Abstract

Background

Early infantile epilepticencephalopathy-9 (EIEE9) is an X-linked genetic disorder characterized by the onset of seizures during infancy. Mutations in PCDH19 are the main causes of EIEE9. The subject of our study is a child who presented with recurrent epileptic seizures and findings of abnormal synchronous discharges on electroencephalography. Our study aimed to identify causative variants of pathogenic genes and to confirm the diagnosis of epilepsy.

Case presentation

The proband is a girl who had been experiencing recurrent epileptic seizures for more than 2 years. The electroencephalography features strongly supported the diagnosis of epileptic encephalopathy. Whole-exome sequencing (WES) was conducted on the proband and parents to identify potential genetic variants. Candidate pathogenic variants were validated using Sanger sequencing. A pathogenic heterozygous variant was observed in PCDH19 (c.695A > G). Two likely benign variants, c.71C > T in GABRD and c.G1691A in SCN1A were also identified, including a paternally inherited nonsense heterozygous variant.

Conclusions

Based on clinical phenotyping and genetic analyses, the patient was diagnosed with PCDH19-female limited epilepsy. The application of WES to reveal the pathogenic variant was a crucial step in arriving at an accurate diagnosis. Genetic testing improves the diagnoses of PCDH19-related epilepsy manifesting with typical symptoms and provides valuable evidence that may enhance genetic counseling and clinical treatment guidance.

1. Introduction

Epilepsy is a recurrent chronic neurological disorder caused by sudden abnormal discharge of brain neurons, resulting in short-term brain dysfunction. A meta-analysis study estimated that the prevalence of epilepsy in China increased from 1.99‰ to 7.15‰ between 1990 and 2015 (1). Improved diagnostic methods have led to an increase in the number of reported epilepsy cases. Early diagnosis is crucial for proper management, given that nearly 30% of patients exhibit resistance to antiepileptic drugs. (2, 3).

The main genetic cause of infantile epilepsy is variability in the sodium voltage-gated channel alpha subunit 1 gene (SCN1A), and mutations in the protocadherin 19 gene (PCDH19) constitute the second most common cause (4). Mutations in the SCN1A gene have been associated with various seizure disorders, such as genetic epilepsy with febrile seizures plus (GEF+) and Dravet syndrome (5). PCDH19, a member of the cadherin super family, is mainly expressed in the nervous system. PCDH19 is located on chromosome Xq22, and its mutation leads to early infantile epileptic encephalopathy-9 (EIEE9, OMIM# 300088) (3).

EIEE9 is also called epilepsy and mental retardation limited to females (EFMR, MIM# 300088) and PCDH19-related epilepsy. It is an X-linked genetic disorder that affects heterozygous females and is transmitted through unaffected males. It is characterized by the onset of seizures during infancy sans cognitive impairment, various intellectual disturbances, and autistic features (3). Typical clinical features of PCDH19-related epilepsy include generalized or focal seizures, high sensitivity to fever, and occurrence in clusters (6).

Since the PCDH19 mutation was first reported to be related to EIEE9, many studies have expanded the clinical spectrum associated with variations in PCDH19(7–9). These studies demonstrated that next generation sequencing has the potential to elucidate the pathogenic mechanism underlying EIEE9 and yield information on gene mutation sites. This information can be useful for diagnosing individuals suspected with epilepsy and provide the basis for personalized treatment, genetic counseling, and disease risk assessment.

Our study aimed to identify causative variants of pathogenic genes and to confirm the diagnosis of epilepsy. In the current study, we identified a pathogenic de novo mutation with a high degree of association with the phenotype of PCDH19-related epilepsy in a girl. Our study introduces a pathogenic variant of EIEE9. This finding may contribute to more efficient diagnosis and treatment of this form of epilepsy.

Case presentation

Clinical phenotype and physical examination

The proband is a girl, 3 years and 10 months old, who had been experiencing recurrent epileptic seizures for more than 2 years. She was admitted to the pediatric ward of Shenzhen People’s Hospital in July 2020. At the age of 18 months, she had 3-day-long episodes of clustered febrile seizures characterized by loss of consciousness, eyes rolling upwards, drooling, and shaking limbs. Each episode lasted approximately 30 s to 1 min. Febrile convulsions occurred 2–3 times per year during the 2018–2019 period. In the same period, she also experienced recurrent febrile seizures, with such seizures lasting for 2–7 days. Although the patient did not exhibit any signs of intellectual disability (ID) or autism, she was inattentive and impulsive. Her parents were nonconsanguineous and had no family history of febrile seizures or epilepsy. Clinical data and physical examination did not indicate any signs of abnormalities. Head magnetic resonance imaging (MRI) findings were normal(Fig. 1). Serum ceruloplasmin and electrolyte levels were normal, and infection markers and autoimmune encephalitis antibodies were negative.

Electroencephalographic pattern and epilepsy diagnosis

Electroencephalography (EEG) was performed. The resting awake state (eyes closed) was characterized by a well-formed and fairly sustained posterior dominant rhythm of 5–6Hz of moderate amplitude, which was attenuated with eye opening. The frequency amplitude gradient was moderately well organized, and the background consisted of minimal delta, moderate theta, and decreased alpha and beta frequencies. No significant regional asymmetries of background activity were noted (Fig. 2A). The interictal period showed a few high-amplitude, generalized spike-and-wave or sharp-wave discharges (2–3.5 Hz) in the awake period, whereas a few high-amplitude spikes/sharps and spike/sharp waves were prominent in bifrontal areas in the awake and sleeping periods (Fig. 2B-D).

Seizure onset was signaled by a sudden jerk of the body while sleeping, eye opening, staring to the right, raised right upper limb, stiffness of the extremities, posture maintenance, and slight clonus. The synchronous EEG was characterized by generalized spike-and-wave or sharp-wave discharges, or by triphasic waves intermixed with low-amplitude fast activity. Generalized low-amplitude fast activity appeared in the initial portion of the epoch; activity gradually increased in frequency and amplitude and developed into continuous generalized spike waves. The high-frequency activity lasted for approximately 50–60 s and overlapped with many muscle artifacts recorded simultaneously in the electromyogram (EMG) bursts (Fig. 2E-H). The EEG features strongly supported the diagnosis of epileptic encephalopathy.

2. Materials And Methods

2.1 Whole-exome sequencing and data analysis

Whole-exome sequencing (WES) of the samples from the proband and her parents was conducted using the NovaSeq platform (Illumina, San Diego, CA).Variants were analyzed using a Genome Analysis Tool Kit (GATK) (9). Annotation was conducted by ANNOVAR(5), and an in-house pipeline was used to filter the common variants. Rare coding variants were filtered using minor allele frequency (MAF) in the 1000 Genome Project and gnomAD databases of the eastern Asian population (cut-off frequency, 0.001). Only rare and coding variants were considered as potential genetic variants. Tools from MutationTaster, Likelihood Ratio Test (LRT), and PolyPhen-2 (Polymorphism Phenotyping HVAR) were used to evaluate pathogenic variants. The American College of Medical Genetics and Genomics (ACMG) guidelines were used for variant interpretation and pathogenicity assessments(5).

2.2 Sanger sequencing validation

Blood samples were obtained from the patient and her parents. Genomic DNA was extracted from peripheral blood using the QIAamp DNA Blood Kit (Qiagen, Hilden, Germany), following the manufacturer’s protocol. The primers were designed using online tools of the IDT PrimerQuest software (https://sg.idtdna.com/Primerquest/Home/Index)(10). The primer sequences of GABRD and PCDH19 were as follows: GABRD-F, 5-CCAGAACAGTGCTGCATCC; R, 5-CCTCACCTCCGATGCCA; and PCDH19-F, 5-TGGATCGCTGGCGTTGA; R, 5-GAGACGCAGTCGCACTACA, respectively. All candidate gene segments were amplified via standard polymerase chain reaction. Candidate rare variants among subjects were genotyped using the Sanger sequencing platform. Sequences were analyzed using the Chromas software.

3. Results

3.1 Whole-exome sequencing revealed a pathogenic PCDH19 c.695A > G missense variant

To identify the relevant genetic variants of candidate genes obtained from the online database MalaCards, WES was performed on the proband and her parents. An in-house variant filtering pipeline was used, as described in the Methods section. Three coding variants were identified in epilepsy-associated candidate genes. One heterozygous variant was observed in exon 1 of PCDH19 (c.695A > G) of the proband (Fig. 3; Table 1). This variant arose de novo. ACMG interpretation indicated that it was a likely pathogenic variant, while the Clinvar database demonstrated that it was associated with EIEE9, with a pathogenic prediction. The proband carried a nonsense heterozygous variant in exon2 of GABRD (c.71C > T), which is a paternally inherited variant and considered benign (Fig. 3). One rare variant of spectrin alpha, non-erythrocytic1 (SPTAN1), was likely benign (c.G1691A, Table 1). Based on the non-typical familial inheritance mode and the known association between PCDH19 and EIEE9, the PCDH19 (c.695A > G) variant was identified as the most promising pathogenic variant. The clinical history of recurrent epileptic seizures and abnormal synchronous EEG discharges, combined with the discovery of the PCDH19 variant led to the diagnosis of PCDH19-related epilepsy.

Table 1

Rare variants of epilepsy associated genes identified in the proband.
Chromosome	Start	refGene	Alteration	avsnp147	gnomAD _EAS	ClinVar CLNSIG	P/M/L^a	ACMG
chr1	1956383	GABRD	NM_000815:c. 71 C > T:p.A24V	rs748341188	0.0001	NA	D/D/D	Uncertain significance
chrX	99662901	PCDH19	NM_001105243: c. 695 A > G:p.N232S	rs587784299	NA	Pathogenic	D/D/D	Likely_pathogenic
chr9	131345013	SPTAN1	NM_001130438: c. 1691 G > A:p.R564H	rs201168391	0.0003	Likely_benign	B/N/N	Likely_benign
^aMutationTaster (M), LRT (L), PolyPhen-2 (P) pathogenic evaluation results of variants.

3.2 Treatment of seizures

The disease phenotype was classified as EIEE9. After hospital admission, the patient continued to experience frequent convulsions, which manifested as clustered seizures 6–10 times/day, mostly while crying or sleeping. Phenobarbital sodium and a low dose of mannitol were administered to treat and suppress brain edema, respectively, followed by the administration of vitamin B6 and other treatments. This approach reduced the frequency of convulsions. A 15 hour video revealed EEG abnormalities in the child. Levetiracetam tablets (10 mg/kg/day) were prescribed as anti-epileptic treatment starting from July 06, 2020, and the dose was gradually increased to 40 mg /kg/day. Seizures did not recur, even during fever episodes. Since her discharge, the patient has been regularly attending the pediatric outpatient clinic and was seizure-free until the last follow-up in May 2022 at the time of preparing this report.

4. Discussion And Conclusions

This study describes a typical case of epilepsy in a girl presenting with recurrent seizures and abnormal synchronous EEG discharges. Further genetic studies identified rare mutations in three genes: a de novo pathogenic variant in PCDH19 (c.695A > G) and a benign variant each in SPTAN1 (c.G1691A) and GABRD (c.71C > T), respectively. The missense variant in PCDH19 (c.695A > G) has been previously reported in a patient with generalized tonic-clonic seizures(11). Based on the clinical phenotype and genetic findings, the patient was diagnosed with PCDH19-related epilepsy.

In the past few years, much progress has been made in clarifying the etiology of epilepsy. Some forms of epilepsy can be divided into genetic syndromes: Dravet syndrome (caused by variants in SCN1A (12)), CDKL5/STK9 Rett-like epileptic encephalopathy (13, 14), and EFMR associated with variants in PCDH19 (15). Many reports highlight that PCDH19 plays an important role in the disease process and diagnosis of EFMR. PCDH19 contains six exons that encode a protocadherin protein and contribute to the function of cadherin (3). Mutations in PCDH19 have been reported to cause sporadic infantile-onset epileptic encephalopathy in females, mostly occurring de novo(6). Although the physiological role and pathogenic mechanisms of PCDH19 remain unclear, a previous study suggested a function in establishing specific neuronal connections (16).

Epilepsy related to heterozygous female children with clinical manifestations of PCDH19 mutation has diverse phenotypes. Mutations in PCDH19 of heterozygous female patients and somatic mosaic male patients have been shown to cause EIEE9, demonstrating ID and autistic features (17). The onset age of PCDH19-related epilepsy ranges from 1 to 70 months, with a median of 10 months (15). Mental retardation is not a definite clinical feature of EFMR. Some patients with PCDH19 mutation present with certain clinical manifestations that overlap with Dravet syndrome without SCN1A mutations (4). PCDH19 plays a major role in infantile-onset familial or sporadic epilepsy in female patients (18, 19). In our study, clinical manifestations typical of PCDH19-related epilepsy were recorded in the patient, including a combination of fever-induced seizures, EEG abnormalities, and genetic defects. However, it is worth noting that our patient had normal neurodevelopment and speech in the 3 years follow-up. Despite no signs of cognitive impairment or behavioral problems,the clinical features, genetic results, and the unique mode of inheritance, confirmed the diagnosis with PCDH19-related epilepsy due to a variant in PCDH19 (c.695A > G).

According to a previous study, GABRD is a susceptible site for mutations underlying systemic epilepsy, and it contributes to common generalized epilepsies (20). Mutations in GABRD and GABRG2 have been shown to cause epilepsy by haploinsufficiency in GEF+ (21). In the present patients, the GABRD mutation was a benign variant, inherited from a healthy father. We also identified another variant of SPTAN1, c.G1691A. Several variants in SPTAN1 associated with early-onset epileptic encephalopathies, have been identified. In-frame duplications or deletion alterations in the SPTAN1 gene can cause EIEE type 5 (EIEE5) (22), and missense variants lead to neurodevelopmental disorders (23). Thus, owing to specific mutations, SPTAN1 encephalopathy has distinct clinical and neuroradiological features (24). Pathogenic evaluation according to the ACMG guidelines indicated that SPTAN1 (c.G1691A) was likely a benign variant. Based on these outcomes, our study indicated that the mutation in PCDH19 was the main genetic factor responsible for the phenotype of our patient.

Epilepsy tends to have an early onset in infancy, with or without fever sensitivity, cognitive impairment, and resistance to antiepileptic treatment (2). Of the many factors affecting epilepsy, genetic factors are essential. At present, early diagnosis and the implementation of effective treatment strategies to improve the clinical management of patients with PCDH19-related epilepsy pose a challenge. Thus, accurate diagnosis of this complex heterogeneous disease as early as possible is of utmost importance, and this can be achieved using genetic testing that reveals the genetic architecture and phenotypic and genetic complexities of epilepsy.

In conclusion, our study identified a typical case of epilepsy in a girl with a pathogenic de novo variant, c.695A > G in PCDH19, using WES. Further clinical and EEG analysis confirmed the diagnosis of EIEE9. NGS in genetic analyses may help improve the diagnosis of PCDH19-related epilepsy. High-throughput genetic analyses can be expected to facilitate more effective genetic counseling and clinical treatment guidance for genetically determined epilepsy.

Declarations

Ethics approval and consent to participate

Ethical approval was obtained from the ethics committee of the Shenzhen People’s Hospital.

All methods were performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and its later amendments or comparable ethical standards.

Informed consent to the research and publication of results was obtained from the parents.

Consent for publication

Written informed consent was obtained from the parents for publication of this case report and accompanying images.

Availability of data and material

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare no conflicts of interest.

Funding

The study was supported by the Guangdong Provincial Natural Science Foundation (2021A1515012325), the Guangdong Provincial Science and Technology Project (2017A020214016) , and Shenzhen Science and Technology Project (JCYJ20170413093032806).

Authors' Contributions

YY and KZ prepared the project proposal and study design. YY, DY, and LC conducted bioinformatics and statistical analysis of sequencing data. YC conducted sample collection and Sanger sequencing validation. YC and ZL conducted clinical diagnosis of epilepsy. DY assisted with the prepared and revised manuscript. All the authors have read and approved the final manuscript.

Acknowledgments

Thanks to the support from the platform of Key Laboratory of Shenzhen Respiratory Diseases (ZDSYS201504301616234).

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Table

Table1. Rare variants of epilepsy associated genes identified in the proband.

Chromosome	Start	refGene	Alteration	avsnp147	gnomAD _EAS	ClinVar CLNSIG	P/M/L^a	ACMG
chr1	1956383	GABRD	NM_000815:c. 71 C >T:p.A24V	rs748341188	0.0001	NA	D/D/D	Uncertain significance
chrX	99662901	PCDH19	NM_001105243: c. 695 A >G:p.N232S	rs587784299	NA	Pathogenic	D/D/D	Likely_pathogenic
chr9	131345013	SPTAN1	NM_001130438: c. 1691 G >A:p.R564H	rs201168391	0.0003	Likely_benign	B/N/N	Likely_benign

^aMutationTaster (M), LRT (L), PolyPhen-2 (P) pathogenic evaluation results of variants.

Identification of a pathogenic mutation in PCDH19-related female-limited epilepsy