Molecular Etiology Of Common Inner Ear Malformations And The Application Of CfBEST In Noninvasive Prenatal Detection Of Hereditary Deafness

Deafness is a major global health concern at present and in the future.Inner ear malformation(IEM)is one of the most important causes of deafness.Based on 2017 Sennaroglu Classification,there are altogether eight subsypes of IEM,among which incomplete partition of the cochlea(IP)and enlarged vestibular aqueduct(EVA)account for more than 80%.However,due to the extremely complex anatomy of the inner ear,along with insufficiency of both high-resolution computed tomography(CT)equipment and physicians skilled in diagnostic imaging,many cases are misdiagnosed or remain undiagnosed in local clinics.Therefore,it is of great significance to identify a new or complementary approach to improve the diagnosis of inner ear malformations.Along with the technologic progress of molecular diagnosis,scientists have found that the occurrence of IEM is closely related to genetic factors.Whether gene diagnosis can be a new diagnostic method of IEM has become our research goal.Part ?.Molecular etiology of common inner ear malformationsPurpose:To further broaden the gene mutation spectrum of inner ear malformations,fill the gap in the pathogenesis of IEM and establish the concept and method that gene diagnosis can precede imaging diagnosis Methods:From 2018 to 2020,77 unrelated patients with characteristic high-resolution computed tomographic images showing EVA,IP-? or IP-? were enrolled in this study.Targeted next-generation sequencing(TS)and Nanopore long-read sequencing were used to analyse the genetic etiology.Results:Firstly,in the molecular analysis of 65 patients with EVA or IP-?,we found that:1.SLC26A4 gene mutation was the most important molecular cause,accounting for 96.92%(63/65).A total of 29 pathogenic sites were found,of which c.919-2a>G was the hot spot mutation.2.The patients with SLC26A4 biallelic mutations were 100%associated with enlarged vestibular aqueduct.3.A new mutation c.304+941C>t was found by the third generation sequencing,and the functional experiment revealed that it would change gene splicing.4.The genotype combination did not cause the difference between inner ear malformation phenotype and hearing threshold phenotype(P>0.05);5.The mutation spectrum of EVAs gene in Fujian was drawn for the first time.Secondly,in the molecular etiology analysis of 12 patients with IP-?,it was found that POU3F4 gene mutation was the main cause,and the most common forms of mutations were point mutation and small INDELs.Among six patients with negative NGS results,three patients were volunteered to choose further Nanopore sequencing and were all found to have structural variations(SVs).Conclusions:The next generation sequencing technology greatly improves the diagnosis rate of molecular etiology in common IEM,thus establish the concept and methods which enable genetic diagnosis can precede that of imaging.Part ?.Application of the cfBEST technology in noninvasive prenatal testing for hereditary deafnessPurpose:Concerning with the prevention and control of hereditary deafness(HD),a clear molecular etiology is a prerequisite and prenatal diagnosis which is an important solution,but presently prenatal diagnosis of hereditary deafness is mainly achieved through invasive procedures,where some huge risks lurk.During the past ten years,Noninvasive prenatal testing(NIPT)for common aneuploidy has become into the routine check.However,concerning the test for but for monogenic disorders,even in the most recent studies,there are still limitations.The purpose of this study is to overcome these limitations:complex procedures,low detection accuracy and the need to know the parent genotype or haplotype in advance.Methods:This study actively explored noninvasive prenatal test(NIPT)of hereditary deafness based on a cell-free DNA(cfDNA)allelic molecule counting system termed cfDNA barcode-enabled single-molecule test(cfBEST)for speculating the genotypes of fetal.Results:In a double-blind evaluation of 20 singleton pregnancies with a risk of birthing a deaf child,the sensitivity,specificity,and diagnostic accuracy of cfBEST was 100%,99.78%,and 90%,respectively.Conclusions:As the cfBEST technology allows precise detection of maternal-fetal genotype combinations in cfDNA,it will have a broad application prospect in non-invasive prenatal detection(NIPT)of HD and will greatly reduce the economic and mental pressure of pregnant women.