A Decade of Trials and Tribulations of PCDH19 Research to Make a Difference.

Jozef Gecz

Adelaide Medical School and the Robinson Research Institute, the University of Adelaide, SA; and Women and Kids Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia.

The PCDH19 gene is a well-established neurodevelopmental disability (NDD) gene facilitating precise molecular diagnosis of thousands of patients world-wide and irrespective of their genetic or ethnic background. It has been just over a decade since its first implication in familial as well as isolated cases of a seizure disorder currently referred to as girls clustering epilepsy (GCE). The early studies, which also led to the mapping and the identification of the gene involved primarily families with affected girls. By now we know, that males can also be affected, and there is more than a dozen of such males recorded. These males ought to carry a postzygotic somatic DNA variant of the PCDH19 gene making them cellular mosaics, i.e. mix of cells with wild type PCDH19 and cells with altered PCDH19 or absent altogether, similar to females where such cellular mosaicism is a consequence of X- chromosome dosage compensation, that is X-inactivation (XI). XI is a mechanism which silences one copy of the PCDH19 gene (and many other genes) on one X-chromosome of a female in a given cell.

PCDH19-GCE is a unique genetic disability. Its incomplete penetrance, that is not always present clinical-level disability, which is at around 80%, makes it even more challenging to study and understand. A good example are a few known cases of monozygotic, that is genetically identical female twins, with PCDH19 gene variation, which are different to each other in their clinical presentations or cases of sisters or mother-daughter pairs with PCDH19 variants and with or without disability.

It has only been ten years since the initial identification of the PCDH19 DNA variation as the primary cause of these disabilities. We have since witnessed some major advancements in our understanding of different aspects of the clinical presentations, structural and functional as well as basic biology of PCDH19 GCE. GCE is known as a drug resistant seizure disorder, but we know by now that is not just a seizure disorder, but a rather complex neurocognitive and neuropsychiatric disability with autism, executive function deficits, hyperactivity and aggression, obsessive compulsive features and other comorbidities, which are often individual patient specific and variable. Current systematic studies of GCE show that onset of seizures can be a strong predictor of the overall clinical outcome of GCE, that is seizures and neuro-cognitive or neuro-psychiatric outcomes. While the seizures can become less frequent with age, unfortunately, psychiatric symptoms often exacerbate with age and can become the most disabling aspect of GCE.

PCDH19 is a molecule originally known to facilitate cell-cell interaction for the purpose of cell communication, differentiation or movement. Our knowledge of PCDH19 also dramatically evolved in this regard. While PCDH19 still holds certain neurons in bundles to wire the brain, it also appears to work in gene regulation as well as at the synapse, which is the essential unit of neuronal cell communication and as such seizures and/or cognition and behaviour. PCDH19 is now known as a multitasker protein with discrete roles across the development of the brain. Most intriguingly, its complete loss is tolerated, as judged by males with complete loss of function of PCDH19 who are not affected. Several animal models have also been generated and have helped to untangle the fundamental biological principles of PCDH19 GCE and its underlying molecular and cellular mechanisms for the purpose of better treatment and better management.

Sadly there is no robust anti-epilepsy or anti neuro-behaviour therapy as yet available for a broad range of PCDH19 GCE individuals, but there is a promise that there will be one and sooner rather than later. Multiple clinical trials are currently underway, more and more informed and/or stratified by the genetic as well as metabolic or clinical findings of individuals with PCDH19 GCE. Global access to and sharing of information on PCDH19 has also made a significant difference. Any, even minor advancements, even of a single GCE case can be captured and assessed on their merit and inform the next best step in our research and treatment decision-making and direction.

Bringing families, carers, researchers, doctors, pharmaceutical industry, philanthropy as well as policy makers together at meetings like this is a must to facilitate holistic and multidisciplinary care for this disability. Sharing is caring! Your research, your experience is the major force in our fight for better life of all individuals living with PCDH19-related neurodevelopmental disability. Thank you to everyone for participating to this meeting and sharing your knowledge, wisdom and your experience!

Modeling PCDH19-Related Epilepsy in human cortical organoids

Wei Niu, Lu Deng, Xixi Du, Elmira Jalilian, Sandra Mojica-Perez, Andrew Tidball, Jack Parent

Department of Neurology, University of Michigan, Ann Arbor, MI  48109

PCDH19-Related Epilepsy (PRE) is caused by mutations of the PCDH19 gene on the X-chromosome and exclusively affects females and mosaic males while male carriers are spared. Mosaic expression of PCDH19 due to random X-inactivation is thought to cause impaired cell-cell interactions between mutant and wild type PCDH19-expressing cell populations to produce the disease phenotype. However the precise function of PCDH19 during human cortical development and how the mosaic expression of PCDH19 leads to PRE remain unclear. We are using CRISPR genome editing and stem cell approaches, including human cortical organoids (hCOs), to interrogate the function of PCDH19 and how its mutations lead to seizure-like activity in developing human brain in vitro. To this end, we used CRIPSR/Cas9 to generate in-frame epitope tagged PCDH19 H9 female human pluripotent stem cells (hPSCs) that allow us to use a standard antibody against the epitope to detect PCDH19 expression (most antibodies to date have shown non-specific labeling). We found that PCDH19, along with N-Cadherin, is localized to the apical lumens of neural rosettes in both 2D adherent cultures and 3D hCOs derived from the tagged hPSCs, consistent with its high mRNA expression level at this stage. Using CRISPR/Cas9, we also established homozygous PCDH19 knockout hPSCs and generated a "virtual PRE patient" model in which isogenic lines with a HA-FLAG-tagged PCDH19 allele are mixed with knockout cells, providing a reliable system to model the mosaic expression of PCDH19 that occurs in vivo due to random X-inactivation. We used these lines to generate single rosette hCOs that model early human cortical development. We observed altered N-Cadherin+ apical lumens, altered PCDH19 subcellular localization and abnormal cell segregation in hCOs derived from the "virtual PRE patient" model. Moreover, the migration of deep layer cortical neurons was disrupted. Our results using a 3D model of human cortical development suggest that PCDH19 acts as a critical cell-cell adhesion molecule through its interactions with other cadherin proteins, and that mosaic loss of PCHD19 function leads to aberrant cell sorting and impaired neuronal migration.

Biomarker-stratified Phase 3 Study Evaluating Ganaxolone in Children with PCDH19-Related Epilepsy

Alex Aimetti,

Marinus Pharmaceuticals, Inc.

Ganaxolone (GNX) is a synthetic analog of endogenous allopregnanolone and acts on GABAA receptors in the brain to promote inhibitory signaling. GNX was studied in a Phase 2 open-label study in PCDH19-related epilepsy. Data from this study yielded preliminary evidence of a predictive biomarker that could potentially be used to enhance the treatment effect in a refined patient group. These findings were used to guide the design and initiation of a larger Phase 3 biomarker-stratified study (the Violet Study) to further evaluate the potential utility of the biomarker as well as the safety and efficacy of GNX.

Investigation of the mechanism by which mosaic expression of Protocadherin 19 leads to PCDH19-Girls Clustering Epilepsy

Stefka Tasheva1,2, Claire Homan1, Jozef Gecz1,2 and Paul Thomas1,2

1School of Medicine, University of Adelaide, Adelaide, Australia 

2South Australian Health and Medical Research Institute, Adelaide, Australia 

PCDH19-Girl Clustering Epilepsy (PCDH19-GCE) is a rare syndrome that affects females, caused by heterozygous mutations of the X chromosome gene PCDH19. While there is compelling evidence that this disorder stems from mosaic expression of PCDH19 (due to X-inactivation in the developing brain), the pathological mechanism underpinning PCDH19-GCE remains unclear. 

We have shown previously that PCDH19 contributes to adhesion specificity in a combinatorial manner such that mosaic expression of Pcdh19 in heterozygous female mice leads to striking sorting between cells expressing wild-type (WT) PCDH19 and null PCDH19 in the developing cortex, correlating with altered network activity. More recently we have shown that endogenous PCDH19 is located in 25-30% of synapses in primary hippocampal neurons, suggesting a role for PCDH19 in synaptogenesis. To investigate how mosaic PCDH19 expression impacts synaptogenesis and neuronal behaviour, we used CRISPR/Cas9 technology to knock out PCDH19 in primary hippocampal neurons derived from our PCDH19-HA-FLAG tagged mouse. Quantitative analysis of synaptic formation revealed that PCDH19-null cells, in contact with PCDH19-positive cells, display a significant reduction in the number of excitatory synapses and Synapsin1 puncta compared to controls. We also observed reduction of synaptic contacts between PCDH19-positive and -null cells when we overexpressed PCDH19 in individual neurons of hippocampal primary cultures from PCDH19 KO mice. Multi electrode array (MEA) analysis of hippocampal and cortical neurons from PCDH19-KO and WT mice revealed changes in the neuronal network activity in the mixed population cultures compared to homogeneous controls. These observations point to the possibility of synaptic dysregulation in GCE. Interestingly, morphometric analysis of PCDH19-null neurons in mixed cultures (null/positive) showed an increase in the total neurite length compared to controls (PCDH19-positive/positive and -null/null). Taken together, our results suggest that the mosaic expression of PCDH19 causes a disruption of the physiological neurite communication between PCDH19-positive and -null neurons leading to abnormal brain activity, a hallmark of PCDH19-GCE.

PCDH19-related disorder: neuroimaging study of a cohort of patients evaluating morphological brain abnormalities, cortical thickness and gyration index.

Edoardo Canale, Carla Marini 

Neurology Unit and Laboratories, A. Meyer Children's Hospital, Florence, Italy.

Objective: PCDH19-related syndrome is an infantile genetic disorder manifesting with clusters of seizures triggered by fever associated in 70% of patients with intellectual disability and autism spectrum disorder. The PCDH19 gene encodes a protein involved in neuronal circuit formation during development and in the maintenance of normal synaptic circuits in adulthood. The aim of this study is to describe the neuroradiological features and to evaluate the clinical outcome of a cohort of patients with PCDH19 gene pathogenetic mutation.

Methods: The study includes 20 patients with PCDH19-related disorder. We performed 3 Tesla neuroimaging studies using specific MRI protocols to evaluate morphological brain abnormalities, cortical thickness and gyrification index. To evaluate the clinical outcome we analyzed clinical data of these patients subdivided in 4 epochs due to the variable ages of subjects at the time of the study: 1) 0-5 years; 2) 6-12 years; 3) 13-19 years and 4) older than 20 years.

Results: Single-subject and group morphometric analysis disclosed increased cortical thickness involving predominantly the temporal regions of the brain. In most patients gyrification index was decreased in the fronto-temporal, hippocampal regions and cingulate gyrus but the group analysis didn't reveal a specific pattern of gyrification in the cortex when compared to an healthy control group. Clinical data analysis showed that PCDH19-related disorder has a semi-progressive course until puberty, an increasing proportion of patients evolves to develop intellectual disability and psychiatric disorders including autism, whereas seizure clusters decrease progressively. Approximately 30% of the patients are seizure-free for more than two years after puberty.

Significance: The present neuroradiological study suggests that PCDH19 mutations can associate to sub-microscopical abnormalities in fronto-temporal regions, hippocampal regions and cingulate gyrus confirming the role of PCDH19 in brain development. 

Gene Editing and PCDH19

Frullanti Elisa1, Diana Alaverdian1, Katia Capitani1, Alessandra Renieri1,2

1 Medical Genetics, University of Siena, Siena Italy;

2 Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italia;

PCDH19 loss of function mutations cause Early Infantile Epileptic Encephalopathy type 9 (EIEE9), an X-linked, female-limited form of infant-onset epilepsy associated with intellectual disability and autistic features. Rare EIEE9 males are somatic mosaics. To explain this pattern, the “cellular interference” model has been proposed: random chromosome X-inactivation leads to tissue mosaicism in females, with part of the cells expressing wild-type and part mutant PCDH19. The coexistence of these two cell types is proposed to scramble cell-to-cell communication. No cure is presently available for EIEE9, since the currently available treatments are purely symptomatic.

Our study aims to develop as novel therapeutic strategy based on CRISPR/Cas9 technology able to rescue the “cellular interference” in vitro and in vivo. Building on our availability of induced-Pluripotent Stem Cells (iPSCs) and encouraging preliminary gene editing results, our CRISPR/Cas9 approach will be used to efficiently delete the Pcdh19 gene. This strategy is expected to generate a homogeneous population of PCDH19-negative cells, mimicking PCDH19-mutated healthy males and thus rescuing the pathological PCDH19 mosaicism. Rescue of PCDH19-related phenotypes will be evaluated on patients’ cell lines and in mouse model.

The epilepsy-related protein PCDH19 regulates tonic inhibition, GABAAR kinetics and the intrinsic excitability of hippocampal neurons

Giulia M. Serratto1#, Erika Pizzi2#, Michele Mazzanti2, Maria Passafaro1, Silvia Bassani1*

1Institute of Neuroscience, CNR, Milan 20129, Italy

2Department of Bioscience, University of Milan, Milan 20133, Italy

#These authors contributed equally

Protocadherin-19 (PCDH19) is a cell- adhesion molecule encoded by PCDH19 gene (Xq22.1), whose mutations cause epileptic encephalopathy, early infantile, 9 (EIEE9, OMIM #300088), characterized by early-onset epilepsy, intellectual disability and autism spectrum disorder (Dibbens et al., 2008). Recently, we reported that PCDH19 is a binding partner of GABAA receptors (GABAARs) alpha subunits and is able to regulate GABAAR surface expression and currents (Bassani et al., 2018).

Given the role of GABAARs in setting neuronal excitability, here we investigated whether PCDH19 regulatory function might extend to the extrasynaptic receptors pool of GABAARs that mediate tonic currents. By whole-cell and cell-attached patch- clamp recordings, we provided a functional characterization of rat primary hippocampal neurons in which PCDH19 was downregulated. The shRNA-mediated downregulation of PCDH19 reduced GABAARs-mediated tonic current, measured by baseline noise analysis and current shift. Furthermore, single channel recordings of GABAARs showed that PCDH19 modulates the kinetics of the receptors without altering their conductance. Indeed, following PCDH19 downregulation, GABAARs preferentially exhibit brief openings at the expense of long ones, indicating a flickering behavior. Finally, we demonstrated that PCDH19 downregulation reduces the rheobase and increases action potential frequency, revealing a hyperexcitability phenotype. Ongoing experiments aim at reconfirming these results in acute brain slices from mice injected with adeno-associated vectors (AAVs)-shRNA.

Altogether, these data suggest that PCDH19 is a critical determinant of tonic inhibitory transmission and GABAARs gating, thus providing the first mechanistic insights into PCDH19-related hyperexcitability.


  1. Dibbens LM, Tarpey PS, Hynes K et al. (2008) X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment. Nat Genet 40(6): 776-781.

  2.  Bassani, S., Cwetsch, A.W., Gerosa, L., Serratto, G.M., Folci, A., Hall, I.F., Mazzanti, M., Cancedda, L., and Passafaro, M. (2018). The female epilepsy protein PCDH19 is a new GABAAR-binding partner that regulates GABAergic transmission as well as migration and morphological maturation of hippocampal


Towards the in vitro understanding of PCDH19-epilesy

Borghi R.1,2, Magliocca V.1,2, Petrini S.3, Zanni G.1, Tartaglia M.4, Bertini E.1, Moreno S.2, Compagnucci C.1, 4

1 Dept. of Neuroscience, Lab. of Molecular Medicine IRCCS, Children’s Research Hospital Bambino Gesù, Rome, Italy

2 Dept. of Science-LIME, University Roma Tre, Rome, Italy

3Confocal Microscopy Core Facility, Research Laboratories, Bambino Gesù Children's Hospital, Rome, Italy

4 Genetics and Rare Diseases Research Division, Children’s Research Hospital Bambino Gesù, Rome, Italy

Introduction. PCDH19 (Protocadherin 19), a member of the cadherin superfamily, is involved in the pathogenic mechanism of an X-linked model of neurological disease. The biological function of PCHD19 in human neurons and during neurogenesis is poorly known. Therefore, we decided to use the model of the induced pluripotent stem cells (iPSCs) to characterize the molecular and cellular phenotype of PCDH19 mutated neurons derived from patients’ iPSCs. Previous data (Compagnucci et al., 2015) show that PCDH19 is expressed in pluripotent cells, before differentiation, in a homogeneous pattern, despite its localization is often limited to one pole of the cell. During neuronal differentiation, positional information on the progenitor cells assumes an important role in acquiring polarization. The proper control of the cell orientation ensures a fine balancing between symmetric (giving rise to two progenitor sister cells) versus asymmetric (giving rise to one progenitor cell and one newborn neuron) division. This process results in the polar organization of the neural tube with a lumen indicating the basal part of the polarized neuronal progenitor cell; in the iPSC model the cells are organized in the ‘neural rosette’ and interestingly, PCDH19 is located at the center of the rosette, with other well-known markers of the lumen (N-cadherin and ZO-1).

Results. The data obtained suggest us to pursue the investigation of the plane of cellular division in neuronal precursors at the stage of the neural rosette. With this aim we used iPSCs derived from patients’ fibroblasts obtained from skin biopsies. Importantly, we found alterations in the plane of division of neuronal precursors in the cell cultures derived from patients’ iPSCs. The observed disruption of the plane of cell division possibly accounts for the accelerated rate of neuronal differentiation observed in the patients’ neuronal cultures. In order to test the effect of PCDH19 reduction on cell division, we silenced PCDH19 in control iPSCs (derived from a healthy individual) and analyzed the percentage of altered dividing cells (with centrosome hyper-amplification). To confirm an effect of mutated PCDH19 protein on dividing cells, we focused on the dividing cells at the stage of proliferating iPSCs finding numerous cells with centrosome hyperamplification in iPSCs silenced for PCDH19.

Discussion. Our data support the use of iPSCs for in vitro modeling of PCDH19 syndrome, highlighting that PCDH19 has a role in instructing the apico-basal polarity of the progenitor cells, thus regulating the development of a properly organized neuronal network. The alterations in the cell division observed in cells silenced for PCDH19 and in PCDH19-mutated patients may account for an altered control of the symmetric versus asymmetric cell division.

PCDH19: Proteolytic processing and gene regulatory functions

Sylvia A Newbold, James Wilding, Isabel Martinez-Garay

Division of Neuroscience, School of Biosciences, Cardiff University

Mutations in the X-linked gene PCDH19 lead to epilepsy with cognitive impairment in heterozygous females. Although the gene codes for a cell adhesion protein of the cadherin superfamily expected to localize at the cell membrane, recent reports have implicated PCDH19 in the regulation of gene expression and have identified the protein in the nucleus.

Our aim is to determine if PCDH19 undergoes proteolytic cleavage resulting in the generation of an intracellular fragment (PCDH19-ICD) that translocates to the nucleus to regulate gene expression. In addition, we want to identify which genes are regulated by PCDH19 in neural progenitors and neurons.

To test this hypothesis, we have analysed how the absence, inactivation or overexpression of specific proteases affects the generation of PCDH19-ICD in heterologous cells. We have also generated neurons from embryonic stem cells to analyze PCDH19 processing in neurons, including the possible role of neuronal activity. To determine potential transcriptional targets of PCDH19, we have created a mouse embryonic stem cell line that overexpresses the cytoplasmic domain of PCDH19 from the Rosa26 locus (CYTO), and an isogenic PCDH19-knockout line (KO). This is allowing us to compare the transcriptional profiles of these embryonic stem cell-derived neuronal progenitors and neurons in the presence or absence of PCDH19, as well as under conditions of PCDH19-ICD overexpression.

We believe that PCDH19 is capable of transducing information about events happening at the membrane to the nucleus to elicit appropriate cellular responses and expect that our RNAseq analysis will provide new mechanistic insights into the function of PCDH19 both during neurogenesis and in post-mitotic neurons.



Duyen H Pham1,2, Melissa R Pitman4, Renee Schulz1, Alison Gardner1, Sarah E Heron1,2, Mark A Corbett1,2, Kavitha Kothur8,9, Deepak Gill8,9, Sulekha Rajagopalan11, Kristy Kolc1, Benjamin J Halliday10, Stephen P.Robertson10, Brigid Regan12, Heidi E Kirsch14, Samuel F Berkovic12, Ingrid E Scheffer12,13, Stuart M. Pitson4,1,3, Slave Petrovski5,6, Jozef Gecz1,2,3,7

1 Adelaide Medical School, The University of Adelaide, Adelaide 5000, Australia.

2 Robinson Research Institute, The University of Adelaide, Adelaide 5006, Australia.

3 School of Biological Sciences, The University of Adelaide, Adelaide 5000, Australia.

4 Centre for Cancer Biology, University of South Australia and SA Pathology, Adelaide, South Australia, Australia.

5 Centre for Genomics Research, Precision Medicine and Genomics, IMED Biotech Unit, AstraZeneca, Cambridge, UK.

6 Department of Medicine, University of Melbourne, Parkville, VIC, Australia.

7 South Australian Health and Medical Research Institute, Adelaide 5000, Australia.

8 Kids Neuroscience Centre, The University of Sydney.

9 TY Nelson Department of Neurology and Neurosurgery, The Children’s Hospital at Westmead, Sydney, NSW 2145, Australia.

10 Department of Women's and Children's Health, Dunedin School of Medicine, University of Otago, Dunedin 9016, New Zealand.

11 Department of Clinical Genetics, Liverpool Hospital, Liverpool, NSW 1871, Australia.

12 Departments of Medicine, Austin Health, The University of Melbourne, Melbourne, VIC Australia.

13 Department of Paediatrics, The University of Melbourne, Royal Children’s Hospital, Melbourne, Florey and Murdoch Institutes, Melbourne, Australia.

14 Department of Neurology, University of California, San Francisco, CA 94122, USA.

Background: Assessment of variants currently classified as variants of unknown significance (VOUS) in genes known to be associated with disease is an increasingly important task, particularly where precision medicine trials are underway or planned. However, determining the clinical relevance of these variants can be challenging, particularly when functional data for the affected protein is scarce. While the clinical interpretation of DNA variation has improved significantly through the implementation of high international standards in the field, there are still numerous VOUS, which need ongoing and often very specialized assessment. Pathogenic variants in PCDH19 coding for the cell adhesion/estrogen receptor transcription coregulatory protocadherin 19 cause girls clustering epilepsy (PCDH19-GCE). PCDH19-GCE is an early-onset neurodevelopmental disorder (NDD) of drug-resistant epilepsy, intellectual disability, autism spectrum disorder and other neuropsychiatric disturbances. Here we aimed to systematically assess reported and novel missense variants in PCDH19.

Methods: We evaluated the performance of 16 selected publicly available in silico prediction tools including MutPred2 (URL:http://mutpred.mutdb.org/) and ANNOVAR tool-package (URL: http://wannovar.wglab.org/) to assess the pathogenicity of 322 PCDH19 missense variants selected from two datasets. The first dataset included 238 PCDH19 missense variants selected from the general population (female only) gnomAD database (URL:http://gnomad.broadinstitute.org/). The second dataset included 84 reported PCDH19-GCE disease-causing missense variants. We then performed integrative in silico (topthree bioinformatics tools and protein structure modelling) and experimental evaluation (using in vitro reporter assays) together with InterVar (according to 2015 American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines) to interpret the clinical significance of 45 PCDH19 variants.

Results: MutPred2, MutationAssessor and GPP were the top-three performing in silico tools alone or in combination. The prediction accuracy was 100% for published PCDH19-GCE variants and 78% for population variants (gnomAD allele frequency >1x10-4). Fifty-six percent of VOUS (gnomAD allele frequency ≤1x10-4) tested were classified as deleterious. The assessment toolbox was validated on a set of recently published, novel PCDH19 variants with high reliability. Further testing and validation of the toolbox for novel unpublished PCDH19 variants and also for variants identified in other genes involved in NDDs such as, TBLXR1 and PCDH12 demonstrated a high utility and accuracy.

Conclusion: We have developed a toolbox for the accurate assessment of PCDH19 variant pathogenicity. This is the crucial next step in readiness for precision medicine to address the severe seizure and behavioural disorders that occur in females and mosaic males with PCDH19 disease.

Cortical excitability in a conditional model of PCDH19 Epilepsy

Didi Lamers1, Roberta Mezzena1, Silvia Landi2 , Maria Passafaro3, Silvia Bassani3 , Gian Michele Ratto1

1 NEST, Scuola Normale Superiore and Instituto Nanoscienze Consiglio Nazionale delle Ricerche (CNR), Pisa, Italy.

2 Institute of Neuroscience, Pisa, Italy.

3 Institute of Neuroscience, Milan, Italy

PCDH19 Epilepsy is characterized by epileptic seizure onset in early infancy and is frequently associated with intellectual disability and autism spectrum disorder1,2,3. The disease has been attributed to mutations in the X chromosomal Protocadherin 19 (PCDH19) gene1, encoding a Ca2+-dependent cell-cell adhesion protein. Interestingly, only heterozygous females and males with somatic mutations are affected4, leading to the hypothesis that the disease is caused by a mosaic PCDH19 expression in the brain5. However, the mechanism by which PCDH19 mosaicism causes epilepsy and cognitive impairment is unknown.

Here, we employed a novel mouse model of PCDH19 Epilepsy and performed in vivo electrophysiological and imaging studies. We obtained a focal mosaic loss of PCDH19 by the injection of an AAV vector carrying CRE-EGFP in the visual cortex. The opposite hemisphere served as internal control in the electrophysiological experiments. Local field potential recordings in anesthetized animals demonstrate that mosaic PCDH19 patches in the brain have disrupted slow wave activity and show transient episodes of hyperexcitability and hypersynchronous activity. Intriguingly, in-depth analysis of the slope of slow wave activity and single unit statistics, suggest that the local network has an overall reduced firing rate. These findings are consistent with the attenuation of slow wave activity but are at odds with the onset of transient episodes of hyperexcitability.

To further explore the neuronal activity underlying the observed phenotype, we performed combined LFP recordings and two photon calcium imaging of PCDH19 positive and negative neurons in a mosaic brain in vivo. Preliminary data suggest that some mosaic animals displayed an increased calcium activity likely to be associated to the transient hyperexcitability. However, more data are needed to confirm this indication. No difference was observed between PCDH19 positive and negative neurons in the mosaic animals.

Given the importance of slow wave activity for both the homeostatic and memory functions of sleep, and observations that PCDH19 patients demonstrate abnormal sleep patterns6, our data could be relevant to patients to increase understanding of the underlying mechanisms of sleep disturbances in PCDH19 Epilepsy.


1. Dibbens, L. M. et al. X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment. Nat. Genet. 40, 776–781 (2008).

2. Marini, C. et al. Protocadherin 19 mutations in girls with infantile-onset epilepsy. Neurology 75, 646–53 (2010).

3. Scheffer, I. E. et al. Epilepsy and mental retardation limited to females: An under-recognized disorder. Brain 131, 918–927 (2008).

4. de Lange, I. M. et al. Male patients affected by mosaic PCDH19 mutations: five new cases. Neurogenetics 18, 147–153 (2017).

5. Depienne, C. et al. Sporadic infantile epileptic encephalopathy caused by mutations in PCDH19 resembles dravet syndrome but mainly affects females. PLoS Genet. 5, e1000381 (2009).

6. Smith, L. et al. PCDH19-related epilepsy is associated with a broad neurodevelopmental spectrum. Epilepsia 59, 679–689 (2018).

Genetic silencing of PCDH19 as treatment for Early Infantile Epileptic Encephalopathy type 9 (EIEE-9)

Giorgia Giansante1, Elisa Giorgio2, Marta Ferrero2, Elisa Pozzi2, Luca Murru1, Silvia Bassani1, Alfredo Brusco2, Maria Passafaro1

1 Institute of Neuroscience, National Research Council (CNR), Milan 20129, Italy

2 University of Torino, Dept. Medical Sciences, Torino 10126, Italy

Early Infantile Epileptic Encephalopathy type 9 (EIEE-9) is a form of epilepsy characterized by a spectrum of neurodevelopmental disorders with autistic features. Mutations in PCDH19 gene cause a loss of homophilic cell-cell interactions (Hayashi et al., 2017), axon guidance and dendrite self-avoidance (Pederick et al., 2018). The proposed pathogenic mechanism is called “cellular interference”, which is generated by a mosaic expression of PCDH19 in brain cells. In fact, EIEE-9 mainly affects females with heterozygous PCDH19 mutations and the identification of some male patients with mosaics expression of PCDH19 strongly supports the cellular interference hypothesis. In this study we investigated whether PCDH19 mosaic expression is a critical determinant of altered neuronal activity in cultured neurons and in acute brain slices. Moreover, we evaluated if genetic rescue of cellular interference mediated by RNA interference might represent a promising therapeutic option for EIEE-9.

Methods: In order to obtain different levels of PCDH19 mosaic expression in vitro, we infected primary cortical and hippocampal neurons obtained from PCDH19 floxed mice at postnatal day (P)0 with increasing amount of adenoviral particles expressing the Cre recombinase (AAV-Cre). The extent of PCDH19 mosaicism has been evaluated by immunocytochemistry and western blot. Subsequently, we studied extracellular spontaneous activity of cultured neuronal at two weeks in vitro. Neurons infected with a multiplicity of infection chosen to reproduce the 50:50 mosaic condition in vitro were analyzed by Micro-Electrode Array (MEA) device. In parallel, MEA has been used to record spontaneous electrical activity in acute cortical hippocampal slices from PCDH19 conditional KO mice.

With the aim of identifying genetic modulators of PCDH19, we have designed and tested three different siRNAs targeting both the human and the murine PCDH19 gene in human

and murine cell lines. Cells have been transfected with selected PCDH19 siRNAs, a siRNA against GAPDH (C+) and a scramble siRNA (C-) at 40nM and 100nM concentrations. Forty-eight hours post transfection cells have been harvested and splitted to extract RNA and proteins. PCDH19 levels were evaluated by real-time PCR and western blot analysis.

Results: Preliminary results suggested an increase in the duration of the mean burst activity of cortical neuronal networks in PCDH19 mosaic expression recorded in spontaneous conditions respect to cortical neurons expressing PCDH19. Furthermore, we observed an increase in the main firing and burst parameters of the network recorded in cortico-hippocampal slices from PCDH19 conditional KO mice compared to wild-type littermates.

Finally, we have identified two efficient siRNAs able to efficiently silence the human and murine PCDH19 gene and to use for future therapeutic approaches.

Conclusions: The preliminary data obtained suggest a possible role of the PCDH19 mosaicism in the functioning of brain circuitry in vitro and identified two RNA molecules capable of abrogating PCDH19 expression, opening the way for future therapeutic approaches.

PCDH19 EPILEPSY: preliminary analysis of 55 cases included in RESIDRAS

Ragona Francesca and Darra Francesca on behalf of RESIDRAS Network (N. Specchio, E. Fontana, T. Granata, R. Guerrini, L. Giordano, M. Nosadini, N. Zamponi, A. Pini, D. Battaglia, C. Fusco, C. Zucca, M.P. Canevini)

The Italian Registry of Dravet Syndrome and other syndromes correlated with genes on SCN1A and PCDH19 currently includes 55 females affected by PCDH19 epilepsy enrolled by the following Centers: Roma OPBG (15 cases), Verona (9 cases) Milano Besta (8 cases), Firenze Meyer (5 cases), Brescia (5 cases), Padova (3 cases), Ancona (3 cases), Bologna (2cases2 cases), Roma Gemelli (2 cases) Reggio Emilia (1 case), Bosisio Parini (1 case), Milano S.Paolo (1 case).

The geographical distribution of patients is the following: Northen Italy 28, Central-South Italy 25, Switzerland 1 and Romania 1.

The age at last observation ranges from 1 to 27 years (median 10 years); the present age being from 5 to 32 years (median 13 years).

One patient is carrying a deletion, in 2 cases the result of genetic analysis is missing, the remaining 52 patients carry a PCDH19 gene mutation The age of seizures onset is within the 6th month of life in 10 cases, between 7 and 12 months in 27 cases; between 12 and 24 months in 13 cases and after 2 years of age in 5 patients.

The age at diagnosis is within 2 years from seizures onset in 22 subjects. In 13 subjects the disease was diagnosed very late (after the age of 10 years). The first seizure appears in a febrile condition in 28/55 cases. Seizure semeiology at onset is reported as: Generalized tonic clonic in 30 cases, Focal motor in 9, Focal non motor in 3, massive myoclonia in 2 and Undefined Clustered seizures in 11.

The psychomotor development at onset is within the normal range in 49 subjects, delayed or clearly impaired in 6 cases. At last follow-up the seizures persist in 26/50 subjects; respectively in 17/29 of those aged less than 12 years and 9/21 of the older subjects.

All subjects but one are taking chronic antiepileptic treatment (13 mono-therapy, 37 polytherapies).

At last follow-up 15 subjects still have a normal cognitive functioning, 7 patients present a borderline functioning, 28 patients have an Intellectual Disability of various degree (mild in 11, moderate in 11 and severe in 6 – these data are missing in 5 subjects). Twenty three patients suffer of behavioral disorders: autistic features in 6, oppositive disorder in 13, attention deficit in 10, impulse control disorder in 13, obsessive-compulsive disorder in 4.

The AA will discuss the correlations between the early clinical features and the global clinical outcome (epilepsy, cognitive and behavioral outcome).

Targeting the Gut Microbiota to Treat Epilepsy

Antonio Leo1, Emilio Russo1.

1 Department Science of Health, School of Medicine and Surgery, University of Catanzaro, Italy.

More than 65 million people worldwide are affected by epilepsy, “a heterogeneous group of neurological diseases characterized by an enduring predisposition to generate spontaneous recurrent seizures (SRSs) that are often accompanied by neuropsychiatric comorbidities”, according to International League Against Epilepsy (ILAE) definition [1]. Accordingly, there is an urgent need for innovative more effective and better tolerated therapies able to manage epilepsy and the issues related to it. Regarding to this aim, it is vital to both better understand the mechanisms underlying seizures onset and to identify patients at risk to develop epilepsy [2,3]. Very recently, there has been increasing interest on the role of peripheral inputs (stimuli) that can interfere with neurodevelopment and be thus possibly involved in brain diseases such as epilepsy. In tandem, there is a growing realization of the role of the gut-microbiota, a complex group of symbiotic microorganisms colonizing the gastrointestinal tract, in many aspects of brain and behaviour. Moreover, the microbiota represents one of the major sources of these peripheral stimuli [4]. Recently, due to the progress in sequencing technology, it has also been possible to recognize a bidirectional link between the microbiota and brain. This link is globally named the microbiota-gut-brain (MGB) axis, including neural, endocrine, metabolic and immune system/pathways. Interestingly, through this link, brain and gut produce signals influencing each other to coordinate functions in health and disease. Therefore, the gutmicrobiota complex might be involved in brain disorders including epilepsy by mediating the proexcitatory effect of peripheral inflammation through immune system activation (e.g. release of inflammatory mediators), modulating neural networks by production of neurotransmitters, SCFAs and key dietary amino acids and acting consequently on the excitation and inhibition (E/I) balance. Based on this background, a rationale to study the MGB axis and its potential role in epilepsy exists.

Nowadays, currently available preclinical and clinical studies, in spite of some limitations, seem to support the link between microbiota and epilepsy [5,6]. Regarding to this, a study performed in rats, reported the effect of a probiotic mixture (Lactobacillus rhamnosus, Lactobacillus reuteri, and Bifidobacterium infantis) administration in a pentylenetetrazole (PTZ)-induced model of kindling, substantively reduces seizure severity and epileptic activity compared to controls. The intervention had also a positive effect on the cognitive performances in all experimental groups. At odds, the concentrations of oxidant factors, nitric oxide and malondialdehyde, were reduced in the probioticadministered groups. Lastly, elevated levels of GABA have been found in brain, but only in the group with probiotics administration during kindling [7]. Regarding clinical evidence, few comparative and interventional studies have been so far performed on the link between gut microbiota and epilepsy.

Recently, it has been demonstrated a clear difference in gut microbiota composition between drug-resistant and drug-sensitive patients with epilepsy (PWE). In details, α-diversity was increased and gut microbiota was enriched in rare microbes in the drug resistant group (i.e. Verrucomicrobia), whereas bacteria population and α-diversity was similar between control and drugs-sensitive patients [8]. Accordingly, it could be hypothesized that the gut microbiota and its mediators could have a role in drug-resistant mechanisms. Surprisingly, it has also been shown that the ketogenic diet, used in the pediatric population with drugs-resistant epilepsy, changes the composition and function of the gut microbiome both in animal model of epilepsy and in PWE. Likewise, studies in mice model of epilepsy have demonstrated that the gut microbiota was necessary for the therapeutic effect of this diet [6]. Regarding the use of pre- or probiotics in PWE, one study has evaluated the effects of 4-months probiotic supplementations (a mixture containing 8 different bacterial subspecies of Lactobacillus, Bacteroides and Streptococcus species) in 45 patients with drug-resistant epilepsy. The study revealed that probiotics supplementation decreased seizure number and improved the Quality of Life score. However, authors did not perform any microbiota analysis before and after supplementation [9]. Furthermore, doubtful evidence also exist for the association between seizure incidence and antibiotic treatment [10]. Overall, multiple and not yet completely understood mechanisms could be involved in this bidirectional link and, although alterations in the gut microbiota and the epileptic process have not been directly investigated, the nature of these connections suggest their potential significance to epilepsy. Accordingly, several future studies are mandatory to establish whether microbe-based treatments can be effectively and securely used for clinical improvement of seizure frequencies, severity and epilepsy-related disorders. Likewise, future researches should be aimed at understanding whether the epilepsy itself shapes first the microbiota, or whether differences in microbiota-composition (during lifetime) can be the cause of seizures onset and maintenance.

Elucidating the relationship between the MGB axis and epilepsy could also lead to the discovery of innovative therapies as well as useful biomarkers of illness advancing the knowledge on the complex mechanisms underlying epileptogenesis and epilepsy themselves.


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