactyly (Figure 1A and Table 1). Brain MRI and muscle biopsy showed no abnormalities. Other features include scoliosis, lymphomatoid papulosis, testicular ectopia and recurrent alternation of constipation/diarrhea (Table 1). At the age of 21, the patient is 1m69 tall, he weighs 47 kg and has a head circumference of 60.5 cm (within the normal range). He still lacks speech capacities and exhibits a friendly behavior with no signs of aggressivity. He eats alone but cannot dress himself and is not toilet trained. He has sleep disturbance.

Identification of a CSDE1 c.362C>A p.(Ser121*) heterozygous de novo variation in the patient with severe ID

As no molecular abnormalities were found in TCF4, the gene involved in the Pitt-Hopkins syndrome, whole-exome sequencing was performed and identified the de novo heterozygous nonsense variation c.362C>A, p.(Ser121*) in CSDE1 (NM_001242891). The identified variation has not been described in sequence-variant databases, such as the genome aggregation database (gnomAD).

Sanger sequencing confirmed that the c.362C>A variation is carried by the patient and absent in his parents (Figure 1B). Another de novo missense variation c.616C>T, p.(Leu206Phe) was identified in the GNB2L1 gene (NM_006098). Taking into account the previously reported involvement of CSDE1 in ID-ASD [START_REF] Guo | Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission[END_REF] and the nature of the identified variation (nonsense), we sought to further study the functional impact of this gene variation. As the identified variation leads to a premature termination codon (PTC) in the 4 th exon of CSDE1, which contains 21 exons, it is expected to result either in the production of a truncated protein or in the absence of protein production through activation of the nonsense-mediated mRNA decay (NMD) pathway. Indeed, mutant PTC-containing mRNAs are expected to be degraded through the NMD signaling pathway, unless the PTC is located more than 50-55 nucleotides upstream of the last exon-exon junction [START_REF] Hsu | NMD Classifier: A reliable and systematic classification tool for nonsense-mediated decay events[END_REF][START_REF] Brogna | Nonsense-mediated mRNA decay (NMD) mechanisms[END_REF]. To discriminate between these two possibilities, we sequenced the CSDE1 transcripts obtained from the patient's fibroblasts before and after treatment with cycloheximide, an inhibitor of the NMD pathway. The patient's fibroblasts expressed only the normal CSDE1 allele, whereas cycloheximide led to accumulation of the mutated CSDE1 transcripts (Figure 1C). These data indicate that NMD plays an active role in the degradation of the mutated CSDE1 transcripts that contain a premature stop codon. Consistent with this observation, we found a ~50% decrease of CSDE1 transcripts from total RNA extracted from the patient's skin fibroblasts as compared to healthy individuals (Figure 1D), leading to a ~40% decrease in CSDE1 protein levels (Figure 1E). Taken together, these data demonstrate the deleterious character of the identified c.362C>A variation, which leads to CSDE1 haploinsufficiency.

Recurrent phenotypic features in patients upon partial loss of CSDE1

With the aim to identify the main phenotypic features associated with a partial loss of CSDE1, we analyzed the phenotype of our patient and compared it to the phenotypes of the previously reported individuals carrying a deletion encompassing CSDE1 (located on the short arm of chromosome 1, 1p13.2) [START_REF] Bisgaard | Interstitial deletion of the short arm of chromosome 1 (1p13.1p21.1) in a girl with mental retardation, short stature and colobomata[END_REF][START_REF] Fitzgibbon | Haploinsufficiency of the nerve growth factor beta gene in a 1p13 deleted female child with an insensitivity to pain[END_REF] and the recently reported ASD patients carrying truncating CSDE1 mutations [START_REF] Guo | Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission[END_REF]. All phenotypic features are summarized in Table 1. Noticeably, the patients share some common features among which the most recurrent clinical symptoms are language impairment (20/20), motor developmental delays (18/20), intellectual disabilities (17/19), and autistic features (12/16).

Deregulation of major cellular pathways upon partial loss of CSDE1 in the patient's fibroblasts

CSDE1 interacts with a large number of transcripts to regulate multiple cellular pathways. In order to identify the pathways hindered upon partial loss of CSDE1 in the patient, we performed an RNA-Seq transcriptomic analysis on fibroblasts obtained from the patient and three healthy individuals. This analysis showed that the steady-state of 1,131 transcripts is significantly modified in the patient's fibroblasts compared to the healthy individuals (545 upregulated genes and 586 down-regulated genes), with a cut-off of 0.05 for the unadjusted p-value and a minimum fold change of 1.5 (Table S1 -Data deposited at Gene Expression Omnibus GEO, Accession code GSE162954). Gene ontology (GO) biological process term analysis was performed to identify the key pathways related to the differentially expressed genes using Metascape (http://metascape.org, [START_REF] Zhou | Metascape provides a biologist-oriented resource for the analysis of systems-level datasets[END_REF]). The statistically enriched terms identified were hierarchically clustered into a tree based on the similarities among their gene memberships. The GO enrichment analysis revealed that the most significantly upregulated genes were associated with the regulation of the cell cycle and cell division (e.g. CDC25A, CDC20, CHEK1, E2F1) (Figure 2A -left panel and Table S1). Noteworthy, the down-regulated genes were essentially involved in the homophilic cell adhesion via plasma membrane adhesion molecules, mainly belonging to the cadherin and protocadherins family e.g. CDH4, CDH6, CDH10, PCDH7, PCDHA9, PCDHB5 (Figure 2A -right panel and Table S1).

In order to further characterize the specific contribution of the CSDE1 loss-of-function mutation in these transcriptomic data, we focused our analyses on genes containing CSDE1 binding motifs. To this end, we conducted a comparative study of our results with those obtained by Ju Lee et al. in their transcriptomic analysis of genes containing at least one consensus CSDE1 binding motif upon CSDE1 knockdown in human embryonic stem cells [START_REF] Lee | A posttranscriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells[END_REF]. Furthermore, the identification of deregulated genes common to fibroblasts and embryonic stem cells allowed us to palliate the bias linked to a cell-type specific analysis. The comparison of the list of deregulated genes detected in our study with the list of Ju Lee et al. (531 up-regulated genes and 676 down-regulated genes retained on the basis of a log2-fold change and at a False Discovery Rate (FDR) < 0.05) identified 30 and 33 common genes that are up-and down-regulated, respectively (Figure 2B & 2C, top panel). We conducted GO enrichment analyses on these genes and all statistically enriched terms were converted into a network layout (Figure 2B

Inhibition of the Wnt/β-catenin pathway in the fibroblasts of the patient with CSDE1 haploinsufficiency

Our RNA-seq analysis revealed that APCDD1 (APC Down-Regulated 1, MIM #607479), a component of the Wnt/β-catenin pathway is upregulated in the patient's fibroblasts as compared to three healthy individuals (Table S1). As shown in Figure 3A, this significant increase in the expression levels of APCDD1 was confirmed by RT-qPCR. Western blot analysis on protein samples obtained from fibroblasts of the patient and healthy individuals showed a 2-fold increase of the APCDD1 protein in the patient (Figure 3B). APCDD1 is a direct target of the Wnt/β-catenin pathway and the corresponding protein is an essential inhibitor of this pathway, thus exerting a negative feedback control [START_REF] Shimomura | APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex[END_REF][START_REF] Takahashi | Isolation of a novel human gene, APCDD1, as a direct target of the beta-Catenin/T-cell factor 4 complex with probable involvement in colorectal carcinogenesis[END_REF].

In order to evaluate the state of the Wnt/β-catenin signaling in the patient's fibroblasts, we first assessed through western blot analysis the expression levels of β-catenin and showed a ~25% decrease in the β-catenin levels in the patient as compared to healthy individuals (Figure 3C), whereas β-catenin transcripts were unchanged compared to healthy individuals as shown by our RNA-seq analysis. We further quantified the Wnt/β-catenin signaling in fibroblasts from the patient and from healthy individuals using a TOPFlash/FOPFlash luciferase reporter assay that provides a precise measurement of the canonical Wnt-specific transcriptional activity (Figure 3D). Results of this assay showed a significant inhibition of this pathway in the patient, in keeping with the identified decrease of β-catenin protein levels. Altogether, these data show an increase of APCDD1 transcripts and protein levels and a subsequent inhibition of the Wnt/β-catenin pathway in the patient carrying the CSDE1 null-mutation.

Enhanced substrate adhesion faculties of the fibroblasts of the patient with CSDE1 haploinsufficiency

As our RNA-seq analysis suggested an alteration of the overall adhesion capacities of the cells of the patient through deregulation of the expression of cadherins and integrins (Figure 2B & 2C and Table S1), we sought to assess cellular adhesion in the patient's fibroblasts. We first confirmed by RT-qPCR the significant increase, in the patient's fibroblasts, of the expression levels of CDH2 (cadherin 2 or ncadherin, MIM #114020), a pivotal actor in cell-cell adhesion as well as of SERPINE1 (Serpin Family E Member 1, MIM #173360) and ITGA5 (Integrin Alpha-5, MIM #135620), two genes involved in cellsubstrate adhesion (Figure 3E). Noteworthy, CDH2 is also a regulator of the Wnt/β-catenin pathway.

To further investigate the cell-substrate adhesion abilities of the fibroblasts obtained from the patient, we conducted a cell-substrate adhesion assay using a collagen I -coated surface. We observed significant enhanced adhesion abilities of the fibroblasts of the patient as compared to those from healthy individuals (Figure 3F). Taken together, these data reveal enhanced cell-substrate adhesion faculties directly through the modulation of CSDE1 target genes.

DISCUSSION

In this study, we provide a thorough description of the disease phenotype of a patient with severe ID-ASD carrying a heterozygous de novo nonsense variation in CSDE1 and followed from birth to the age of 21 years. We performed functional studies aiming at deciphering the CSDE1-dependent pathways involved in the pathophysiology of this highly heterogeneous disease condition. The study of the patient's primary fibroblasts, through a combination of various approaches, indeed revealed impairment of the Wnt/β-catenin pathway and of the cellular adhesion abilities that result from a deregulated expression of two specific CSDE1 targets, namely APCDD1 and CDH2. Overall, these data support a pathophysiological model of the disease in which reduced levels of CSDE1 impair the regulation of two of its target transcripts (APCDD1 and CDH2) leading to abnormal levels of the corresponding proteins and therefore deregulation of the associated pathways (Figure 4).

CSDE1 and ID-ASD -

The in-depth description of the patient's phenotype, which includes severe developmental delay with no malformations, cognitive impairment, seizures and distinctive morphological characteristics at different stages of his life, represents a major asset for the diagnosis of other CSDE1-related ID-ASD cases. By reviewing the phenotype of the patients so-far reported with a CSDE1 loss-of-function variant, we noticed a variable expressivity of this autosomal dominant disorder, with severity ranging from isolated language impairment in some parents of the patients carrying an inherited CSDE1 mutation to severe ID [START_REF] Guo | Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission[END_REF]. Such large phenotypic spectrum suggests a potential role of environmental or modifying factors. In this regard, our patient carries another de novo missense variation c.616C>T p.(Leu206Phe) in the GNB2L1 gene. Despite the fact that GNB2L1 is not involved in human ID-ASD pathology, we cannot exclude the possibility that this GNB2L1 variation may have an impact on disease expressivity in our patient. Implication of the homolog of GNB2L1 (Rack1) was suggested in axon guidance in C.elegans [START_REF] Demarco | RACK-1 acts with Rac GTPase signaling and UNC-115/abLIM in Caenorhabditis elegans axon pathfinding and cell migration[END_REF], in neurite extension of neuronally differentiated rat cells [START_REF] Dwane | RACK1 promotes neurite outgrowth by scaffolding AGAP2 to FAK[END_REF], and in axon outgrowth of mouse primary cortical neurons [START_REF] Kershner | RACK1 is necessary for the formation of point contacts and regulates axon growth[END_REF].

Among the commonly used patient-derived cellular models for studying neurological disorders (e.g. human dermal fibroblasts, induced pluripotent stem cells, neural cells induced from iPSC or directly from postmitotic somatic cells, immortalized lymphocytes and peripheral leukocytes), we have chosen to conduct our investigations using dermal fibroblasts derived from the patients as they represent a relevant patient-derived cellular model for the study of ID-ASD. Indeed, they have been widely used as patient-derived cellular models for studying several disease conditions affecting the central nervous system (CNS) function as they share molecular similarities with CNS cells and express molecular factors that are highly similar to cells of neuro-ectodermal origin [START_REF] Kálmán | Human dermal fibroblasts in psychiatry research[END_REF][START_REF] Rieske | Human fibroblast-derived cell lines have characteristics of embryonic stem cells and cells of neuro-ectodermal origin[END_REF]. Our study establishes the role of CSDE1 in the onset of this ID-ASD in humans based on data obtained from the patient's fibroblasts. So far, the involvement of Csde1 in neurodevelopment has been previously suggested by two studies performed in mouse and drosophila models. Csde1 has been implicated in neuronal and synapse development including synapse transmission [START_REF] Guo | Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission[END_REF] and has been shown to be crucial for the proper control of the migration of precerebellar neurons [START_REF] Kobayashi | The control of precerebellar neuron migration by RNA-binding protein Csde1[END_REF]. Furthermore, our study highlights the role of CSDE1 in human neuronal development as our transcriptomic analysis reveals a significant deregulation of the expression of NEFM (Neurofilament medium polypeptide) in the patient's fibroblasts, a protein belonging to GO: actin cytoskeleton organization and involved in neuron projection development.

CSDE1 is a key regulator of the Wnt/β-catenin pathway -Our study shows that the Wnt/β-catenin pathway is deregulated upon partial loss of CSDE1 through a mechanism at least partly explained by the modulation of APCDD1 and CDH2 expression by CSDE1. Several of our data strongly support a downregulation of the Wnt/β-catenin pathway in the patient's cells. First, we show that among the diverse CSDE1 direct transcript targets that belong to the Wnt/β-catenin network, APCDD1 and CDH2 are significantly upregulated, APCDD1 being the transcript with the most deregulated expression as compared to healthy individuals. APCDD1, a target of the Wnt/β-catenin pathway also acts as an inhibitor of this pathway by preventing the formation of the Wnt-Fzd-Lrp5/6 (LDL Receptor Related Protein 5/6) receptor complex, thus creating a negative feedback loop on Wnt/β-catenin signaling [START_REF] Shimomura | APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex[END_REF][START_REF] Takahashi | Isolation of a novel human gene, APCDD1, as a direct target of the beta-Catenin/T-cell factor 4 complex with probable involvement in colorectal carcinogenesis[END_REF]. CDH2 regulates the availability of key factors of the Wnt/β-catenin signaling like the core protein β-catenin or the Wnt co-receptor Lrp6 [START_REF] Revollo | N-cadherin restrains PTH activation of Lrp6/β-catenin signaling and osteoanabolic action[END_REF][START_REF] Shin | Dominant negative Ncadherin inhibits osteoclast differentiation by interfering with beta-catenin regulation of RANKL, independent of cell-cell adhesion[END_REF]. Second, our data show decreased levels of the β-catenin protein in the patient's fibroblasts although the levels of β-catenin transcripts are not deregulated, thereby supporting a posttranslational role of CSDE1 in the regulation of β-catenin.

Accordingly, decreased levels of the β-catenin protein with no deregulation of the β-catenin transcripts was observed in Csde1 knock-out mice [START_REF] Guo | Disruptive variants of CSDE1 associate with autism and interfere with neuronal development and synaptic transmission[END_REF] as well as after CSDE1 silencing in human melanoma cells [START_REF] Wurth | UNR/CSDE1 Drives a Post-transcriptional Program to Promote Melanoma Invasion and Metastasis[END_REF]. The discrepancy we observe between the decreased levels of β-catenin protein and the unchanged levels of the corresponding transcripts is in accordance with the well-established posttranslational regulation of the β-catenin protein through modulation of its phosphorylation and ubiquitination states (reviewed in ( 57)). Third, the observed decreased levels of β-catenin are indeed followed by an overall inhibition of the pathway as shown by our luciferase assay. Overall, CSDE1, which acts as an upstream modulator of the expression of individual effectors of the Wnt/β-catenin pathway, plays therefore a pivotal role in the regulation of this pathway.

Alteration of the fine-tuning of the Wnt/β-catenin pathway in ID-ASD -The spatiotemporal regulation of the evolutionarily conserved Wnt/β-catenin pathway is crucial for correct neurodevelopmental and post-neurodevelopmental processes; actors of this pathway are involved in regionalization of nervous system, differentiation of neural progenitor cells, axon guidance, dendritic development, as well as neuronal migration and plasticity (reviewed in [START_REF] Kumar | Impaired neurodevelopmental pathways in autism spectrum disorder: a review of signaling mechanisms and crosstalk[END_REF][START_REF] Kwan | Wnt signaling networks in autism spectrum disorder and intellectual disability[END_REF]). In consistence with these functions, our study highlights the involvement in ID-ASD pathophysiology of three key players of the Wnt/β-catenin signaling pathway: the β-catenin core element and two modulators of the pathway, APCDD1 and CDH2. Accordingly, specific gene mutations in β-catenin (CTNNB1) [START_REF] Dubruc | A new intellectual disability syndrome caused by CTNNB1 haploinsufficiency[END_REF][START_REF] Kuechler | De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum[END_REF][START_REF] Dong | Deletion of CTNNB1 in inhibitory circuitry contributes to autism-associated behavioral defects[END_REF] or CDH2 [START_REF] Accogli | De Novo Pathogenic Variants in N-cadherin Cause a Syndromic Neurodevelopmental Disorder with Corpus Collosum, Axon, Cardiac, Ocular, and Genital Defects[END_REF] have been reported in ID. Furthermore, APCDD1 has been involved in neurological disorders through its roles in oligodendrocyte differentiation and regenerative myelination in the central nervous system [START_REF] Lee | Apcdd1 stimulates oligodendrocyte differentiation after white matter injury[END_REF] as well as in the generation of neurons from progenitors [START_REF] Shimomura | APCDD1 is a novel Wnt inhibitor mutated in hereditary hypotrichosis simplex[END_REF]. Whereas our data show that the Wnt/β-catenin pathway is downregulated in the ID-ASD patient, both hyperand hypoactivity of this pathway have been shown to alter cognitive functions (reviewed in ( 62)).

Indeed, the deregulated expression of the β-catenin protein illustrates this duality: several patients with cognitive impairments including ID and ASD present β-catenin haploinsufficiency [START_REF] Dubruc | A new intellectual disability syndrome caused by CTNNB1 haploinsufficiency[END_REF][START_REF] Kuechler | De novo mutations in beta-catenin (CTNNB1) appear to be a frequent cause of intellectual disability: expanding the mutational and clinical spectrum[END_REF] whereas others exhibit increased β-catenin levels due to the deletion of the gene encoding the APC (Adenomatous polyposis coli) protein that mediates β-catenin degradation [START_REF] Barber | Adenomatous polyposis coli and a cytogenetic deletion of chromosome 5 resulting from a maternal intrachromosomal insertion[END_REF]. Modulation of the Wnt/β-catenin pathway can also occur in a cell-specific manner as illustrated by the regulation mediated by CHD8 (chromodomain helicase DNA binding protein 8), a protein involved in ASD pathogenesis [START_REF] Bernier | Disruptive CHD8 mutations define a subtype of autism early in development[END_REF] which acts as a positive regulator of the Wnt β-catenin pathway in neural progenitor cells [START_REF] Durak | Chd8 mediates cortical neurogenesis via transcriptional regulation of cell cycle and Wnt signaling[END_REF], and as a negative regulator of this pathway in non-neuronal cell lines.

Overall, these data raise the larger question of the mechanisms by which the altered spatiotemporal fine-tuning of the Wnt/β-catenin pathway contributes to the occurrence of ID-ASD. Several nonexclusive answers can be put forward based on studies performed in mouse and rat models: (i) the Wnt/β-catenin pathway plays a key role in the regulation of neuronal polarity and axonal outgrowth through the ligand Wnt3a (66); (ii) it is also important for dendritic morphogenesis via the β-catenin protein (67); (iii) the Wnt/β-catenin pathway is also pivotal for the proper functioning of the neuronal network through the modulation of the neural glucose metabolism. Indeed, by means of a mechanism dependent on the ligand Wnt3a and the metabolic sensor Akt, the Wnt/β-catenin pathway regulates the glucose intake in neurons as well as the activity of the hexokinase and the glycolytic rate (68). In keeping with this impact of the Wnt/ β -catenin pathway on metabolism, our RNA-seq analysis shows a deregulated expression of genes involved in the carbohydrate metabolism.

Enhanced cellular adhesion in a neurodevelopmental disease -Our results unveil a role for CSDE1 in enhanced cellular adhesion in the context of ID-ASD. The implication for CSDE1 in cellular adhesion has so far been reported in the context of cancer progression in melanoma cell models [START_REF] Wurth | UNR/CSDE1 Drives a Post-transcriptional Program to Promote Melanoma Invasion and Metastasis[END_REF], but not in the context of ID-ASD. Most importantly, our transcriptomic analysis performed in patient's primary fibroblasts shows the modulation of the expression of genes belonging to the cadherin/protocadherin superfamily and integrin families that control both cell-cell and cell-matrix adhesion. On the one hand, cell-cell adhesion -essentially ensured by molecules belonging to the cadherin and protocadherin superfamily -regulates the formation of both adherens and synaptic junctions in the central nervous system [START_REF] Redies | Cadherins and neuropsychiatric disorders[END_REF]. CDH2, a gene known to be essential for proper axon guidance [START_REF] Accogli | De Novo Pathogenic Variants in N-cadherin Cause a Syndromic Neurodevelopmental Disorder with Corpus Collosum, Axon, Cardiac, Ocular, and Genital Defects[END_REF], is one of the cell-cell adhesion genes whose expression was found deregulated in our patient. On the other hand, cell-matrix adhesion -under the control of proteins mainly belonging to the integrin family -affects cell migration, axon guidance and synapse formation in the central nervous systems [START_REF] Ringer | Sensing the mechano-chemical properties of the extracellular matrix[END_REF][START_REF] Reichardt | Extracellular matrix molecules and their receptors: functions in neural development[END_REF][START_REF] Schmid | Role of integrins in the development of the cerebral cortex[END_REF]. Among the integrins, ITGA5 -significantly deregulated in the fibroblasts of our patient -is known to contribute to the survival and proliferation of glial progenitor cells [START_REF] Haack | Integrin receptors are required for cell survival and proliferation during development of the peripheral glial lineage[END_REF].

Moreover, the integrin-mediated cell interactions are tightly regulated by plasminogen activators like SERPINE1 (74) that is significantly upregulated in the fibroblasts of our patient. Neurological impairments can result from deregulation of cell-cell or cell-substrate adhesion (reviewed in [START_REF] Redies | Cadherins and neuropsychiatric disorders[END_REF][START_REF] Jaudon | Integrin adhesion in brain assembly: From molecular structure to neuropsychiatric disorders[END_REF].

Overall, our data support a key role for CSDE1 in the regulation of these two cell adhesion types, a result that is in line with the wide-ranging neurological phenotype observed in the patient (epilepsy and cognitive, motor, behavioral impairments).

In summary, our work identifies the Wnt/β-catenin signaling and cellular adhesion as two major deregulated pathways in an ID-ASD patient carrying a monoallelic null mutation in CSDE1 through mechanisms relying on APCDD1 and CDH2. In addition, by providing an in-depth description of the phenotype of the patient at different stages of his life, our study should help identifying patients with ID-ASD who could benefit from a screening for CSDE1 mutations. C. Analysis of the common downregulated genes from our transcriptomic study and Ju Lee et al. [START_REF] Lee | A posttranscriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells[END_REF].

Network of enriched terms colored by cluster identity, where nodes that share the same cluster ID are typically close to each other. The p-value corresponding to each enriched term is indicated in brackets; terms containing more genes tend to have a more significant p-value. All graphs were generated by Metascape (http://metascape.org, (42)) and were modified graphically only. Lower panel -In the ID-ASD patient, the heterozygous nonsense mutation in CSDE1 leads to mRNA decay of the transcripts that carry it. This results in CSDE1 protein haploinsufficiency which will compromise the regulation of the target transcripts (e.g. APCDD1 and CDH2) and consequently the levels of the corresponding proteins. Increased levels of APCDD1 and CDH2 proteins lead to the downregulation of the Wnt/β-catenin pathway and the increase of cellular adhesion.

TABLE LEGEND

Phenotype and features of the individual II.I carrying the c.362C>A, p.(Ser121*) variation in CSDE1 compared to patients lacking the chromosomal region that contains CSDE1 (del 1p13.1p13.2, [START_REF] Fitzgibbon | Haploinsufficiency of the nerve growth factor beta gene in a 1p13 deleted female child with an insensitivity to pain[END_REF] and del 1p13.1p21.1 [START_REF] Bisgaard | Interstitial deletion of the short arm of chromosome 1 (1p13.1p21.1) in a girl with mental retardation, short stature and colobomata[END_REF]) and patients presenting mutations in CSDE1 (7 frameshift, 6 stopgain, 3 splicing mutations (36)); n.d. : not defined; § : The molecular anomalies are present at the heterozygous state.

  & 2C, bottom panel). We observed a significant upregulation of genes involved in the negative regulation of Wnt signaling (CDH2, GRB10, DACT1, APCDD1, APCDD1L, E2F1), cell-substrate adhesion (COL8A1, ITGA5, LGALS1, SERPINE1, ZYX, ACTA2, CDH2, TGFBI), smooth muscle contraction (ACTA2, ADRA1D, CNN1, MSN, CDC42EP1), p53 downstream pathway (E2F1, SERPINE1, STEAP3), regulation of cell shape (MSN, CDC42EP1, SHROOM3, ACTA2, CDH2, SERPINE1), tissue morphogenesis (ACTA2, ITGA5, MSN, DACT1, SHROOM3, COL8A1, LGALS1), actin cytoskeleton organization (CNN1, INPPL1, ZYX, CDC42EP1, SHROOM3, NEFM), and cell junction organization (CDH2, ITGA5, KIFC3) (Figure2B, bottom panel). Downregulated genes belong to five main pathways: regulation of reproductive process (BMP4, GJA1, MSX2, SERPINF1, CSDE1, FZD5, CD9, PRKAR2B, CDA, CREG1), cellular response to hormone stimulus (BMP4, GJA1, MSX2, SERPINF1, PRKAR2B, PCSK9, KLF15, KCNS3), cell-cell junction assembly (CD9, GJA1, FZD5, MSX2, PRKAR2B, KCNS3, GNPTAB, SERPINF1), ECM (extracellular matrix) regulators (SERPINF1, SERPINB9, PAMR1) and cellular carbohydrate metabolism process (FAM3C, GNPTAB, PPP1R3B) (Figure2C, bottom panel). Overall, these transcriptomic analyses unveil that decreasing CSDE1 levels lead to a deregulated expression of genes involved in several pathways, among which, most importantly, the Wnt/β-catenin pathway and cellular adhesion exhibit the lowest p-values.

Figure 1 .

 1 Figure 1. The patient's clinical features and the molecular bases of the CSDE1 haploinsufficiency A. Phenotypic features of the patient -Front and profile view of individual II.1 at 5 (a,b) and 21 years (c,d) of age showing the distinct facial features. Photos of the patient at age 21 showing the (e) pectus excavatum, (f) his hand and (g,h) his foot. B. Genealogical tree of the patient with ID-ASD (Individual II.1) and his parents (Individuals I.1 and I.2). Electropherograms from Sanger sequencing showing the heterozygous c.362C>A p.(Ser121*) mutation only in individual II.1 and not in his parents. C. Sanger sequencing of cDNA obtained from fibroblasts of healthy individuals and individual II.1 upon cycloheximide treatment (30 μg/ml, for 4 hours) showing accumulation of the mutated CSDE1 transcripts. D. Quantification of CSDE1 transcripts from fibroblasts of three healthy individuals and individual II.1 by RT-qPCR. The transcript levels of CSDE1 were normalized to the housekeeping gene ERCC3. The results are shown as the mean of three independent experiments ± SEM and plotted relative to the healthy controls conditions. E. CSDE1 protein expression in fibroblasts of healthy individuals and individual II.1 -Equal amounts of protein extracts were subjected to SDS-PAGE and immunoblotted with CSDE1 antibody. The CSDE1 signal was quantified with ImageJ software and normalized to the amount of GAPDH used as a loading control. The results are shown as the mean of three independent experiments and are plotted as a percentage of the healthy controls condition. For all experiments, asterisks indicate that the mean is significantly different; *: t test p value < 0.05, **: t test p value < 0.01, ***: t test p value < 0.001.

Figure 2 .

 2 Figure 2. Transcriptomic analysis in the patient's fibroblasts A. Enrichment analysis of RNA-seq data sets -Bar graph of enriched terms across input gene lists, colored by p-values and showing biological processes enrichment analysis of up-regulated genes (left panel) and for down-regulated genes (right panel).B. Analysis of the common upregulated genes from our transcriptomic study and Ju Lee et al.[START_REF] Lee | A posttranscriptional program coordinated by CSDE1 prevents intrinsic neural differentiation of human embryonic stem cells[END_REF].

Figure 3 .

 3 Figure 3. Deregulation of the Wnt/β-catenin pathway and cellular adhesion in the patient A. Quantification of APCDD1 transcripts from fibroblasts of three healthy individuals and individual II.1 by RT-qPCR. The transcript levels of APCDD1 were normalized to the housekeeping gene ERCC3. The results are shown as the mean of three independent experiments ± SEM and plotted relative to the healthy controls conditions. B. APCDD1 protein expression in fibroblasts of three healthy individuals and individual II.1 -Equal amounts of protein extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibody. The APCDD1 signal was quantified with ImageJ software and normalized to the amount of GAPDH used as a loading control. The results are shown as the mean of three independent experiments and are plotted as a percentage of the healthy controls condition. Note: Samples from figure 1E and 3B were loaded on the same gel and different parts of the membrane were revealed with different antibodies, therefore, the GAPDH loading control is the same for figure 1E and 3B. C. β-catenin protein expression in fibroblasts of three healthy individuals and individual II.1. -Equal amounts of protein extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibody. β-catenin signal was quantified with ImageJ software and normalized to the amount of βtubulin used as a loading control. The results are shown as the mean of three independent experiments and are plotted as a percentage of the healthy controls condition. D. Quantification of the Wnt/β-catenin signaling in fibroblasts from the patient and from healthy individuals using a TOPFlash/FOPFlash luciferase reporter assay -Fibroblasts of healthy individuals and individual II.1 were transfected with TOP or FOP plasmids then 20 hours later treated with 20 mM LiCl for 6 hours. Luciferase activity was normalized to total protein amounts and plotted as 4 independent experiments. E. Quantification of CDH2, SERPINE1 and ITGA5 transcripts from fibroblasts of three healthy individuals and individual II.1 by RT-qPCR. The transcript levels of the mentioned genes were normalized to the housekeeping gene ERCC3. The results are shown as the mean of three independent experiments ± SEM and are plotted relative to the healthy controls conditions. F. Cell-substrate adhesion assay in fibroblasts of healthy individuals and individual II.1 -Fibroblasts were harvested on Collagen-I coated plates and the number of cells was quantified by MTT assay. The results are shown as the ratio of number of adherent cells on the number of total cells seeded and are plotted as five independent experiments.

Figure 4 .

 4 Figure 4. Pathophysiological model of the CSDE1-haploinsufficiency-related diseaseUpper panel -In healthy individuals the levels of CSDE1 mRNA and its corresponding proteins are normal. CSDE1 proteins interact with the IRES, the 5'UTR or the 3'UTR of their target transcripts to regulate their translation or their stability. In the case of APCDD1 and CDH2, this interaction would lead to the regulation of the transcripts abundance. Normal amounts of APCDD1 and CDH2 proteins control the homeostasis of specific cellular pathways.

Figures

  Figures

TABLE Table 1

 Table Study

		Our study	Bisgaard et. al 2007	Fitzgibbon et al. 2009	Guo et. al 2019
	Molecular anomalies §	c.362C>A p.(Ser121*) in CSDE1 c.616C>T p.(Leu206Phe) in GNB2L1	del 1p13.1p21.1	del 1p13.1p13.2	7 frameshift, 6 stopgain, 3 splicing mutations in CSDE1
	Gender, age	Male, from birth to 21 years old	Female, 13 years old Female, 5 years old	11 males, 6 females 3 to 19 years old
	Height	-2SD	<5th centile	n.d.	8/15: short (7), tall (1)
	Weight	-2SD	Weight-for-height : 50th centile	n.d.	6/15: overweight (3), lean (3)
	Head Circumference 0SD	<3rd centile	n.d.	6/14: macrocephaly
	Neurodevelopmental
	features
					3/17: below average
	Intellectual disability Severe	Severe	Severe	or learning disability, 14/17: mild to severe
					ID
	Language impairment Yes	Yes	Yes	17/17
	Motor development delay	Walked at 8 years	Yes	Yes	15/17
	Neurological features
	Epilepsy	Yes	Yes	n.d.	7/16
	Brain MRI abnormalities	No	Delayed myelination n.d.	7/14 (delayed myelination :1)
	Facial features
	Telecanthus	Yes	n.d.	Mild	n.d.
	Long eyebrows	Yes	Yes	Yes	n.d.
	Epicanthic folds	Yes	No	No	n.d.
	Short palpebral fissures	Yes	No	No	n.d.
	Short/broad nose	Yes	Yes	Yes	n.d.
	Macrostomia	Yes	Yes	No	n.d.
	Philtrum	Short	Short	Long	n.d.
	Low set ears	Yes	Yes	n.d.	n.d.
	Musculoskeletal
	abnormalities
	Amyotrophy	Yes	n.d.	n.d.	n.d.
	Hypotonia	Yes	Yes	Yes	6/13
	Pectus excavatum	Yes	n.d.	n.d.	n.d.
	Genu flexum	Yes	n.d.	n.d.	n.d.

ACKNOWLEDGEMENTS

We thank the patient and his family as well as control individuals for their cooperation. We thank Virginie Bordereau and Patrick Raymond for their technical contributions and Marthe Rizk-Rabin and Bruno Ragazzon for the material and advice on the functional assessing of the Wnt/β-catenin pathway. This work was supported by grants from the Foundation of rare diseases (GIS -Institut des Maladies Rares).

CONFLICT OF INTEREST

The authors have no conflict of interest to declare.


P Muller

G Sudre

O Théodoly

Wetting transition on hydrophobic surfaces covered by polyelectrolyte brushes

Keywords: polyelectrolyte, wetting, Young-Dupré equation, polymer brush, interfacial tension

We study the wetting by water of complex "hydrophobic-hydrophilic" surfaces made of a hydrophobic substrate covered by a hydrophilic polymer brush. Polystyrene PS substrates covered with polystyrene-block-poly(acrylic acid) PS-b-PAA diblock copolymer layers were fabricated by Langmuir-Schaefer depositions and analyzed by AFM and ellipsometry. On bare PS substrate, we measured advancing angles θ A = 93 ± 1° and receding angles θ R = 81 ± 1°. On PS covered with poorly anchored PS-b-PAA layers, we observed large contact angle hysteresis, θ A ≈ 90° and θ R ≈ 0° that we attributed to nanometric scale dewetting of the PS-b-PAA layers. On well-anchored PS-b-PAA layers that form homogeneous PAA brushes, a wetting transition from partial to total wetting occurs versus the amount deposited: both θ A and θ R decrease close to zero. A model is proposed, based on the Young-Dupré equation, that takes into account the interfacial pressure of the brush Π, which was determined experimentally, and the free enthalpy of hydration of the polyelectrolyte monomers hyd PAA G ∆ , which is the only fitting parameter. With ⋅ ∆ hyd PAA G

≈ -1300 J/mol, the model renders the wetting transition for all samples and explains why the wetting transition depends mainly on the average thickness of the brush and weakly on the length of PAA chains.

Introduction

Practical and durable hydrophilization of hydrophobic substrates is a on-going field of research motivated by academic and industrial interests. Non-woven polypropylene tissues are naturally hydrophobic and have to be hydrophilized to absorb water (wipes), or let water go through (diapers). For surface cleaning (windshield, glasses, windows, cars), hydrophilizing the surface permits to avoid, upon drying, the formation of drops that let unaesthetic stains. A simple solution to hydrophilize a surface consists in depositing amphiphilic molecules. However, small-molecule surfactants have the disadvantage of being easily washed-out upon first contact with water. Polymeric amphiphilic molecules are therefore an interesting alternative to achieve more durable hydrophilizing treatments at low cost. The quality of such treatments will depend on the stability of the polymer layer at the surface (against washing, drying, rubbing, aging, etc…), and of the efficiency of the hydrophilization, i.e. the increase of the surface energy. Several routes permit to improve the stability of the polymer layer. One route relies on adhesion, which is maximized with a higher affinity between the surface and the hydrophobic polymer moieties and also with polymers of larger molar mass. Another route consists in anchoring the polymeric layer to the surface, either physically or chemically. For the hydrophilization efficiency, one may expect that covering a hydrophobic substrate with a highly hydrophilic layer is enough to make the surface totally wetting. However, a hydrophilic layer is by essence not waterproof, and does not hamper the contact between the hydrophobic substrate and water. It is not obvious to infer what shall be the thickness and/or density of the hydrophilic layer necessary to overcompensate the presence of the underlying hydrophobic layer. The question tackled in this paper can be stated as: what governs the wetting by water of a complex surface made of a hydrophobic substrate covered by a hydrophilic polymer? A pioneer theoretical work was published in 1984 1 , and later improved [START_REF] Leibler | Ordering in Macromolecular Systems[END_REF] , on the wetting of a pure non volatile solvent on a solid coated by an uncharged polymer. In good solvent conditions, it is predicted that the entropy of dissolution of the polymer for usual brushes (polymerization length 100, grafting density 0.01 chain.Å -2 ) drives the spreading parameter, leading to conditions of total wetting. Later, experimental and theoretical works have treated the case of a polymer brush in contact with a liquid polymer made up of the same monomers as the brush [START_REF] Maas | [END_REF]4,5,6,7 , with a mixture of solvents 8 , and with a nematic fluid 9 . The wetting by a simple fluid (water) of a hydrophilic uncharged brush grafted on a hydrophobic substrate was only considered recently 10 . It was demonstrated that a transition to complete wetting could not be obtained with poly(ethylene oxide) PEO brushes. This result was attributed to a bridging of the solvent-vapor interface by the grafted layer. In the present study, we consider the wetting by a simple volatile fluid (water) of a polyelectrolyte brush. We use model hydrophobic substrates made of polystyrene spin-coated on atomically flat silicon wafers. The grafted hydrophilic polymer brushes are made with diblock copolymers of polystyrene PS and poly(acrylic acid) PAA. PS permits a physical anchoring of the chains to the substrate, whereas PAA is a strongly hygroscopic polyelectrolyte, which is routinely used for super-absorbent properties. The surface treatments are performed by the Langmuir-Schaefer technique (transfer from air/water to PS-water interface) followed by an annealing procedure to anchor the brush. The state of the brushes is characterized by ellipsometry and AFM. The contact angles with water are finally measured versus the amount of PAA deposited and the length of the PAA chains. A thermodynamic model is then proposed to interpret the wetting properties of complex surfaces made of a hydrophobic substrates covered by a hydrophilic polymer brush.

Experimental Section

Materials. Silicon wafers (thickness 281µm +/-25µm, orientation (100), no dopant, RMS roughness measured by AFM at 5 Å on 1x1 mm images) were purchased from Silicon Inc., Hexamethyldisilasane (HMDS) from Gelest, Inc., polystyrene (Primary Standard polystyrene M w =350,000g/mol) from TSK, and polystyrene-blockpoly(acrylic acid) PS-b-PAA samples from Polymer Source Inc. The PS-b-PAA samples used in this study have molar masses of 1.8K-6K, 1.5K-44K, 4.5K-19.3K and polydispersity indexes lower than 1.1.

Preparation of PS substrates. Silicon wafers are first cleaned in a UV/O 3 chamber for 15 min and immediately silanized with HMDS in vapor phase for 2 hours at ambient pressure and temperature. This pre-treatment is required to avoid problems when the PS layer is put in water solutions. Without this pre-treatment, small pockets of water appear between the PS layer and the hydrophilic wafer surface. PS layers are then spin-coated at 5,000 RPM for 1 min from a PS solution in toluene filtered at 0.2 µm and at concentration 2.5 wt%. The layers are finally annealed overnight at 80 ºC. The average thickness of the PS layer is measured by ellipsometry around 120 nm. The average RMS roughness measured by AFM is around 0.6 nm for 1 micron square.

Langmuir trough isotherms.

Compression isotherms of PS-b-PAA copolymers at the air/water interface have been measured on a commercial Langmuir trough (model NIMA 611). A known amount of PS-b-PAA samples is dissolved at a concentration of 1 mg/g in a solvent made of 1,4-dioxane with HCl at 2 wt%. This solvent is a good compromise between good spreading properties at the air/water interface, which is easily achieved with apolar solvents, and good solvency properties for PAA-rich diblock samples, which can be achieved with polar solvents. A few microliters of diblock solutions are spread drop by drop onto the water surface with a micro syringe (Hamilton). Since dioxane is miscible with water, part of the material may sink in the water subphase. The absolute amount deposited has to be measured a posteriori by ellipsometry.

Langmuir-Schaefer depositions. The monolayers are transferred from the air/water interface to the PS substrate by the Langmuir-Schaefer technique [START_REF] Roberts | Langmuir-Blodgett Films[END_REF] , which consists in stamping the PS substrate face-down across the air/water interface. The monolayer at the air/water interface is removed by aspiration in order to avoid the deposition of a second layer on the substrate during its removal from the trough. The samples with the depositions are stored in water. Caution was taken to maintain the sample wet and avoid strong shear flows during all manipulations to avoid damaging the deposited monolayers. Each transferred layer is identified by the surface pressure at which the transfer is performed. As explained above, the area per molecule calculated based on the amount of material dissolved in the spreading solution is subject to uncertainty, whereas a given surface pressure Π characterizes a unique surface layer concentration. For very dilute layers, when the slope of the surface pressure versus the surface concentration is small, the control of the amount of transferred copolymer per unit area at a given surface pressure Π is less precise. For this reason, we decided to proceed differently for depositions at surface pressures Π lower than 2.5 mN/m. A layer was first compressed to a surface pressure of 2.5 mN/m, then the area was increased by a factor Y. Deposition prepared by this method are labeled 2.5mN/m:Y. Three dilute layers have been prepared, 2.5mN/m:3, 2.5mN/m:5, and 2.5mN/m:8.

Anchoring of deposited layers. Two methods have been used to anchor the deposited PS-b-PAA monolayers on the PS substrates. The toluene swelling method consists in adding toluene to the water in which the samples are stored. In order to control the amount of toluene in water, we prepared mixtures of pure water and water saturated with toluene. We call X the mass percent of toluene-saturated water in a mixture. The samples with a deposited diblock monolayer were let for 1 min in a water-toluene solution with X = 40%. The watertoluene solution was then exchanged by pure water and the samples were finally dried with a gentle flow of pure nitrogen. Alternatively, the temperature annealing method consisted in increasing the water temperature in which the samples are stored. The samples were left in water at 60 ºC for 19 hours, then cooled down and dried with nitrogen.

Contact angle measurement.

Side pictures of drops on the substrates were taken on a home-made goniometer and contact angles were measured by fitting the edges of drops with the DropImage software (Rame Hart, Mountain Lake, NJ). The precision is of ±1° and the lowest measurable angle is around 10°. The volume of the drop was manually tuned with a precision syringe in order to measure the advancing contact angle θ A (highest stable angle observed when the drop volume is increased) and the receding contact angle θ R (lowest stable angle observed when the drop volume is decreased).

Ellipsometry. We used a multi-wavelength ellipsometer MOSS model ESVG (SOPRA, France). Measurements were taken at wavelengths between 250 nm and 600 nm, and at an incidence angle of 70°. For analysis, we used the WinElli software (SOPRA, France), that considers flat and homogeneous multilayers. The refractive index and thickness of the native silica layer on the silicon wafers is measured on a bare substrates. The refractive index as function of the wavelength for PAA and PS materials were determined experimentally on thin layers of homopolymers PAA and PS spin coated on a silicon wafer. The initial thickness of the substrates of PS is measured for each sample, prior to the Langmuir-Schaefer deposition. These data are introduced as fixed parameters in the subsequent multilayer models used to determine the thickness of deposited PS-b-PAA monolayers. All ellipsometric measurements are performed in the dry state. AFM. A Nanoscope III AFM (from Digital Instrument, now Veeco) in the tapping mode was used to investigate the structure of the deposition in air. We used Silicon cantilevers NSC35/AIBS/50 from Micro Mash.

Results and Discussion

Isotherms. The isotherms for all the diblock copolymers on pure water are reported on Figure 1. The increase of pressure with surface density is smooth, which is an indication that the hydrophobic PS block does not participate to the surface pressure. Homopolymer of PS isotherms are typically flat up to a limiting area, with a sudden pressure rise from that point. They are also characterized by irreversible collapse. The small mass fraction of PS in the copolymer studied insure that the hydrophobic block never completely cover the air/water interface. It is thus natural to assume that the surface pressure is only due to the hydrophilic part of the diblock. The area per molecule is calculated from the concentration of the solution deposited, the volume of solution deposited, and the surface of the air/water interface. Several consecutive compressions and expansions for a given deposition superpose on a single curve, which means that diblock copolymers are well anchored at the air/water interface. Figure 1 reports isotherms for different deposited amounts. Between measurements taken with different deposited amounts, a multiplicative factor is applied to the abscises in order to merge all data corresponding to a same copolymer on a single curve. The need of a multiplicative factor shows that the deposition of material at the air/water interface is not exactly reproducible. The reason comes from the use of dioxane as spreading solvent. Dioxane is miscible with water at all concentration so that some material is lost in the subphase upon deposition. The calculated area per molecule on Figure 1 is only defined within a multiplicative constant. The absolute area per molecule has to be determined by ellipsometry measurement done after transfer onto solid substrate. These absolute values are also needed for a precise physical understanding of the isotherms characteristics, which is out of the scope of the present work and is presented elsewhere [START_REF] Gareil | Compression isotherms of charged diblock copolymers[END_REF] . Isotherms are used here as a mean to prepare polymer monolayer on solid substrates in a controlled manner.

Anchoring. A gentle flow of water is sufficient to desorb the monolayer transferred on PS substrates. This is detected by contact angle measurements (contact angles with water increase with the number of rinsings) and ellipsometric measurements (the deposited layer thickness falls down to zero after rinsing). We also remarked that drying the samples without any anchoring procedure was damaging for the copolymer monolayers and changed the wetting properties. This has two implications. First, anchoring the brush to the PS substrates is a prerequisite to wetting measurement with water. And second, since samples after Langmuir-Schaefer deposition are wet and drying damages the samples, the anchoring method must be performed in the wet state. The idea of the toluene method is to swell the PS sublayer and allow the PS block of copolymers to penetrate the PS sublayer 13 , as cartooned on Figure 2-a-b. When toluene is removed, the PS sublayer contracts back and traps the diblock chains. Note that cartoons of Figure 2-a-b illustrate the interpenetration of PS chains and does not pretend to represent the organization of the PAA chains 14 . In order to optimize the toluene method, the PS layer thickness versus the content of toluene was measured by ellipsometry in immersed conditions (Figure 2-b). The thickness increases by less than 5% for toluene-water solutions with X lower than 50%, and diverges for X above 60% (the PS layer is dissolved). In this work, mixtures at X = 40% were used for anchoring the diblock copolymers. The PS layers swell by 3.5% without damage for the PS layer, as checked by AFM imaging before and after treatment (average RMS roughness is 0.6 nm in both cases). With the diblock monolayer, we found great improvement of the anchoring against drying. Figure 3 compares AFM images of dry PS-b-PAA 4.3k-19.5k layers deposited at Π = 4.5mN/m., for different anchoring conditions. The average thickness of the layers measured by ellipsometry are comparable for the three samples. The anchored layers (Figure 3-b andc) appear featureless and have an average RMS roughness of 0.85 nm. On the contrary (Figure 3-a), the non-anchored layer shows heterogeneities and has an average roughness of 1.5 nm. Differences are also detected by contact angle measurements that lead to lower angles and smaller hysteresis for the anchored samples. Anchoring strength is also improved against rinsing. Repetitive contact angles give higher and higher values on non treated samples, indicating a washing of the deposited diblock layer, whereas constant values are found on treated samples for at least 5 consecutive measurements. Note that the toluene anchoring method works only for a narrow window of toluene concentration (around X = 40%) and treatment time (around 60 s). For lower X values or shorter treatment time, anchoring is too weak, whereas for higher X values or longer treatment time, the depositions layers were damaged. Although the toluene swelling method was found efficient for most deposition conditions, we found that they induce instabilities of deposited layers at low surface concentration. Figure 4 shows AFM pictures of deposited layers that have been treated by the toluene method and that present typical patterns of a monolayer dewetting. This interpretation is confirmed by the thickness difference between the dark and bright zones on the picture (4-5 nm) that correspond to the average layer thickness measured by ellipsometry (3.5-4.7 nm). For low concentration depositions, we therefore preferred the temperature annealing method, at 60 ºC for 19 hours. With this method, all deposited layers at 4.5 mN/m are featureless and have an average RMS roughness of 0.7 nm.

Ellipsometric calibration of deposited monolayers.

The actual amount deposited on the wafer was systematically determined by ellipsometry. Two fitting models have been tried. Model 1 considers the diblock layer as a homogeneous PAA layer, whose thickness h 1 is the only fitting parameter. Model 2 considers the PS subphase and the deposited diblock layer as two superposed layers of PS and PAA of thicknesses h . An example of ellipsometric measurement before and after deposition of a layer of PS-b-PAA 1.8k-6k deposited at 21 mN/m is presented on Figure 5. The one parameter fit for the thickness of the PS substrate gives 118.7 nm. Models 1 and 2 lead to fittings of equivalent quality and yield consistent results in terms of total thickness. Table 1. Model 1 is reported on Figure 5 and used for all results presented in the following.

Ellipsometry thickness e ellipso of deposited layers for the three diblock samples deposited at different surface pressures are reported on Figure 6 versus the langmuir thickness e Langmuir calculated based on the number of molecule per angstrom square used to plot the isotherms of Figure 1. The dependence is remarkably linear for the three samples. This proves that the effective transfer rate upon the Langmuir-Schaefer transfer and the anchoring process is constant, otherwise, the data of Figure 6 would be random. On the other hand, the ellipsometric amounts are 2.59, 2.86 and 3.40 times smaller for respectively PSb-PAA 1.8k-6k, 4.3k-19.5k, and 1.5k-44k, as compared to the amounts deposited on the Langmuir trough. This is explained by a loss of material upon deposition of copolymer with dioxane solutions. In any case, ellipsometry permits to access the exact amount of diblock present on the solid substrates and to recalibrate the areas of the isotherm data. We have checked that the correction does not change the relative position of the isotherms for the three diblocks on Figure 1. One can conclude that the pressure increases expectedly with the molar mass of the PAA block at a given area per molecule [START_REF] Gareil | Compression isotherms of charged diblock copolymers[END_REF] .

AFM imaging of deposited monolayers. The topography of deposited and anchored monolayers was systematically checked by AFM. For depositions made at surface pressures above 4.5 mN/m, the samples are generally flat and featureless with RMS values around 0.5-0.7 nm, as shown on Figure 7. Macroscopic cracks are occasionally observed in deposition made at the pressures larger than 20 mN/m. These defects, visible with the naked eye, are an opportunity to get more insight into the structure of the layers via AFM imaging. Figure 8-a presents the image of a crack in a PS-b-PAA 4.3k-19.5k deposited at 25 mN/m and Figure 8-b presents a height profile taken on the same image. One can clearly identify three different levels. As cartooned on the profile, these levels correspond to the bare PS substrate, the diblock monolayer, and a diblock trilayer formed by local collapse of the monolayer. The cracks and collapses probably form during the Langmuir-Schaefer transfer. The monolayer zone is very flat (RMS = 0.27 nm), which shows that the carpet of PAA chains is dense and homogeneous. We note also that the thickness of the monolayer measured by AFM (10.5 nm) is remarkably consistent with the ellipsometric measurement (10.3 nm). The case of depositions made at pressures lower than 4.5 mN/m is different. Figure 4 shows that layers deposited at 4.5 mN/m and anchored by the toluene method present topographic heterogeneities that result from a dewetting of copolymer monolayer. At the opposite, Figure 9-a shows that the same deposition at 4.5 mN/m anchored by the temperature method are flat and homogeneous. The temperature method is clearly more appropriate to anchor the low density layers. Nevertheless, at 2.5mN/m (Figure 9-b), heterogeneities appear at the surface even with the temperature annealing method. On samples 2.5mN/m:3 (Figure 9-c) and 2.5mN/m:5 (Figure 9-d), we observe circular objects of diameter around 50 nm, which are attributed to surface micelles. At 2.5mN/m:8 (not shown) there are no features anymore. It is out of the scope of this paper to find out if the micelles observed on the transferred layers are due to the transfer/anchoring process or to organizations already present at the air/water interface. This study of the spontaneous formation of micelles at the air/water interface is presented elsewhere 14 . . Figure 10 presents the contact angle versus the thickness of acrylic acid anchored e AA for all samples with homogeneous PSb-PAA layers, i.e. for depositions made at pressures above 4.5 mN/m and annealed by the "toluene route". The thickness e AA of dry acrylic acid layer for a PS-b-PAA λk-µk layer of ellipsometric thickness e ellipso is calculated as µ + λ µ ⋅ = ellipso AA e e (where λ and µ are the molar masses in kg/mole of the PS and PAA block respectively), which assumes comparable densities for the two polymers. All receding angles are smaller than the experimental limit of 10° and all the samples remain wet after complete aspiration of a drop. For the advancing angles, there is a transition from very low advancing angles (15-25º), that form a plateau at high PAA thickness, to high advancing angles (50-60°) at low PAA thickness. The hysteresis between the advancing and receding contact angles is important. It is interesting to remark that hysteresis of contact angles have also been measured with PS-b-PAA layers that are not properly anchored. However, wetting behavior on such substrates is quite different. The receding contact angles can be close to zero for high PAA thickness, whereas the advancing contact angles are always close to 90°.

Wetting

Dewetting of the PS-b-PAA layer into small caps of 20-50 nm distant by 50-100 nm have been clearly identified by AFM imaging of dry samples. The hysteresis in this case is due to the chemical heterogeneity of the surface, the areas of bare PS explaining the high advancing contact angles. In the case of Figure 10, the contact angle hysteresis can not be attributed to chemical heterogeneities because AFM characterizations have shown that the surface of dry samples consist of dense and homogeneous carpet of PAA. On Figure 10, the PAA thickness at which the transition of advancing angles occurs is indicated by horizontal bars a, b, and c for respectively PS-b-PAA 1.8k-6k, 4.3k-19.5k, and 1.5k-44k. The edges of bars a, b and c correspond to the highest e AA with θ A above the plateau of small angles, and the lowest e AA with θ A at the plateau of small angles. Despite the scarcity of data point and the width of the bars, it appears that the transition of advancing angles occurs at higher PAA thickness for copolymers of longer PAA blocks. This suggests that for a given amount of PAA, PAA carpets with dense and short hair induce less hysteresis than carpets with scarce and long hair. It is then tempting to attribute this hysteresis to surface heterogeneities at the molecular level. Let us now present the contact angle measurements for PS-b-PAA 4.3k-19.5k for all depositions, either annealed by the temperature or the toluene route (Figure 11). We decided to present these data on a separate plot because they correspond to some layers that are homogeneous and others that are heterogeneous. Still, it appears that there is a good continuity between results for all layers. A clear wetting transition from partial wetting to total wetting is visible on both advancing and receding angles around 3.5 to 4.5 nm of PAA thickness.

Model. Figure 12 presents a cartoon of a water drop on a hydrophobic substrate covered with a polyelectrolyte brush. The brush is collapsed and dry when exposed to air and hydrated and swollen underneath the drop. Following the reasoning of the Young-Dupré equation [START_REF] Young | [END_REF] , [START_REF] Dupré | Théorie mécanique de la chaleur[END_REF] , [START_REF] Adam | [END_REF] , we evaluate the infinitesimal work dW involved in the displacement of the triple contact line on such a complex surface. One can express dW as:

Eq. 1

⋅ γ - + θ γ + σ - γ - γ = - - - - dA ). W cos . ) 1 )( (( dW air PAA hyd PAA air O H PAA PS O H PS 2 2
where γ PS-H2O is the interfacial energy between PS and water, γ H2O-air between water and air, γ PAA-PS between PS and PAA and γ PAA-air between PAA and air. The term 1-σ describes the reduction of contact area between PS and external phase (water underneath the drop and PAA in the dry area) due to surface occupied by the grafting sites. These interfacial tension contributions are classically found in the Young-Dupré equation. The additional term in Eq. 1 is the work hyd PAA W per unit area needed to bring a PAA chain from a dry state to its hydrated state in the brush. hyd PAA W can be expressed as the sum of two components hyd PAA W = W 1 + W 2 , where W 1 corresponds to the hydration of a dry PAA chain by water at infinite dilution, and W 2 corresponds to the transfer of PAA chains from a solution at infinite dilution to a solution at the concentration of the brush. It is interesting to realize that W 2 is equivalent to the surface pressure of the corresponding diblock layer measured with the Langmuir trough at the deposition, i.e.W 2 = Π. As for W 1 , it depends on the enthalpy of hydration of PAA and on the density of monomers per unit area as:

Eq. 2 AA AA hyd PAA 1 M e d G W ⋅ ∆ = where hyd PAA G ∆
is the free enthalpy of hydration of PAA per mole of monomer (at infinite dilution), M AA is the molecular weight of a AA monomer, e AA is the thickness of the PAA layer, and d is the density of PAA. Setting dW to zero in Eq. 1 and solving for θ leads the expression of the contact angle as:

Eq. 3                 Π - ∆ - γ + σ - γ - γ γ = θ - - - - AA AA hyd PAA Air PAA O H PS PS PAA O H Air M e d G ) 1 )( ( 1 arccos 2 2
Total wetting is obtained if the work is negative even for θ = 0°.

Eq. 4

(

) where S m ≈ V m 2/3 is the area occupied by a monomer, and V m is the molecular volume of a monomer. χ PS/PAA is established in the literature at 0.4 21 . Based on a density of PS of 1.05 [START_REF] Brandrup | Polymer Handbook 4th edn[END_REF] , the molecular volume for PS is V m ≈ 165 Å [START_REF] Maas | [END_REF] . For dry PAA in the protonated form, it is established in the literature that V m ≈ 74 Å 3 . Eq. 5 supposes that PS and PAA have the same monomer size. Taking a median value of 120 Å 3 , we find γ PS/PAA ≈ 13.5 mN/m. By comparison with other acrylate polymers, e.g. poly(nbutylacrylate) PBA (γ PBA-air ≈ 31 mN/m 18 ), or poly(diethyleneglycol ethylether acrylate) PDEGA (γ DEGA-air ≈ 33-35 mN/m 23 ), the interfacial tension between PAA and air γ PAA-air can be estimated around 35 mN/m. In Eq. = -1300 J/mol. The results of the calculation that are reported on Figure 10. the model shows that the wetting is hardly dependant on the molar mass of the sample used. The curves for 4.3k-19.5k and 1.5k-44k are even undistinguishable. This is in good agreement with experimental observations. We have also checked that the role played by the area fraction σ occupied by the grafting site in the area of the transition is totally negligible. The maximum value of σ below the wetting transition for all samples remains below 0.04. In the calculation, the small influence of the molar mass on the wetting transition against the molar mass of the PAA chains comes from the internal surface pressure Π inside the brush. The term

O H Air Air PAA O H PS PS PAA AA AA hyd PAA 2 2 ) 1 ( M e d G - - - - γ - γ + σ - γ - γ ≤ Π + ∆ Π,
O 2 H Air Air PAA O 2 H PS PS PAA - - - - γ - γ + γ - γ
≈ -55 mN/m in Eq. 3 is much larger than the interfacial pressure Π at which the wetting transition occurs, i.e. around 3-4 mN/m. This explains why the transition is hardly dependant on the length of PAA chains. With a less hydrophilic polymer than PAA, wetting transitions obviously occur at higher amounts of polymer deposited. The relative importance of grafting sites area and internal pressure Π in Eq. 3 would then be larger, and the model predicts an important effect dependence of the molar masses of the grafted polymer on the wetting transition with e AA . With a less hydrophilic polymer, other effects may have to be taken into account in the wetting properties. Cohen Stuart et al. 10 have studied the wetting of PEO brushes. They found an increase the hydrophilicity of substrates by PEO brushes but they never obtained a transition to total wetting. The authors have attributed this weak hydrophilization effect of PEO brushes to a bridging between the substrate and the air/water interface by grafted PEO chains. Indeed, PEO chains are well known for anchoring at an air/water interface 25 . In their view, this bridging effect retards spreading. Homogeneous and dense grafted monolayers of moderately hydrophilic chains that are able to promote a transition to total wetting would be a nice system to test further our model and the hypothesis of air/water surface bridging. We also note that our model predicts that layers of short molar masses PAA are less wetting than layers of large molar masses PAA, for a given PAA amount. This is opposite to the tendency observed with the advancing angles. This proves that the effect of the PAA molar mass on the advancing angles is not due to an average thermodynamic effect. The idea of a surface heterogeneity effect at the molecular scale, i.e. a carpet of short and dense hair against a carpet of scarce and long hair, is somehow comforted. It is interesting to comment our data in the light of Halpering and de Gennes 1 predictions. We confirm that a transition to complete wetting can be induced by grafting a polymer brush in good solvent conditions to a hydrophobic substrate. Whereas the transition in their case of neutral polymer was driven by the entropy of dissolution of the polymer, the transition with a polyelectrolyte is driven by both the entropy of dissolution of the polymer chains and the counter ions [START_REF]The ionization state of PAA chains in a brush exposed to pure water is intermediate between the state in a brush exposed to pH 11 aqueous solution (full ionization) and pH 2 aqueous solutions (no ionization)[END_REF] . In spreading conditions, they predict the wetting film thickness versus the spreading coefficient S. However, they consider the case a nonvolatile solvent and their predictions are not adapted to water, which is highly volatile. Experimentally, the study of the equilibrium thickness of a wetting film in total wetting conditions, would require a precise control of air humidity content, which was not done in this work. From a fundamental point of view, a wetting study of the same "hydrophobic-hydrophilic" surface by non volatile solvent would therefore be enlightening. One can think of a hydration transition versus e AA in a water saturated atmosphere, since the physical condition driving hydration and wetting of a complex "hydrophilichydrophobic" surface are similar. A hydration study by X-ray reflectivity would permit to characterize the hydration properties and the eventual formation of hydration gradients in the polymer brush. By the same token, in order to complement the wetting study, it would be interesting to investigate the structure of the drop edge in the partial wetting conditions at a microscopic scale. It can not be discarded that a hydrated film forms ahead of the macroscopic edge, its growth being frustrated by evaporation.

Conclusion.

We have presented an experimental and theoretical study of the wetting by water of complex "hydrophobichydrophilic" surfaces made of a hydrophobic substrate covered by a highly hydrophilic polymer brush. For the preparation of model samples, we used polystyrene layers spin-coated on silicon wafers and covered with polystyrene-b-poly(acrylic acid) PS-b-PAA. The diblock copolymers layers were fabricated by Langmuir-Schaefer depositions and analyzed by AFM and ellipsometry. On poorly anchored layers, we observed large contact angle hystereses that have been attributed to nanometric scale dewetting of the polymer monolayer. On well-anchored monolayers, that form dense and flat brushes in the dry state, a wetting transition occurs versus the amount deposited. This confirms the prediction by Halperin and de Gennes 1 that a brush in good solvent condition can overcompensate the hydrophobicity of the underneath substrate and create total wetting conditions . We propose a model, that takes into account the finite concentration of the hydrophilic block inside the brush, through the air/water interfacial pressure Π of the brush and the free enthalpy of hydration the polyelectrolyte monomers hyd PAA G

∆

. The originality and strength of the model is that all interfacial tensions between PS, PAA, water and air as well as the pressure in the brush Π can be measured or calculated. hyd PAA G ∆ is the only fitting parameter and is the same for all grafting conditions (polymer length, grafting density). With hyd PAA G ∆ = -1300 J/mol, the model renders the wetting transition for all samples and explains why, with PAA as hydrophilic agent, the wetting transitions depend mainly on the average thickness of the brush and weakly on the length of chains. We also found that this value of hyd PAA G ∆ is remarkably consistent with determination based on PAA hydration experiments against water partial pressure, which reinforce the validity of the wetting model. In future work, it would be interesting to test the model with less hydrophilic polymer than PAA, for which the model predicts a significant (and therefore detectable) effect of the molar mass of the polymer on the wetting transition. The region in the vicinity of the edge drop remains to be investigated at the microscopic scale in order to get a better insight into the eventual hydration of the brush ahead of the drop and the role of evaporation of the solvent from this hydrated thin film.

dx x p p 1 x x RT p p Ln RT x 1 x G ∂ ∂ - +                 - = ∆ ∫ →
With a first order development of the logarithm term , we finally obtain : Eq. 10

( ) ( )         ∂ ∂ - + ∂ ∂ - = ∆ ∫ → 1 1 1 1 x 0 1 1 1 1 1 X 1 0 1 1 hyd PAA dx x p p 1 x x x p / p RT G hyd PAA G ∆
appears the sum of two terms, the left one being the contribution of water and the right term the contribution of PAA. Strictly speaking, at the limit x 1 →1, the term =-1500 J/mol for a sample of molar mass M= 4.10 3 g/M (Figure 13). , and the interfacial pressure inside the brush Π.

Acknowledgements

Figure 13: Experimental measurements of the molar fraction x 1 of water in PAA versus the partial pressure p 1 of water in the gas phase for a PAA of molar mass M= 4,000 g/M. These data have been published by Safronov et al. [START_REF] Safronov | [END_REF]

  data. The measured contact angles on the bare PS substrates

  only unknown. On Figure 11, the model fits satisfactorily the experimental data with a value hyd PAA G ∆ = -1200 to -1400 J/mol depending on the interfacial value O 2 H / PS γ that we use. These values corresponds to approximately ½ kT per monomer. As a comparison, it is possible to calculate hyd PAA G ∆ from experimental data 24 of the molar fraction x 1 of water in PAA versus the partial pressure p 1 of water in the gas phase. The calculation is detailed in the annex and lead to hyd PAA G ∆ = -1500 J/mol for a PAA sample of molar mass 4.10 3 g/mol. Given the imprecision in the determination of hyd PAA G ∆ from the wetting data and from the hydration data, the consistency is quite remarkable. As hyd PAA G ∆ is only dependant on the nature of the hydrophilic block and not on the molar mass of the sample, it is then interesting to apply the model to all polymer samples with hyd PAA G ∆

  null for the integral to converge and the left term due to water in Eq. 10 is necessarily null. In practice, we apply Eq. 10 and we calculate the derivative ( )

Figure 1 :

 1 Figure 1: Isotherms of compression on pure water of PS-PAA diblock copolymers 1.6k-6k (green), 4.3k-19.5k (blue) and 1.5k-44k (orange). The amount of spreading solution is either 50 µL (light colors ) or 100 µL (dark colors). The area indicated correspond to rw areas calculated from the amount of copolymer deposited.

Figure 2 :

 2 Figure 2: Schematic of PS-b-PAA copolymer chains a) deposited on PS without annealing, the chains can detach or move, b) after annealing, the chains are entangled and immobilized in the PS sublayer. c) PS sublayer thickness versus toluene composition X of toluene in water.

Figure 3 :

 3 Figure 3: AFM images of PS-b-PAA 4.3k-19.5k monolayers deposited at 4.5mN/m.a) without annealing treatment -RMS Roughness = 1.50 nm, b) annealed by the toluene method-RMS Roughness = 0.86 nm, c) annealed by the temperature method -RMS Roughness = 0.67nm.

Figure 4 :

 4 Figure 4: AFM images of PS-b-PAA monolayers deposited at 4.5mN/m and annealed by the toluene method: a) 1.8k-6k, holes: 3-5nm e ellipso =4.72 nm, b) 4.3k-19.5k, holes: 4-5nm e ellipso = 3.59 nm, and c) 1.5k-44k, holes : 3-4nm e ellipso = 3.66

Figure 5 :

 5 Figure 5: Ellipsometric data cos∆ (squares) and TanΠ (dots) before (hollow symbols) and after (dark symbols) deposition of a PS-b-PAA 1.8k-6k monolayer at Π = 21 mN/m. The lines corresponds to fits without (---) and with (--) the monolayer. The thickness of the PS substrate is found at 120 nm and of the PS-b-PAA monolayer at e = 7 nm.

Figure 6 :

 6 Figure 6: Ellipsometric thickness e ellipso versus Langmuir thickness e Langmuir calculated from the amount of copolymers spread at the air/water interface.

Figure 7 :

 7 Figure 7: AFM images of PS-b-PAA 4.3k-19.5k monolayers after deposition and annealing a) deposition at Π = 21 mN/m, Rms Roughness = 0.27 nm, b) deposition at Π = 13 mN/m, Rms Roughness = 0.33nm.

Figure 8 :

 8 Figure 8: a) AFM image of PS-PAA 4.3k-19.5k layer deposited at 25 mN/m, 5µmx5µm and cracked, b) profile and cartoon of the organization of the copolymer chains at the surface.

Figure 9 :

 9 Figure 9: AFM images of PS-PAA 4.3k-19.5k layers deposited at a) 4.5mN/m, b) 2.5mN/m, c) 2.5mN/m:3, d) 2.5mN/m:5.

Figure 10 :

 10 Figure 10: Water contact angles versus PAA thickness e AA with samples annealed by the toluene method for PS-b-PAA 1.8k-6k (triangles▲ ∆), 4.3k-19.5k (squares □ ■) and 1.5k-44k (dots • o ). Hollow symbols correspond to advancing angles and full symbols to receding angles. The horizontal bars materialize the approximate position of the wetting transition observed with the advancing angles in the case of PS-b-PAA a) 1.8k-6k b) 4.3k-19.5k and c) 1.5k-44k. The lines correspond to fits by Eq. 5 with hyd PAA G ∆ = -1300 J/mol for PS-b-PAA 1.8k-6k (---), 4.3k-19.5k (--) and 1.5k-44k ( ).

Figure 11 :

 11 Figure 11: Wetting results -Contact angles versus PAA thickness for PS-PAA 4.3k-19.5k -Hollow symbols correspond to advancing angles and full symbols to receding angles. The data correspond either to samples annealed by the temperature method (circles) or the toluene method (triangles). The solid line corresponds to fits by Eq. 5 with hyd PAA G ∆ = -1300 J/mol.

Figure 12 :

 12 Figure 12: Illustration of the thermodynamic model, which takes into account the interfacial energies γ PS/H2O , γ H2O/air , γ PS/PAA , γ PAA/air , the enthalpy of hydration of AA monomers hyd PAA G ∆, and the interfacial pressure inside the brush Π.

  e AA and σ are accessible experimentally for each sample by Langmuir trough and ellipsometry measurements. The interfacial tension

																	Eq. 5	γ	PS	/	PAA	=	2χ	PS	/	PAA	k	B	T	/	S	m
																	γ	air	/	water	= 72 mN/m
	is well established. The interfacial tension of polystyrene
	with water and air is subject to some uncertainty. We
	found	γ	PS	/	air	≈ 40 mN/m and	γ	PS	/	H	2	O	≈ 32 mN/m in one set
	of publication 18,19 and	γ	PS	/	air	≈ 29 mN/m and	γ	PS	/	H	2	O	≈ 24
	mN/m in an other recent publication 20 . Note that the two
	set of values are consistent with the Young-Dupré
	equation for a water drop on bare PS making a contact
	angle with water of 83.5-86°. Since PS and PAA are
	strongly immiscible polymer, the interfacial tension
	γ PS/PAA can be estimated from the Flory parameter χ PS/PAA
	between PS and PAA via the formula:

:

  We want to thank Rhodia, Inc. for financial and technical support, Thomas Gillet and Florence Coignet for participation to some of this work,

					JP	Chapel	for	help	with	ellipsometry.
	Tables
		h PS	1 h	h	PS 2	h	PAA 2	Total thickness
	Model 1	118.7	7.1	-		-	125.8
	Model 2	118.7	-	121.3	4.0	125.3

Table 1 :

 1 Thicknesses determined by ellipsometry of the PS subphase (h PS ) and of the deposited diblock layer by model 1

	( 1 h ) and model 2 ( PS 2 h and PAA 2 h ) for a PS-b-PAA 1.8k-6k layer deposited at 21 mN/m.

Theodoly, O.; Checco, A.; Ocko, B.; Muller, P. A GISAXS study of PS-b-PAA at the air/water interface, to be submitted.

Annex

The free Gibbs enthalpy of hydration of PAA at infinite dilution per mole of monomer hyd PAA G ∆ can be extracted from experimental measurements of the molar fraction x 1 of water in PAA against the partial pressure p 1 of water in the gas phase. The chemical potential of the water is equal to : Eq. 6 ( )

where 0 1 µ is the chemical potential of pure water. By application of the Gibbs-Duhem equation, the chemical potential of PAA 2 µ can be expressed as :

Eq. 7

( )

where 0 2 µ is the chemical potential of pure PAA. The change of Gibbs energy free when N 1 moles of water and N 2 moles of PAA are mixed is equal to : Eq. 8 ( ) ( )

, one has to divide Eq. 8 by N 2 and take the value at x 1 ≈ 1. This leads to: Eq. 9