Study On BAFME Causative Gene Identification And Secondary Findings Mining Of NDD Disease Cohort

Part 1Purpose:Benign adult familial myoclonic epilepsy（BAFME）is an epilepsy syndrome with an autosomal dominant inheritance pattern.In order to clarify the genetic etiology and provide a new reference for elucidating the pathogenesis of BAFME,we aimed to analyze the clinical characteristics of two newly collected Chinese BAFME pedigrees in detail,and use various sequencing technologies to identify their causative genes.Methods:Whole exome sequencing（WES）was performed on the two BAFME pedigrees respectively,and the variants were screened based on the frequency of public databases such as the 1000 Genomes Database,variant type,inheritance pattern,and harmfulness prediction.Sanger sequencing was performed to validate the candidate variants.Whole Genome Sequencing（WGS）was performed on three members of family1,and Nanopore Sequencing on one member of family 2.Repeat-primed PCR（RP-PCR）was used to validate of the identified variants within two families.Results:All patients had a benign non-progressive disease course,and the core symptoms showed obvious genetic anticipation at the age of onset.The candidate variants obtained by second-generation sequencing did not co-segregate with the phenotype in the family,and the pathogenic gene of BAFME was not successfully identified.The third-generation sequencing successfully identified the TTTTA/TTTCA pentanucleotide located in the intron region of the SAMD12 gene.The average number of the abnormal repeat is（TTTCA）₂₁₉（TTTTA）₅₈₅.RP-PCR found（TTTCA）_npentanucleotide insertions co-segregate with phenotypes in both families,while aberrant expansion of（TTTTA）_n was found in three healthy individuals.Conclusion:BAFME is a benign non-progressive epilepsy syndrome.Abnormal expansion and insertion of（TTTCA）_n in the intron region of SAMD12 gene is the pathogenic variant of these two BAFME pedigrees from Hunan,China.Our study expands the BAFME pedigree and genetic spectrum,providing a new reference for elucidating the pathogenesis of BAFME.The third-generation sequencing technology represented by Nanopore sequencing is a reliable method to identify complex structural variants.Part 2Purpose:While next-generation sequencing is performed on patients,in addition to detecting variants related to the main clinical symptoms of patients,some variants that are not related to the main phenotype but have medical value may also be detected,which are called secondary finding.Based on the ACMG SF v2.0,we intended to conduct secondary findings study in a cohort of 1330 participants which consists of neurodevelopmental disorders and their relatives to explore the secondary findings of the Chinese population,which will provide new evidence for how to interpret secondary findings.Methods:Whole-exome sequencing was performed on all participants,and variants in 59 secondary-discovery-related genes within the ACMG recommendations were extracted from the sequencing data.After quality control of the extracted variants,variants defined as DM or DM?by the HGMD database and P or LP variants identified according to ACMG guidelines were selected.Further screening was performed based on allele frequency and sequencing depth to obtain possible disease-associated variants.Families with these variants were followed up comprehensively and verified by Sanger sequencing for reliable clinical and genetic evidence.Results:A total of 297 variants were screened,including 149pathogenic variants（DM）in the HGMD database,94 suspected pathogenic（DM?）variants,and 16 pathogenic variants（P）determined according to the ACMGP Genetic Variation Interpretation Guidelines.181 and possibly pathogenic variants（LP）.Through further follow-up and verification,9 secondary variants from 10 families with credible clinical and genetic evidence carrying secondary variants were finally identified,and they were associated with oncogenic diseases,cardiogenic diseases,hypercholesterolemia,and connective tissue disorders.Conclusions:Our study identified 9 secondary variants from 10families with neurodevelopmental disorders.For the first time,we provided convincing clinical evidence for a variant associated with the rare connective tissue disease vascular Ehlers-Danlos syndrome in a secondary finding study.Next-generation sequencing technology can discover medically actionable secondary variants.Identifying and reporting these secondary variants can help in the early prevention and treatment of related diseases,thereby benefiting patients,which is of great positive significance.