Chinese cases of early infantile epileptic encephalopathy: a novel mutation in the PCDH19 gene was proved in a mosaic male- case report

Epilepsy and mental retardation limited to females (EFMR; MIM 300088), first reported by Juberg et al. (1971), affects females but is transmitted through unaffected males, following an unusual X-linked form of inheritance [1]. EFMR, also known as early infantile epileptic encephalopathy-9 (EIEE9, OMIM# 300088), is a genetic disorder, of which the de novo or familial heterozygous variation of the PCDH19 gene (OMIM 300460) is forecasted to be the second most common cause in addition to variations of the sodium voltage-gated channel alpha subunit 1 (SCN1A) gene [2‐5]. Since the initial description of the PCDH19 gene, the evidence of a link between the clinical features of epilepsy and de novo or familial mutations in the PCDH19 gene has increased. These links include infantile onset of multiple seizure types, such as Dravet syndrome, generalized or focal epilepsy with or without fever sensitivity, and epilepsy of variable severity in females with or without intellectual disability, behavioural disturbance or psychiatric symptoms [2, 6‐8].

The PCDH19 gene is located on the X chromosome (Xq22.1) and encodes protocadherin-19. This is a calcium-dependent adhesion molecule belonging to the δ2-protocadherin (pcdhs) subclass of the cadherin (cdhs) superfamily [9]. The PCDH19 gene shows an unusual X-linked form of inheritance affecting only females but being transmitted via asymptomatic males, which implies that it is pathogenic in heterozygous mutated females but non-pathogenic in hemizygous mutated males [6, 10]. PCDH19 has been shown to be expressed in the central nervous system and is possibly involved in a cellular interference mechanism that affects brain development, neuronal connections, and intracellular signal transductions at the synaptic membrane [3, 11, 12].

Variations in the PCDH19 gene were first reported by Dibbens et al. (2008) in several families with the EFMR disorder [10]. Depienne et al. (2009) discovered that subjects with PCDH19-related disease might be normal during early development but experience seizure onset occurring in clusters with fever sensitivity, an observation similarly seen with Dravet syndrome (DS) [6]. An increasing number of female epilepsy patients with PCDH19 mutations have been reported over recent years, and the clinical spectrum of diseases associated with PCDH19 gene mutations has also expanded, including patients displaying focal or generalized seizures recurring in clusters or isolation with or without febrile trigger, ranging from mild to severe, with or without cognitive impairment, behavioural disturbance, and psychiatric symptoms [2, 4, 11, 13].

Up to now, the molecular genetic study for PCDH19-related epilepsy has been focused on females. Male subjects were generally excluded because of the unusual X-linked inheritance and the pathogenic hypothesis associated with the cellular interference mechanism. However, following the report of a male with a mosaic PCDH19 deletion [6], a small number of recent studies have described several alterations of the PCDH19 gene in mosaic male patients in succession [14‐17].

Here, we present evidence of three mutations of the PCDH19 gene in a mosaic male and two unrelated females in Chinese individuals with epilepsy, further contributing to the clinical understanding of EFMR in relation to the PCDH19 gene.

We recruited three Chinese children with epilepsy treated in the Qingdao Women & Children’s Hospital between March 2016 and April 2017. The clinical diagnosis was made by clinical geneticists. Additional clinical information relevant to the diagnosis was provided by the parents and the caregivers. The CARE guidelines were followed in reporting these cases. Two millilitres of venous blood was collected from each patient and each parent of the subject upon signed informed consent. Then, the blood samples were sent to Beijing Kangso Medical Inspection for gene testing: nervous system diseases “big bag” for patient 1 (a total of 1906 genes) and epilepsy and related paroxysmal diseases for patient 2 and patient 3 (a total of 518 genes). Genomic DNA from blood samples was extracted using the Qiagen FlexiGene DNA kit (Qiagen, Germany) following the manufacture’s guidelines. Candidate genes were screened for mutations in the coding exons and intron–exon boundaries using PCR of genomic DNA followed by sequencing. Primer sequences and PCR conditions are available upon request. The variations were sequenced with next-generation sequencing and verified with first-generation sequencing. Detailed experimental methods can be found in Additional file 1.

We identified three mutations of PCDH19-related epilepsy in a cohort of Chinese children. The clinical data and epileptogenic characters of the three patients carrying PCDH19 variations are described in Table 1.

In a 2016 study, Terracciano et al. revealed a male patient with the nonsense mutation c.918C > G (p.Tyr306Ter), the same position as patient 3 in our cohort. Here, we compared the findings from our study with those of Terracciano et al. [14]. Of the four patients, seizures started within the first year of life for two patients and after the first year of life for the other two, with an average age of onset of 12 months (6–20 months). All patients had generalized seizures or focal seizures with secondary generalization (SG), characterizing the onset of epilepsy. Cluster seizures occurred in all subjects, and patient 4 had confirmed epileptic status. Two patients had seizures associated with fever sensitivity at onset, and the other two were afebrile onset. EEG was normal in patients 2 and 4 but showed abnormality in patients 1 and 3. Brain MRI had no special findings. Psychiatric symptoms were absent from all the patients. However, patients 1 and 4 developed intellectual impairment and behavioural disturbance. Patient 1 had recurrent epilepsy despite being treated with multiple anti-epileptic drugs, suggesting that the seizures related to PCDH19 variations were difficult to control to a certain degree.

Despite the many clinical manifestations of PCDH19-related epilepsy, the disease shares some common characteristics, namely, onset at the age of infancy and babyhood, generalized seizures and cluster seizures [3]. Generalized tonic seizures (TS), tonic-clonic seizures (GTCS) or focal seizures with SG occurring in clusters with or without fever sensitivity are the common seizure types. Other seizure types, such as myoclonic seizures, atonic seizures and atypical absences, are rare. Intellectual development abnormalities vary from normal to severe impairment. Patients may or may not display psychiatric symptoms and behavioural disturbance [4, 13].

Of the four patients, three genetic alterations were de novo, and one was inherited from her asymptomatic father. PCDH19 mutations in most patients are sporadic and de novo, whilst some are of paternal inheritance. This makes it difficult to recognize the pattern of inheritance [18, 19]. To determine the complete feature of EFMR, more patients need to be studied.

With the progress of genetic detection technology, thus far, over 150 PCDH19 mutations have been identified, featuring small deletion, small insertion, nonsense, missense, microduplication, intragenic deletion, splice site mutation and whole gene deletion. The great majority of the variations (over 90%) are located in exon 1, the largest exon encoding the extracellular cadherin domain [5]. In our study, we report three mutations located in exon 1, including a frameshift mutation, a missense mutation and a nonsense mutation. One mutation was novel, and two were reported recently. The frameshift alteration c.1508_1509insT (p.Thr504HisfsTer19) was identified in a mosaic male, which was a novel and de novo mutation. This discovery expands the spectrum of PCDH19-related epilepsy in males. A brief review of mosaic males in terms of the PCDH19 gene are as follows: Stosser et al. (2017) identified five males with mosaic pathogenic variants presenting with epilepsy with or without signs of developmental delay, similar to female patients with PCDH19 pathogenic variants [20]. Other researchers also reported several variants in mosaic males, and these male patients with mosaic mutations showed similar clinical features of epileptic encephalopathy to those presented in PCDH19 affected females. The previously described pathogenic mutations in mosaic males contain frameshift mutation, nonsense variant, missense mutation of exon 1 and alteration located in the splice site of intron 1 [14, 16, 17, 20]. Nevertheless, here, we attest the first male with the PCDH19 gene pathogenic variant c.1508_1509insT, which contributes to further explanation of this complex and an interesting genetic disorder.

The missense mutation c.1681C > T (p.Pro561Ser) was reported in a female patient by Carvill in 2013 [21] and the nonsense mutation c.918C > G (p.Tyr306Ter) in a mosaic male patient by Terracciano in 2016 [14]. These reports together with our findings collectively suggest that PCDH19 gene mutations have a significant association with epilepsy. The missense alteration was de novo. The nonsense mutation was transmitted from an asymptomatic father, but the transmitting male showed no abnormalities because of the unusual X-linked inheritance of PCDH19.

The full-length mRNA of the PCDH19 gene is 9765 nucleotides, encoding a 1148 amino acid protein. The PCDH19 gene consists of six exons; exon 1 is the largest exon encoding a signal peptide, the whole extracellular domain and a short transmembrane domain (TM), including six repeats of EC cadherin regulating cell-cell interactions [10]. All three mutations we identified in the present study were found in exon 1 of the PCDH19 gene, with the nonsense mutation c.918C > G (p.Tyr306Ter) in EC3 and both the frameshift mutation c.1508_1509insT (p.Thr504HisfsTer19) and the missense mutation c.1681C > T (p.Pro561Ser) in EC5 (Fig. 4). The nonsense and missense mutations were predicted to be deleterious in our report and were previously reported [14, 21]. Although the frameshift alteration was novel, the frameshift nature of c.1508_1509insT and the predicted introduction of a termination codon of p.Thr504HisfsTer19 are predicted to be harmful. Moreover, the extracellular domains of six EC cadherin repeats were highly conserved in the amino acid sequence and shown to be critical for normal protein function. Hence, our study added further evidence to the pathology of these mutations.

In conclusion, we report three mutations in the PCDH19 gene associated with early infantile epileptic encephalopathy. Of the three mutations, one variation was in a mosaic male and is a novel finding, the missense variation is de novo and is the second female case reported, and the nonsense variation is of paternal inheritance and is the first report in a female. Our report provides further proof and opens a new point of view for the molecular genetic diagnosis of patients with a special type of epilepsy. Perhaps more significantly is that the new mutation discovered in the mosaic male would expand the current knowledge about EIEE9 and guide diagnosis and targeted therapy in male patients. Finally, there are notable limitations to this study, including the relatively small sample size. However, this study has certainly opened a new frontier for further investigation into early infantile epileptic encephalopathy related to the PCDH19 gene in the future.