Novel and de novo mutation of PCDH19 in Girls Clustering Epilepsy

Abstract Background PCDH19 has become the second most relevant gene in epilepsy after SCN1A. Seizures often provoked by fever. Methods We screened 152 children with fever‐sensitive epilepsy for gene detection. Their clinical information was followed up. Results We found eight PCDH19 point mutations (four novel and four reported) and one whole gene deletion in 10 female probands (seven sporadic cases and three family cases) who also had cluster seizures. The common clinical features of 16 patients in 10 families included fever‐sensitive and cluster seizures, mainly focal or tonic‐clonic seizures, and absence of status epilepticus, normal intelligence, or mild‐to‐moderate cognitive impairment, the onset age ranges from 5 months to 20 years. Only four patients had multiple or focal transient discharges in interictal EEG. Focal seizures originating in the frontal region were recorded in four patients, two from the parietal region, and one from the occipital region. Conclusion PCDH19 mutation can be inherited or de novo. The clinical spectrum of PCDH19 mutation includes PCDH19 Girls Clustering Epilepsy with or without mental retardation, psychosis, and asymptomatic male. The onset age of PCDH19 Girls Clustering Epilepsy can range from infancy to adulthood. Sisters in the same family may be sensitive to the same antiepileptic drugs. And our report expands the mutation spectrum of PCDH19 Girls Clustering Epilepsy.

with an early seizure onset and cognitive impairment. Seizures often occur in clusters and are often provoked by fever. With the expansion of phenotypic spectrum in PCDH19 mutation patients, some patients do not have severe intellectual disability. Subsequent studies highlighted that most patients had focal epilepsy clusters triggered by fever, so "PCDH19 girls clustering epilepsy" (Homan et al., 2018;Vlaskamp et al., 2019) (PCDH19-GCE) was proposed as a name to facilitate clinical identification of this disorder.
The reported PCDH19 mutations were mostly located at exon 1 (Depienne et al., 2011;Leonardi et al., 2014), which encodes the entire extracellular domain. About one-half of the reported mutations leading to PCDH19-related diseases are nonsense, frameshift, and splicing mutations, which severely truncate the protein protocadherin 19 (PCDH19). The remaining are missense mutations, and the missense mutations are concentrated in the extracellular domain of the protein (Kolc et al., 2019). The extracellular domain is essential for the normal function of the original cadherin function (Gerosa, Francolini, Bassani, & Passafaro, 2019). PCDH19 is mainly expressed in nerve tissues at different developmental stages, but its specific function is still unclear.
Studies have shown that the function of PCDH19 may be related to neuronal connections and signal transduction on synaptic membranes (Duszyc et al., 2015). PCDH19 mutation may lead to protein dysfunction.
In this study, we have screened 152 children with fever-sensitive epilepsy and found PCDH19 mutation in 10 female probands who also had cluster seizures with or without cognitive impairment or mental retardation in order to further understand the clinical and mutational features of PCDH19-GCE.

| Subjects
We analyzed fever-sensitive epilepsy children with the onset age 0-14 years between 2015 and 2019 in the Pediatrics Department of Qilu Hospital Affiliated to Shandong University and Linyi people's Hospital Affiliated to Shandong University, China. Exclusion criteria included seizures caused by nongenetic factors, such as an acquired brain injury (including traumatic brain injury, encephalitis, vasculitis, hypoxia, tumors, metabolic disorders, and toxicity); blood and urine screening indicate a metabolic disease; chromosome disease and clinically phenotypically defined monogenic diseases (e.g., tuberous sclerosis complex). Their clinical information was retrospectively col- The junction sequences were trimmed, and the contamination or low-quality reads were filtered for the raw data. Then, the clean data were aligned to the human reference genome sequence

| Genetic analyses
We screened 152 children with fever-sensitive epilepsy for gene detection (85 male and 67 female). We found eight PCDH19 point mutations and one whole gene deletion, four novel and four reported mutations in 10 female probands who also had cluster seizures (10/152, 6.57%) ( Table 1 and Figure 1). Seven mutations were located in exon 1 and one in exon 6. Two missense mutations

| Clinical features
The follow-up period ranged from 2 months to 4 years. The onset age of 152 children ranged from 6 days after birth to 7 years old. Twenty  found. The genetic pattern was consistent with the hypothesis of cell interference mechanism.

| D ISCUSS I ON
PCDH19-GCE is caused by PCDH19 mutations, and the protein PCDH19 which encoded by PCDH19 is expressed in various embryonic and human tissues (Cooper et al., 2015), including kidney, lung, and trachea, but it is significantly expressed in the nervous system, especially in limbic system (such as amygdala, hippocampus, and ventral hypothalamus) and cortex (Hertel, Redies, & Medina, 2012;Kim et al., 2010;Pederick et al., 2016;Schaarschuch & Hertel, 2018), and acts on the proliferation of progenitor cells and formation of nerve circuits and regulation of nerve activity (Bassani et al., 2018;Fujitani, Zhang, Fujiki, Fujihara, & Yamashita, 2017;Hayashi et al., 2017). In neurons, this regulation is the basis for the transmission and integration of synaptic inputs, and establishes adequate responses for the development, plasticity, and survival of neurons (Compagnucci et al., 2015;Kurian et al., 2018;Pederick et al., 2018). subunit can regulate the availability of receptor surface which may be involved in the regulation of intracellular transport of GABA A R (Bassani et al., 2018;Gerosa et al., 2019). Other studies have found that the level of allopregnanolone (AP) in PCDH19 female epilepsy patients was decreased (Tan et al., 2015), and AP is the most effective positive regulator of GABA A R which mediates fast inhibitory neurotransmission in the brain, so PCDH19 as a key modulator of GABAergic transmission and may suggest new pathogenic mechanisms. Six mutations were found in EC1-4 (6/8, 75%), including two missense mutations, which are highly conserved in orthologs and in paralogs of PCDHs. The EC1-4 repeats of PCDH19 have been identified as the minimal adhesive unit involved in the generation of a trans adhesive interface. These repeats interact in antiparallel PCDH19 dimer (forearm handshake model) to play the role of adhesion and mediation (Cooper, Jontes, & Sotomayor, 2016).
In this study, Seven PCDH19 mutations of the 10 probands were de novo, three mutations were inherited from fathers, and sporadic new mutations accounted for the majority, which was the main mutation type, consistent with the literature (Duszyc et al., 2015).
And, our report found four novel mutations which expand the spectrum of PCDH19 mutations associated with epilepsy in females. All the patients in the study were female, with onset age ranging from 5 months to 20 years old, later than the 6 months to 3 years old reported in the literature (Smith et al., 2018). In our study, the patients with the onset age older than 3 years (5/16, 31.25%) were mainly the first and second generations of the families. So PCDH19-GCE does not only onset in infancy, but it also can occur late in adulthood, and the late-onset patients may have mild clinical phenotype.
Corticosteroids have been reported to be effective in the treatment of acute cluster termination in a patient with PCDH19-GCE, then the hypothesis that blood-brain barrier dysfunction exists in PCDH19-GCE patients has been proposed (Bertani et al., 2015;Higurashi et al., 2015). However, remission is only temporary and epileptic seizures recur. After discovering that the level of allopregnanolone and steroidogenesis decreased (Tan et al., 2015;Trivisano et al., 2017), clinical trials began with ganaxolone, a synthetic analog of isoprogesterone (Tan et al., 2015). Surgical excision can reduce seizures in patients with cortical dysplasia (Kurian et al., 2018). Children with PCDH19-GCE had better responsiveness to benzodiazepines in the acute stage, and midazolam could control epileptic seizures. Eight patients needed midazolam to help control the acute cluster seizures in the study; however, soon after midazolam was reduced or discontinued, the seizures resurged. Considering the unique pattern of epilepsy, even in the case of continuous seizures, added multiple AEDs quickly should be avoided.
In this group of patients, except for patient No. 12, seizures were not sensitive to AEDs. Family No. 1 and No. 2 had two affected sisters separately. The sisters in the same family have the same reactivity to AEDs. This may suggest that if we met a PCDH19 family in clinic, the medication characteristics of proband in the family can guide the treatment of other patients in the same family. But the number of samples is too small, so that is just our guess. During the recent follow-up, the seizure-free interval ranged from 3 months to 13 years. Two patients had remission time longer than 3 years, with remission ages of adolescence and adulthood, respectively. Specchio et al. (2011) believed that AEDs could not control the cluster seizures in children, and the decrease in seizures with age may be related to the decrease in febrile diseases.
In conclusion, PCDH19 mutations can be inherited or de novo.
PCDH19 mutations-related epilepsy has incomplete penetration rate and phenotypic heterogeneity. The phenotypes of PCDH19 mutations include PCDH19-GCE with or without mental retardation, psychosis, and male asymptomatic carriers. PCDH19-GCE is characterized by clustered transient GTCS and focal seizures, and fever sensitivity. The onset age of PCDH19-GCE can vary from childhood to adulthood. And, our report expands the spectrum of PCDH19 mutations associated with epilepsy in females. We thank the patients and their family members for taking part in this study.

CO N FLI C T O F I NTE R E S T
All authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.