Introduction

Early infantile epileptic encephalopathy-9 (MIM 300088), also known as epilepsy and mental retardation limited to females (EFMR), is caused by mutations in the X-linked PCDH19 gene. It is characterized by seizures with an onset in infancy and cognitive impairment. Seizures often occur in clusters and are often provoked by fever. Nevertheless, the phenotypic spectrum is heterogeneous and can fluctuate between severe generalized or multifocal epilepsy, resembling Dravet syndrome and more benign focal epilepsies with normal intelligence [1–21]. So far over 100 females and one mosaic male with a PCDH19 mutation have been reported [6]. The X-linked inheritance pattern is remarkable: Heterozygous females are affected while transmitting hemizygous males do not show epilepsy or mental retardation. However, transmitting males in familial EFMR were reported to have controlling, rigid personalities [4]. We screened a large cohort of female patients with EFMR or a Dravet-like phenotype and describe 15 new cases with a PCDH19 mutation. Clinical characteristics and genetic findings were compared with patients reported in literature. We also hypothesized that PCDH19 mutations may play a role in normal intelligent males with autism spectrum disorders (ASD) and analysed the PCDH19 gene in 20 high-functioning males with ASD.

Methods

Selection of female patients for PCDH19 screening

Analysis of the PCDH19 gene was performed in two patient cohorts: a cohort of 120 females with features of Dravet syndrome originally referred for analysis of the SCN1A gene but without mutation and a cohort of 136 patients referred for analysis of the PCDH19 gene because of features of EFMR. Clinical data of the patients with a PCDH19 mutation were collected from the medical records and through a questionnaire completed by the treating physicians. We collected data on age at seizure onset, seizures types, seizure frequency, seizure offset and results of EEG and magnetic resonance imaging (MRI). We also collected data on psychomotor development, behavioural features and the level of intellectual disability, assessed clinically.

Selection of males with ASD

At the department of Child and Adolescent Psychiatry of the UMC Utrecht, a repository of genomic DNA and phenotypic data on patients with ASD has been collected over the past years for research purposes. The parents or caretakers have given their informed consent for these data storage and DNA banking. We selected a total of 20 sporadic male patients with ASD and an intelligent quotient above 75. Nine of these patients had Asperger syndrome, pervasive developmental disorder—not otherwise specified was diagnosed in six, four patients had a diagnosis of multiple complex developmental disorder and autistic disorder was diagnosed in one patient.

PCDH19 mutation analysis

Genomic DNA was extracted from whole blood cells using standard procedures. PCR amplification of DNA was done using specific primer pairs for the six exons and adjacent intron–exon boundaries of the PCDH19 gene (reference sequence EF676096.1). Primer sequences and PCR/sequencing conditions are available on request. Sequence products were run on an ABI 3730 automated DNA sequencer. Mutation analysis of the coding exons and intron/exon boundaries was undertaken using suitable mutation analysis software. The analysis of larger structural variants (deletions/duplications) was performed using MLPA (MRC Holland probe kit nr. P330-A1) or Multiplex Amplicon Quantification (http://www.multiplicom.be).

Females with a PCDH19 mutation reported in literature

To find reports in literature on females with a PCDH19 mutation, we performed a Medline search from 2008 onwards using the key words EFMR, PCDH19, epilepsy female mental retardation and protocadherin 19.

Results

Screening results and clinical characteristics

A PCDH19 mutation was detected in seven out of 136 (5.1 %) EFMR suspected females, eight out of 120 (6.7 %) Dravet-like females and one out of 20 (5 %) ASD males. Only three mutations were inherited, and the remaining 12 were confirmed de novo mutations. Table 1 summarizes the clinical characteristics and PCDH19 gene mutations. Patients 2, 3, 4, 5, 10, 11, 12 and 15 were originally referred for analysis of the SCN1A gene. The other patients were referred for analysis of PCDH19 based on a clinical phenotype thought to be compatible with EFMR.

Table 1 Clinical characteristics and PCDH19 mutations in 15 females
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Table 2 summarizes the clinical characteristics of all 125 patients with PCDH19 mutations reported in the literature thus far [4, 6–12, 14–16, 18, 19, 21], including the 15 patients identified in our study. One study was excluded because part of all patients in this study had been reported earlier and because the focus of this study was on seizure types and EEG characteristics. The overall clinical characteristics were only summarized for the whole group of patients and not for each individual patient [20].

Table 2 Summary of the clinical characteristics of all 125 female patients with PCDH19 mutations
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Epilepsy course

In all reported patients, seizure onset occurred at the end of the first year of life. The most common seizure type at onset was febrile or afebrile generalized tonic–clonic seizures. In 34 % of patients, the first seizures occurred in a cluster. Different seizure types followed, but a high prevalence of fever sensitivity (in 54 % of patients), generalized tonic–clonic seizures (in 80 %) and occurrence of seizures in clusters (in 62 %) remained. In a recent study on seizure semiology in 35 patients, “fearful screaming” in the context of focal seizures was reported as a predominant ictal sign, occurring in 70.5 % of patients [20].

In our patients, the frequency of febrile seizures (100 %), tonic seizures (60 %), myoclonic seizures (40 %), complex partial seizures (67 %) and seizures occurring in clusters (100 %) was high compared with the frequency of these seizures in previously reported patients. Clusters of seizures lasted several days or weeks. In ten of our 15 patients (67 %), the seizure-free interval in between clusters ranged from 2 to 10 months during the first 4 years of life. For many of the previously published patients, data on seizure frequency and seizure free intervals are not available. Data on seizure offset were available for patients from four studies [4, 7, 12, 14] and for our own patients. Long seizure-free intervals may hamper a reliable assessment of the percentage of patients with seizure offset. Therefore, we decided to include only patients who were seizure free for at least 2 years. Two of 20 patients (10 %) aged under 10 years, three of eight patients (38 %) aged 10 to 15 years and 24 of 30 patients (80 %) older than 15 years were seizure free for at least 2 years. The mean age of seizure offset was 12 years (range 1, 8 to 30 years).

Intellectual disability and psychiatric disorders

Thirteen of our 15 patients (87 %) showed developmental delay or intellectual disability, which is comparable with the proportion in reported patients (78 %). Half of the reported patients had mild ID while the other half had moderate to severe ID. Most patients had normal development prior to seizure onset. After seizure onset, development remained normal, showed slowing, stagnation or regression. In four patients from one family, development was slow form birth [4]. Behavioural disturbances, including autistic features, aggression and hyperactivity, were present in 73 % of our patients versus 46 % of reported patients.

EEG

Ictal or peri-ictal EEGs of our patients showed focal, multifocal or bilateral synchronous discharges, and background activity was either normal or showed slowing. For 12 patients, interictal EEGs were available and were reported to be normal.

The results of EEGs of patients reported in literature are also heterogeneous [4, 5, 7–16, 20]. Many patients had normal interictal EEGs, but a variety of EEG abnormalities have been described. Unfortunately, reports did not always specify whether these abnormal EEGs were recorded within a cluster of seizures. Background activity has been reported to be normal or showed diffuse or focal slowing. Focal, multifocal and generalized epileptic discharges have all been described. A photoparoxysmal response was seen in only two patients [7, 12], and in one patient, electrical status epilepticus in slow wave sleep was present [11]. In a series of 35 patients, the electroclinical pattern of ictal EEG data was interpreted as suggestive of an early involvement of the frontotemporal limbic structures in 74 % of patients [20].

The mutation spectrum

Table 3 gives an overview of the mutations identified in our patients and of all mutations reported in literature. Nine of the mutations identified in our patients (60 %) and 56 of the reported mutations in the literature (49 %) were missense mutations. All missense mutations were located in the extracellular domains and were never present in the cytoplasmic domain. Nonsense, frameshift and splicing mutations have been identified in all protein domains.

Table 3 PCDH19 gene mutations reported in females
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In our patient series, we identified three novel variants which were classified as rare polymorphisms based on the results of in silico analyses and segregation analyses: c.2598C > T (p.Asn866Asn), c.1294A > G (p.Met432Val) and c.769 G > C (p.Val257Leu).

Inheritance of mutations

For two of our patients, the inheritance of the mutation is unknown. In 11 of the other 13 patients (85 %), the mutation arose de novo. For 27 of all 130 mutations reported in Table 3, the inheritance of the mutation is unknown. For the other 103 mutations, 68 were de novo (66 %), 12 maternally inherited (12 %), 13 paternally inherited (13 %) and 10 (10 %) were familial (indicating extended families with multiple affected females). Mosaicism was demonstrated in three transmitting mothers, one affected and two asymptomatic [11, 21]. In sporadic patients, 68 out of 93 mutations (73 %) arose de novo.

Recurrent mutations and genotype–phenotype correlation

Two recurrent mutations are relatively frequent. The p.Asn340Ser mutation has been identified in 16 independent probands, including one of our patients. They showed variable phenotypes, ranging from generalized tonic–clonic convulsions only with normal development to an explosive onset of epileptic encephalopathy with developmental regression and electrical status epilepticus in slow wave sleep on EEG. Seven probands had the c.1091dupC mutation and one the c.1091delC mutation. Both the insertion and the deletion result in a frameshift. Detailed clinical data were available for three of these probands who all had fever sensitive epilepsy, behavioural disturbances and moderate to severe ID.

Intrafamilial variability and penetrance

The clinical phenotype in affected females from three of the four extended families described by Scheffer et al. [4] showed wide intrafamilial variability. Dibbens et al. reported on the identification of PCDH19 mutations in the same four families and in three additional extended families [5]. Penetrance in these seven families was high: 66 out of 68 carrier females (97 %) were classified as affected.

Our patient 12 comes from a three generation family of African ancestry with a phenotype compatible with EFMR (Fig. 1). All female mutation carriers presented seizures in childhood, and although detailed information is lacking for the first generation, most of them had febrile and afebrile seizures from childhood, often occurring in clusters. Cognitive impairment was very variable within the family and ranged from moderate ID to borderline intelligence.

Fig. 1
figure 1

Phenotypes and genotypes in a three generation family with EFMR

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PCDH19 mutations in males

Epilepsy has not been reported in male carriers of a PCDH19 mutation, except for one male patient with clinical features of Dravet syndrome who proved to be mosaic for a PCDH19 gene deletion [6]. One father who carried the mutation identified in his affected daughter had moderate ID without epilepsy. His mother and sister also had ID without epilepsy. They were not tested for the presence of the mutation [6]. Scheffer et al. observed that five obligate male carriers from four unrelated families with EFMR had obsessive, controlling, rigid, inflexible personalities [4].

Males with an autism spectrum disorder

Sequence analysis of PCDH19 in the 20 males with ASD revealed one missense mutation: c.2359C > T in exon 3 in a 14-year-old boy diagnosed with Asperger syndrome at the age of 8.5 years. His family members were not affected by autism or epilepsy. This mutation results in the replacement of an arginine amino acid by a cysteine (p.Arg787Cys) in the cytoplasmic domain of the protein. The mutation affects a highly conserved amino acid with a Grantham score of the amino acid change of 180. A score higher than 100 indicates radical amino acid changes [22]. Two bioinformatic prediction programs show that the p.Arg787Cys is probably damaging (Polyphen2) and not tolerated (SIFT). The inheritance of the mutation is unknown.

Discussion

Clinical characteristics

We present an overview of phenotypes and the mutation spectrum in females with EFMR caused by PCDH19 mutations. For this purpose, we included 13 case series [5, 7–10, 12–16, 18–21], two papers on families with a PCDH19 mutation [4, 11], and we report 15 new cases with a PCDH19 mutation. The criteria for inclusion of patients for analysis of the PCDH19 gene varied: Some studies only included patients with SCN1A-negative Dravet syndrome [6, 14, 19], whereas others included females with various epilepsy syndromes including patients without ID [7, 12]. Our own patients contributed to both categories. The clinical picture associated with PCDH19 mutations emerging from the individual case series reflects to some extent the inclusion criteria. In studies including mainly Dravet syndrome patients, onset was relatively early and seizure types other than generalized tonic–clonic seizures occurred more frequently compared with studies including patients without ID or familial cases. The combined data from these studies give an overall picture of the highly variable clinical characteristics associated with PCDH19 gene mutations (Table 2). Most patients have febrile and afebrile, tonic–clonic seizures with onset in infancy. Focal seizures with or without impaired consciousness or status epilepticus occur in about one third of all patients. A very characteristic and discriminating feature is the occurrence of (febrile) seizures in clusters with seizure-free intervals that may last for months. Seizures remit in the majority of patients during teenage years. Interictal EEG is usually normal, and (peri-)ictal EEG shows variable abnormalities.

A subgroup of patients with a PCDH19 mutation has a phenotype resembling Dravet syndrome. Most patients with Dravet syndrome carry a de novo mutation in the SCN1A gene [23] and have refractory seizures, (severe) ID and poor outcome [24–27]. PCDH19-positive patients differ from the classical SCN1A-positive Dravet patients though, first of all because prognosis seems more favourable. Seizure onset is somewhat later, seizures occur more in clusters and status epilepticus and myoclonic seizures occur less frequently. Moreover, in SCN1A-related Dravet syndrome, treatment with carbamazepine and lamotrigine is contraindicated as it may aggravate seizures [28, 29]. It remains to be seen if this also holds true for patients with Dravet syndrome features and a PCDH19 mutation.

Mutation spectrum

Most mutations are found in the large exon 1, corresponding to the extracellular cadherin domains, which are pivotal for normal function. In exons 3 to 6, encoding the cytoplasmic domain, a relatively small number of only truncating mutations are found. Amino acid substitutions in the cytoplasmic domain appear to have less or no deleterious effects on protein function. The truncating mutations in the cytoplasmic domain may result in nonsense mediated decay of the mRNA and thereby cause loss of function of the whole protein. The extracellular domain being involved in all cases supports the view that adverse interaction between genetically different cell populations, expressing one or the other allelic protein, is the pivotal mechanism of disease [30].

Clinical genetic aspects

Both truncating and missense mutations are associated with variable phenotypes. Among non-related patients with the same mutation and among affected relatives, there is variable expression, which may be explained by different patterns of X-inactivation in brain, the influence of genetic modifiers or mosaicism for the mutation in the brain of patients with de novo mutations. This variable expression makes it difficult to predict the clinical course for unborn or young children. From extended families, the penetrance in females appears to be high [4].

About 35 % of probands have an inherited mutation, with an inherent high recurrence risk for the probands’ sisters and possibly also for other female relatives. Because of the unusual X-linked mode of inheritance with asymptomatic transmitting males, it is highly important to recognize the clinical picture of EFMR in probands and to screen the parents once a PCDH19 mutation is found, also when they are asymptomatic. When providing genetic counselling, the possibility of gonadal mosaicism of the PCDH19 mutation in one of the parents should be taken into account [11].

Males with a PCDH19 mutation

Males carrying PCDH19 mutations are generally unaffected, although they seem to have a more rigid personality. Nevertheless, PCDH19 mutations in the mosaic state may cause EFMR. In males with clinical characteristics highly suggestive of EFMR, analysis of PCDH19 in different tissue samples to detect such a mosaicism might be considered.

In one out of 20 high-functioning males with ASD, we identified a missense variation in exon 3. The pathogenicity of this variation is unclear. On the one hand, the affected amino acid is highly conserved and has a high Grantham score. On the other hand, it affects the cytoplasmic domain of the protein, in which no pathogenic missense mutations have been found in females with EFMR. Previously, PCDH19 mutations have been reported in a male with ASD [31], in a male with schizophrenia [31] and in two males with intellectual disability [32]. Larger cohorts of males with ASD, but also of males with a broader spectrum of psychiatric and neurological phenotypes, need to be tested to further assess the significance of PCDH19 gene mutations in these disorders.