Novel and de novo point and large microdeletion mutation in PRRT2‐related epilepsy

Abstract Background Point and copy number variant mutations in the PRRT2 gene have been identified in a variety of paroxysmal disorders and different types of epilepsy. In this study, we analyzed the phenotypes and PRRT2‐related mutations in Chinese epilepsy children. Methods A total of 492 children with epilepsy were analyzed by whole exome sequencing (WES) and low‐coverage massively parallel CNV sequencing (CNV‐seq) to find the single nucleotide variants and copy number variations (CNVs). And quantitative polymerase chain reaction was utilized to verify the CNVs. Their clinical information was followed up. Results We found PRRT2‐related mutations in 19 patients (10 males and nine females, six sporadic cases and 13 family cases). Twelve point mutations, four whole gene deletion, and three 16p11.2 deletions were detected. The clinical features of 39 patients in 19 families included one early childhood myoclonic epilepsy (ECME), one febrile seizure (FS), two infantile convulsions with paroxysmal choreoathetosis (ICCA), six paroxysmal kinesigenic dyskinesias (PKD), 12 benign infantile epilepsy (BIE), and 17 benign familial infantile epilepsy (BFIE). All patients had normal brain MRI. Interictal EEG showed only one patient had generalized polyspike wave and five patients had focal transient discharges. Focal seizures originating in the frontal region were recorded in one patient, two from the temporal region, and two from the occipital region. Most patients were treated effectively with VPA or OXC, and the child with myoclonic seizures was not sensitive to antiepileptic drugs. Conclusion PRRT2 mutations can be inherited or de novo, mainly inherited. The clinical spectrum of PRRT2 mutation includes BIE, BFIE, ICCA, PKD, FS, and ECME. The PRRT2‐related mutations contained point mutation, whole gene deletion and 16p11.2 deletions, and large microdeletion mutations mostly de novo. It is the first report of PRRT2 mutation found in ECME. Our report expands the mutation and clinical spectrum of PRRT2‐related epilepsy.

Therefore, in this study, we conducted whole exome sequencing and copy number variation sequencing (CNV-seq) on 492 epilepsy children who had benign or refractory epilepsy, in order to find new epileptic phenotypes related to PRRT2 and verify the positive prediction results of PRRT2-related CNV by qPCR, so as to further clarify the role of 16p11.2 deletion and further understand the clinical and mutational features of PRRT2-related epilepsy.

| Subjects
We analyzed 492 epilepsy children with the onset age of 0-14 years between 2016 and 2019 in the Pediatrics Department of Qilu Hospital Affiliated to Shandong University and Linyi people's Hospital Affiliated to Shandong University, China. Their clinical information was retrospectively collected and followed up, including seizure types, onset age, treatment process, growth and development history, previous disease history, family history, intellectual test, cranial magnetic resonance imaging (MRI), and video-EEG, antiepileptic drugs (AEDs), and age of epilepsy remission. The patients were followed up by phone or visit clinic every three months. Exclusion criteria included seizures caused by nongenetic factors, such as an acquired brain injury, metabolic disease, and clinically phenotypically defined monogenic diseases (e.g., tuberous sclerosis complex).
The study protocol was approved by the ethical committee of the Qilu Hospital Affiliated to Shandong University  (027)) and Linyi people's hospital Affiliated to Shandong University (No.13003).
All guardians signed informed consent forms.

| Next-generation sequencing (NGS) and DNA sequence analysis
Informed consent and blood samples were obtained from all the participants in the families. Genomic DNA was extracted using QIAamp DNA Blood Mini Kit (Qiagen), according to the manufacturer's protocol. Each DNA sample is quantified by agarose gel electrophoresis and Nanodrop 2000 (Thermo). Libraries were prepared using Illumina standard protocol. The amplified DNA was captured with whole exome sequencing. The capture experiment was conducted according to manufacturer's protocol. 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 (hg19) by Burrows-Wheeler Alignment.
Single nucleotide variation (SNV) and insertion deletion mutation (InDel) were called by Genome Analysis Toolkit. Then, all SNVs and InDels were annotated by ANNOVAR (RRID: SCR_012821). The mutation sites with frequencies less than 0.05 in the normal population database were screened out, including the 1,000 genome pro- PRRT2 mutation found in ECME. Our report expands the mutation and clinical spectrum of PRRT2-related epilepsy.
The selected mutation sites were verified by Sanger sequencing. The analysis of deletions or duplications was performed using low-coverage massively parallel CNV sequencing (CNV-seq). After sequencing, the raw data were saved as a FASTQ format, then followed the bioinformatics analysis: First, Illumina sequencing adapters and low-quality reads (<80bp) were filtered by Cutadapt (1.16) software (RRID: SCR_011841). After quality control, the clean reads were mapped to the UCSC hg19 human reference genome using BWA (0.7.12) software (RRID: SCR_010910). Only uniquely mapped reads were selected. Then, we use GATK (4.0.8.1) (RRID: SCR_001876) Mark Duplicates to remove duplicated reads. Mapped reads were classified into adjustable sliding windows, which were 50 kb in length with 5 kb increments. The coverage of each window was calculated by the read amount and underwent two-step bias correction (GC correction and population-scale normalization). After correction, we use the binary segmentation algorithm to localize the segment breakpoints to identify the candidate CNV regions and determination CNV genotype. Then, we use U test and Parallelism test to estimate the genotype and significance of each segment. All the obtained suspected missing repetitive regions were compared with OMIM (RRID: SCR_006437), GeneReviews (RRID: SCR_006560), Decipher (RRID: SCR_006552), ClinVar (RRID: SCR_006169), Database of Genomic Variants (DGV, RRID: SCR_007000) and other databases. CNV-related genes will also be searched in the Human Phenotype Ontology (HPO, RRID: SCR_006016) database to match similar phenotypes. After the analysis, the data were analyzed for advanced manual analysis, and the suspicious mutation fragments that were highly similar to the clinical phenotype of the proband were selected. Then, real-time quantitative PCR detecting system (qPCR) experiment was conducted to verify this section, so as to exclude false positive of second-generation sequencing and ensure the accuracy of the results.

| Genetic analyses
We screened a cohort of 492 children with epilepsy for mutations in the PRRT2 gene using whole exome sequencing (WES) and low-coverage massively parallel CNV sequencing (CNV-seq) (281 male and 211 female). We found heterozygous PRRT2-related mutations in 19 patients (19/492, 3.86%), six sporadic cases and 13 family cases.

| Clinical features
The onset age of 492 children ranged from one day after birth to eight years. Thirty-two (32/492, 6.5%) patients were diagnosed as BIE, 61 as Febrile convulsion plus, 59 as West syndrome, 41 as Dravet syndrome, 12 as Ohtahara syndrome, four as Lennox-Gastaut syndrome, three as West syndrome evolving to Lennox-Gastaut syndrome, four as Doose syndrome, three as childhood absence epilepsy, two as benign childhood epilepsy with central temporal spikes, and one as early childhood myoclonic epilepsy (ECME). Twenty-nine patients were diagnosed as unclassified epileptic encephalopathy, and 241 patients were diagnosed as unclassified epilepsy. We found PRRT2-related mutations in 19 patients. Among the 19 probands, 10 were males and nine were females. The onset age of the 19 probands ranged from 3 months to 3 years and 2 months. The mode of these 19 patients is six months old, and the median is six months old too.
There were 39 patients in these 19 proband families with PRRT2 mutations. The common clinical features of the 39 patients included one ECME, one febrile seizure (FS), six paroxysmal kinesigenic dyskinesias (PKD), two infantile convulsions with paroxysmal choreoathetosis (ICCA), 12 benign infantile epilepsy (BIE), and 17 benign familial infantile epilepsy (BFIE). The clinical information of 39 patients with PRRT2 mutations in 19 proband's families is summarized in Table 3.
The proband with ECME was a boy aged four years and seven months old, the only child of nonconsanguineous parents.    (2017)), we diagnosed the child as ECME. This is the first report of PRRT2 F I G U R E 3 Quantitative PCR validation of whole PRRT2 gene deletion. Y-axes represent Log R ratio; the X-axis indicates the exon on PRRT2 hearing loss, and cardiac defects (Li et al., 2018b;Yang et al., 2015).

F I G U R E 4
In our study, all three BIE patients with 16p11.2 deletion had focal motor seizures, only one with the largest 16p11.2 deletion of 863kb  We had not found the autism or intellect regression in the other two children with 16p11.2 deletions of our study who had excellent seizure-free and developmental outcomes as followed up at 18 months to 31 months old. But, the patients with 16p11.2 deletions still need long-term clinical follow-up.

| CON CLUS IONS
In conclusion, PRRT2 mutations can be inherited or de novo. ZR2016HB38). We thank the patients and their family members for taking part in this study. Thanks to Rowan Coates for his guidance on English grammar.

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

F I G U R E 6
Ictal EEG and EMG tracing of patient II1 in family 1. High to very high 3-4Hz generalized polyspike waves with time-locked relation between muscle activation corresponding to a sudden shake of the whole body

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 on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.