Patient case: 58-year-old male with gastrointestinal stromal tumor; genetic feature: KIT (exon 9 502_503 duplication)

Clinical trial: Etiology and Treatment of Neonatal Seizure

Summary: Genetic diagnosis for neonates suffering from epilepsy has important implications for treatment, prognosis, and development of precision medicine strategies. Investigator performed exome sequencing (ES) or targeted sequencing on neonates with seizure onset within the first month of life. Investigator subgrouped our patients based on the onset age of seizure into neonatal and before 1 year (1-12 months), to compare the clinical and genetic features and treatment strategies.

Detailed description: Seizure is one of the most common neurological conditions in neonates, and has substantial impact on patients'quality of life and social integration. Epileptic encephalopathy is characterized by refractory seizures, cognitive dysfunction, and poor prognosis. Despite the recent progress in technology, molecular diagnosis of neonates suffering from possible epileptic seizures can be challenging, due to genetic and phenotypic heterogeneities. A large number of specific pathogenic variations have been related to various forms of epilepsies. Next-generation sequencing (NGS) has significantly improved the molecular diagnosis for rare diseases. NGS focusing on genes known to be associated with human diseases is a practical approach as a first-tier assessment for patients with heterogeneous genetic background. In addition, currently medical therapy for seizure is not based on the etiology, but the clinical manifestations, and the main purpose is not to rescue the underlying diseases process, but just to reduce the likelihood of seizures occurrence. In this study, Investigator performed NGS on neonates with seizure onset before 1 year of age, to detect and quantify genetic variants, and assess existing therapeutic effects. Our findings will have important implications for the development of precision medicine strategies. Patients are enrolled between January 1, 2014, and December 31, 2021 with the following inclusion criteria: (1) severe seizures in neonates or generalized seizure or intractable seizure in infancy, (2) seizures onset before 1 year of age, and (3) epileptic syndromes/epileptic encephalopathies with unknown etiology. Patients were excluded if they had traumas, central nervous system infections, hypoxic-ischemic encephalopathy, vascular events, systemic infections, and diagnosed metabolic disorders, and pathogenic copy-number variants were identified using array-based comparative genomic hybridization (CGH). Clinical manifestations, laboratory results including head computed tomography (CT) and magnetic resonance imaging (MRI), application of antiepileptic drugs, and prognosis were reviewed. Median follow-up duration after was 6 months. Seizure outcome was assessed by using 3 categories: seizure free, seizure reduction, and no change in seizure frequency. All samples in this study were collected with appropriate informed consent and approval of the ethics committee of Neonates's Hospital, Fudan University. The methods used in this study were carried out in accordance with the approved guidelines. Genomic DNA fragments of patients were enriched for panel sequencing using the Agilent (Santa Clara, CA, USA) ClearSeq Inherited Disease panel kit or for exome sequences using the Agilent SureSelectXT Human All Exon 50-Mb kit. DNA fragments were ligated with adaptors and two paired-end DNA libraries with insert size of 500 bp were formed for all samples. DNA libraries after the enrichment by polymerase chain reaction (PCR) were sequenced on the HiSeq2000/2500 sequencer according to the manufacturer's instructions (Illumina, San Diego, CA), resulting in the 90-bp paired-end sequencing reads with at least 100-fold average sequencing depth for each sample. Alignment and mapping Reads with adaptors, reads in which unknown bases (Ns) are more than 10%, and low-quality reads (the percentage of lowquality bases is over 50% in a read, and the low-quality bases are those whose sequencing quality is no more than 5) were discarded from raw data to generate clean reads. Clean reads were aligned to the reference human genome (UCSC hg19) by Burrows-Wheeler Aligner (BWA) (v.0.5.9-r16). Subsequent processing of sorting, merging, and removing duplication for the BAM files were performed by using SAMtools and Picard (http://picard.sourceforge.net/index.shtml). Variant calls, which differed from the reference sequence, were obtained with the use of GATK. Variant annotation and filtering Variants with suboptimal quality scores were removed from consideration. Remaining variants were annotated by ANNOVAR and VEP software6,7 and compared computationally with the list of reported pathogenic variations from the Human Gene Mutation Database (HGMD, professional version). Variants in HGMD were retained. For changes that are not in the HGMD, synonymous variants, intronic variants that were more than 15 bp from exon boundaries (which are unlikely to affect messenger RNA splicing), and common variants (minor allele frequency >1%) were also discarded. Missense variants were assessed with SIFT, PolyPhen-2, and MutationTaster. Criteria for classifying pathogenic and likely pathogenic variants Pathogenic variants: (1) this variant would likely explain the indication for testing and may be responsible for this individual clinical presentation; (2) same nucleotide and amino acid change as a previously established pathogenic variant from both published studies and internal database. Likely pathogenic variants: (1) this variant would likely explain the indication for testing and may be responsible for this individual clinical presentation; (2) same amino acid change as a previously established pathogenic variant regardless of nucleotide change; or null variant (nonsense, frameshift, canonical +/-1 or 2 splice sites, initiation codon, single or multiexon deletion) in a gene where loss of function (LOF) is a known mechanism of disease; (3) de novo (both maternity and paternity confirmed) in the proband in the negative family history; or inherited from the affected parents. Positive results: (1) one heterozygous pathogenic or likely pathogenic variant on an autosomal dominant, or X-linked dominant gene that explains the indication for testing; (2) one homozygous or two heterozygous pathogenic or likely pathogenic variants (compound heterozygous) on an autosomal recessive gene that explains the indication for testing; (3) one pathogenic or likely pathogenic variant on an Xlinked recessive gene in a male patient that explains the indication for testing.

Eligibility: Inclusion Criteria:
  -  (1) severe seizures in neonates or generalized epilepsy or intractable epilepsy in infancy with generalized tonic-clonic seizures,
  -  (2) seizures onset before 1 year of age,
  -  (3) epileptic syndromes/epileptic-encephalopathies with unknown etiology.
  Exclusion Criteria:
  -  Patients were excluded if they had traumas, central nervous system infections, hypoxic-ischemic encephalopathy, vascular events, systemic infections, and diagnosed metabolic disorders, and pathogenic copy-number variants were identified using array-based comparative genomic hybridization (CGH).

Relevance to patient case: not relevant