A Multiplex PCR Amplicon Sequencing Assay To Screen Genetic Hearing Loss Variants In Chinese Newborns

BackgroundHearing loss is a common sensory dysfunction and one of the most common birth defects.About two-thirds of Hearing loss are related to genetic factors.Undetected hearing loss may cause delays in language acquisition,behavior,and learning.Three genes(GJB2,SLC26A4,MT-RNR1)contribute the most deafness cases.Genetic screens for hearing loss can reveal the etiology of deafness,compensate the physical newborn hearing screening,and informing at-risk newborns and their maternal relatives of increased susceptibility to ototoxicity.Multiplex PCR amplicon sequencing is a technique to enrich and sequence targeted regions.It uses multiplex PCR to amplify DNA fragments in targeted regions,then constructs the library,and finally performs massively parallel sequencing.Multiplex PCR amplicon sequencing has the advantages of a high sensitivity and specificity,high throughput,and low cost.With such advantages,the multiplex PCR amplicon sequencing assay is a reliable test to detect variants in genetic hearing loss genes in a large number of samples rapidly.ObjectiveIn order to develop a fast,efficient,high-throughput,cost-effective test to screen variants in genetic hearing loss genes.In this study,we developed a multiplex PCR amplicon sequencing assay to sequence all the coding regions of the GJB2 gene,the most pathogenic variants of the SLC26A4 gene,and hotspot variants of the MT-RNR1 gene.MethodsIn this study,the method of multiplex PCR amplicon sequencing analysis was adopted.Firstly,this assay was validated by positive samples with known genotypes.The process includes DNA extraction,multiplex PCR amplification,library construction,massively parallel sequencing and bioinformatics analysis.After evaluating the sensitivity,specificity and stability of this method,the clinical anonymous newborn samples were finally screened to evaluate its clinical value.Results1.This study included 94 variants of GJB2 gene,79 variants of SLC26A4 gene,3variants of MT-RNR1 gene,and a total of 176 variants.2.192 positive samples of whole blood with known genotypes,92 positive samples of dried blood with known genotypes were tested by using the multiplex PCR amplicon sequencing assay.268 variants from 192 whole blood samples and 101 variants from the 92 dried blood samples were all detected.The results are completely consistent with the Sanger sequencing results.3.To validate the assay,we analyzed the test-retest reliability and internal consistency reliability.Specifically,31 samples were tested for test-retest reliability,and 14 samples were tested for the internal consistency reliability.The reads ratio was1 for variants in homozygous state and 0.5 for heterozygous states.There is no significant difference between the reads ratio of the test results.4.171 out of 1490 newborns(11.48%)carried at least one screened variants in genetic hearing loss genes.Among them,150 cases were heterozygous,5 cases were homozygous,15 cases were homoplasmy,and 1 case was heteroplasmy.The carrier rate was 7.52%(112/1490),2.89%(43/1490),and 1.07%(16/1490)for the GJB2,SLC26A4,MT-RNR1 genes,respectively.ConclusionsIn this study,we developed a multiplex PCR amplicon sequencing assay to sequence the full coding region of the GJB2 gene,the most pathogenic variants of the SLC26A4 gene,and hotspot variants in the MT-RNR1 gene.We then validated and piloted the genetic test in a newborn population.The multiplex PCR amplicon sequencing assay is an accurate and reliable test to detect hearing loss variants in the GJB2,SLC26A4,and MT-RNR1 genes.It can be used to screen genetic hearing loss in newborns.