Immediate suppression of seizure clusters by corticosteroids in PCDH19 female epilepsy

Open ArchivePublished:February 16, 2015DOI:https://doi.org/10.1016/j.seizure.2015.02.006

      Highlights

      • Corticosteroids efficiently suppressed seizure clusters in PCDH19 female epilepsy.
      • The clinical improvements were obtained immediately.
      • Neuronal antibodies were nonspecifically detected in many patients.
      • Blood–brain barrier vulnerability may possibly underlie disease pathogenesis.

      Abstract

      Purpose

      The pathomechanism and treatment of PCDH19 female epilepsy (PCDH19-FE) remain unclear. Here, we report that corticosteroids are effective for control of the seizure clusters or other acute symptoms of PCDH19-FE and argue for the possible involvement of a compromised blood-brain barrier (BBB) in its pathogenesis.

      Methods

      The efficacy of corticosteroids was retrospectively reviewed in five Japanese patients with PCDH19-FE. The results of antibody assays against the N-methyl-d-aspartate-type glutamate receptor (abs-NR) in serum/cerebrospinal fluid were also compiled.

      Results

      Corticosteroid treatments significantly improved the acute symptoms, including seizure clusters, in all cases, most often immediately after the initial administration. However, the effect was transient, and some seizures recurred within a few weeks, especially in association with fever. Serum and/or cerebrospinal fluid abs-NR were detected in all patients. Target sequences of the detected antibodies were multiple, and the titers tended to decrease over time. In one patient, immunohistochemical analysis using rat hippocampal slices also revealed serum antibodies targeting an unknown epitope in neuronal cytoplasm.

      Conclusion

      Our findings imply an involvement of inflammatory processes in the pathogenesis of PCDH19-FE and therapeutic utility for corticosteroids as an adjunctive option in acute treatment. PCDH19 is well expressed in brain microvascular endothelial cells and thus its impairment may cause BBB vulnerability, which may be ameliorated by corticosteroids. The abs-NR detected in our patients may not indicate an autoimmune pathomechanism, but may rather represent non-specific sensitization to degraded neuronal components entering the general circulation, the latter process facilitated by the BBB vulnerability.

      Keywords

      1. Introduction

      A heterozygous defect in the gene encoding protocadherin 19 (PCDH19) causes early-onset intractable epilepsy in females (i.e., PCDH19-related female epilepsy, PCDH19-FE, or previously, epilepsy and mental retardation limited to females, EFMR) [
      • Dibbens L.M.
      • Tarpey P.S.
      • Hynes K.
      • Bayly M.A.
      • Scheffer I.E.
      • Smith R.
      • et al.
      X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment.
      ]. PCHD19, an adhesion molecule of the δ2-protocadherin subclass of the cadherin superfamily, is highly expressed in the vertebrate brain. δ-Protocadherins are intimately involved in brain development and neural functions, as well as in many neurological diseases [
      • Kim S.Y.
      • Yasuda S.
      • Tanaka H.
      • Yamagata K.
      • Kim H.
      Non-clustered protocadherin.
      ]. However, the homophilic adhesion capacity of PCDH19 by itself is low, and its exact function remains unclear.
      The hallmark clinical feature of PCDH19-FE is recurrent seizure clusters consisting of brief focal seizures and/or generalized convulsions, which can be triggered by febrile or afebrile illnesses [
      • Higurashi N.
      • Shi X.
      • Yasumoto S.
      • Oguni H.
      • Sakauchi M.
      • Itomi K.
      • et al.
      PCDH19 mutation in Japanese females with epilepsy.
      ]. The seizures do not recur regularly, but once they recur, the cluster continues for days to weeks despite multiple treatments. Conventional antiepileptic drugs fail to control or prevent most of these seizures. Ictal symptoms and EEG findings indicate that the seizures mainly involve the limbic system and medial frontal region [
      • Marini C.
      • Darra F.
      • Specchio N.
      • Mei D.
      • Terracciano A.
      • Parmeggiani L.
      • et al.
      Focal seizures with affective symptoms are a major feature of PCDH19 gene-related epilepsy.
      ].
      The clinical features indicate a possible immune/inflammation involvement in seizure generation, which could be a non-genetic modifier of the disease phenotype. In agreement with this, we have previously reported patients showing excellent efficacy of corticosteroids for seizure clusters [
      • Higurashi N.
      • Nakamura M.
      • Sugai M.
      • Ohfu M.
      • Sakauchi M.
      • Sugawara Y.
      • et al.
      PCDH19-related female-limited epilepsy: further details regarding early clinical features and therapeutic efficacy.
      ]. We have also encountered cases having antibodies to the N-methyl-d-aspartate (NMDA)-type glutamate receptor (abs-NR) in the serum or cerebrospinal fluid (CSF). Abs-NR cause limbic encephalitis, predominantly in young women (anti-NMDA receptor encephalitis) [
      • Dalmau J.
      • Gleichman A.J.
      • Hughes E.G.
      • Rossi J.E.
      • Peng X.
      • Lai M.
      • et al.
      Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.
      ], but may also appear secondarily and non-specifically in various neurologic diseases including epilepsy [
      • Ekizoglu E.
      • Tuzun E.
      • Woodhall M.
      • Lang B.
      • Jacobson L.
      • Icoz S.
      • et al.
      Investigation of neuronal autoantibodies in two different focal epilepsy syndromes.
      ]. In the latter case, abs-NR are not significantly involved in the disease pathogenesis.
      This study aims to explore whether corticosteroids have an ability to improve the seizures in PCDH19-FE and if any immune mechanism is involved. We retrospectively reviewed and summarized the clinical results of corticosteroid treatments as well as the results of an assay for abs-NR in Japanese patients. The potential significance of these findings with regard to the pathomechanisms of this disorder is also discussed.

      2. Methods

      2.1 Patients

      Japanese patients with PCDH19-FE who received corticosteroid treatments and/or underwent the ab-NR assay were retrospectively studied. They were genetically diagnosed at Fukuoka University5 and clinical details were collected from their doctors in charge. Since the patients were children, the doctors obtained written informed consent from the parents before the blood and/or CSF samples were drawn for genetic analysis of PCDH19 and/or for assay for ab-NR. Genetic analysis of PCDH19 was approved by the ethics committee of Fukuoka University. The ab-NR assay was approved by the National Epilepsy Center, Shizuoka Institute of Epilepsy and Neurological Disorders.

      2.2 ab-NR assay

      The ab-NR assay was performed at National Epilepsy Center, Shizuoka Institute of Epilepsy and Neurological Disorders. Experimental details have been described elsewhere [
      • Fujita K.
      • Yuasa T.
      • Takahashi Y.
      • Tanaka K.
      • Sako W.
      • Koizumi H.
      • et al.
      Antibodies to N-methyl-d-aspartate glutamate receptors in Creutzfeldt-Jakob disease patients.
      ]. Briefly, serum and CSF samples were examined by enzyme-linked immunosorbent assay (ELISA) or, in one patient, by immunoblot analysis targeting GluN2B. The ELISA target peptides were the extracellular N-terminal (NT) and/or intracellular C-terminal (CT) regions of the GluN2B, GluN1, and GluD2 subunits (Supplementary Table 1). Titers were determined by comparing the optical densities of our patients with those of 35 patients with non-inflammatory focal epilepsy, who served as controls. The results were expressed as number of standard deviations of the controls (SD) from the mean of the controls and were considered positive when ≥2 SD.

      2.3 Immunohistochemical analysis for anti-neuronal autoantibodies

      In two patients, the presence of anti-neuronal autoantibodies was further examined immunohistochemically using rat hippocampal slices exposed to serum or CSF from the patients. Experimental details are described in Supplementary information.

      3. Results

      Five patients (Patients 1–5) received corticosteroid treatments mainly during the acute phase before and/or after the diagnosis of PCDH19-FE. Abs-NR were examined in these patients mostly before corticosteroid administration and in four other patients (Patients 6–9). These tests were performed because autoimmune or inflammatory processes were clinically suspected in the pathogenesis of the epilepsy, although none of these patients except Patient 4 showed pleocytosis or an elevation of CSF protein level. Outlines of Patients 1, 2, and 5–9 have been described previously [
      • Higurashi N.
      • Shi X.
      • Yasumoto S.
      • Oguni H.
      • Sakauchi M.
      • Itomi K.
      • et al.
      PCDH19 mutation in Japanese females with epilepsy.
      ,
      • Higurashi N.
      • Nakamura M.
      • Sugai M.
      • Ohfu M.
      • Sakauchi M.
      • Sugawara Y.
      • et al.
      PCDH19-related female-limited epilepsy: further details regarding early clinical features and therapeutic efficacy.
      ].

      3.1 Efficacy of corticosteroid treatments

      Treatment details and results are summarized in Table 1. The details of the clinical courses are described in Supplementary information. Overall, corticosteroids dramatically improved acute neurological symptoms: ongoing seizure clusters in Patients 1–3 and 5, and an acute encephalopathic episode that developed after a seizure cluster in Patient 4, were controlled. In most cases, the improvement was achieved after the first administration. In the cases of seizure cluster, the initial administration was conducted well in advance of the expected time of spontaneous remission of the cluster. Furthermore, in Patient 3, corticosteroids were initiated on the second day of each cluster to confirm that the cluster continued for more than 1 day despite midazolam administration. Regarding drugs and dosages, four young patients (Patients 1–4) received an intravenous drip infusion of 10–30 mg/kg methylprednisolone once daily, for up to 3 days. Patient 5 received an intravenous infusion of 0.35 mg/kg prednisolone once or twice, depending on the cluster, followed by oral administration of prednisolone at 1 mg/(kg·day) at age 11. However, as observed in Patients 1, 3, and 5, the effect was fundamentally transient; seizure clusters often recurred within a few weeks, especially when fever appeared.
      Table 1Details and efficacy of corticosteroid therapy.
      Pt noPCDH19 mutationAge at onset (m)Age at CS TXCSRoute & doseTarget symptomSimultaneous TXUsual duration of Sz clusterResultPresent intellect
      1p.L719*132y4mmPSLIV, 30 mg/kg, 3dSz clusterMDL CBZ CZP VPA LTG LEVDays ∼2 wkDisappeared after 1st IVNormal

      5y1m
      2y10mmPSLIV, 30 mg/kg, 3dSz clusterDisappeared after 1st IV

      Recurred in 2 wk w/fever
      Noted when seizures recurred within 3 weeks after corticosteroid administration.
      2y11mmPSLIV, 30 mg/kg, 3dSz clusterDisappeared after 1st IV

      Recurred in 1wk w/fever
      3y0mmPSLIV, 10 mg/kg, 3dSz clusterDisappeared after 1st IV
      3y4mBETOral, 0.01 mg/kg, 3d

      When fever appeared
      Sz preventionNo or mild recurrence
      4y1mPSLOral, 1–1.5 mg/kg, 3d When fever appearedSz preventionNo or mild recurrence
      2p.K120Rfs*31010mmPSLIV, 30 mg/kg, 3dSz clusterMDL PB ACV IVIG EDVDisappeared after 1st IV

      Recurred in 1wk
      Moderate delay 3y
      3p.D417H p.D596Y51y11mmPSLIV, 20 mg/kg, 2dSz clusterMDL fPHT CLB LEV KBr DZPDays ∼2wkDisappeared after 1st IVNormal

      2y8m
      2y1mmPSLIV, 20 mg/kg, 3dSz clusterDisappeared after 1st IV
      2y2mmPSLIV, 20 mg/kg, 2dSz clusterDisappeared after 1st IV
      2y5mmPSLIV, 10 mg/kg, 1d

      fol. by 20 mg/kg, 1d
      Sz clusterDisappeared after 2nd IV
      2y7mmPSLIV, 20 mg/kg, 1dSz clusterDisappeared after 1st IV

      Recurred in 9d w/flu
      2y7mmPSLIV, 20 mg/kg, 2dSz clusterDisappeared after 1st IV
      4p.D596G61y0mmPSLIV, 30 mg/kg, 3dEncephalopatic symptomsCBZ fPHT LDC PB1dDisappeared after 1st IVHyperactive

      1y6m
      5p.D45Gfs*43811y5mPSLIV, 0.35 mg/kg x1

      fol. by Oral, 1 mg/kg
      In Patient 5, oral prednisolone was gradually tapered off. Pt no, Patient number; m, month(s); CS, corticosteroid; TX, treatment; Sz, seizure; y, year(s); mPSL, methylprednisolone; BET, betamethasone; PSL, prednisolone; IV, intravenous route; d, day(s); MDL, midazolam; CBZ, carbamazepine; CZP, clonazepam; VPA, valproic acid; LTG, lamotrigine; LEV, levetiracetam; wk, week(s); PB, phenobarbital; ACV, acyclovir; IVIG, intravenous immunoglobulin; EDV, edaravone; fol. by, followed by; fPHT, fosphenytoin; CLB, clobazam; KBr, potassium bromide; DZP, diazepam; LDC, lidocaine.
      Sz clusterKBr CZPHalf a dayDisappeared after 1st IVModerate delay

      11y8m
      11y6mPSLIV, 0.35 mg/kg x1

      fol. by Oral, 1 mg/kg
      Sz clusterDisappeared after 1st IV

      Recurred in 1wk w/fever
      11y6mPSLIV, 0.35 mg/kg x1

      fol. by Oral, 1 mg/kg
      Sz clusterDisappeared after 1st IV
      11y8mPSLIV, 0.35 mg/kg x2

      fol. by Oral, 1 mg/kg
      Sz clusterDisappeared after 2nd IV
      a Noted when seizures recurred within 3 weeks after corticosteroid administration.
      b In Patient 5, oral prednisolone was gradually tapered off. Pt no, Patient number; m, month(s); CS, corticosteroid; TX, treatment; Sz, seizure; y, year(s); mPSL, methylprednisolone; BET, betamethasone; PSL, prednisolone; IV, intravenous route; d, day(s); MDL, midazolam; CBZ, carbamazepine; CZP, clonazepam; VPA, valproic acid; LTG, lamotrigine; LEV, levetiracetam; wk, week(s); PB, phenobarbital; ACV, acyclovir; IVIG, intravenous immunoglobulin; EDV, edaravone; fol. by, followed by; fPHT, fosphenytoin; CLB, clobazam; KBr, potassium bromide; DZP, diazepam; LDC, lidocaine.
      For Patient 4, methylprednisolone was used at age 1 for an encephalopathic episode with decline of consciousness and systemic weakness, which abruptly developed 3 days after the termination of a one-day seizure cluster. EEG showed an increase of δ-waves, but no ictal activity. Mild CSF pleocytosis (85 cells/μL) was identified a week before the episode. These symptoms completely disappeared immediately after the initial administration of methylprednisolone.
      For Patient 1, corticosteroids were administered prophylactically after age 3, with 3 days of oral betamethasone or prednisolone administration at times of fever appearance. After starting this treatment, no or only mild recurrences (not requiring hospitalization) were observed, even during fever.

      3.2 ab-NR and further anti-neuronal autoantibody assays

      Eight of the nine patients who underwent the assay showed positivity to multiple epitopes in the serum or CSF (Patients 1–7 and 9, 88.9%, Table 2). The epitopes included GluN1-NT, which has been reported to be critical for the emergence of neuropsychiatric symptoms in anti-NMDA-receptor encephalitis [
      • Dalmau J.
      • Gleichman A.J.
      • Hughes E.G.
      • Rossi J.E.
      • Peng X.
      • Lai M.
      • et al.
      Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.
      ]. Patients 1 and 2 had high CSF titers of antibodies during the acute phase (>10 SD). Patient 6 showed positivity in the CSF at onset and in serum half a year later. Patients 3, 5, and 9 underwent follow-up assays, and their titers were found to decrease over time. In Patient 5, immunohistochemical analysis during seizure recurrence at age 11, using serum drawn before prednisolone administration, revealed autoantibodies to the cytoplasm of hippocampal neurons, as demonstrated in hippocampal slices taken from rats (Supplementary Fig. b). The assay failed to identify the epitope. These results suggest that following seizure clusters, an immune reaction occurs non-specifically to degraded neuronal proteins, including NMDA-type glutamate receptor, inside and subsequently outside the brain. Such reactions appear to be strong at early ages, but do not show a uniform pattern.
      Table 2Results of assay for antibodies to N-methyl-d-aspartate-type glutamate receptor.
      Pt noPCDH19 mutationAge at onset (m)Age at assaySerumCerebrospinal fluid
      GluN2BGluN1GluD2GluN2BGluN1GluD2
      NTCTNTNTCTNTCTNTNTCT
      1p.L719*132y5m1.613.121.930.4514.1015.1010.808.10
      2p.K120Rfs*31011m15.42
      4y2m2.211.533.583.63
      3p.D417H

      p.D596Y

      Heterozygous
      511m2.172.943.622.85
      1y0m3.393.282.812.35
      1y7m2.652.212.171.95
      1y10m1.230.370.720.99
      4p.D596G61y0m2.672.391.511.68
      5p.D45Gfs*4389m++
      3y5m
      6Whole del1011m0.571.700.46(0.34)1.612.731.340.75
      1y5m2.332.732.480.920.461.181.130.70
      7p.Asn340Ser98y1m3.492.079.551.87
      8p.R198L76y5m1.700.901.781.68
      9p.Y366Lfs*1056y0m9.5212.509.20−0.88−1.37−1.08
      8y5m0.60(0.35)0.250.37
      Results are expressed as standard deviation (SD, enzyme-linked immunosorbent assay) or plus/minus sign (immunoblot, Patient 5 only). Bold values denote positivity (≥2 SD or +). Blanks are unexamined. *Patients who received corticosteroid treatments. Pt no, Patient number; m, month(s); NT, N-terminal; CT, C-terminal; y, year(s); Whole del, Whole PCDH19 deletion.

      4. Discussion

      This study revealed the therapeutic potency of corticosteroids for acute symptoms in PCDH19-FE. The rapid and efficient response was remarkable and might be a useful indicator for this disease. The cases of Patients 1 and 9 suggested that oral corticosteroids taken during interictal periods might exert some prophylactic effects, but further assessments are necessary to establish this. Despite such efficacy, short-term administration of corticosteroids provided only transient effects and had no potential for preventing further seizure clusters. Therefore, the clinical benefit will be limited for cases with frequent recurrences such as Patients 3 and 5. Moreover, excessive amounts of corticosteroids may have adverse effects, actually increasing seizure-proneness as described below. These findings suggest that the attending physician should consider discontinuing corticosteroid administration for acute treatment soon after seizure disappearance. Indication for treatment should be based on various patient conditions such as age, seizure severity, and comorbid infections.
      Corticosteroids may exert primarily excitatory/pro-convulsive actions on brain neurons both in vitro and in vivo, especially under chronic stress conditions such as epilepsy [
      • Castro O.W.
      • Santos V.R.
      • Pun R.Y.
      • McKlveen J.M.
      • Batie M.
      • Holland K.D.
      • et al.
      Impact of corticosterone treatment on spontaneous seizure frequency and epileptiform activity in mice with chronic epilepsy.
      ]. Despite this, the therapeutic efficacy of corticosteroids is well established in many intractable epilepsies such as West syndrome, Landau-Kleffner syndrome, and autoimmune epilepsy/encephalitis [
      • Gupta R.
      • Appleton R.
      Corticosteroids in the management of the paediatric epilepsies.
      ]. In these diseases, clinical improvements after corticosteroid administration are usually delayed and are probably mediated by mechanisms such as immunosuppression and feedback inhibition of corticotropin-releasing hormone secretion. In our patients, however, the clinical effects appeared quickly, usually immediately after the initial administration. Moreover, significant brain inflammation was not found, suggesting a particular pathogenesis and mechanism of action of corticosteroids in PCDH19-FE.
      Such a mechanism may be restoration of blood-brain barrier (BBB) integrity [
      • Forster C.
      • Waschke J.
      • Burek M.
      • Leers J.
      • Drenckhahn D.
      Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific.
      ]. This is known to be a crucial action of corticosteroids, the underlying molecular basis of which has been partially elucidated [
      • Salvador E.
      • Shityakov S.
      • Forster C.
      Glucocorticoids and endothelial cell barrier function.
      ]. Seizures are easily triggered by a mild breakdown of brain homeostasis due to a compromised BBB, which can be prevented or alleviated by corticosteroid administration [
      • Marchi N.
      • Granata T.
      • Freri E.
      • Ciusani E.
      • Ragona F.
      • Puvenna V.
      • et al.
      Efficacy of anti-inflammatory therapy in a model of acute seizures and in a population of pediatric drug resistant epileptics.
      ]. Although PCDH19 is abundantly expressed in brain neurons, it is unclear how the heterozygous mutation in PCDH19 leads to epilepsy in females. “Cellular interference,” that is, the presence of somatic mosaicism in PCDH19 expression between normal and abnormal neurons, is the currently proposed explanation for such sex-specific pathogenesis, but the concept is very theoretical and has yet to be established. However, PCDH19 is also expressed in the BBB. In mouse, the BBB-specific transcriptome included PCDH19, and PCDH19 expression in microvascular endothelial cells was significantly higher in the brain than in the liver or lung [
      • Daneman R.
      • Zhou L.
      • Agalliu D.
      • Cahoy J.D.
      • Kaushal A.
      • Barres B.A.
      The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells.
      ]. Although currently no human evidence exists, this study suggests that PCDH19 plays a role in the BBB, and speculatively, that PCDH19 mutation leads to a functional BBB vulnerability, that underlies the pathogenesis of PCDH19-FE.
      Interestingly, PCDH19 expression may be significantly altered in the BBB during systemic inflammation, which is the predisposing factor for seizure recurrence in PCDH19-FE. In cultured mouse brain microvascular endothelial cells, treatment with lupus serum or an activated complement, C5a, significantly down-regulated miR-320a expression [
      • Eadon M.T.
      • Jacob A.
      • Cunningham P.N.
      • Quigg R.J.
      • Garcia J.G.
      • Alexander J.J.
      Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells.
      ]. PCDH19 is one potential target of this microRNA. Although systemic inflammation will impair BBB function to some extent [
      • Marchi N.
      • Fan Q.
      • Ghosh C.
      • Fazio V.
      • Bertolini F.
      • Betto G.
      • et al.
      Antagonism of peripheral inflammation reduces the severity of status epilepticus.
      ], the impairment may be exacerbated in patients with PCDH19-FE, probably due to PCDH19 insufficiency. The resulting seizure clusters will further exacerbate the BBB dysfunction by inducing brain inflammation. Corticosteroids may ameliorate such dysfunction and thus the acute neurological symptoms.
      Other aspects of PCDH19-FE and the findings of this study are also consistent with the BBB hypothesis: The seizures mainly involve the limbic system, which is anatomically close to some of the periventricular regions that lack a BBB due to their endocrine roles; seizure occurrence and remission are strongly age-related, and BBB integrity also develops age-dependently; abs-NR in our patients covered multiple epitopes of various subunits, and Patient 8 had antibodies to some neuronal cytoplasmic component(s) as well. These results indicate that anti-neuronal antibodies may be produced in PCDH19-FE non-specifically and commonly. Various neuronal proteins will be degraded by recurrent seizures and the compromised BBB may then facilitate leakage of such degraded proteins into the bloodstream. This may induce non-specific sensitization to them outside the brain, resulting in the high rate of anti-neuronal autoantibody positivity seen in our patients. Thus, the abs-NR found in our patients does not represent an autoimmune pathogenesis.
      However, a possible modifying effect of the abs-NR on the neurological phenotype in our patients with PCDH19-FE can also be considered. The seroprevalence of abs-NR has been found to be identical between patients with psychiatric diseases, including schizophrenia, and healthy individuals, but the disease phenotypes were more severe in patients with compromised BBBs than in those without [
      • Hammer C.
      • Stepniak B.
      • Schneider A.
      • Papiol S.
      • Tantra M.
      • Begemann M.
      • et al.
      Neuropsychiatric disease relevance of circulating anti-NMDA receptor autoantibodies depends on blood-brain barrier integrity.
      ]. Serum abs-NR could have passed the compromised BBB and worsened the psychiatric symptoms of these patients. In anti-NMDA-receptor encephalitis, a primary involvement of antibodies to GluN1-NT in causing its neuropsychiatric symptoms has been suggested [
      • Dalmau J.
      • Gleichman A.J.
      • Hughes E.G.
      • Rossi J.E.
      • Peng X.
      • Lai M.
      • et al.
      Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.
      ]. Therefore if BBB compromise exists in PCDH19-FE, the anti-GluN1-NT could be partially responsible for the neuropsychiatric symptoms also seen in this disease.
      This study proposes corticosteroid treatment as an efficacious adjunctive treatment for the acute symptoms of PCDH19-FE and suggests BBB involvement in this disease. Although PCDH19-FE is rare, future multicenter clinical trials should be conducted to verify the acute and long-term efficacy of corticosteroid treatment and to define the therapeutic indications of such treatment, since the present study is only a retrospective review of 5 patients. In addition, reliable animal models should be devised to elucidate the entire molecular pathogenesis of PCDH19-FE. Importantly, not only must neurons be studied, but also other actors such as the BBB and the inflammatory system as well.

      Conflict of interest

      All authors wish to confirm that there are no known conflict of interest associated with this publication.

      Acknowledgments

      The authors are indebted to all members of the study family for their helpful cooperation. This work was supported by Grants-in-Aid for Scientific Research (A) (24249060) to SH, (C) (21591342, 23591238 and 24591537) to YT, and (C) (26461552) to NH, a Grant-in-aid for Challenging Exploratory Research (25670481) to SH, Bilateral Joint Research Projects to SH, and a Grant-in-aid for Young Scientists (B) (24791095) to NH from the Japan Society for the Promotion of Science (JSPS); by Grants for Scientific Research on Innovative Areas (221S0002 and 25129708) to SH from the Ministry of Education, Culture, Sports, Science and Technology (MEXT); by a MEXT-supported Program for the Strategic Research Foundation at Private Universities 2013–2017 to SH; by a Grant-in-aid for the Research on Measures for Intractable Diseases (No. H26-Nanji-Ippan-49 and 51) to SH, and Comprehensive Research on Disability Health and Welfare and Research on Rare and Intractable Diseases to YT from the Ministry of Health, Labor and Welfare (MHLW); by an Intramural Research Grant (24-7) for Neurological and Psychiatric Disorders of NCNP to SH; by the Joint Usage/Research Program of Medical Research Institute, Tokyo Medical and Dental University to SH; by research grants from The Mitsubishi Foundation to SH, from Takeda Scientific Foundation to SH, from Kiyokun Foundation to SH, from The Japan Epilepsy Research Foundation to YT, and from Kaibara Morikazu Medical Science Promotion Foundation to NH.

      Appendix A. Supplementary data

      The following are Supplementary data to this article:

      References

        • Dibbens L.M.
        • Tarpey P.S.
        • Hynes K.
        • Bayly M.A.
        • Scheffer I.E.
        • Smith R.
        • et al.
        X-linked protocadherin 19 mutations cause female-limited epilepsy and cognitive impairment.
        Nat Genet. 2008; 40: 776-781
        • Kim S.Y.
        • Yasuda S.
        • Tanaka H.
        • Yamagata K.
        • Kim H.
        Non-clustered protocadherin.
        Cell Adh Migr. 2011; 5: 97-105
        • Higurashi N.
        • Shi X.
        • Yasumoto S.
        • Oguni H.
        • Sakauchi M.
        • Itomi K.
        • et al.
        PCDH19 mutation in Japanese females with epilepsy.
        Epilepsy Res. 2012; 99: 28-37
        • Marini C.
        • Darra F.
        • Specchio N.
        • Mei D.
        • Terracciano A.
        • Parmeggiani L.
        • et al.
        Focal seizures with affective symptoms are a major feature of PCDH19 gene-related epilepsy.
        Epilepsia. 2012; 53: 2111-2119
        • Higurashi N.
        • Nakamura M.
        • Sugai M.
        • Ohfu M.
        • Sakauchi M.
        • Sugawara Y.
        • et al.
        PCDH19-related female-limited epilepsy: further details regarding early clinical features and therapeutic efficacy.
        Epilepsy Res. 2013; 106: 191-199
        • Dalmau J.
        • Gleichman A.J.
        • Hughes E.G.
        • Rossi J.E.
        • Peng X.
        • Lai M.
        • et al.
        Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies.
        Lancet Neurol. 2008; 7: 1091-1098
        • Ekizoglu E.
        • Tuzun E.
        • Woodhall M.
        • Lang B.
        • Jacobson L.
        • Icoz S.
        • et al.
        Investigation of neuronal autoantibodies in two different focal epilepsy syndromes.
        Epilepsia. 2014; 55: 414-422
        • Fujita K.
        • Yuasa T.
        • Takahashi Y.
        • Tanaka K.
        • Sako W.
        • Koizumi H.
        • et al.
        Antibodies to N-methyl-d-aspartate glutamate receptors in Creutzfeldt-Jakob disease patients.
        J Neuroimmunol. 2012; 251: 90-93
        • Castro O.W.
        • Santos V.R.
        • Pun R.Y.
        • McKlveen J.M.
        • Batie M.
        • Holland K.D.
        • et al.
        Impact of corticosterone treatment on spontaneous seizure frequency and epileptiform activity in mice with chronic epilepsy.
        PLoS One. 2012; 7: e46044
        • Gupta R.
        • Appleton R.
        Corticosteroids in the management of the paediatric epilepsies.
        Arch Dis Child. 2005; 90: 379-384
        • Forster C.
        • Waschke J.
        • Burek M.
        • Leers J.
        • Drenckhahn D.
        Glucocorticoid effects on mouse microvascular endothelial barrier permeability are brain specific.
        J Physiol. 2006; 573: 413-425
        • Salvador E.
        • Shityakov S.
        • Forster C.
        Glucocorticoids and endothelial cell barrier function.
        Cell Tissue Res. 2014; 355: 597-605
        • Marchi N.
        • Granata T.
        • Freri E.
        • Ciusani E.
        • Ragona F.
        • Puvenna V.
        • et al.
        Efficacy of anti-inflammatory therapy in a model of acute seizures and in a population of pediatric drug resistant epileptics.
        PLoS One. 2011; 6: e18200
        • Daneman R.
        • Zhou L.
        • Agalliu D.
        • Cahoy J.D.
        • Kaushal A.
        • Barres B.A.
        The mouse blood-brain barrier transcriptome: a new resource for understanding the development and function of brain endothelial cells.
        PLoS One. 2010; 5: e13741
        • Eadon M.T.
        • Jacob A.
        • Cunningham P.N.
        • Quigg R.J.
        • Garcia J.G.
        • Alexander J.J.
        Transcriptional profiling reveals that C5a alters microRNA in brain endothelial cells.
        Immunology. 2014; 143: 363-373
        • Marchi N.
        • Fan Q.
        • Ghosh C.
        • Fazio V.
        • Bertolini F.
        • Betto G.
        • et al.
        Antagonism of peripheral inflammation reduces the severity of status epilepticus.
        Neurobiol Dis. 2009; 33: 171-181
        • Hammer C.
        • Stepniak B.
        • Schneider A.
        • Papiol S.
        • Tantra M.
        • Begemann M.
        • et al.
        Neuropsychiatric disease relevance of circulating anti-NMDA receptor autoantibodies depends on blood-brain barrier integrity.
        Mol Psychiatry. 2014; 19: 1143-1149