Molecular Diagnostics And Variant Interpretation Of The SLC26A4 Gene In Hearing Impairment Patients With Enlarged Vestibular Aqueduct

BackgroundDeafness is one of the most common human disabilities that leads to malfunctional recognition and communication.It accounts for 33% of all types of disabilities and causes a huge spiritual and economic burden on patients,families and society.Enlarged vestibular aqueduct(EVA)is the most common malformation of the inner ear among children in China.SLC26A4 is the second most frequently mutated gene found in Chinese patients with hearing loss,which accounts for 5-19% of the patients with NSHL.The SLC26A4 gene is the only gene identified so far that highly specific for the EVA.In previous studies,approximately 90% of EVA patients can be diagnosed by detecting the mutations on the SLC26A4 gene.ObjectivesThis study aims to develop a rapid,low-cost,and high-throughput panel for SLC26A4 detection based on multiplex PCR amplification and next-generation sequencing.Through multi-approaches to analyze the SLC26A4 gene in EVA patients,we expectall patients can obtain genetic diagnosis.Furthermore,genetic counseling is provided for patients and their families to reduce secondary damages.Methods1.In this study,we designed a new molecular diagnosis panel for EVA based on Multiplex PCR Enrichment and Next-Generation Sequencing of the exon and flanking regions of SLC26A4.Multiple pairs of primers are tested to determine the optimal reaction conditions and used to amplify the target area,construct the library,and perform High-throughput sequencing.2.This study recruited families of EVA patients,used this detection method for genetic testing,and used the custom analysis pipeline for SNP/Indel analysis.The detected variants are verified by Sanger sequencing,and the detected rare variants are interpreted according to the ACMG guidelines.3.For the undiagnosed(only one or none mutation in SLC26A4 gene)EVA patients,we performed CNV analysis by mosdepth software,and further used longdistance PCR and Sanger sequencing or the Third-Generation Sequencing technology to verify the CNVs breakpoint.Results1.Our results showed that 107/112(95.54%)families carried SLC26A4 biallelic mutations,4/112(3.57%)carried monoallelic variants,and 1/112(0.89%)had none variant,resulting in a diagnostic rate of 95.54%.The results are verified by Sanger sequencing.This detection method has been applied for an invention patent.2.In addition,we interpreted the pathogenicity of the rare variants detected in the SLC26A4 gene according to the ACMG guidelines.Among the 30 rare mutations,26 variants were Pathogenic,4 as Likely Pathogenic.Compared with the Clinvar database,20 variants were update from LP/VUS/Not include to Pathogenic.3.Copy number variations were detected in five of ten families(two families are from 112 families mentioned above),a 1845 bp heterozygous deletion of SLC26A4 gene exons5-6 was detected in two families,and a homozygous deletion was detected in one family.A 7666 bp heterozygous deletion of SLC26A4 gene exons1-3 was detected in one family and a heterozygous deletion of SLC26A4 gene exons9-10 was detected in one family.The test results of 112 EVA families showed that the diagnosis rate was95.54%.Combined with CNV diagnosis,the final diagnosis rate was 97.32%.Conclusion1.In this study,we designed a new molecular diagnosis panel for EVA based on Multiplex PCR Enrichment and Next-Generation Sequencing of the SLC26A4.The assay is fast and low-cost,which can be completed in thirty-six hours.This panel greatly improved the genetic diagnosis rate of EVA patients and the highest diagnosis rate compared with similar products at home and abroad.Comparison experiments and Sanger sequencing have also verified that the accuracy of this detection method is100%.2.We interpreted the genetic variants identified in the SLC26A4 gene according to the standards and guidelines for interpreting genetic variants proposed by the ACMG,improved the pathogenicity of rare mutations,provided more reliable test results for patients,and also provided a reference for other researchers.3.We also detected CNVs in 5 families,which may be an important cause of EVA.Therefore,it is highly recommended that the genetics analysis should be performed on both the EVA patients and their parents,which is a better strategy for screening and identifying the pathogenic variation of the patients.In addition,we also detected two novel mutations,which are SLC26A4: c.2162C>A and SLC26A4: Exons9-10 deletion.