Whole Exome Sequencing Identifying Causative Gene In A Migraine Without Aura Pedigree

Objective: In the current study,we apply the high throughput sequencing platform and one generation sequencing on the collected pedigree to obtain the new mutated genes,to explore its correlation with pathogenesis,to enrich the etiology of migraine without aura(MO),and to provide the basis for the treatment of MO.Methods: We collected the propositus in the epilepsy center of Jiamusi Central Hospital,diagnosed as MO.There have eight MO patients in her pedigree by inquiring about her family history.First,the propositus?s small family was selected,containing three patients.Genomic DNA were extracted from peripheral blood samples,using a Flexi Gene DNA Kits(Qiagen,Germany).The genomic DNA library was prepared using the SureSelect Target Enrichment System(Agilent,USA)in accordance with the manufacturer's instructions.Using Sure Select Human All Exon Kit version 2,genomic DNA was randomly fragmented and hybridized with probes for capture and enrichment.The enriched DNA samples were sequenced via using a NextSeq 500 Sequencing System(Illumina,USA).During the process of analysising the whole exon group sequencing information,Align analysis,SNP analysis and DIP analysis were performed to identify missense mutations.Annotating all the variants,excluding the reported variants in OMIM,ACMG and 1000 human genome databases,we screened out mutations that cause protein truncation,splicing region mutation,and missense mutations in highly conserved sequence regions.Unreported non-synonymous amino acid variants were analyzed by Mutation Taster,Polyphen-2 and SIFT,in order to assess any potentially damaging effects.And the probability of the qualitative prediction results.Finally,The mutational sites that need to be further verified are obtained.For the selected gene mutation sites,Sanger sequencing was used to verify all the samples collected in the family.Results: WES was preceded in the small proposita family,containing three MO patients and one non-MO patients.By further data analysis,the missense mutations of the preliminary statistics were respectively 378394382 and 428 in the ?3??2??3 MO patients and ?4 non-MO patient.Removing the four common missense mutations,three patients shared 62 common missense mutations.After the final database filter,finally selected 11 new heterozygous single nucleotide mutation sites were DSCAML1 c.4829G>A(p.Arg1610His),IFIH1 c.1899G>T(p.Glu633Asp),CAPN2 c.1253G>A(p.Arg418Gln),MYRF c.649G>A(p.Ala217Thr),CGREF1 c.185A>T(p.Glu62Val),USP34 c.9448C>G(p.His3150Asp),SORBS2 c.1423G>A(p.Val475Ile),DUOX1 c.3312G>A(p.Met1104Ile),TBC1D8 c.2566A>G(p.Ile856Val),DNTT c.1382G>A(p.Arg461His),SSPO c.13733G>A(p.Arg4578Gln).Verified by Sanger sequencing,the variant point of the CAPN2 gene existed in the remaining three patients;the variant points of the MYRF gene,SORBS2 gene,DNTT gene and SSPO gene existed five patients in the pedigree.the variant points of the DSCAML1 gene,IFIH1 gene,CGREF1 gene and DUOX1 gene existed in the remaining one patients;the variant points of the USP34 gene and TBC1D8 gene didn't exist in the remaining patients.Conclusions: Eleven novel rare non-synonymous mutations for migraine without aura are identified by whole exome sequencing.The CAPN2 mutation gene is involved in six patients in the family,accounting for 75%.MYRF mutation gene,SORBS2 mutation gene,DNTT mutation gene and SSPO mutation gene are involved in five patients in the family,accounting for 62.5%.IFIH1 mutation gene,DSCAML1 mutation gene,CGREF1 mutation gene and DUOX1 mutation gene are involved in four patients in the family,accounting for 50%.USP34 mutation gene and TBC1D8 mutation gene are involved in three patients in the family,accounting for 37.5%.