• Modeling PCHD19 Epilepsy with Patient-Derived Cells and Rat In Utero Electroporation – Jack Parent

PCDH19-Related Epilepsy is caused by loss-of-function mutations in the PCDH19 gene located on the X-chromosome. The disorder manifests with seizure onset in infancy or early childhood as well as cognitive impairment. This X-linked condition has an unusual inheritance pattern as it only affects females while carrier males are spared. This inheritance pattern has been postulated to arise from random X-inactivation in females where mutant and wild type PCDH19-expressing cells interact abnormally to produce the disease phenotype. This cellular interference theory is supported by the single reported case of PCDH19-Related Epilepsy in a mosaic male, as well as a similar inheritance pattern in another disorder, craniofrontonasal syndrome (CFNS).

Although very little is known about 
the function of PCDH19, it likely plays an important role in neural development. PCDH19 also binds to N-cadherin (NCAD) during development to form a complex with homophilic binding activity. To better understand the mechanisms underlying PCDH19-Related Epilepsy, we generated induced pluripotent stem cells (iPSCs) from affected subjects and differentiated them into neural progenitors and neurons. We found abnormal neural rosette formation and aberrant expression of developmental transcription factors in neural progenitors and cortical-like excitatory neurons derived from iPSCs with mutant PCDH19. Differentiated neurons also appeared to have aberrant neurite patterning and altered NCAD subcellular localization. To complement studies of patient-derived cells with an animal model, we are using in utero electroporation (IUE) combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas) genome editing to generate a mosaic deletion of Pcdh19 in rat embryonic forebrain ventricular zone progenitors.

This work should 
advance our understanding of the role that PCDH19 plays in neural development and epileptogenesis, and provide both cell-based and animal model platforms to test novel treatments for preventing seizures and intellectual disability in PCDH19-Related Epilepsy.

  • Molecular characterization of PCDH19 in rat hippocampal neurons - Maria Passafaro

Female-limited Epilepsy (FLE), also known as Early Infantile Epileptic Encephalopathy-9 (EIEE9), is a debilitating neurological condition characterized by early onset seizures followed by intellectual disability and autistic features. Recently PCDH19, which encodes for protocadherin-19 (PCDH19), has been identified as the causative gene. The protocadherin family is the largest group within the cadherin superfamily of calcium-dependent cell adhesion molecules.

PCDH19 is present in rat brain and, consistent with its role in cognitive function and epilepsy aetiology, is highly expressed in cortex and in the hippocampus. In hippocampal neurons, PCDH19 localizes at synapses and is detectable both on the plasma membrane and in intracellular compartments. Similarly, when overexpressed in COS-7 cells PCDH19 distributes in vesicle-like structures inside the cells and is recruited at cell-cell contacts. By yeast two-hybrid screening we found a direct interaction between the intracellular C-terminal tail of PCDH19 and the alpha1 subunit of GABAA receptor (GABAAR). Interestingly, several pathogenic mutations identified so far in patients are predicted to generate truncate variants of PCDH19, in which the region required to bind GABAAR alpha1 subunit is loss.  

PCDH19 and alpha1 overexpressed in HEK293 cells coimmunoprecipitate and colocalize in vesicle-like structures. In hippocampal neurons PCDH19 and GABAAR alpha1 subunit partially colocalize along dendrites and coimmunoprecipitate. Notably, PCDH19 shRNA-mediated downregulation reduces GABAAR alpha1 surface expression.

We hypothesize that PCDH19 is involved in GABAAR alpha1 subunit trafficking and that PCDH19 mutations might compromise inhibitory synapse transmission.

  • PCDH19 and the establishment of planar cell polarity in iPS-derived developing neurons in a X-lonked model of Epilepsy - Claudia Compagnucci

PCDH19 (Protocadherin 19), a member of the cadherin superfamily, is involved in the pathogenic mechanism of a X-linked model of Epilepsy. The biological function of PCHD19 in human neurons and during embryogenesis is currently unknown. Therefore, we decided to use the model of the induced pluripotent stem cells (iPSCs) to characterize the location and timing of expression of PCDH19 during cortical neuronal differentiation. We used human iPSCs, obtained from skin fibroblasts of healthy subjects, and iPSC-derived neurons to recapitulate the role of PCDH19 in neuronal differentiation. In particular we developed adherent culture protocols leading to the formation of Neuronal Stem Cells (NSC) arranged radially around a lumen termed “neural rosettes”. Importantly the structure of the neural rosette resembles the polar organization of the neural tube in the developing human embryo (with a lumen indicating the basal part of the polarized neuronal progenitor cell). Interestingly PCDH19 has a polarized location in the rosette and in mature neuronal cultures. Our data suggests that PCDH19 might have a role in instructing apico-basal polarity of the progenitor cells, thus guiding the development of a properly organized human brain.

  • Mosaic PCDH19 point mutation in male patients

Alessandra Terracciano, Marina Trivisano, Raffaella Cusmai, Lucia Fusco, Claudia Compagnucci, Enrico Bertini, Federico Vigevano, Nicola Specchio.

PCDH19 gene mutations have been recently associated with an epileptic syndrome characterized by focal and generalized seizures. PCDH19 gene, located on the X chromosome (Xq22.1), has an unusual X-linked inheritance with a selective involvement for female subjects. A cellular interference mechanism has been hypothesized and males can manifest epilepsy only in the case of a mosaicism.

So far about one hundred female patients, and only one symptomatic male have been 
described. Using targeted Next Generation Sequencing (NGS) approach we found a PCDH19 gene point mutation in two male patients; the system allowed us to verify that the two c.1352 C>T; p.(P451L) and c.918C>G; p.(Y306Ter) variants occurred in mosaic status. In both cases the minor allele, preliminary filtered out by alignment software, accounted for about 10% of total DNA. Both mutations were confirmed by Sanger sequencing, with the detection of a low electropherographic peak, close to the minimum resolution level. Up to now, the traditional molecular screening for PCDH19-related epilepsy has been targeted to all females with early-onset epilepsy with or without cognitive impairment and family history. Male patients were generally excluded. By NGS approach, we were been able to detect, mosaic PCDH19 point mutation in two male patients with a clinical picture suggestive of PCDH19 related epilepsy. This finding expands mutational spectrum of the disease and opens new chances for the molecular diagnoses in patients with a peculiar type of epilepsy that remains undiagnosed in male.

  • PCDH19; from mutation to molecular mechanism(s) to treatment. Are we there yet? J Gecz & collaborators 

Robinson Research Institute and School of Paediatrics and Reproductive Health, The University of Adelaide, Adelaide, Australia

Female epilepsy due to mutations of the PCDH19 gene (PCDH19-FE) has been recognized as a clinical entity since 2008/2009 when the PCDH19 gene disease causing mutations were identified in familial and sporadic cases. By now PCDH19-FE encompasses a broad clinical spectrum from infantile epileptic encephalopathy resembling Dravet syndrome to epilepsy with or without intellectual disability and autism spectrum disorders. PCDH19-FE is clinically variable and some girls with PCDH19 mutations are not presenting with the disorder. Until now there was no test available to determine if a particular DNA variant in PCDH19 is disease causing or not. We have used multiple approaches to study molecular pathology of PCDH19-FE. We found that PCDH19 regulates nuclear hormone receptors and as such it may regulate gene expression. This also allowed us to evaluate the functional consequence of various PCDH19 DNA and protein variants and as such test for PCDH19 protein function. This novel ‘gene expression regulatory’ function of PCDH19 together with its proposed role in cell-cell adhesion provided a possible molecular mechanism of PCDH19-FE. Among the genes we found originally altered in patients with PCDH19-FE were the ARK1C genes, coding for enzymes involved in the production of neurosteroids allopregnanolone, estradiol and testosterone. Based on this research a small scale open label clinical trial using ganaxolone, a synthetic analog of allopregnanolone, has been designed by Marinus Pharmaceuticals Inc. (USA) and is currently (June 2015) ongoing. Interestingly, ganaxolone is also being trialed in the fragile X syndrome, where in the animal model it ablated seizures after one administration. We hope that these clinical trials and ongoing research will together significantly improve our understanding of the underlying mechanism(s) of the PCDH19-FE and open up opportunities for effective treatment.

This work is supported by NHMRC Program grant 628952 and NHMRC senior principal research fellowship 1041920 to JG, and Insieme per la Ricerca PCDH19 – ONLUS Foundation grants (Italy).

  • Neurosteroids as endogenous modulators of epileptic seizures 

Giuseppe Biagini1,2, Cecilia Rustichelli3, Chiara Lucchi1,2, Stefano Meletti1,2 

1Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy; 2Department of Neurosciences, NOCSAE Hospital, AUSL Modena, Modena; 3Department of Life Sciences, University of Modena and Reggio Emilia, Modena.

Neurosteroids are a family of steroidal molecules which produce a variety of biological effects, mainly by interacting with the γ-aminobutyric acid receptor A (GABAA). Among neurosteroids, allopregnanolone and tetrahydrodeoxycorticosterone are positive modulators of GABAA receptors which were shown to counteract seizures in animal models. However,  it is still to be defined the role of neurosteroids both in patients affected by epilepsy and in animal models, in which epileptic seizures can be reproduced. By using finasteride, an irreversible inhibitor of the enzyme 5α-reductase, we found that allopregnanolone cerebral levels were significantly reduced in rats treated with the convulsant pilocarpine. Interestingly, this decrease was followed by enhanced epileptogenesis. We also described a case of antiepileptic drug resistance in a patient that was maintained on finasteride to treat a dermatological disorder: seizures ceased by withdrawing finasteride. These findings point to a modulatory role of allopregnanolone and other neurosteroids on epileptic seizures.

  • Introduction of ganaxolone as a treatment for PCDH19 female pediatric epilepsy - Albena Patroneva

Marinus is conducting a multicenter, open-label proof-of-concept trial of ganaxolone in children with PCDH19 Female Pediatric Epilepsy.  Ganaxolone is a synthetic analog of allopregnanolone, an endogenous allosteric modulator of GABAA receptors in the central nervous system. Ganaxolone has potency and efficacy comparable to allopregnanolone in activating synaptic and extrasynaptic GABAA receptors at a site distinct from the benzodiazepine site.  Protocadherin 19 is a transmembrane protein of calcium-dependent cell-cell adhesion molecules that is strongly expressed in neural tissue, and which appears to be related to synaptic transmission and formation of synaptic connections during brain development.  Research suggests impairment of GABAergic signaling both at the agonist and receptor levels is present in girls with PCDH19 mutations and pediatric-onset epilepsy.

This Phase 2 study is designed to enroll approximately 10 female pediatric patients with a confirmed PCDH19 genetic mutation. After establishing baseline seizure frequency, patients will be treated with ganaxolone administered as either oral liquid suspension or capsules for up to 26 weeks.  Those who respond to treatment with ganaxolone will be offered additional 1 year open label treatment. The objective of the study is to evaluate the efficacy of ganaxolone to reduce seizure frequency per 28 days relative to baseline. Preliminary results from the first group of patients will be discussed.

  • Potential New Treatment Options for PCDH19 epilepsy - Joseph Sullivan

The variable phenotype of girls with PCDH19 mutations results in some patients having pharmacoresitant epilepsy that does not respond to conventional anti-epileptic drugs.  An open-label trial with Ganaxolone in children with medically refractory epilepsy caused by PCDH19 mutations is underway and preliminary observations from this trial will be discussed.

  • Cannabidiol in Treatment resistant epilepsy in children.  Can we extrapolate the data to children with PCDH19? - Stephen Wright, Chief Medical Officer, GW Pharmaceuticals.

The development of cannabidiol in the treatment of several drug-resistant epilepsies of childhood is  based both on empirical evidence of its effectiveness when used as an extract of cannabis 1,2 but also as a consequence of a substantial body of pre-clinical data.  Its mechanism of action seems to be related to its ability to inhibit neuroexcitation through a mechanism that involves calcium flux in the pre-synaptic neurone3, by its ability to inhibit adenosine re-uptake with a consequent anti-inflammatory effect in the CNS, and possibly by its antagonism at the GPR55 receptor. These multiple mechanisms of action may allow for efficacy across a number of seizure and epilepsy types.  

In addition, CBD shows very little if any capacity to produce cognitive or motor impairment in animal models of epilepsy. Initial therapeutic interest in CBD has focussed on Dravet syndrome.   PCDH19 epilepsy shows some similarities with Dravet syndrome, although it is clear that the syndrome is distinct, and defined by mutations in the PCDH19 gene.  

CBD is effective across a range of pre-clinical models of epilepsy and is currently being used by a number of young patients with treatment-resistant epilepsies in a compassionate use (Expanded Access INDs) in the USA. To date, 137 patients (mean age 11 years [range 2-26]), with a variety of diagnoses, have received at least 12 weeks of continuous treatment with CBD.  The mean baseline seizure frequency was 29 seizures per month, and  patients were taking a mean of 2.8 AEDs.   After 12 weeks of treatment, monthly seizure frequency reduction of 54% was seen across the whole population, with a reduction of 65% in 25 children with Dravet syndrome.  In this program, Epidiolex has been well tolerated, with a withdrawal from treatment due to adverse events of less than 5%.  There is preliminary evidence of improvement in cognition, especially in a subset of children with tuberous sclerosis complex4.

The observation that Epidiolex has shown evidence of efficacy across a range of seizure types, and has shown improvements in cognitive function, suggests that it might have therapeutic effects in other treatment-resistant epilepsies.   


1.   Porter B, Jacobson C.  Report of a parent survey of cannabidiol-enriched cannabis use in pediatric treatment-resistant epilepsy.  Epilepsy Behav.  2013; 29: 574-7

2. Pelliccia, A., Grassi, G., Romano, A., & Crocchialo, P. (2005). Treatment with CBD in oily solution of drug-resistant paediatric epilepsies. Congress on Cannabis and the Cannabinoids, Leiden, The Netherlands: International Association for Cannabis as Medicine, p14.

3. Ryan,D. et al.(2009) Cannabidiol targets mitochondria to regulate intracellular Ca2+ levels. J. Neurosci>29, 2053-2063.

4. Geffrey AL, Pollack SF, Paolini JL, Bruno PL, Thiele EA.   Cannabidiol (CBD) Treatment for Refractory Epilepsy in Tuberous Sclerosis Complex (TSC).   December 2014.  At The American Epilepsy Society annual meeting, Seattle, USA.

  • Alternative treatments for Drug-Resistant Epilepsy - Pasquale Striano

Complementary and alternative medicine (CAM) has become much in vogue, and CAM practitioners have increased in tandem with this. The trend of using CAM for treating epilepsy does not differ from that in other medical conditions, with nearly one half of patients using CAM. In this article we review the major complementary and alternative medicines used for treatment of epilepsy. They include mind-body medicines such as reiki and yoga; biologic-based medicine such as herbal remedies, dietary supplements, and homeopathy; and manipulative-based medicine such as chiropractic. In the available literature, there is a sense of the merit of these therapies in epilepsy, but there is a paucity of research in these areas.

Individualized therapies such as homeopathy and reiki cannot be compared with medicines in a conventional pharmaceutical model. More effort needs to be put into future trials, with the assistance of qualified CAM professionals to ensure conformation to their therapeutic principles.