Genetic Etiology Study In 208 Families With Epilepsy

Background Epilepsy is a chronic disease characterized by recurrent spontaneous episodes,usually beginning in childhood,with a prevalence of approximately 7.1‰ in children.70%-80% of epilepsy is related to genetic mutation.At present,epilepsy diseases are roughly divided into two categories according to their symptoms: one is idiopathic epilepsy,which is only clinically manifested as epilepsy;the other is associated symptoms of other neurological diseases,called syndromic epilepsy.Genetic factors that cause epilepsy include chromosomal aneuploidy,copy number variations(CNVs),single-nucleotide variants(SNVs)in specific genes,small insertion or deletion variants(In Dels)and abnormal amplification of tandem repeats.Epilepsy caused by chromosomal abnormalities and chromosomal microdeletions and microduplications is usually accompanied by severe phenotypes such as special facial features,intellectual developmental disabilities,and multiple malformations.More than 900 genes have been reported to be associated with epileptic seizures,among which more than 80 pathogenic genes with epilepsy as the main manifestation.Epilepsy belongs to a large class of diseases with strong phenotypic and genetic heterogeneity,and it is easy to miss diagnosis if only a single gene or mutation hotspot is detected.With the clinical application of Whole Exon Sequencing(WES)technology,the detection of epilepsy The drop rate increased from 5-12% to22-59%.However,until now,there is no uniform standard for genetic testing strategies for epilepsy patients.There are few studies on the genetic etiology of epilepsy patients with large samples and in-depth discussions on the genotype-phenotype of patients in China.In addition,for the variant sites of unknown significance detected by the WES method,various functional studies are also needed to provide more pathogenic evidence.Object To study the genetic etiology of 208 epilepsy families,and to analyze the genotype-phenotype relationship of patients by consulting databases and literature,aiming to discover new gene-disease associations.In vitro study was performed on some of the detected variants of unknown significance to clarify the pathogenicity of the variants.Methods 208 families with epilepsy and epilepsy children with pregnancy history were collected,clinical data were improved,and peripheral blood was collected from patients and family members for genetic testing of probands or core members;suspicious variants were screened according to mutation frequency and prediction of harmfulness of mutant proteins;Family mutation co-segregation and mutation source verification by Sanger sequencing;combined genotype-phenotype analysis,pathogenic interpretation of potential variants;for splice site mutations without a clear splicing pattern,peripheral blood leukocytes or transgenic cells are extracted from patients.HEK293T/He La cells were transfected with minigene RNA for reverse transcription PCR(RT-PCR).At the same time,for variants of unknown significance,HEK293T/He La cells were transfected by constructing wild-type and mutant plasmids,and real-time fluorescence was used.Quantitative PCR technology(Real-time quantitative PCR,q PCR)was used to analyze the changes of transcription level,Western blot was used to study the effect of mutation on protein expression,dual luciferase was used to analyze the effect of mutation on transcriptional regulation,and EMSA experiment was used to further explore the cause of Causes of impaired transcriptional regulation.Results(1)CNVs were detected in 84 families in this study,and pathogenic copy number variants were found in 22 families,with a diagnosis rate of 26.2%.87 families were tested for WES,of which 36 were probands with negative copy number variation test(singleton-WES)test,14 families obtained diagnostic results,the diagnostic rate was 38.9%;51 were not tested In the family WES(trio-WES)test for copy number variation detection,diagnostic results were obtained in 30 families,with a diagnostic rate of 58.9%.There were also 48 pedigrees with a clinical diagnosis,and single gene-targeted Sanger sequencing was directly performed,and 31 pedigrees obtained diagnostic results,with a diagnostic rate of 64.6%.A total of 97 families were identified in this study,and the overall diagnosis rate was 46.6 %.A total of 19 new mutations that were not included in the database and literature were found in the study.Trio-WES showed additional advantages in detecting the genetic etiology of epilepsy.(2)Genotype-phenotype association analysis showed that pathogenic mutations in SBDS may lead to epilepsy phenotype in patients.The discovery of the c.1937＿1944dup mutation in the MED13 L in patients with Dravet syndrome is the first report that a pathogenic mutation in the MED13 L is associated with Dravet syndrome.(3)The RT-PCR results of 8 splicing site mutations with no clear splicing pattern showed that: c.998+3＿998+6del of MFSD8 resulted in complete deletion of exon 10;c.343+1G >A of RDH12 leads to the retention of 15 bp bases at the 3’ end of exon 5,resulting in the insertion of 5 amino acids at positions 115-119 of the protein-coding amino acids;ATP7A gene c.1870-13T>G leads to complete deletion of exon 8;CHD7 gene c.8077-1G>A resulted in the deletion of 1 bp base at the 5’ end of exon 38,resulting in the mutation of the 2697 th amino acid into a stop codon,and the length was shortened by 301 amino acids;the c.199+6T>C of the FGF12 may be Does not affect splicing;three splice site variants in the COQ4 gene:c.402+1G>C,c.402+1G>T,c.402+1G>A all lead to the retention of intron 4,and in A premature stop codon(PTC)was generated at amino acid position 164,terminating translation 102 amino acids earlier.(4)The 3 missense mutations of unknown significance screened by the above trio-WES assay were studied.The results showed that the expression level of the COQ4 gene c.550T>C mutant protein was lower than that of the wild-type protein;FGF12 gene c.563G>A was no significant difference compared with wild type;DEAF1 gene c.825C>G mutant protein could not bind to target DNA,thereby affecting the transcriptional regulation function of the protein.Conclusion: 1.In this study,208 clinically diagnosed epilepsy patients were screened for related gene variants by various genetic methods,and a total of 97 families were identified as the etiology.2.A total of 19 new mutations that were not included in the database and literature were found in this study.3.This study is the first to report the epilepsy phenotype of patients with SBDS pathogenic mutations.4.An unreported pathogenic mutation in the MED13 L was found in the diagnosis of Dravet syndrome,which not only enriched the phenotype of the disease caused by the MED13 L,but also expanded the genotype of Dravet syndrome.5.This study confirmed that MFSD8,RDH12,ATP7 A,CHD7,and COQ4 splicing site variants can cause the production of abnormally spliced transcripts,and preliminarily clarified the pathogenicity of the seven splicing site variants at the gene transcription level.6.This study confirmed for the first time that pathogenic mutations in the zinc-finger region of DEAF1 cause pathogenicity by affecting transcription by failing to bind to target DNA;the decrease in transcription level may be one of the pathogenic causes of missense mutations in COQ4.