Welcome Speech - Francesca Squillante (Onlus Insieme per la Ricerca PCDH19)

Expanding the Clinical phenotype - Moderator Bernardo dalla Bernardina (University of Verona) - 9.45 Renzo Guerrini (Meyer Children Hospital, Firenze) - Clinical Update on

Antisense RNA silencing as a possible therapeutic option for EIEE-9.

Alfredo Brusco
Associate Professor in Medical Genetics
Department of Medical Sciences
University of Turin

Our group has recently worked on antisense RNA silencing therapy applied to neurodegenerative disorders. This technology is able to modulate the expression of genes, altering the amount of their proteins.
We propose a proof of concept approach for the use of antisense RNA silencing as treatment for Early Infantile Epileptic Encephalopathy type 9 (EIEE-9) which is associated with mutations in protocadherin 19 (PCDH19).
EIEE-9 is a paradoxical X-linked disorder in which females are severely affected and males with the same mutation are healthy. An explanation for this unusual mode of inheritance is “cellular interference”, which postulates that random inactivation of the X-chromosome in mutant females generates tissue mosaicism, which is pathogenic by altering cell-cell interactions. Our rationale is that gene silencing will mimic the PCDH19-knockout state of healthy mutant males, reverting the epilepsy and associated phenotypes.
Our work may provide novel insights into the pathogenic mechanisms of EIEE-9, and open the way for an innovative therapeutic approach for EIEE-9.

Stress and seizures: adrenal function in patients affected by PCDH19 mutation

Giuseppe Biagini, MD
Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, Modena, Italy.

Patients affected by protocadherin 19 (PCDH19)-female limited epilepsy (PCDH19-FE) were found to present reduced cortisol and pregnenolone sulfate serum levels when examined after puberty (Trivisano et al. 2017. Specifically, basal cortisol levels were approximately one-fourth of those found in age-matched controls, but they increased in a manner similar to that observer in controls when the adrenal gland was stimulated with adrenocorticotropic hormone (ACTH). A defect in cortisol production has been observed also in pre-pubertal girls. In this case, basal cortisol levels were similar to those measured in controls, but cortisol production was blunted under ACTH stimulation in patients affected by PCDH19-FE (approximately -25% vs controls). It is a common observation that exposure to stress may facilitate the occurrence of seizures, especially in patients affected by epilepsy (van Campen et al. 2014). It has recently been shown that patients who present an association between acute stress and seizures are characterized by reduced adrenal response when compared to those less sensitive to stress (van Campen et al. 2015). Thus, a reduced capability to produce cortisol apparently results in enhanced seizure susceptibility to environmental challenges. Although studies on the adrenal response to acute stress in patients with PCDH19-FE are lacking, the observed response to ACTH in pre-pubertal PCDH19-FE girls suggests that also this type of epilepsy may be aggravated by exposure to stressors, thus indicating that attainment of a normal adrenocortical function could be a therapeutic goal in this condition.

Trivisano M., Lucchi C., Rustichelli C., et al. Reduced steroidogenesis in patients with PCDH19-female limited epilepsy. Epilepsia 2017, 58: e91-e95.

van Campen JS, Jansen FE, de Graan PN, et al. Early life stress in epilepsy: a seizure precipitant and risk factor for epileptogenesis. Epilepsy Behav 2014; 38: 160-171.

van Campen JS, Jansen FE, Pet MA, et al. Relation between stress-precipitated seizures and the stress response in childhood epilepsy. Brain 2015; 138: 2234-2248.

Altered sleep in a conditional mouse model of PCDH19 loss: an EEG study

S. Landi, D. Lamers, V. Pillai, M. Passafaro, S. Bassani, G.M. Ratto. NEST, Istituto Nanoscienze CNR and Scuola Normale Superiore, Pisa, Italy. Istituto di Neuroscienze CNR, Milano, Italy.

The gene PCDH19 encodes protocadherin 19, a protein whose function is basically unknown. In heterozygous females, inherited or de novo mutations of this gene can result in a syndrome with seizure onset in infancy and mild to severe intellectual impairment, at times accompanied by autistic features (Depienne et al., 2009; Dibbens et al., 2008; Marini et al., 2010). Affected females exhibit a wide clinical spectrum that often includes sleep disorders with difficulties in sleep onset and frequent awakenings. Although sleep impairment severely affects the quality of life of patients and caretakers and negatively impacts cognitive functions, it has received relatively little attention at the levels of both basic and clinical research. Sleep is an active process gradually moving along different phases that can be recognized by specific electroencephalographic signatures. Deep sleep (slow wave sleep) is characterized by the lack of REM activity and by a very synchronized EEG producing slow waves in a frequency range of 0.4-4 Hz (Greene and Frank, 2011). These oscillations of the extracellular field potential correspond to the synchronized transition of neurons from a hyperpolarized potential (down states) - corresponding to periods of abolished activity and neuronal resting - to a depolarized potential (up states), when neurons have a larger probability of firing. Slow wave sleep is important for memory consolidation (Walker, 2009) and essential for processes leading to synapse normalization.

Given this background, we consider essential to understand whether the loss of PCDH19 affects slow wave activity in the murine model. In this study we have performed the first in vivo electrophysiological recordings of a conditional mouse model for PCDH19 and, specifically, we studied the structure of sleep induced by urethane anesthesia that is known to maintain slow wave oscillations (Sanchez-Vives and McCormick, 2000; Steriade et al., 1993). In these experiments,  we created a limited cortical territory characterized by mosaic expression of PCDH19 by injecting at P1 an adeno-associated viral vector carrying CRE and a floxed fluorescent reporter. This treatment generated a normal brain that contained a patch of cortex where a fraction of infected neurons carried the PCDH19 loss of function mutation. The electrophysiological phenotype of these mice was assessed at P30 or P60 by means of simultaneous extracellular recordings from the mutated cortical patch and the corresponding area in the opposite hemisphere. Since slow wave activity is strongly synchronized between the two hemispheres (Petrucco et al., 2017), we could compare directly the local field potential (LFP) recorded simultaneously in the normal and mutated region. Spectral analysis of the LFP demonstrated that the power of the slow wave band was affected by the loss of PCDH19, and that up states were shorter. Occasionally, we observed episodes of hypersynchronous activity focally centered in the PCDH19 mutated territory. Finally, about 50% of recorded mice presented interruption of slow wave sleep in the injected cortex, while it continued in the control side. During these episodes, power of γ activity (30-80 Hz) in the injected side increased substantially, indicating a state of localized wakefulness.

Our study is the first attempt at studying the cellular and neurophysiological basis of hyper-excitability

and sleep in parallel in mouse and pave the way for a similar exploratory effort to be performed in patients. In perspective, we expect that these data might lead to a mechanistic understanding of the sleep phenotype of PCDH19 patients and its relationship with cognitive impairment.

Human Neuronal Model of PCDH19-linked EIEE9

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Jack M. Parent, Xi Plummer, Andrew Tidball, Wei Niu
Department of Neurology, University of Michigan Medical Center

Protocadherin-19 (PCDH19)-associated Female Limited Epilepsy (FLE), also known as Early Infantile Epileptic Encephalopathy type 9 (EIEE9), is a pediatric epilepsy that is caused by mutations in the PCDH19 gene on the X-chromosome. PCDH19 is a member of the cadherin superfamily of cell-cell adhesion molecules that has been shown to participate in homophilic binding. While the normal function of PCDH19 remains unknown, evidence suggests that other members of the protocadherin family are critical for neural development. A key feature of PCDH19 FLE is its unusual inheritance pattern. Females who carry heterozygous PCDH19 mutations are affected with seizures and cognitive impairment while mutation-carrying males are largely asymptomatic. Some affected males with mosaic mutations have also been reported. This phenomena support the cellular interference hypothesis, which posits that X-inactivation in females with heterozygous PCDH19 mutations leads to the expression of two different cell populations, one expressing wild type and the other mutant PCDH19, and that these two cell populations interact aberrantly to cause the disorder. To test this hypothesis and explore EIEE9 mechanisms in human neurons, we generated induced pluripotent stem cells (iPSCs) from two female patients with clinical EIEE9 and associated pathogenic PCDH19 mutations. We found that cortical-like excitatory neurons differentiated from EIEE9 patients have atypical outgrowth of processes and accelerated maturation, as well as increased excitability on multielectrode array (MEA) recordings. We also found that inhibitory interneurons derived from EIEE9 patient iPSCs have atypical morphology and increased MEA activity. We generated additional iPSCs from an unaffected male carrier and also used CRISPR gene editing to generate a PCDH19-null male iPSC line with isogenic control. We mixed these lines with wildtype (WT) PCDH19-expressing controls to recapitulate the mosaic male phenotype in vitro. When we differentiated mixed WT/PCDH19-null or carrier male iPSCs into cortical-like excitatory neurons, we found atypical segregation of the two cell populations. Ongoing work involves growing 3-D cerebral organoid cultures and generating a tagged PCDH19 allele in human PSCs using CRISPR. Our results support the cellular interference hypothesis and suggest that atypical cell-cell interactions of developing cortical neurons alter brain development and contribute to epileptogenesis in EIEE9.

The dual role of the female epilepsy protein pcdh19 at the synapse and the nucleus

Silvia BASSANI – CNR Istituto di Neuroscienze, Milano

Mutations in the PCDH19 gene on chromosome X (Xp22.1) cause a female-limited epilepsy (PCDH19 Female Epilepsy, PCDH19-FE) that is frequently associated with intellectual disability and autism.

Epilepsy affects heterozygous females and spares hemizygous males, with the exception of few mosaic males. In adulthood, the non-epileptic symptoms become the most disabling issues.

Since the discovery of its involvement in PCDH19-FE in 2008, PCDH19 has rapidly become the second most clinically relevant gene in epilepsy after the Dravet Syndrome causative gene SCN1A. To date, more than 100 inherited or de novo mutations have been reported in PCDH19, including point mutations and partial or whole gene deletions. Despite this, a comprehensive understanding of PCDH19 biological function as well as its role in the PCDH19-FE pathogenesis is lagging behind.

PCDH19 encodes for protocadherin-19 (PCDH19) that is a calcium-dependent cell-adhesion molecule belonging to the non-clustered elta2-protocadherin subclass of the cadherin superfamily. PCDH19 has six conserved extracellular cadherin repeats, a transmembrane region and an intracellular C-terminus (CT).

We found that PCDH19 is expressed at both excitatory and inhibitory synapses of hippocampal neurons and regulates neuronal excitability via two distinct mechanisms: by modulating GABAAR transmission at synapses and gene expression in the nucleus.

Our data indicate that PCDH19 CT interacts with the GABAAR alpha subunits; upon PCDH19 shRNA-mediated downregulation, GABAAR surface expression is reduced and fast GABAergic transmission impaired. Since PCDH19 expression increases throughout embryonic development and peaks in the first postnatal days, when GABA signaling orchestrates neuronal migration and arborization, we downregulated PCDH19 via shRNA in utero electroporation in rat hippocampus. Consistently with an impairment of GABAergic transmission, PCDH19 downregulation during brain development affects the migration and morphological maturation of pyramidal neurons and increases rat’s seizure susceptibility.

In addition, upon sustained NMDAR activation, PCDH19 CT is cleaved by gamma secretase and enters the nucleus. In the nucleus, PCDH19 CT associates with the CoREST complex and represses the transcription of immediate early genes (IEGs), which are key regulators of neuronal plasticity and excitability. Conversely, PCDH19 shRNA-mediated downregulation increases IEGs transcripts. Notably, PCDH19 cleavage occurs in vivo upon epileptogenic stimuli, as demonstrated by CT generation in hippocampal homogenates from mice that experienced pilocarpine induced-seizures. We hypothesize that PCDH19 cleavage might represents a homeostatic mechanism in response to strong neuronal activation that prevents IEGs overactivation.

Finally, we generated a conditional PCDH19 KO mouse model by using LoxP-Cre technology. Our preliminary data suggest that Cre-mediated PCDH19 inactivation is associated with an epileptic phenotype that we will investigate in detail with a focus on mosaic PCDH19 expression and its role on PCDH19-FE pathogenesis.

Altogether, PCDH19 emerges as a new GABAAR binding partner that controls GABAergic transmission and simultaneously exerts a homeostatic control of excitability via IEGs expression regulation, thus suggesting new pathogenic mechanisms for PCDH19-FE. 

Abnormal cell sorting underlies the unique X-linked inheritance of PCDH19 Epilepsy

Daniel T. Pederick1, Kay L. Richards2, Sandra G. Piltz1, Simone A. Mandelstam3, 4, 5, Russell C. Dale6, Ingrid E. Scheffer2, 7, Jozef Gecz1, 8, 9, Steven Petrou2, James N. Hughes1 and Paul Q. Thomas1, 9*

*Presenting author

1School of Biological Sciences and Robinson Research Institute, The University of Adelaide, Adelaide, South Australia 5005, Australia

2Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Melbourne, Victoria 3010, Australia

3Department of Paediatrics, The University of Melbourne, Melbourne, Victoria 3010, Australia

4Department of Radiology, The University of Melbourne, Melbourne, Victoria 3010, Australia

5Department of Medical Imaging, Royal Children's Hospital, Florey Neurosciences Institute, Parkville, Victoria 3052, Australia

6Institute for Neuroscience and Muscle Research, University of Sydney

7University of Melbourne, Austin Health and Royal Children’s Hospital, Victoria, 3084, Australia

8School of Medicine, The University of Adelaide, Adelaide, South Australia 5005, Australia

9 South Australian Health and Medical Research Institute, Adelaide, South Australia 5000, Australia

X-linked diseases typically exhibit more severe phenotypes in males than females. In contrast, Protocadherin 19 (PCDH19) mutations cause epilepsy in heterozygous females but spare hemizygous males. The cellular mechanism responsible for this unique pattern of X-linked inheritance is unknown. We show that PCDH19 contributes to highly specific combinatorial adhesion codes such that mosaic expression of Pcdh19 in heterozygous female mice leads to striking sorting between WT PCDH19- and null PCDH19-expressing cells in the developing cortex, correlating with altered network activity. Complete deletion of PCDH19 in heterozygous mice abolishes abnormal cell sorting and restores normal network activity. Furthermore, we identify variable cortical malformations in PCDH19 epilepsy patients. Our results highlight the role of PCDH19 in determining specific adhesion codes during cortical development and how disruption of these codes is associated with the unique X-linked inheritance of PCDH19 epilepsy. 

Jozef Gecz (University of Adelaide) - Cell-based modelling of PCDH19 epilepsy

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Ganaxolone, a novel investigational treatment for PCDH19 Epilepsy: Results of a phase 2 open-label study

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Lorianne K. Masuoka, MD, Julia Tsai, PhD, Jaakko Lappalainen, MD
Marinus Pharmaceuticals

Background and Mechanistic Rationale:

PCDH19 pediatric epilepsy is a rare, severe and debilitating epilepsy characterized by early-onset cluster seizures, cognitive and sensory impairment, and behavioral disturbances, with no approved treatments. Children with PCDH19 epilepsy have reduced expression of the AKR1C3 gene and associated low levels of the naturally occurring brain neurosteroid allopregnanolone. Ganaxolone is a modified version of allopregnanolone, in which the addition of a methyl group prevents it from back converting to hormonally active progesterone, enabling chronic use. This is unlike allopregnanolone, which readily back converts to progesterone, making it a poor candidate for chronic use. Like allopregnanolone, the role of ganaxolone is to positively modulate both specific synaptic and extra-synaptic GABA-A receptors in the brain to greatly decrease excitability of neurons. Unlike the benzodiazepine GABA receptor, the neurosteroid receptors do not downregulate or internalize, therefore, tolerance does not develop and there is no loss of effect or need for increasing doses on the basis of receptor density loss. This gives ganaxolone (GNX) the potential for providing durable responses.

GNX has anticonvulsant activity with an acceptable safety and tolerability profile in doses of 900 to 1800 mg in adults and children. We hypothesized that GNX could reduce the frequency of seizures in children with treatment-refractory PCDH19 epilepsy, and potentially increase seizure free days.


Eleven PCDH19 epilepsy patients (aged 5-16 years, median age 8) with severe, treatment-resistant seizures, all receiving stable doses of AEDs were enrolled at 7 centers in the US and Italy. Patients received oral GNX, either titrated to a maximum of 63 mg/kg/day if under 30kg, or 1800 mg/day on an open label basis. The primary objective was to study safety and tolerability of GNX and the primary efficacy endpoint was median percentage change in 28-day seizure frequency at 26 weeks. The secondary efficacy endpoint was median percentage change in 28-day seizure free days at 26 weeks. The efficacy analyses were by modified intention to treat (>= 1 seizure during baseline). Patients with a good response to treatment were given the option to enroll into a 52-week open label extension period.


At baseline, the patients had a median 28-day seizure rate of 19, but this was highly variable (range 5-112). At 26 weeks, the patients experienced a median 26% reduction in seizures. The baseline 28-day seizure free rate was 20 days (range 2-26 days) with a median increase in 14%, representing an increase in 4 seizure free days per 28 days. The difficulty in this measure is that the patients generally had their seizures in clusters and mostly experienced seizure-free days.

Treatment with GNX was generally safe and well tolerated. One subject developed a rash that was possibly associated with GNX. The most common, non-epilepsy related adverse events, included somnolence (36%), headache (27%) and fatigue (27%).

Response to ganaxolone was highly variable with 4 subjects achieving a very high response, and 5 subjects with a poor (no or worsening) response

Conclusions and Discussion

Treatment with ganaxolone resulted in excellent reduction in seizure frequency in approximately half the cohort. Most responders experienced long term (6 month) treatment effect. The durability of response further validates a lack of tolerance to treatment effect, despite the GABA-A stimulation mechanism of action. The drug was very reasonably well tolerated, and the degree of treatment effect warrants further evaluation.

The principal barriers to further study in children with PCDH19 epilepsy is the high degree of variability of the seizures in these children and the extreme rarity of the condition. As seen with the dramatic improvement in seizure reduction rate when focal seizures are eliminated from consideration, proper selection of patient demographics will be extremely important for improving sensitivity in a study necessarily enrolling a relatively small number of subjects. The availability of a patient registry with longitudinal data that enable clinical researchers to understand the natural history of the disease as well as identify the locations of potential study patients is critical to developing new therapeutics for children with PCDH19 epilepsy.


Pham D.1, 2, Schulz R.1, Kolc K.1, Corbett M.1, 2, Epi4k Consortium6, Epilepsy Phenome Genome Project7, Pitson S.3, Petrovski S.4, Pitman M.3 and Gecz J.1, 2, 5

1Adelaide Medical School, The University of Adelaide, Adelaide 5005, Australia. 2Robinson Research Institute, The University of Adelaide, Adelaide 5006, Australia. 3Centre for Cancer Biology, University of South Australia, Adelaide 5000, Australia. 4Department of Medicine, Austin Health and Royal Melbourne Hospital, The University of Melbourne, Melbourne 3050, Australia. 5South Australian Health and Medical Institute, Adelaide 5000, Australia. 6 7

Mutations in the cell adhesion/estrogen receptor transcription co-regulator molecule protocadherin 19 (PCDH19) cause girls clustering epilepsy (GCE) and is the second most frequent single gene cause of epilepsy. The genetics of this disorder is unusual. PCDH19 is on the human X-chromosome. Heterozygous females are affected, while hemizygous males are not, contradicting normal X-linked disorders. Males with a postzygotic somatic mutation in PCDH19 are also affected. Cellular mosaicism due to X-chromosome inactivation in females is the likely driver of the disorder. Our research shows that estrogen receptor alpha (ERα) is involved in the pathogenesis of PCDH19-GCE. We performed integrative in silico (26 different bioinformatics tools, protein structure modelling) and experimental (ERE-LUC reporter assays, RT-qPCR, Western blotting of ERα endogenous gene targets e.g. OXTR, AKR1C3) evaluation of the functional impact of 31 different PCDH19 missense variants. These included published PCDH19-GCE disease-causing variants (n=9), benign higher frequency (ExAC or gnomAD) variants (n=7) and variants of unknown significance (VOUS) (n=15). Out of 31 variants, 21 with protein structure availability were tested using all pathogenicity assessment tools. We achieved accurate prediction for 17/21 variants including 78% (7/9) published PCDH19-GCE, 100% (3/3) frequent variants and 78% (7/9) VOUS in the concordance of in silico and experimental results. By integrating selected in silicoand experimental tools, we can now significantly improve the interpretation of disease-relevance of PCDH19 missense variants. This is crucial for clinical trials and personalised medicine for PCDH19-GCE patients.

Neuropsychiatric profile of PCDH19 epilepsy: a systematic review and meta-analysis

Kristy Kolc -  Neurogenetics Laboratory - Adelaide Medical School - University of Adelaide

Epilepsy and Mental Retardation Limited to Females (EFMR) is an infantile onset disorder characterized by clusters of seizures. EFMR is due to mutations in the X-chromosome gene PCDH19 and is underpinned by cellular mosaicism due to X-chromosome inactivation in females or somatic mutation in males. This review characterizes the neuropsychiatric profile of the disorder and examines the association of clinical and molecular factors with neuropsychiatric outcomes. Data were extracted from 38 peer-reviewed original articles including 271 individual cases. We found that seizure onset ≤ 12 months (M = 2.31, SE = 0.12) was significantly associated (p = 9.501x10-7) with more severe intellectual disability compared with onset > 12 months (M = 1.17, SE = 0.20). We identified two recurrent variants p.Asn340Ser and p.Tyr366Leufs*10, occurring in 25 (17 unrelated) and 30 (11 unrelated) cases, respectively. PCDH19 mutations were associated with psychiatric comorbidities in approximately 60% of females, 80% of affected mosaic males, and reported in nine hemizygous males. Hyperactive, autistic, and obsessive-compulsive features were most frequently reported. There were no phenotype-genotype associations in the individuals with recurrent variants or the group overall. Age at seizure onset can be used to provide more informative prognostic counselling. 

Human induced pluripotent stem cell model of PCDH19-girls clustering epilepsy reveals roles for this protocadherin in regulation of neuronal polarity and differentiation. 

Claire Homan1,2, Lachlan Jolly2,4, Stanley Tan2,4, Paul Thomas1,2, Ernst Wolvetang3 and Jozef Gecz1,2,4,5 

1 Medicine, The University of Adelaide, Australia, 2 Robinson Research Institute, The University of Adelaide, Australia, 3 The University of Queensland, Australia, 4 South Australian Health and Medical Research Institute, Adelaide, Australia 

Human induced pluripotent stem cells (hiPSC) provide a unique opportunity to study neurological disorders using disease relevant cells usually unattainable from the patients. We have generated hiPSCs from PCDH19-Girls Clustering Epilepsy (PCDH19-GCE) patient skin fibroblasts. PCDH19-GCE is a female specific epilepsy associated with a spectrum of neurodevelopmental and behavioural problems. It is caused by a variety of loss of function mutations in an X-chromosome gene, Protocadherin19 (PCDH19), with 100s of cases reported to date. PCDH19-GCE is a disorder of cellular mosaics, that is females who undergo X-inactivation and males with somatic mosaicism (3 cases reported). In addition to generating PCDH19-GCE hiPSC we also developed an optimised protocol of cortical development based on a model of dual-SMAD inhibition, which we found reproducible across multiple PSC lines. Using this protocol and PCDH19-GCE hiPSCs, we modelled PCDH19-GCE by replicating the cellular mosaicism of the patient brain. We found that PCDH19 is important for the maintenance of neural stem cell polarity during cortical development, with loss-of-function mutation in PCDH19 being able to form neural rosettes, but unable to properly maintain these structures as evidenced by a 46% decrease in lumen size and a 63% decrease in the number of polarised structures/rosette area, compared to wildtype. A significant increase in the number of neurons at the edge of the rosettes was also observed suggesting increased neuronal differentiation. We also found that PCDH19 regulates axonal extensions with mutant neurons having a 60% increase in primary neurite length. Taken together this work potentially identifies novel roles for PCDH19 in neuronal polarity during cortical development and neuronal differentiation and morphology. 

Round Table on Cognitive and associated behavioral disturbances - Raffaella Cusmai (OPBG, Roma), Bernardo dalla Bernardina (University of Verona), Nilika Singhal (UCSF, San Francisco)

Round Table on Therapeutic Treatments (Anecdotal and hard evidence) Carla Marini (Meyer Children Hospital, Firenze), Francesca Ragona (C. Besta, Milano), Marina Trivisano (OPBG, Roma)