Tangui Le Guen

Yann Fichou

Juliette Nectoux

Nadia Bahi-Buisson

Francois Rivier

Nathalie Boddaert

Bertrand Diebold

Delphine Heron

Jamel Chelly

Dr Thierry Bienvenu
email: thierry.bienvenu@inserm.fr

Le Guen

François Rivier

Delphine Héron

A missense mutation within the fork-head domain of the Forkhead box G1 (FOXG1) gene affects its nuclear localization

Keywords: Rett syndrome, FOXG1, Microcephaly, Nuclear speackles FOXG1, Rett syndrome, Microcephaly, Encephalopathy, Nuclear speckles . Human Mutation

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

The mammalian forkhead family belongs to the large family of over 100 known forkhead genes in animals [Solomon et al., 2003a]. All forkhead family members contain the ~100 amino acid, monomeric DNA "wingedhelix" binding domain of the founding members: Drosophila forkhead [ [START_REF] Lee | Survey of forkhead domain encoding genes in the Drosophila genome: Classification and embryonic expression patterns[END_REF] and hepatocyte nuclear factor 3a [START_REF] Tao | Telencephalon-restricted expression of BF-1, a new member of the HNF-3/fork head gene family, in the developing rat brain[END_REF]. Forkhead gene products play pivotal roles in organogenesis, including patterning and morphogenesis, through the regulation of the proliferation and cell fate specification [Solomon et al., 2003b].

The neocortex develops from the progenitor cells of the rostral neural plate, the telencephalic neuroepithelial cells. Following a period of uniform proliferation, cerebral cortical progenitors generate neurons asynchronously beginning at embryonic day 11 (E11) in mouse. Progenitors undergo asymmetric cell divisions in which one daughter cell differentiates while the other divides. The proportion of cells with asymmetric divisions steadily increases over a period of several days. At the end of the neurogenetic period (E17), both daughter cells differentiate, resulting in the depletion of the progenitor pool. The duration of the neurogenetic period is a critical determinant driving the quantity of neurons generated within the cerebral cortex. Three factors have a major influence on the cellular output from the proliferative zones: the rate of cell proliferation, the rate of cell differentiation, and the rate of cell death. The progenitor cells of the telencephalon are identifiable as early as the eight-somite stage (E8.5) by the expression of one of the members of the forkhead box family, the FoxG1 transcription factor, also known as the winged-helix (WH) protein brain factor (BF-1) [START_REF] Murphy | Human brain factor 1, a new member of the fork head gene family[END_REF].

FoxG1 (MIM 164874) is a DNA-binding transcription factor with a forkhead binding domain (FBD; amino acids (AA) 181-275), which represses target genes during brain development by recruiting transcriptional corepressor proteins via two protein-binding domains: the Groucho-binding domain (GBD; AA 307-317) and the JARID-1B binding domain (JBD;.

In a recent series of articles, several groups identified ten different mutations in FOXG1 as the cause of the congenital form of Rett syndrome (RTT, OMIM 312750) [START_REF] Ariani | FOXG1 is responsible for the congenital variant of Rett syndrome[END_REF][START_REF] Philippe | Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females[END_REF][START_REF] Mencarelli | Novel FOXG1 mutations associated with the congenital variant of Rett syndrome[END_REF][START_REF] Bahi-Buisson | Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant[END_REF]. All patients fulfilled the criteria of the congenital variant of RTT except one presenting a classical form of Rett syndrome [START_REF] Philippe | Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females[END_REF]. The congenital variant of RTT, which was initially described by [START_REF]Rett syndrome: report of eight cases[END_REF][START_REF]Rett syndrome: report of eight cases[END_REF], involves girls that present with a normal perinatal <Running Title> 3 period followed by a phase of developmental regression at the age of 3-6 months. At birth, head circumference is normal while a deceleration of growth is observed soon afterwards, resulting in a severe microcephaly. Patients with FOXG1 mutations usually exhibit a phenotype reminiscent of the congenital RTT variant. Motor development is severely impaired and voluntary hand-use is absent. In contrast with classic RTT, patients show poor eye contact. Typical stereotypic hand movements with hand-washing and hand-mouthing activities are observed continuously. Several patients show abnormal tongue movements, as well as jerky movements of the limbs. Brain MRI typically shows corpus callosum hypoplasia, abnormal ventricles, and abnormal white matter in most cases, while epilepsy is a variable sign [START_REF] Bahi-Buisson | Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant[END_REF]. Scoliosis and autonomic neurovegetative symptoms typical of Rett syndrome are frequently present [START_REF] Philippe | Phenotypic variability in Rett syndrome associated with FOXG1 mutations in females[END_REF].

To date, only a few missense mutations have been reported in the FOXG1 gene. In this report, the gene was screened by direct sequencing in more than 150 sporadic cases of girls with microcephaly, stereotypic hand movements and jerky movements. A novel missense mutation within the DNA-binding domain was identified and subsequent studies by genetic and cellular approaches showed that the nuclear localization of the protein is impaired. These results provide new insights into the understanding of FoxG1 dysfunction and its associated phenotype. This last heterogeneous group of patients included patients with mental retardation, autistic features, stereotypic hand movements, progressive microcephaly, epileptic encephalopathy, and/or severe congenital encephalopathy without recognizable aetiology. In all selected individuals, neither point mutation nor large rearrangement in the MECP2 and CDKL5 genes had been identified. Methylation studies for Angelman syndrome and chromosome analysis were also normal.

MATERIALS AND METHODS

Patients

DNA mutation analysis

DNA was extracted from peripheral blood using standard methods. All blood samples were obtained after provision of informed consent. DNA samples were screened for mutations in FOXG1 (GenBank reference NG_009367.1)) using PCR amplification and direct sequencing. Primer sequences and PCR conditions are available upon request to the corresponding author. Sequencing reactions were carried out with the BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, Courtaboeuf, France) and loaded on the ABI 3100 Genetic Analyzer (Applied Biosystems). Nucleotide numbering reflects cDNA numbering with +1 corresponding to the A of the ATG initiation codon (codon 1) in the reference sequence, according to journal guidelines (www.hgvs.org/mutnomen). Mutation nomenclature follows the recommended guidelines of the human genome Primers were designed with Primer Express software and are available upon request to the corresponding author.

After PCR amplification, the purity of products was determined by a melting-curve analysis, and all samples indicating a single amplification product were considered positive for further analysis. The identity and size of the single PCR product were also confirmed by agarose gel electrophoresis. For each sample, a relative quantification of the copy number of the FOXG1 gene was calculated by the ∆Ct method as described by the manufacturer using the ALB gene as the calibrator for normalization.

Plasmid construction and production

The human FOXG1 open-reading frame was extracted from the BC035020 clone (cDNA clone IMAGE: Taq DNA Polymerase. Cycle conditions were: an initial denaturation step at 95°C for 2 min; followed by 40 cycles of denaturation at 95°C for 45 sec, annealing at 62°C for 30 sec, extension at 68°C for 1 min 45 sec; and a final extension step at 68°C for 2 min. PCR products were loaded on a 2% agarose gel and purified with the QIAquick Gel Extraction Kit (Qiagen, Courtaboeuf, France). The purified fragments were subsequently cloned within the pCR4 ® -TOPO ® (Invitrogen) vector according to the protocol described by the manufacturer. The cloning product was transformed into XL1-Blue Supercompetent Cells (Stratagene, purchased from Agilent Technologies, Massy, France) cultured in a selective medium. The reported G>C substitution at codon 82, which converts a proline to an arginine, was found in the clone and reversed by directed mutagenesis using the QuikChange ® Site-Directed Mutagenesis Kit (Stratagene) with oligonucleotides 5'-CCCCCGGCACCGCAGCCCCCC-3' (F) and 5'-GGGGGGCTGCGGTGCCGGGGG-3' (R) (converted codon underlined; converted nucleotide in bold) according to the manufacturer's instructions.

To generate the expression vectors, the TOPO-TA cloning product containing the human FOXG1 sequence, as well as the pEGFP-C1 Vector (Clontech, purchased from Ozyme, Saint-Quentin-en-Yvelines, France) were digested with both the BglII and BamHI restriction enzymes, and the linearized pEGFP-C1 Vector was treated with Alkaline Phosphatase (New England Biolabs, purchased from Ozyme, Saint-Quentin-en-Yvelines, France).

Digestion products were loaded on a 0.8% agarose gel and extracted as described above. Insert and vector were

Cell fractionation extracts and western blot analysis

Cell fractionation to isolate the nuclear proteins from the cytosolic proteins was carried out as described before [START_REF] Regad | The neural progenitor-specifying activity of FoxG1 is antagonistically regulated by CKI and FGF[END_REF]. All fractions were suspended with Laemmli Sample Buffer 2X (BioRad, Richmond, CA, USA), and equivalent cell ratios (1:1) of nuclear vs cytosolic proteins were loaded on a SDS-polyacrylamide gel to estimate the enrichment of the fusion proteins in either subcellular compartment. After gel electrophoresis, proteins were transferred on a Hybond-C nitrocellulose membrane (GE Healthcare), which was subsequently blocked with a TBS-Tween 20 0.05%/BSA 5% solution, and incubated overnight at 4°C with a rabbit anti-GFP polyclonal antibody (Santa Cruz Biotechnology, Inc.; dilution 1:1000). The membrane was further extensively washed and incubated with a horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (dilution 1:5000), and developed with the enhanced chemiluminescence method (ECL; GE Healthcare). The membrane was reprobed for β-actin (Santa Cruz Biotechnology, Inc.; dilution 1:10000) for fractionation checking.

Cell culture, transfection, and RT-PCR analysis

The human neuroblastoma SH-SY5Y cell line was cultured in 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium (Sigma) containing 100 U/ml penicillin, 100 mg/ml streptomycin and 10% fetal calf serum (FCS) and incubated at 37°C, 5% CO 2 in a humidified atmosphere. Five hundred thousand cells were seeded in 100 mm collagen-coated cell culture dishes. After 24 hours, the medium was replaced by a 1:1 mixture of DMEM and Ham's F12 medium with 0.5% FCS and retinoic acid (RA; final concentration 5 µM) for differentiation. After five days in these conditions, cells were seeded in 6-well plates to reach 50-70% confluency. Cells were then transfected with either FOXG1 constructs (see the cell culture, transfection and immunofluorescence section) using Lipofectamine 2000 (Invitrogen, CA) as the transfection reagent according to the manufacturer's instructions, and incubated for 4-6 hours. The transfection medium was then replaced by the complete growth medium. Cells were cultured for 48 hours, and (GFP+)-transfected cells were collected and selected using a FACScan flow cytometer. Total RNA was extracted from the sorted SH-SY5Y cells by Trizol Reagent (Invitrogen) according to the manufacturer's instructions, and converted to cDNA prior to quantitative, real-time PCR analysis carried out with the SYBR Green PCR Master Mix (Applied Biosystems) on an ABI Prism 7500 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions. CDKN1A expression was assessed by quantitative Reverse Transcriptase (RT)-PCR (primer sequences available upon request to the corresponding author). After PCR amplification, a dissociation protocol was performed to determine the melting curve of the PCR product. Reactions with melting curves indicating a single amplification product were considered positive for further analysis. The identity and expected size of the single PCR product were also confirmed by agarose gel electrophoresis. For each sample, a relative quantification of the expression of each individual gene of interest was calculated by the comparative ∆∆Ct method as described by the manufacturer, using HPRT as the calibrator for normalization. Statistical analysis was carried out using the nonparametric Mann-Whitney test. A p-value <0.05 was considered as significant.

RESULTS

Molecular analysis and case report

The whole coding sequence of the FOXG1 gene was screened by direct sequencing in 150 clinically wellcharacterized girls with severe microcephaly. Two missense mutations were identified: the c.326C>T transition and the c.730C>T transition, which are predicted to result in p.P109L and the p.R244C changes at the amino acid level, respectively. The first variant was subsequently identified in her healthy father at the heterozygous state, suggesting that it is a non-pathogenic polymorphism.

Conversely, the c.730C>T transition is a de novo mutation (data reviewed but not shown) that was not detected in more than 100 control female individuals of different ethnic origins. The patient carrying this mutation is an 8-year-old girl, third child of non-consanguineous healthy parents. Prenatal ultrasound at 33 weeks of gestation showed a relative microcephaly (10 th percentile) without cortical malformation. She was born full-term by spontaneous delivery, with normal neonatal parameters but relative small head circumference (OFC) (33 cm; 10 th percentile). On first examination at 4 months old, deceleration of head growth (3 rd percentile) with good eye contact and smile, moderate axial hypotonia and limb hypertonia were noted. First brain MRI performed at 7 months old showed frontally predominant pachygyria with thick frontal gyri and reduced white matter (Fig. 2 A-D). Initially she was suspected to present with cortical malformation and was referred for anterior pachygyria. At 7 months old, she developed hyperkinetic and dyskinetic movement disorder but neither prehension, nor babbling. In addition, sleep disorders and intermittent unexplained crying were reported. She never presented myoclonia or seizures. EEG was normal, as well as fundoscopy. Progressively, she developed severe mental retardation although she made constant progress. Secondary deceleration of head growth leads to absolute microcephaly. At last evaluation at 8 years of age, microcephaly was severe (45.5 cm; < 3 rd percentile), she was able to walk with aid, presented hyperkinetic and choreic movements, and dyskinetic tongue movement and hand stereotypies. Hand stereotypies were variable, but hand-mouthing stereotypies were predominant. She also presented pyramidal signs with mild spasticity. Eye contact dramatically improved while verbal language did not. Brain MRI performed at 6 years old showed frontal gyral simplification without pachygyria without hypomyelination, and mildly reduced white matter (Fig. 2 E-H). Altogether, the girl presented a phenotype reminiscent of the congenital variant of Rett syndrome. The resulting change at the amino acid level is predicted to replace an arginine by a cysteine residue (p.R244C) in the highly evolutionarily conserved DNA-binding forkhead domain of the protein (Fig. 2), and may thus be critical for the function of the protein.

The p.R244C mutation affects the localisation of the FOXG1 protein

To better understand the pathogenic effect of this mutation, we first investigated the subcellular distribution of the overexpressed FoxG1 mutant protein fused to GFP in HeLa and neuronal DAOY cells (Fig. 3). While the wild-type protein shows a homogeneous nuclear pattern excluding the nucleoli (Fig. 3A,B), the p.R244C mutant appears to aggregate as nuclear foci (Fig. 3E,F). The respective subcellular distribution of the FOXG1 mutant protein was confirmed by western-blot analysis of cell fraction protein extracts (data reviewed but not shown).

Ricciardi and colleagues recently showed that the product of the CDKL5 gene, which is involved in the early onset seizure variant of Rett syndrome, localizes in nuclear speckles that are involved in the pre-mRNA processing in cells [START_REF] Weaving | Mutations of CDKL5 cause a severe neurodevelopmental disorder with infantile spasms and mental retardation[END_REF][START_REF] Ricciardi | CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery[END_REF]]. We then stained our cell models with an antibody directed against this structure (i.e. splicing factor SC-35) (Fig. 3C,G). We clearly observed that the FOXG1 mutant protein extensively, although not fully, localizes in nuclear speckles (Fig. 3H, white arrowheads), while the wild-type FOXG1 protein is more widely dispersed throughout the nucleus (Fig. 3D). This result suggests that the specific

The p.R244C mutation affects the CDKN1A expression

To investigate whether the p.R244C mutation located within the DNA-binding forkhead domain affects the expression of target genes, we studied expression of the cyclin-dependent kinase inhibitor (CKI) p21 CDKN1A

gene in transfected wild-type FOXG1 or mutated FOXG1 neuronal SH-SY5Y cells. After transfection and collection of (GFP+)-transfected using a FACScan flow cytometer, total RNAs were extracted, converted to cDNA, and CDKN1A expression was studied by real-time RT-PCR. As reported in previous reports [START_REF] Seoane | Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation[END_REF][START_REF] Kawauchi | Foxg1 promotes olfactory neurogenesis by antagonizing Gdf11[END_REF][START_REF] Chan | Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer[END_REF]], we first observed that overexpression of the wild-type protein FOXG1 in cells results in a decrease (~40%) of CDKN1A expression as compared with untransfected cells (data not shown). Secondly, we showed that overexpression of the p.R244C mutant results in an increase (Fig. 4; 31±0.2%) in CDKN1A expression, suggesting that in contrast to the wild-type FOXG1 protein, this mutant loses its ability to repress CDKN1A expression, at least partially, and that this misregulation may likely be due to its abnormal concentration in the nuclear speckles.

DISCUSSION

In this report, we describe two female patients with novel missense FOXG1 mutations. One of these variants, the p.P109L mutation, was also identified in her healthy father at the heterozygous state, suggesting that it is a nonpathogenic polymorphism. The other variant, the p.R244C mutation was not detected in more than 100 control female individuals of different ethnic origins, and FOXG1 parent's screening and paternity testing using microsatellite markers suggest that the p.R244C mutation is a de novo mutation. This raises the total number of reported pathogenic missense mutations to three (p.F215L, p.P227K, and p.R244C). All of these mutations are located within the highly evolutionarily conserved DNA-binding forkhead domain of the protein (amino acids 181 to 275). However, up to now, their disruptive effect on the function of the FOXG1 protein has not been clearly

shown. This novel mutation was identified in a girl with a combination of postnatal microcephaly, dyskinetic movement disorder with hand stereotypies, and relative preserved nonverbal communication. These clinical features are similar to those previously described in patients with the congenital variant of Rett syndrome [START_REF] Bahi-Buisson | Revisiting the phenotype associated with FOXG1 mutations: two novel cases of congenital Rett variant[END_REF]]. Moreover, a simplified gyral pattern, as well as a delayed myelination, two features previously reported in FOXG1 mutation patients, are observed in our patient suggesting that these MRI abnormalities are key features to identify patients likely to carry FOXG1 mutations.

In our study, the overexpressed wild-type protein is localised throughout the nucleus excluding the nucleoli, as previously observed. Conversely, the p.R244C FoxG1 mutant aggregate as nuclear foci, and interestingly colocalizes with the splicing factor SC-35, a marker of the nuclear speckles. FoxG1 is a nuclear protein bearing a Finally, it is well established that FoxG1 acts as a transcriptional repressor, with both direct and indirect mechanisms, in both DNA-binding dependent and independent manners. Target genes include cell cycle inhibitors, such as the cyclin-dependent kinase inhibitor 1A (p21, Cip1) (CDKN1A) gene [START_REF] Seoane | Integration of Smad and forkhead pathways in the control of neuroepithelial and glioblastoma cell proliferation[END_REF][START_REF] Kawauchi | Foxg1 promotes olfactory neurogenesis by antagonizing Gdf11[END_REF][START_REF] Chan | Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer[END_REF]]. FoxG1 is known to be a potent inhibitor of the transforming growth factor (TGF) βregulated signalling by inhibiting the TGFβ-dependent transcription of the CDKN1A gene, encoding a cell cycle protein that may induce cell cycle exit in neural precursors [START_REF] Siegenthaler | Transforming growth factor beta 1 promotes cell cycle exit through the cyclin-dependent kinase inhibitor p21 in the developing cerebral cortex[END_REF]. In this study, we showed that overexpression of FOXG1 in neuroblastoma cells reduces CDKN1A expression. This inhibitory role of FoxG1 on TGFβ-induced CDKN1A expression has already been described in earlier reports [START_REF] Adesina | FOXG1 dysregulation is a frequent event in medulloblastoma[END_REF][START_REF] Chan | Overexpression of FOXG1 contributes to TGF-beta resistance through inhibition of p21WAF1/CIP1 expression in ovarian cancer[END_REF]]. The p.R244C FoxG1 mutant loses its effect, suggesting that this mutation affects the expression of CDKN1A at the transcriptional level in SH-SY5Y cells. During brain development, this mutant may induce cell cycle exit in neural precursors leading to severe microcephaly observed in mutated FOXG1 patient, in adult Foxg1

haploinsufficient mice [START_REF] Shen | FoxG1 haploinsufficiency results in impaired neurogenesis in the postnatal hippocampus and contextual memory deficits[END_REF], and in Fgf8 hypomorphic and null mice, which is likely due, at least in part, to alterations in Foxg1 expression [START_REF] Storm | Dose-dependent functions of Fgf8 in regulating telencephalic patterning centers[END_REF].

In conclusion, we identified a novel missense mutation within the DNA-binding forkhead domain of FoxG1 in an 8-year-old girl, who presented the combination of postnatal microcephaly, dyskinetic movement disorders with hand stereotypies, relative good eye contact and frontal gyral simplification and myelination delay. The observation that the FOXG1 mutation associated with the disease state causes mislocalization of the protein in the nucleus offers an additional possible explanation for the loss of function of FoxG1 in mutated affected patients.

This loss of function is associated with the loss of repressive activity against CDKN1A expression. Additional

  As part of the Rettsearch program (SYRENE program), 150 female patients with a clinical diagnosis of severe congenital encephalopathy with microcephaly were selected for the study. The cohort included nine females with the classical form of Rett syndrome, 19 patients with the early seizure onset variant, 7 patients with the congenital variant of Rett syndrome, and 115 patients showing some of the features observed in Rett syndrome.

  www.HGVS.org). Nucleotide numbering is based on cDNA reference sequences GenBank accession number NM_005249.3. Detection of large rearrangements of the FOXG1 gene was assessed by quantitative, real-time PCR carried out with the SYBR Green PCR Master Mix (Applied Biosystems) on an ABI Prism 7000 Sequence Detection System (Applied Biosystems) according to the manufacturer's instructions.

FOXG1

  4823883, Thermo Scientific Open Biosystems, Huntsville, AL, USA) by PCR amplification with forward (F) and reverse (R) primers flanked by BglII and BamHI restriction sites, respectively: 5sequences are in bold). This amplification removes the initiation codon ATG from the sequence and allows its subcloning as the C-terminal part of a fusion protein. The PCR was carried out with the PCRx Enhancer System (Invitrogen, Cergy-Pontoise, France) with 10 ng of template plasmid DNA, 1X PCRx Amplification Buffer, 2X PCRx Enhancer Solution, 2 mM MgSO 4 , 250 µM dNTP each, 0.5 µM each primer, and the Platinum ®

  the T4 DNA Ligase (New England Biolabs) overnight at 4°C, and transformed into XL10-Gold ® Ultracompetent Cells (Stratagene) cultured in a selective medium, to generate the pEGFP-FOXG1 recombinant plasmid producing an EGFP-FOXG1 fusion protein. After direct sequencing, this wild-type construct serves as a template to generate the p.R244C mutant (c.730C>T) by directed mutagenesis with the QuikChange ® XL Site-(F) and 5'-TCGTCGTAGTGGCACGGCACCTTCACG-3' (R) (converted codon underlined; converted nucleotide in bold). Sequences of intermediate and final plasmid constructs were systematically controlled by direct sequencing.Cell culture, transfection and immunofluorescenceThe DAOY medulloblastoma cell line and the HeLa human cervix carcinoma cells (ATCC ® Number: CCL-2™) were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics at 37°C, 5% CO 2 under humidified atmosphere. One day before transfection, cells were seeded onto round coverslips and cultured in minimal medium. They were either mock-transfected or transfected with the plasmid encoding an EGFP-tagged fusion protein using Lipofectamine™ 2000 (Invitrogen) as the transfection reagent following the manufacturer's instructions. Forty-eight hours after transfection, cells were fixed with methanol 100%, -20°C for 5 minutes. For direct fluorescence, coverslips were mounted with Vectashield ® Mounting Medium with DAPI (Vector Laboratories, purchased from Abcys, Paris, France) and visualised with a Leica DMRA2 fluorescence microscope. All subsequent steps were carried out at room temperature unless indicated. For indirect immunofluorescence, cells were permeabilized with 0.2% Triton X-100, 15 min, and unspecific sites were blocked with a 5% non-fat milk solution in PBS 1X, 30 min. Cells were incubated with the mouse monoclonal anti-splicing factor SC-35 antibody (Sigma-Aldrich, dilution 1:200), overnight at 4°C, and then with a Texas Red-conjugated secondary anti-mouse IgG (dilution 1:4000) for 1 hour. After extensive washing, coverslips were mounted as described above.

Figure 1 .

 1 Figure 1. A-D Brain MRI at 7 months of age. T1 weighted sequences show frontal pachygyria with thick frontal gyri (A; B). On T2 weighted sequence, mild myelination delay with only internal capsule myelinated (C). T1 sagital section shows thin corpus callosum and normal fossa posterior (D). E-H refer to age-matched control.

Figure 2 .

 2 Figure 2. Arginine 244 (p.R244) is a conserved amino acid residue located within the forkhead domain of the FoxG1 protein. A) Schematic representation of the FoxG1 protein showing the poly-histidine tract (amino acids 47-57) and the relative position of the R244 amino acid. GBD: Groucho-binding domain; JBD: JARID-1B-binding domain; N and C: N-and C-termini, respectively. Alignment of the human FoxG1 protein sequence (ClustalW2: www.ebi.ac.uk/Tools/clustalw2/) in the close vicinity of the R244 residue with B) 15 multi-species orthologous and C) 33 human paralogous protein sequences imported from Ensembl Genome Browser v60 (www.ensembl.org/). Arginine 244 (R244) is indicated by an arrow.

  the p.R244C mutant in nuclear speckles may affect the functions of these nuclear domains, which are known to be involved in the assembly and/or modification of pre-mRNA splicing factors.

Figure 3 .

 3 Figure 3. Immunofluorescence analysis of (HeLa/DAOY) cells transfected by pEGFP-FOXG1 (A-D) and pEGFP-FOXG1_p.R244C (E-H). Fixed cells were mounted with Vectashield mounting medium with DAPI (A, E). Green fluorescence was observed by direct fluorescence (B, F) and SC-35 staining appears in red in immunolabeled cells (C, G). Merge images (green/red fluorescence) are shown (D, H).

Figure 4 .

 4 Figure 4. Real-time quantitation of CDKN1A mRNA levels in (GFP+)-wild-type (WT) and mutated (R244C) transfected cells Total RNA was converted to cDNA for quantitative real-time PCR with SYBR Green as the detection agent with the ABI Prism 7500 Sequence Detection System (Applied Biosystems). CDKN1A expression was studied by quantitative Reverse Transcriptase (RT)-PCR. RT-PCR was carried out in triplicate and expression levels were normalized to HPRT as an endogenous control.

  (His) repeat (11 His, in its N-terminal part. As observed in our work, it has been shown that loss of DNA binding in other poly-Histidine(His)-containing nuclear proteins (such as the transcription factor MEOX2)[START_REF] Salichs | Genome-wide analysis of histidine repeats reveals their role in the localisation of human proteins to the nuclear speckles compartment[END_REF]] with a point mutation is associated with their localization in intranuclear speckles. Recently, Ricciardi and colleagues showed that the product of the CDKL5 gene, which is involved in the early onset seizure variant of Rett syndrome, also localizes in nuclear speckles that are involved in the pre-mRNA processing in cells[START_REF] Ricciardi | CDKL5 influences RNA splicing activity by its association to the nuclear speckle molecular machinery[END_REF]. Because nuclear speckles are thought to be the result of the aggregation of pre-mRNA splicing factors, including ribonucleoprotein particles (snRNPs) and arginin-serine-rich splicing factors, it has been suggested that any CDKL5 dysfunction results in an imbalance of the various components of the spliceosome machinery, which ultimately leads to alterations of the splicing pattern of a number of yet undefined RNA transcripts. Additional studies will be required to elucidate the functional consequences of the localization of mutant FOXG1 in nuclear speckles on the splicing machinery, and the possible link with CDKL5 in the pathological mechanisms of this disease.

  studies are required to elucidate the functional consequences of the nuclear speckle localization of mutant FoxG1 proteins on the splicing machinery and its possible link with CDKL5 in the pathological mechanisms of this disease.

John Wiley & Sons, Inc.Human Mutation

Contract grant sponsor: Institut National de la Santé et de Recherche Médicale (ANR-08-e-rare EuroRETT Network)

John Wiley & Sons, Inc.

Human Mutation


Christel Depienne
email: christel.depienne@upmc.fr

Oriane Trouillard

Delphine Bouteiller

Isabelle Gourfinkel-An

Karine Poirier

François Rivier

Patrick Berquin

Rima Nabbout

Denys Chaigne

Dominique Steschenko

Agnès Gautier

Dorota Hoffman-Zacharska

Annie Lannuzel

Marilyn Lackmy-Port-Lis

Hélène Maurey

Anne Dusser

Marie Bru

Brigitte Gilbert-Dussardier

Agathe Roubertie

Anna Kaminska

Sandra Whalen

Cyril Mignot

Gaetan Lesca

Alexis Arzimanoglou

Eric Leguern

John Wiley

Inc Sons

Mutations and deletions in PCDH19 account for various familial or isolated epilepsies in females

Keywords: PCDH19, Epilepsy, Febrile seizures, microdeletion, cognitive function PCDH19, Epilepsy, Febrile seizures, microdeletion, cognitive function febrile, generalized, in cluster afebrile, generalized tonic atypical absences, tonic

Mutations in PCDH19, encoding protocadherin 19 on chromosome X, cause familial epilepsy and mental retardation limited to females or Dravet-like syndrome. Heterozygous females are affected while hemizygous males are spared, this unusual mode of inheritance being probably due to a mechanism called cellular interference. To extend the mutational and clinical spectra associated with PCDH19, we screened 150 unrelated patients (113 females) with febrile and afebrile seizures for mutations or rearrangements in the gene. Fifteen novel point mutations were identified in 15 female patients (6 sporadic and 9 familial cases). In addition, qPCR revealed two whole gene deletions and one partial deletion in 3 sporadic female patients. Clinical features were highly variable but included almost constantly a high sensitivity to fever and clusters of brief seizures. Interestingly, cognitive functions were normal in several family members of 2 families: the familial condition in family 1 was suggestive of Generalized Epilepsy with Febrile Seizures Plus (GEFS+) whereas all three affected females had partial cryptogenic epilepsy. These results show that mutations in PCDH19 are a relatively frequent cause of epilepsy in females and should be considered even in absence of family history and/or mental retardation. ©2010 Wiley-Liss, Inc.

INTRODUCTION

Mutations in the PCDH19 gene, located on chromosome X and encoding the protocadherin 19, were originally identified in Epilepsy and mental retardation limited to females (or Epilepsy, Female-restricted, with Mental Retardation, EFMR, MIM #300088) (Dibbens, et al., 2008). EFMR is a disorder with a remarkable X-linked inheritance: only females with heterozygous mutations are affected whereas males with hemizygous mutations are unaffected [START_REF] Fabisiak | A familial form of convulsive disorder with or without mental retardation limited to females: extension of a pedigree limits possible genetic mechanisms[END_REF][START_REF] Juberg | A new familial form of convulsive disorder and mental retardation limited to females[END_REF][START_REF] Ryan | Epilepsy and mental retardation limited to females: an X-linked dominant disorder with male sparing[END_REF]. This unusual mode of inheritance is likely to be due to cellular interference, a mechanism assuming that only the co-existence of PCDH19-positive and -negative cells, as a result of random X inactivation in females, is pathogenic [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Johnson | Metabolic interference and the + -heterozygote. a hypothetical form of simple inheritance which is neither dominant nor recessive[END_REF][START_REF] Wieland | Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome[END_REF].

The phenotype of EFMR varies from mild to severe in terms of seizure type and severity as well as in the degree of cognitive impairment (Scheffer, et al., 2008). Seizures begin in infancy or early childhood (6-36 months) and are sensitive to fever in most patients. Patients present with variable seizure types including tonicclonic, tonic, atonic, absences, myoclonic jerks and partial seizures. Behavioral problems are often part of the clinical picture and can manifest as autistic, obsessive or aggressive features [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF]Scheffer, et al., 2008). Recently, we have shown that the clinical spectrum associated with PCDH19 mutations can overlap that of Dravet syndrome [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF]. Intellectual outcome was reported to range from normal intellect to mental retardation within families; however, all patients reported to date either had intellectual disability or belonged to families where cognitive delay was present in other family members (Scheffer, et al., 2008).

In the present study, we have screened 150 unrelated probands with diverse epilepsy subtypes with or without cognitive impairment or mental retardation in order to define the mutational and clinical spectra associated with PCDH19 mutations.

MATERIALS AND METHODS

Patients

We selected 150 unrelated probands (113 females and 37 males) for PCDH19 screening. The referral physicians filled a detailed clinical questionnaire for every patient and clinical reports were also collected for most patients to assess the clinical history of the disease. Ninety-five percent (n=142) of the patients had been initially referred to our diagnosis laboratory for genetic testing of SCN1A based on the association of febrile and afebrile seizures.

Most patients (n=104) had no family history of epilepsy. These sporadic cases include 16 patients with DS in whom SCN1A testing was negative. Forty-six probands had at least one relative with epilepsy. The mode of inheritance was compatible with that of EFMR in 10 families. Eight families were reported as GEFS+ (Generalized Epilepsy with Febrile Seizures Plus) conditions. Eight families had a homogeneous phenotype of generalized idiopathic epilepsy (n= 5) or partial epilepsy (n= 3). Informed written consent was obtained from the patients' parents before blood sampling. This study was approved by the ethical committee (CCPPRB of Pitié-Salpêtrière Hospital, Paris, n°69-03, 25/9/2003).

PCDH19 Screening

The coding sequence of PCDH19 (transcript reference EF676096) was amplified in 11 fragments, and screened using direct sequencing, as already described [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF]. Mutations found in patients were directly searched for in available parents using direct sequencing of the corresponding amplicon. When the mutation was absent from both parents, parental testing was performed using microsatellite markers at the PCDH19 locus to ensure that the mutation occurred de novo. In addition, 190 French controls (95 females and 95 males who were healthy spouses of patients with other autosomal dominant diseases) and 200 Polish controls (118 females and 82 males) were included to test new variants of the PCDH19 gene. Mutation interpretation and amino acid conservation in orthologs and paralogs was assessed using the Alamut 1.31 Software (Interactive Biosoftware) and Clustalw (available at http://bioinfo.hku.hk/services/analyseq/cgi-bin/clustalw_in.pl).

Deletions or duplications involving PCDH19 were searched for using quantitative real-time PCR. Six primer pairs (one per exon) were designed using PrimerExpress 1.5 (sequences available on request). Real-time PCR experiments were performed using 100 ng of genomic DNA, 0.4 µM of each primer and 12.5 µl of Sybr Green PCR master mix (Applied Biosystems) in a total volume of 25 µl. The RNAse P (RNAse P control assay from Applied Biosystems) was used as the reference amplicon. Each sample was run in triplicate on an ABI PRISM 7900 Detection system (Applied Biosystems). Relative ratios were calculated using the formula r = 2 -∆∆Ct with ∆∆Ct = (Ct Mutation -Ct RNAseP ) ind tested -(Ct Mutation -Ct RNAseP ) ind ref

Characterization of PCDH19 deletions using high density SNP arrays

Patients with PCDH19 deletions were analyzed using 370CNV-Duo genotyping BeadChip arrays (Illumina). The Infinium II Genotyping reaction steps were performed according to the manufacturer's specifications. Image data analysis and automated genotype calling was performed using Beadstudio 3.1 (Illumina). Breakpoints of the deletions were defined as the first and last SNP comprised in the region of the deletion, which were homozygous and presented a decreased log ratio (in the order of -0.5).

RESULTS

Genetic analyses

One hundred and fifty unrelated probands (113 females and 37 males) were screened for mutations in PCDH19 by direct sequencing. A total of 15 different mutations, all novel, were identified at the heterozygous state in 15 female patients (Tables 1A, Figure 1 andsupplementary Fig. 1) 2). To confirm that the 15 mutations were pathogenic, we screened 190 healthy French individuals. None was found in the control population, supporting their deleterious role. Since Family 2, in which the c.437C>G / p.Thr146Arg mutation was identified, was of Polish origin, this variant was also tested and not found in 200 healthy Polish Controls.

Parents of mutated probands and affected relatives were tested for the mutation when available. Six out of the 15 probands with PCDH19 mutations were isolated cases (families 3, 5, 6, 11, 13 and 14) but both parents were available in only 3 families. The mutation was absent in both parents in one family out of the 3, indicating that it arose de novo (family 11). Remarkably, in the 2 other families (13 and 14), the mutation was inherited from the asymptomatic mother. In familial cases (families 1, 2, 4, 7, 8, 9, 10, 12 and 15), mutations were inherited from a healthy father in 3 families (families 2, 9 and 12), leading to a recurrent transmission of the mutation to all the daughters. In families 8 and 15, which yet include two affected daughters, the mutations were absent from both parents, suggesting germinal mosaicism in one parent. However, the sister of the proband was unavailable for genetic analysis in both families, so it remains uncertain whether the affected sisters had the same mutation than the proband. In families 1, 4 and 10, the mutation was inherited from an affected mother and segregated with the disease in all affected family members. It was not possible to determine the origin of the mutation in the remaining In order to uncover micro-rearrangements in PCDH19 that could have been missed by direct sequencing, we developed quantitative real-time PCR assays to measure the copy number of each exon of the gene. This analysis identified two whole gene deletions and a partial deletion spanning exons 1 to 3, in 3 additional female patients (Figure 2 and Table 1B). Analysis of the corresponding patients' DNA using high density SNP array revealed that the deletions had different size and breakpoints. The whole gene deletions in patients N07 0897 (family 17) and N07 1329 (Family 18) spanned 0.5 and 6.3 Mb, respectively, and comprises nearby genes (Figure 3). The largest deletion encompasses DIAPH2, a gene possibly involved in oogenesis and associated with premature ovarian failure type 2 [START_REF] Bione | A human homologue of the Drosophila melanogaster diaphanous gene is disrupted in a patient with premature ovarian failure: evidence for conserved function in oogenesis and implications for human sterility[END_REF]. The 0.5 Mb deletion includes TNMD, TSPAN6, SYTL4 and SRPX2, the gene responsible for X-linked recessive rolandic seizures associated with oral and speech dyspraxia and mental retardation [START_REF] Roll | SRPX2 mutations in disorders of language cortex and cognition[END_REF]. Contrary to the two other deletions, deletion of exons 1 to 3 was limited to the PCDH19 gene in patient N08 0125 (family 16). Parents of the proband were unavailable in family 17 but analysis of the parents in families 16 and 18 revealed that the deletions arose de novo.

Summary of clinical features for PCDH19-positive patients

The clinical features of PCDH19-positive patients are summarized in Table 2. The clinical pictures were variable from one patient to another. However, seizures were highly sensitive to fever in most patients, especially in the 2 first years of life and during childhood. Another characteristic feature observed in PCDH19-positive patients was seizures occurring in clusters (2-10 seizures per day, lasting over several days). Remarkably, cognitive functions ranged from normal to severe intellectual deficiency. Indeed, several or all family members of 2 families (families 1 and 2) had no cognitive impairment.

Case reports

Family 1, of French origin, includes 4 female patients. Neonatal periods were uneventful for all patients. The proband (N05 0550) was a 6-year-old girl. Family 4, of French origin, included 3 female patients who all had GTCS but detailed clinical data were available for the proband only (N09 0418). She was born after uneventful pregnancy and had a normal development before the onset of seizures. She experienced a first brief generalized febrile seizure at 13 months. Two weeks later, she had a cluster of brief GTCS without fever and a treatment by VPA and Clonazepam (CLN) was started. At the age of 3 years, she had several generalized seizures in clusters. Seizures were brief (< 2 minutes) but repeated over several days (3-4 days). Afterwards, she had occasional episodes of GTCS occurring in cluster, which were commonly triggered by fever and lasted one or two minutes or even only a few seconds. VPA was replaced by TPM for a better control of the seizures. She never had myoclonia or partial seizures. At the age of 6 year, her language was delayed but she was able to make simple sentences. She was described as an introverted child, who had difficulties to establish social interactions. She was capable of communicating with words but did not use language spontaneously. For instance, she preferred pointing at objects or images rather than naming them. When examined for the last time, at 7 years old, she had a mild cognitive and language delay. Her mother was reported to have generalized seizures between age 2 and 6 years, that were first treated by PB and later on by VPA. At that time, polyspike-wave discharges were observed on EEGs. At the age of 18 years, she became free of seizures without treatment. However, when examined at age 44, she presented with mild intellectual disability. The halfsister of the proband had 3 "Grand Mal" seizures at the age of 5 years old without fever. She became free of seizures when treated with VPA. However, during the adolescence, she presented with important scholar difficulties and psychiatric instability.

Family 12 includes two affected sisters born from non consanguineous parents of French origin. Both sisters have had intra-uterine growth retardation related to a homozygous variant of the methylenetetrahydrofolate reductase gene in the mother. However, they were considered healthy full-term newborns. The proband (N08 0696), who She had about 10 febrile GTCS lasting a few minutes the same day with normal examination in-between, normal interictal EEG and normal brain MRI. Another series of afebrile paroxysmal events, characterized by numerous absences without abnormal movements lasting less than a minute, occurred one week later despite the initiation of VPA therapy. CLB was therefore added to VPA. Three more clusters of 5-10 seizures occurred at the ages of 2, 3 and 4 years, requiring an add-on treatment with LTG. The clinical manifestations were heterogeneous, including absences, clonic and tonic seizures, sometimes associated with eyes turning. Most seizures lasted 1 to 5 minutes, but clusters of 10 to 15 minute-long seizures were also observed. Interictal EEGs remained normal except for occasional posterior slow waves. At the age of 3.5 years, psychomotor retardation and language delay became evident. She also had a mild microcephaly (-2 SD). The youngest sister (N 09 1103) was 2.5 years old at the time of the study. She had a first cluster of ~10 afebrile GTCS at 8.5 months, leading to a treatment by VPA. Brain MRI was normal and interictal EEG revealed bitemporal slow waves but no paroxysmal activity. Another seizure cluster occurred a few days later and VGB was added to VPA. Two more episodes of 8-10 afebrile seizures occurred at the age of 21 months and were characterized by generalized tonic manifestations, sometimes asymmetrical with head turning, lasting a few minutes. EEG recording of a seizure showed generalized discharges from the beginning of the seizure. After replacement of VGB by TPM, no other seizures occurred. Her psychomotor development was slightly retarded, as she sat alone at 13 months, walked independently at 18, but her speech ability was relatively preserved at the age of 2. Her head growth followed the -2 SD curve until 6 months then slowed down to -2.5 /-3 SD.

Family 17 was of French origin and included a single affected female patient (N07 0897) who was 7 years old at the time of the study. Her parents were reported to be healthful and there was no further history of febrile seizures or epilepsy in the family. The proband was born after an uneventful pregnancy and had a normal development before the onset of seizures. At 15 months, while she was febrile, she started having clusters of brief generalized tonic seizures that required hospitalization. A treatment by VPA was set up but tonic seizures lasting 2-3 minutes CLN was replaced by CLB. At the time of the study (11 years), she was free of seizures since age 9 on tritherapy (VPA, CLB, TPM). She was able to read and her language appeared normal. However, she had major behavioural and personality disturbances. She also had difficulties with logical reasoning and graphics. She never had ataxia or myoclonic jerks and her seizures were photoinsensitive.

DISCUSSION

In this study, we report 15 novel point mutations and three deletions in the PCDH19 gene in 18 unrelated female patients, for an overall mutation frequency reaching 16% (18/113). We also tested 37 male patients in this study but no mutation was found. However, we cannot exclude that mosaicism has been missed by screening only DNA extracted from blood cells in these patients.

Although all the previously reported mutations were clustered in exon 1 of PCDH19 (corresponding to the extracellular cadherin domain of protocadherin 19) [START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF]Dibbens, et al., 2008;[START_REF] Hynes | Epilepsy and mental retardation limited to females with PCDH19 mutations can present de novo or in single generation families[END_REF],

we now report a nonsense mutation in exon 4 (p.Arg886X) that segregated in several affected members of family 1. This shows that the PCDH19 mutational spectrum is not limited to mutations in exon 1. Furthermore, two whole gene deletions and one partial deletion were identified using quantitative real-time PCR. These deletions constitute the first micro-rearrangements involving PCDH19 identified in female patients with febrile seizures and epilepsy.

Altogether, these results confirm that mutations in PCDH19 are associated with a loss-of-function of protocadherin 19 in cells that have inactivated the normal X chromosome, and that screening of PCDH19 should include a specific method to search for rearrangements in addition to direct sequencing of the whole coding sequence.

As previously reported, the phenotype of the patients with PCDH19 mutations was highly variable between families but also within affected members of a same family (Table 2). This variability includes the age at onset (mean age at onset: 16 months, ranging from 7 months to 5 years), the occurrence of status epilepticus episodes (42%, 10/24), the response to AEDs (pharmacoresistance in 76% of the cases, 19/25) and the temporal evolution of the patients. However, seizures occurring in cluster (92%, 23/25 patients) and a high sensitivity to fever (90%, 20/22 patients) were almost invariably present in PCDH19-positive patients. All patients have had generalized seizures (although in some cases, the generalized seizures were only in febrile contexts) and 68% exhibited multiple seizure types (17/25) including partial seizures (of frontal or temporal origin) and atypical absences.

Although seizures appeared highly resistant to AEDs during the first years of life, the frequency and pharmacoresistance of seizures tended to decrease over time, and some patients were even free of seizures during adolescence or adulthood on monotherapy. Myoclonic jerks were infrequent in patients with PCDH19 mutations (9%, 2/23) and photosensitivity was reported in only one case out of 25 (<5%). In contrast, a high frequency of behavioral disturbances, including autistic features, was noticeable (28%, 7/25 patients). In some patients, social withdrawal or personality troubles were even the most prominent and disabling feature when the children became older. Interestingly, the phenotypes of the patients with PCDH19 deletions seemed indistinguishable from those with point mutations although this finding needs to be confirmed with a larger number of patients carrying deletions.

Intellectual deficiency was also very variable from one patient to another: cognitive impairment was reported to be mild in 6 patients, moderate in 10, and moderate/severe in 2 patients. One patient was too young to evaluate her cognitive status (family 7). Interestingly, in families 1 and 2, most or all patients presented without cognitive impairment. In family 2 for instance, all the 3 sisters with the p.Thr146Arg mutation had normal cognitive abilities. The history of epilepsy in the 3 sisters was nevertheless similar to other patients with moderate mental retardation and included in particular many seizures in cluster during childhood, persistence of the seizures despite polytherapy and later development of partial seizures of frontal or temporal origin. This highly suggests that the cognitive prognosis is not related to the severity of epilepsy. In family 1, the history of febrile and afebrile seizures in the proband, her mother and her grand-mother was highly suggestive of Generalized Epilepsy with Febrile

Seizures plus (or Genetic Epilepsy with Febrile Seizures plus, GEFS+, MIM #604233) [START_REF] Scheffer | Generalized epilepsy with febrile seizures plus. A genetic disorder with heterogeneous clinical phenotypes[END_REF]. It is unclear so far whether the favorable cognitive outcome in these patients is due to the mutation itself, which could have milder functional consequences on the protocadherin 19. The large intrafamilial variability of the phenotype (in particular in family 1) however suggests that other genetic or non genetic modifier factors could be involved as well. One attractive hypothesis is that this phenotypic variability could be due to random or skewed X inactivation. Cellular interference is based on the random X inactivation resulting in a functional mosaicism in females [START_REF] Compagni | Control of skeletal patterning by ephrinB1-EphB interactions[END_REF][START_REF] Depienne | Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles Dravet syndrome but mainly affects females[END_REF][START_REF] Johnson | Metabolic interference and the + -heterozygote. a hypothetical form of simple inheritance which is neither dominant nor recessive[END_REF][START_REF] Wieland | Mutations of the ephrin-B1 gene cause craniofrontonasal syndrome[END_REF]. A totally skewed X inactivation in neurons would theoretically leads to a homogenous cell population (expressing or not the normal protocadherin 19), corresponding to a non-pathogenic situation. It is possible that a partially skewed X inactivation (such as 60:40, 70:30 or 80:20) could represent situations less severe than a random X inactivation (50:50) where the cellular interference is expected to be the highest. The severity of the epilepsy could therefore be correlated with the relative amount of inactivated neurons for each chromosome in the brain and some mutation carriers, like mutated mothers in family 13 and 14 for example, could even be asymptomatic due to totally skewed X inactivation. [START_REF] Depienne | Mechanisms for variable expressivity of inherited SCN1A mutations causing Dravet syndrome[END_REF][START_REF] Harkin | The spectrum of SCN1A-related infantile epileptic encephalopathies[END_REF]. We have previously reported that the similarity between PCDH19 and SCN1A clinical spectra comprises Dravet syndrome; now, we show that it also extends to that of GEFS+. Indeed, intellectual disability as well as partial epilepsy can also be part of SCN1A-positive GEFS+ families [START_REF] Depienne | Mechanisms for variable expressivity of inherited SCN1A mutations causing Dravet syndrome[END_REF][START_REF] Escayg | Mutations of SCN1A, encoding a neuronal sodium channel, in two families with GEFS+2[END_REF]. Differentiating one condition to the other would therefore be difficult in some cases.

F

However, some clinical features could help prioritizing the analysis: for instance female patients with an age at onset after 12 months and presenting with seizure clusters should be first screened for PCDH19 mutations, especially when the families include several females but no male patients. Regarding Dravet Syndrome, screening for PCDH19 mutations should be performed for female patients when analysis of SCN1A (including a method searching for SCN1A micro-rearrangements) is negative.

Given the unusual mode of inheritance and the wide phenotypic variability associated with PCDH19, genetic counseling appears delicate. In the case of mutations inherited from an asymptomatic father, all the daughters are expected to be affected. Therefore, taking into account the frequent bad cognitive prognosis, it could be possible to offer a prenatal diagnosis using only a fetal sex determination from maternal blood [START_REF] Wright | The use of cell-free fetal nucleic acids in maternal blood for non-invasive prenatal diagnosis[END_REF].

Female patients with PCDH19 mutations have a 50% risk to transmit the mutation but only the females would be affected, for a global risk of 25%. However, in some situations, genetic counseling could be even more difficult, such as in family 17 in which the proband had a PCDH19 deletion encompassing other nearby genes with a recessive X-linked inheritance such as SRPX2. In that case, the patient will have a 25% risk to have an affected daughter and of a 25% risk to have a son affected with a phenotype of variable severity including rolandic epilepsy, oral and speech dyspraxia, mental retardation and perisylvian polymicrogyria.

In conclusion, these results extend the mutational and clinical spectra associated with PCDH19-related epilepsies and show that mutations in PCDH19 are a frequent cause of epilepsy in females and should be considered even in absence of family history and/or mental retardation.

F

  and 7) although the presence of an affected maternal aunt in family 7 argued in favour of paternal inheritance.

Family 2 <

 2 was of polish Ashkenazi origin on the paternal side. There was no family history of seizures or cognitive impairment in the previous generations. The three patients were born after uneventful pregnancies and had normal neonatal periods. None of them had psychiatric features and neurological examination always remained normal.Patient N09 2853 was 24 years old at the time of the study. Since the age of 7 months, seizures occurred in clusters PCDH19 mutations in Female Epilepsy> 9 lasting a few minutes, with symptoms consisting of sudden cry and behaviour changes. There were one or two clusters per months, frequently favoured by hyperthermia. Brain MRI, at 5 years of age showed a frontal median dermoid cyst. She underwent resection of the cyst without any modification of seizure frequency. From the age of 9 years, the seizure semiology was modified, starting with a short yell or the repetition of a word related to the events of the previous days and followed by hyperactivity, fear, pallor and tachycardia. Consciousness was not altered during the shorter episodes. Clusters including more than 10 daily seizure episodes occurred over 2 to 3 days with intervals of 6 weeks to 3 months. They occurred mostly during daytime or as she fell asleep and were currently triggered by fever, emotions or sleep deprivation. She frequently experienced right frontal headache during the days preceding the seizures. At the age of 10 years, stereoencephalography showed rapid spikes followed by slow spike-waves in the left frontal cortex with contralateral propagation. The diagnosis of cryptogenic partial frontal epilepsy was considered. She has been treated with CBZ from the age of three years to the age of nineteen years in addition to other successive drugs (VPA, VGB, LTG, TPM and LEV). Seizure offset occurred when she was 19 years old, as she was treated with CBZ and LEV, whose doses were then progressively decreased. Neuropsychological testing, at the age of 16 years showed a VIQ score of 115 and a PIQ score of 87 with slight impairment in working memory and visual treatment. She is now 23 years old and is attending the courses of French engineering school.Patient N09 2265 was one year younger than her sister. She experienced a first cluster of GTCS lasting one hour, when she was five years old, 12 hours after a febrile episode. At 6 years, she had two similar clusters favoured by hyperthermia as well as several afebrile nocturnal episodes. Neuropsychological testing, when she was 7 years old showed a VIQ score of 117. She continued to experience repeated clusters of seizures with a modified semiology including automatic gestures, chewing and elevation of the left arm. Seizures were mainly triggered by fever. Some nocturnal tonic-clonic seizures also occurred. She was first treated with VPA, which was substituted with CBZ at the age of 10 years. Treatment could be stopped 6 years later. Now aged 22 years, she attends the classes of a French engineering school as her sister.The youngest sister (N09 2851) was 15 at the time of the study. Since 9 months of age, she experienced clusters of GTCS during febrile episodes as well as nocturnal seizures with a semiology consisting of straightening up of the body, incoherent language, hypertonia with hand contractions, deviation of the head (mainly on the right), chewing and bruxism. Seizure semiology was progressively modified with staring, back dropping and yelling, clonic hypertonia with ocular revulsion and chewing. EEG was in favour of a possible frontotemporal origin. She has first been treated with CBZ. LTG was added when she was 7 years old and CBZ was switched to LEV two years later. Seizure offset occurred at 13 years of age. When seen for the last time, she was attending normal school although she displays slight reduced processing speed. She has won several championships of bridge.

  old at the time of the study, experienced her first seizures at 11 months during a febrile otitis episode.

  Identification of 3 families with PCDH19 mutations, in which the majority of patients had normal cognitive functions. A) Pedigrees of the families and segregation analysis of the PCDH19 mutations; m/+ and m indicate individuals heterozygous and hemizygous for the mutation, respectively; +/+ and + indicate individuals homozygous and hemizygous for the wild-type allele, respectively. Squares represent males, circles females; Upper right black corner: Generalized seizures; Upper left black corner: Partial seizures; Lower right black corner: FS; Lower left black corner: intellectual disability; Grey symbols: Epilepsy of unknown type. Dots in the middle of the squares indicate unaffected mutation carriers. The arrows indicate the index cases. B) Sequence electrophoregrams of the mutations. The mutation nomenclature is based on the PCDH19 transcript reference EF676096. Nucleotides are numbered according to the cDNA with +1 corresponding to the A of the ATG translation initiation codon in the reference sequence, according to the guidelines (www.hgvs.org/mutnomen). 283x209mm (300 x 300 DPI) Identification of 3 families with PCDH19 deletions. A) Pedigrees of the families and segregation analysis of the PCDH19 deletions; del/+ indicates individuals heterozygous for the deletion; +/+ and +, individuals homozygous and hemizygous for the wild-type allele, respectively. Upper right black corner: Generalized seizures; Upper left black corner: Partial seizures; Lower right black corner: FS; Lower left black corner: intellectual disability; hatched symbols: patients with rolandic epilepsy. The arrows indicate the index cases. B) Results of copy number dosage for each exon of PCDH19 using real time-Q-PCR. WT1 is a control female; WT2 is a control male. 283x209mm (300 x 300 DPI) Characterization of the size and breakpoints of the PCDH19 deletions using high-density SNP arrays (Illumina). A) Log R profiles of the 3 patients with deletions. The X-axis indicates the position on the chromosome X and the Y-axis indicates the log R. The grey stripes represent the location of the PCDH19 gene. B) Schematic representation of the deletions indicated by double arrows. The size of the deleted regions (in Mb of Kb) is indicated in brackets. 283x209mm (300 x 300 DPI) Pedigrees of the families and segregation analysis of the PCDH19 mutations in families 3 to 15. m/+ and m indicate individuals heterozygous and hemizygous for the mutation, respectively; +/+ and + indicate individuals homozygous and hemizygous for the wild-type allele, respectively. Squares represent males, circles females; Upper right black corner: Generalized seizures; Upper left black corner: Partial seizures; Lower right black corner: FS; Lower left black corner: intellectual disability. Right black half (mother of the proband, family 10): Febrile seizures (FS) and tonic clonic seizures in childhood, normal cognitive abilitie; Grey symbols: Epilepsy of unknown type. Dots in the middle of the squares indicate unaffected mutation carriers. The arrows indicate the index cases. B) Sequence electrophoregrams of the mutations. The mutation nomenclature is based on the PCDH19 transcript reference EF676096. 283x209mm (300 x 300 DPI) Orthologous and paralogous protein alignments, showing the high conservation of each amino-acid altered by missense mutations in vertebrates and in the delta protocadherin paralogous genes. 283x209mm (300 x 300 DPI)

  patient was normal between seizures. Clinical investigations, including search for infectious agents, lumbar puncture and CT scan, were normal. Seizures persisted but became rarer when the patient was treated with sodium valproate (VPA) and Clobazam (CLB), and finally stopped with PB. Later on, GTCS became very rare, occurring most often in afebrile contexts, but absence episodes were sometimes observed. EEGs were normal. Psychomotor development of the patient was normal and she had no school difficulties at 6 years of age. She was still treated by PB at the time of the study. Her mother (N05 0548) was 28 years old at the time of the study. At 18 months of age, while she was febrile, she presented a cluster of 4-5 brief GTCS per day that lasted for several days. Later on, febrile or afebrile GTCS regularly recurred until age 7. VPA appeared inefficient but GTCS finally ceased when she was treated with PB. However, absence episodes, mostly triggered by fatigue were observed. EEGs and

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Her affected mother was treated with Phenobarbital (PB) during her whole pregnancy. GTCS, lasting ~20 seconds and occurring in clusters of 5-10 seizures per day, have appeared at 11 months of age, after a febrile episode. Clusters of seizures have persisted for 10 days. Neurological status of the cerebral MRI were normal. The maternal grandmother of the proband (N05 0549) was 54 years old when examined for the last time. As her daughter, she has had a cluster of GTCS lasting several days at 18 months of age although it remained uncertain whether she was febrile or not at this time. GTCS persisted until age 10, and then absences occurred from age 15. Myoclonic jerks were also noticed during adolescence. VPA was inefficient but PB allowed a reasonable control of the seizures. She was still treated with PB at the time of the study. Her psychomotor development had always been normal and no cognitive deficit was perceptible. EEGs and cerebral MRI remained unavailable. The maternal aunt of the proband (N09 2223), was 36 years old when examined. At 18 months of age, she developed febrile status epilepticus without later motor deficit but secondary psychomotor decline. When she was 10, she developed afebrile GTCS and transient losses of consciousness (suggesting either absences or partial seizures). She always had school difficulties and developed psychotic manifestations. At the time of the study, she presented with moderate intellectual deficiency. EEGs during childhood were unavailable but those during adulthood were normal. MRI was normal. During childhood, she was treated with PB which appeared effective. In adulthood, carbamazepine (CBZ) was ineffective but she finally became seizure-free under VPA. She was still treated with VPA at the time of the study.

  EEG performed at 19 months was normal. At 5 years old, she had a normal neurologic examination but showed a global developmental delay predominating in language and learning (estimated to a one-year delay).The proband of family 18 was born at 36 weeks from a bichorionic gemellar pregnancy. Her parents, her twin sister and a youngest sister were all healthy. Her psychomotor development was normal until the onset of seizures.She experienced a first afebrile generalized convulsive status epilepticus at the age of 14 months. Investigations including brain imaging, lumbar puncture, toxic and metabolic screenings were normal. EEG was normal. She was treated with CLN and VPA. She started walking at 16 months, almost two months after her twin sister. She had new episodes of febrile GTCS at 1.5, 2.5 and 3.5 years. Behavioural disturbances and language delay became obvious around 3 years old. A decrease in CLN dosage at the age of 4 years led to a new status epilepticus and LTG was added to CLN and VPA. Likewise, a decrease in VPA dosage at the age of 5 years was unsuccessful.

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and repeating during several days recurred at 19 months in a febrile context. Despite the successive introduction of multiple AEDs (CLB, TPM, PB and PHT), brief hypertonic seizures occurring in clusters persisted over time. An From the age of 5 years, she had several clusters of GTC seizures, most of them triggered by mild fever. At the age of 7 years, she had a moderate cognitive impairment including a global executive dysfunction. Karyotyping was normal and search for X-Fragile expansion was negative. Diurnal and nocturnal EEGs and Brain MRI remained normal. A novel attempt to decrease CLN dosage at 7 years led to the recurrence of afebrile seizures in clusters.

  The wide phenotypic expression associated with PCDH19 mutations is very reminiscent of what is observed for patients with SCN1A mutations. It is now well-established that SCN1A mutations are associated with a large clinical spectrum ranging from febrile seizures only to typical or atypical Dravet syndrome

	< PCDH19 mutations in Female Epilepsy> 15
	o
	r
	P
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	R e v i e w

20 10 Afebrile prolonged GTCS Febrile and afebrile GTCS partial sz yes yes no no +++ no moderate words- sentences TPM, LEV, ZNS CBZ, PB, VPA, LTG Behaviour disturbances

															TPM, VPA, CLN	-	-	acquired microcephaly
	13	N09 1090	14	15	Febrile GTCS	brief febrile GTCS, atypic absences Myoclonic jerks	No but prolonged sz (15 min)	yes	yes	yes	+++	yes	moderate/ severe	sentences	VPA, LEV ? LTG, CLB,	-	-	-
														less than				Delayed fine
	14	N08 1063	2	8	febrile, GTCS	Clonic sz Atonic seizures	no	yes	no	no	no	too young ?	moderate	10 isolated words at	VPA, CLB	-	-	motor skills, Delayed
														2y				myelination
	15	N09 1135	6	9	afebrile, generalized	GTCS	yes	yes	no	no	+++	yes	moderate	words-sentences	VPA, CLB, LTG	TPM	CBZ	-
	16 3 17 5 4 18	N08 0125 N09 0176 N07 0897 N09 1326 (DS) N09 0418 N07 1329	17 12 7 7 11	9 11 15 13 14	Generalized o r Generalized Febrile GTCS GTCS febrile, generalized, in cluster Generalized Hypertonic P r yes yes no e e Febrile GTCS GTCS no Afebrile generalized GTCS Yes (first sz)	yes yes yes yes yes	no NA no no no	no NA NA no no	+++ +++ +++ +++ +++	no ? no no no	moderate/s evere mild moderate mild moderate	? words-sentences words-sentences Delayed (sentence s) normal	VGB, TPM, NTZ VPA, CLB VPA, TPM, CLB VPA, CLN, TPM, TPM, CLB, LTG, CLN	--PB, PHT VPA -	-LTG ---	Behaviour disturbances Behaviour -disturbances Delayed fine Behaviour motor skills, Behaviour disturbances disturbances
	6 7 8 9	N09 1568 N09 1461 N05 1365 N09 1515	9 1 13 14	13 8 60 24	NA afebrile GTCS afebrile Febrile generalised Complex partial hemi clonic,	GTCS GTCS Hemiclonic, Partial sz, Absences Motor complex partial 2nd generalised	no R e yes no yes v yes yes i e no NA no w yes yes no	no NA no no	+++ NA + ++	no too young yes subtle	Very mild too young too young NA moderate sentences LTG, CBZ VPA, CLN NA moderate sentences LEV, TPM	-VPA, PHT VPA VGB	---LTG	-Slight dysmorphic features -Behaviour disturbances

Table 2 :

 2 Clinical features of patients with PCDH19 mutations or deletions SE: Status Epilepticus ; ID: Intellectual deficiency; PD: psychomotor delay ; Sz: Seizures; Myocl. : Myoclonic jerks ; Pho Sen. : Seizure photosensitivity ; AEDS : Antiepileptic drugs ; AEDs (+) : AEDs with positive response ; wo: without; AEDs(-) : AEDs with negative response (worsening of the seizures or of the patient state) ; GTCS : Generalized tonic clonic seizures ; NA: Not available ; CBZ : Carbamazepine, CLB: Clobazam, CLN: Clonazepam , LEV : Levetiracetam, LTG: Lamotrigine, NTZ: Nitrazepam, PB: Phenobarbital, PHT: phenytoin, STP : Stiripentol, TPM : topiramate, VGB: Vigabatrin, VPA : Sodium Valproate, ZNS: zonisamide.

John Wiley & Sons, Inc.Human Mutation

Acknowledgements

We thank the families for participating to this study and the cell and DNA bank of the Cricm (UMRS_975) for DNA extraction of some families, Magdalena Nawara for analysis of the Polish Controls and Drs Lionel Thivard, Vincent Navarro, Florence Renaldo and Agnès Guët for referring their patients.

Contract grant sponsor: AP-HP), INSERM and the AFM/Gis maladies rares "Maladies Rares et Anomalies de structure du genome" (Genome structural variation and Rare Diseases); Contract grant number: Programme Hospitalier de Recherche Clinique (n°P020910/AOR02085.

F o r P e e r R e v i e w


Pavel Calda

Michaela Novotná

David Čutka

Miroslav Brestak

Lubomir Haslik

Barbara Goldová

Ivana Vitkova

Manuela Vaněčková

Zdenek Seidl

A case of an

Keywords: epignathus, oral mass, teratoma, ultrasonography, MRI, video clip file -"view these files (e.g. movies) online epignathus, oral mass, teratoma, prenatal, ultrasonography, MRI epignathus, oral mass, teratoma, prenatal, ultrasonography, MRI

des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

An epignathus (also called pharyngeal teratoma) is a rare form of benign congenital teratoma originating from the base of the skull, most commonly the palate or the mandible. Associated malformations may be present and commonly include cleft palate and bifid tongue or nose.

Previous reports have noted the association of epignathus with the duplication of the pituitary gland. [1][2][3][4] Sporadic chromosomal changes have also been reported 5,6 , but usually epignathus is not associated with chromosomal aberrations. Most of these teratomas are unidirectional and protrude more or less through the mouth. Cases with intracranial extension causing destruction of the brain tissue have poor prognosis. 7,8 Prenatal sonographic diagnosis of large protruding oral tumors can be easily made and have been well documented. 9 In most cases, the diagnosis was made in the late second or third trimester. However, one early diagnosis has been described at 15 weeks´gestation 10 and mainly depended on the extend of the protrusion and sonographic appearance of the tumor. We report a case of a small, primarily non-protruding oral mass first visualized at 16w 3d and subsequent management consisting of prenatal 2D and some 3D sonographic images, counseling and postmortem MRI with pathologic correlation).

Case report

A healthy 29-year-old primigravida with no medical history of note underwent a first-trimester ultrasound examination at 12w 3d, with a normal result. An integrated screening test performed by her gynecologist was positive, with an individual Down syndrome risk 1: 230.

(nuchal translucency 2mm, hCG 4.4 MoM, PAPP-A 0.8 MoM, AFP 0.9 MoM, estriol 0.9 MoM) Amniocentesis was therefore performed at 16w 3d with a normal female karyotype. At the Oral teratoma diagnosed at 16 weeks ultrasound examination performed by an expert sonographer prior to amniocentesis, the peculiar finding of a persistently opened mouth was noted, for more than 30 minutes. (Acuson Siemens S 2000, 6C2 and 9L4 probes; Voluson E8, RAB4-8-D probe) With thorough revaluation a 2x2 mm mass inside the mouth was seen. As this finding was very small, re-evaluation in four weeks was recommended. A repeat 2D/3D ultrasound examination at 20w 3d showed an appropriately grown female fetus with a biparietal diameter of 48mm, head circumference of 170mm, abdominal circumference of 152mm, and femur length of 31mm. However, we found a complex mixed echogenic ball-shaped mass, 22x17mm, originating from the mouth, filling the oral cavity (Figure 1a, Figure 1b, Figure 1c). Color Doppler examination revealed hypervascularity within the mass. Intracranial exam showed asymmetry of the lateral ventricles.

Agenesis or partial agenesis of corpus callosum was suspected, but the sonographic appearance did not allow definitive diagnosis (Figure 1d). The placenta was located on the posterior uterine wall and the volume of amniotic fluid was not increased. The fetus did not swallow or close the mouth throughout the whole examination.

The tumor appeared to be growing rapidly and the intracranial changes were quite visible. Our working diagnosis was epignathus with intracranial extension. Although there are case reports of delivery of viable fetuses by ex utero intrapartum treatment (EXIT) of cases with isolated epignathus, 11 in our case, the finding of intracranial extension made the prognosis rather poor.

After non-directive counseling the parents decided to terminate the pregnancy.

A female fetus, weighing 310g had on gross examination a lobulated round vascular mass approximately 2cm in diameter, visible through the open oral cavity (Figure 2a). Histological examination confirmed the presence of a congenital teratoma with neural, myofibrilar and fibrous connective tissue (Figure 3) covered by non-stratified squamous epithelium. The neuroepithelial intracranial cyst measured 13x10x7mm and agenesis of corpus callosum was found (Figure 4a, Figure 4b).

Discussion

Teratomas are tumors derived from pluri-potent cells and made of elements of different types of tissue, from 1 or more of the 3 germ cell layers. Brain tissue is the most frequent component along with cartilage, bronchial epithelium, and ependyma-lined cysts. An epignathus is an extremely rare form of teratoma in which the tumor is attached to the base of the skull, usually the hard palate, or to the mandible. The incidence is approximately of 1 in 35,000 to 1 in 200,000 live births. They are generally considered to occur more frequently in females than males, with a ratio 3:1. 12 Ultrasound can give a reliable diagnosis, when the finding has the Oral teratoma diagnosed at 16 weeks typical appearance. 13 In our case, the lesion was observed from the very early stage, when it was small (e.g. a minimally exophytic oral soft-tissue mass), leading to a wide differential diagnosis. The differential diagnosis of a mass in the fetal or neonatal oral cavity should include teratoma, congenital epulis (rare benign oral cavity tumor originating from anterior alveolar ridge), hemangioma, lymphatic malformation, sincipital cephalocele, dermoid cyst or other benign or malignant soft tissue masses (tongue teratoma). 14 The parasitic twin syndrome has also been described in this location. 11,15,16 Oral teratoma may show heterogeneous signal intensity on MRI due to variable contents such as cyst, calcifications, and fat. Hemangioma presents a solid mass with disorganized vascularization of high flow vessels and intense contrast enhancement on MRI. Lymphatic malformation and cephalocele presents with cystic characteristics. Dermoid has fat intensity signals, with preserved crista galli, contrary to the nasal cephalocele, where crista galli are not preserved.

The clinical presentation of an epignathus varies. The tumor is mainly diagnosed on a routine 2D sonographic examination. Polyhydramnios, hydrops, fetal death and pre-eclampsia can accompany the epignathus. Impaired fetal swallowing can lead to polyhydramnios. The sequence combining fetal hydrops pre-eclampsia, in connection is not quite clear. Epignathus can be accompanied by elevated maternal serum alfa-fetoprotein (AFP), which can mandate a very detailed examination of the fetus. 17 In our case, the AFP level was normal. The size and exact location of the oral mass are important prognostic factors because larger tumors and nasopharyngeal involvement can cause both feeding difficulties and mechanical airway obstruction. 18,[START_REF] Ozeren | Prenatal ultrasound diagnosis of a large epignathus[END_REF] These prognostic factors should be taken into consideration in determination of the optimal management plan. [START_REF] Teal | Fetal teratomas: antenatal diagnosis and clinical management[END_REF] In potentially viable fetuses, when severe impairment is Precise determination of the spread of the tumor to the surrounding tissues is very important, because with intracranial spread, the prognosis is poor i.e. no surviving cases reported in the literature. [START_REF] Shipp | The ultrasonographic appearance and outcome for fetuses with masses distorting the fetal face[END_REF] Termination of pregnancy as an option for management should be discussed, as occurred in our case.

The prognosis of pharyngeal teratoma is very poor. [START_REF] Bornstein | Diagnostic and prognostic aspects in the sonographic evaluation of a fetus with an oral mass[END_REF][START_REF] Tapper | Teratomas in infancy and childhood. A 54-year experience at the Children's Hospital Medical Center[END_REF] Intrauterine fetal demise in up to 50% and neonatal demise of 50% of the survivors have been reported. Only 6 cases of infants surviving a pharyngeal teratoma have been reported over the last 10 years. Some infants required plastic surgery for the cleft palate or lip, while others had just minor repair of luxation of the mandible. Currently there is no evidence to suggest epignathus is caused by environmental influences, Mendelian or polygenic inheritance or chromosomal abnormalities. No cases of recurrent epignathus in one family have been reported in the literature. 1 This information is of essential importance when consulting the parents towards the next pregnancy. Our case illustrates that the suspicions arising from an odd finding (an open mouth) seen early probably should be respected and an appropriate follow-up planned. Previous reports have noted the association of epignathus with the duplication of the pituitary gland. [1][2][3][4] sporadic chromosomal changes have also been reported 5,6 , but usually epignathus is not associated with chromosomal aberrations. Most of these teratomas are unidirectional and protrude more or less through the mouth. Cases with intracranial extension causing destruction of the brain tissue have poor prognosis. 7,8 Prenatal sonographic diagnosis of large protruding oral tumors can be easily made and have been well documented. 9 In most cases, the diagnosis was made in the late second or third trimester. However, one early diagnosis has been described at 15 weeks´gestation 10 and mainly depended on the extend of the protrusion and sonographic appearance of the tumor. We report a case of a small, primarily non-protruding oral mass first visualized at 16w + 3d and subsequent management consisting of prenatal 2D and some 3D sonographic images, counseling and postmortem MRI with pathologic correlation).

F

Case report

A healthy 29-year-old primigravida with no medical history of note underwent a first-trimester ultrasound examination at 12w 3d, with a normal result. An integrated screening test performed by her gynecologist was positive, with an individual Down syndrome risk 1: 230. Oral teratoma diagnosed at 16 weeks Amniocentesis was therefore performed at 16w 3d with a normal female karyotype. At the ultrasound examination performed by an expert sonographer prior to amniocentesis, the peculiar finding of a persistently opened mouth was noted, for more than 30 minutes. (Acuson Siemens S 2000, 6C2 and 9L4 probes, Voluson E8, RAB4-8-D probe) With thorough revaluation a 2x2 mm mass inside the mouth was seen. As this finding was very small, re-evaluation in four weeks was recommended. A repeat 2D/3D ultrasound examination at 20w 3d showed an appropriately grown female fetus with a biparietal diameter of 48mm, head circumference of 170mm, abdominal circumference of 152mm, and femur length of 31mm. However, we found a complex mixed echogenic ball-shaped mass, 22x17mm, originating from the mouth, filling the oral cavity (Figure 1a, Figure 1b, Figure 1c). Color Doppler examination revealed hypervascularity within the mass. Intracranial exam showed asymmetry of the lateral ventricles.

Agenesis or partial agenesis of corpus callosum was suspected, but the sonographic appearance did not allow definitive diagnosis (Figure 1d). The placenta was located on the posterior uterine wall and the volume of amniotic fluid was not increased. The fetus did not swallow or close the mouth throughout the whole examination.

The tumor appeared to be growing rapidly and the intracranial changes were quite highly visible. Our working diagnosis was epignathus with intracranial extension. Although there are case reports of delivery of viable fetuses by ex utero intrapartum treatment (EXIT) of cases with isolated epignathus, 11 in our case, the finding of intracranial extension made the prognosis rather poor. After non-directive counseling the parents decided to terminate the pregnancy.

A female fetus, weighing 310g had on gross examination a lobulated round vascular mass approximately 2cm in diameter, visible through the open oral cavity (Figure 2a). Histological examination confirmed the presence of a congenital teratoma with neural, myofibrilar and fibrous connective tissue (Figure 3) covered by non-stratified squamous epithelium. The neuroepithelial intracranial cyst measured 13x10x7mm and agenesis of corpus callosum was found (Figure 4a, Figure 4b).

Discussion

Teratomas are tumors derived from pluri-potent cells and made of elements of different types of tissue, from 1 or more of the 3 germ cell layers. Brain tissue is the most frequent component along with cartilage, bronchial epithelium, and ependyma-lined cysts. An epignathus is an extremely rare form of teratoma in which the tumor is attached to the base of the skull, usually the hard palate, or to the mandible. The incidence is approximately of 1 in 35,000 to 1 in 200,000 live births. They are generally considered to occur more frequently in females than males, with a ratio 3:1. 12 Ultrasound can give a reliable diagnosis, when the finding has the Oral teratoma diagnosed at 16 weeks typical appearance. 13 In our case, the lesion was observed from the very early stage, when it was small (e.g. a minimally exophytic oral soft-tissue mass), leading to a wide differential diagnosis. The differential diagnosis of a mass in the fetal or neonatal oral cavity should include teratoma, congenital epulis (rare benign oral cavity tumor originating from anterior alveolar ridge), hemangioma, lymphatic malformation, sincipital cephalocele, dermoid cyst or other benign or malignant soft tissue masses (tongue teratoma). 14 The parasitic twin syndrome has also been described in this location. 11,15,16 Oral teratoma may show heterogeneous signal intensity on MRI due to variable contents such as cyst, calcifications, and fat. Hemangioma presents a solid mass with disorganized vascularization of high flow vessels and intense contrast enhancement on MRI. Lymphatic malformation and cephalocele presents with cystic characteristics. Dermoid has fat intensity signals, with preserved crista galli, contrary to the nasal cephalocele, where crista galli are not preserved.

The clinical presentation of an epignathus varies. The tumor is mainly diagnosed on a routine 2D sonographic examination. Polyhydramnios, hydrops, fetal death and pre-eclampsia can accompany the epignathus. Impaired fetal swallowing can lead to polyhydramnios. The sequence combining fetal hydrops pre-eclampsia, in connection is not quite clear.Epignathus can be accompanied by elevated maternal serum alfa-fetoprotein (AFP), which can mandate a very detailed examination of the fetus. 17 In our case, the AFP level was normal. The size and exact location of the oral mass are important prognostic factors because larger tumors and nasopharyngeal involvement can cause both feeding difficulties and mechanical airway obstruction. 18,[START_REF] Ozeren | Prenatal ultrasound diagnosis of a large epignathus[END_REF] These prognostic factors should be taken into consideration in determination of the optimal management plan. [START_REF] Teal | Fetal teratomas: antenatal diagnosis and clinical management[END_REF] In potentially viable fetuses, when severe impairment is Precise determination of the spread of the tumor to the surrounding tissues is very important, because with intracranial spread, the prognosis is poor i.e. no surviving cases reported in the literature. [START_REF] Shipp | The ultrasonographic appearance and outcome for fetuses with masses distorting the fetal face[END_REF] Termination of pregnancy as an option for management should be discussed, as occurred in our case.

The prognosis of pharyngeal teratoma is very poor. [START_REF] Bornstein | Diagnostic and prognostic aspects in the sonographic evaluation of a fetus with an oral mass[END_REF][START_REF] Tapper | Teratomas in infancy and childhood. A 54-year experience at the Children's Hospital Medical Center[END_REF] Intrauterine fetal demise in up to 50% and neonatal demise of 50% of the survivors have been reported. Only 6 cases of infants surviving a pharyngeal teratoma have been reported over the last 10 years. Some infants required plastic surgery for the cleft palate or lip, while others had just minor repair of luxation of the mandible. Currently there is no evidence to suggest epignathus is caused by environmental influences, Mendelian or polygenic inheritance or chromosomal abnormalities. No cases of recurrent epignathus in one family have been reported in the literature. 1 This information is of essential importance when consulting the parents towards the next pregnancy. Our case illustrates that the suspicions arising from an odd finding (an open mouth) seen early probably should be respected and an appropriate follow-up planned.

  at 16 weeks Postmortem MRI was performed the same day (5 hours had elapsed between the termination and before post-mortem examination of the brain) to determine the extent and characteristics of the mass. Imaging consisted of high-spatial-resolution in the three orthogonal planes in T2 weighted images. The post mortem MRI exhibited extended heterogeneous mass distorting the face, with distinct in-homogeneity in signal intensity. The mass was in the anterior oral cavity, prevented the mouth from closing and propagated dorsally, causing a posterior displacement of the tongue. Intracranial extension was not described. (Figure2b). Another finding was mild bilateral ventricular dilatation (Figure2c) caused by corpus callosum agenesis with a paramedian cyst).

  at 16 weeks detected or suspected, the neonatal team should be prepared to rapidly secure the neonate's airway.

Figure 1a :

 1a Figure 1a: Sonogram at 20+3 weeks´ gestation shows a ball shaped oral mass (E) with an opened

Figure 2b :

 2b Figure 2b: Post mortem MRI. Sagittal T2W image: in-homogenous signal intensity of mass in the

Figure 2c :

 2c Figure 2c: Coronal T2W image: the pathologic lobulated mass in the oral cavity and also

Figure 3 :

 3 Figure 3: Histological examination of the teratoma: Cartilage, bone and dermal tissue

(

  nuchal translucency 2mm, hCG 4.4 MoM, PAPP-A 0.8 MoM, AFP 0.9 MoM, estriol 0.9 MoM)

Formatted

  at 16 weeks Postmortem MRI was performed the same day (5 hours had elapsed between the termination and before post-mortem examination of the brain) to determine the extent and characteristics of the mass. Imaging consisted of high-spatial-resolution in the three orthogonal planes in T2 weighted images. The post mortem MRI exhibited extended heterogeneous mass distorting the face, with distinct in-homogeneity in signal intensity. The mass was in the anterior oral cavity, prevented the mouth from closing and propagated dorsally, causing a posterior displacement of the tongue. Intracranial extension was not described. (Figure2b). Another finding was mild bilateral ventricular dilatation (Figure2c) caused by corpus callosum agenesis with a paramedian cyst).

  Impaired fetal swallowing can lead to polyhydramnios. The sequence combining fetal hydrops Deleted: and Deleted: pre-eclampsia, in connection Deleted: with this pathology Deleted: is not quite clear. at 16 weeks detected or suspected, the neonatal team should be prepared to rapidly secure the neonate's airway.

Formatted:Figure 1a :Figure 1b :Figure 1d :

 1a1b1d Figure 1a: Sonogram at 20+3 weeks´ gestation shows a ball shaped oral mass (E) with an opened

Figure 2b :

 2b Figure 2b: Post mortem MRI. Sagittal T2W image: in-homogenous signal intensity of mass in the

Figure 2c :

 2c Figure 2c: Coronal T2W image: the pathologic lobulated mass in the oral cavity and also

Figure 3 :FigureFigure 1a :Figure 1b Figure 1d Figure 2b :Figure 3 :Figure 4a :

 31a1b1d2b34a Figure 3: Histological examination of the teratoma: Cartilage, bone and dermal tissue

John Wiley & SonsJournal of Clinical Ultrasound