High-Throughput Sequencing Combined With Minigene To Verify Gene Detection And Deafness Application Of Pathogenicity Identification Of New Splicing Variants

Objective:To detect the pathogenic genes of congenital deafness patients in Guangxi area through targeted enrichment of deafness genes,and to perform functional verification of new disease-causing sites.Minigene experiments were conducted to verify the pathogenicity of new splicing site variants,and understand the deafness genes of congenital deafness patients in Guangxi.The characteristics of mutation provide a basis for the prevention and treatment of deafness.Method:1.A total of 54 families were selected from special education schools in Guangxi and the immediate relatives of congenital deaf probands and deaf patients who were treated in our department,a total of 153 cases,including 59 cases of congenital deafness,and 5 families with family history of deafness.All patients have undergone medical history collection,physical examination,specialist physical examination,audiology and imaging examinations,and applied deafness gene targeted enrichment high-throughput sequencing technology to the 5 common deafness genes GJB2 and SLC26A4 in these 59 patients with congenital deafness.,MYO7A,MYO15A and mtDNA for detection.We performed bioinformatics analysis on the test data to screen candidate genes,and verified the mutation frequency in the gnomAD population database,and determined the pathogenic site.2.Minigene experimental verification was performed on rare splicing site mutations of newly-onset deafness genes.We transfected the target gene fragments into He La and 293T cell lines through plasmids for cell culture,extracting RNA from cultured cells,and perform RT-PCR In the experiment,gel electrophoresis of the two cell line products verified the molecular weight of the two bands;Sanger sequencing was performed on the recovered products to verify the shearing method,combined with bio-information analysis,software prediction and patient clinical phenotypes,according to ACMG guidelines for deafness The pathogenicity of rare gene splicing site mutations has been preliminarily verified in functional experiments.Result:1.Among 59 patients with congenital deafness,18 were detected with biallelic mutations,and the overall biallelic mutation detection rate was 30.51%(18/59),of which 8 cases were GJB2 gene mutations,biallelic The gene mutation detection rate was 13.56%(8/59),including 4 cases of c.109G>A homozygous mutation,1 case of c.508＿511dup/c.235 del compound heterozygous mutation,3cases of c.235 del C homozygous mutation,and 9 cases SLC26A4 gene mutation,the detection rate of biallelic mutation was 15.25%(9/59),including 1 case of c.919-2A>G homozygous mutation,c.1614+5G>A/c.919-2A>G 2 cases of compound heterozygous mutation,c.697G>C/c.919-2A>G compound heterozygous mutation in 1 case,c.1226G>A/c.1547 dup compound heterozygous mutation in 1 case,c.1079C>T/c.919-2A>G compound heterozygous mutation in 2 cases,c.2086C>T/c.754T>C compound heterozygous mutation in 2 cases,MYO15 A gene mutation in 1 case,the detection rate of biallelic mutation was 1.69%(1/59),including 1 case of c.3524dup/c.419 dup compound heterozygous mutation.MYO7A gene and mtDNA mutation were not detected.2.In this study,25 cases of GJB2 gene positive mutations(including homozygous,compound heterozygous,and single heterozygous mutations,the same below)were detected,and the positive detection rate was 42.47%(25/59).15 cases of SLC26A4 gene positive mutations.The positive detection rate was25.42%(15/59),of which c.1614+5G>A,c.1028T>G and c.431T>G were not reported in databases such as gnomAD,DVD,ClinVar,etc.,which are new mutations.8 cases of MYO15 A gene positive mutation,the positive detection rate was 13.56%(8/59),of which c.9571C>G,c.6692-1G>A were not reported in databases such as gnomAD,DVD,ClinVar,etc.,which are new mutations.3.The SLC26A4 gene c.1614+5G>A detected in the twin family is a new mutation,and there is no related report at home and abroad.The Minigene experiment at this locus indicates that the mutation will change the original shearing method and make the appearance The exon14 jumps and some introns are retained,which makes the length of mature mRNA lengthen,which is a pathogenic variant that affects shearing.Conclusion:1.GJB2 gene and SLC26A4 gene are the two most common pathogenic genes in congenital deafness patients in Guangxi in this study.GJB2 gene c.109G>A,c.235 del C and SLC26A4 gene c.919-2A>G are in this study.Hotspot mutation sites in patients with congenital deafness in the region.2.SLC26A4 gene c.1614+5G>A is a new pathogenic site,and its mutation will lead to large vestibular aqueduct syndrome.The identification of the pathogenicity of this site has expanded the mutation spectrum of SLC26A4 gene,which will be the site in the future.The rapid diagnosis provides a reliable basis.3.Common deafness genes GJB2,SLC26A4,MYO7A,MYO15A and mtDNA cannot fully meet the testing needs of patients with congenital deafness in Guangxi.The scope of testing must be further expanded,but they can still be used as the initial selection of deafness genes for patients with congenital deafness in Guangxi.4.High-throughput sequencing combined with Minigene verification can verify splicing aberrations at new splicing sites,providing reliable evidence support for its pathogenicity.