Analysis Of Genotype-Phenotype Correlations In Deafness Syndrome And Exploration Of Early Non-invasive Prenatal Testing Based On Endocervical Fetal Trophoblast

According to the last update of the World Health Organization,approximately 466 million people worldwide,equaling 5%of the world’s population,suffer from disabling hearing loss(HL).About 60%cases considered to be genetic and approximately 30%of the genetic HL are expected to be syndromic.This study investigated the molecular etiology and genotype-phenotype correlation of Waardenburg Syndrome(WS)and Treacher Collins Syndrome(TCS),which is required for precise diagnosis,genetic counseling,and future treatment with mutation-specific therapy.Prenatal diagnosis of gentic HL is very important bacause of the high prevalence and the potential fatal birth defects.Noninvasive prenatal testing(NIPT)with circulating cell-free fetal DNA is limited by a low fetal DNA fraction.The collection of trophoblast cells from the endocervical canal during early pregnancy is another approach for NIPT.Our study investigated the possibility of recovery fetal trophoblast from endocervix for genetic diagnosis.Part I Analysis of Genotype-Phenotype Correlations in Waardenburg SyndromeWaardenburg syndrome(WS)is an genetically heterogeneous and phenotypically variable disorder characterized by congenital sensorineural hearing loss and pigmentary abnormalities.This study investigated the mutation spectrum and genotype-phenotype correlation of WS in 90 Chinese probands.All 90 probands have hearing loss and at least one type of pigmentary abnormalities including heterochromia(85.6%,77/90),freckles(17.8%,16/90),premature graying of the hair(10%,9/90),white forelock(8.9%,8/90),skin hypopigmentation(5.6%,5/90).Molecular investigation based on target-next generation sequencing(NGS)and Sanger sequencing detected PAX3 mutations in 13(14.4%)probands,MITF mutations in 22(40%)probands,SOX10 mutations in 20(36.4%)probands,and 35(38.9%)probands remain molecularly unexplained.In total,51 heterozygous mutations in PAX3,MITF,and SOX10 were detected.The majority(66.7%,34/51)of the mutations were truncating or null mutations including nonsense mutations(n=17),frameshifting mutations(n=10),synonymous mutation(n=1),splicing site mutations(n=2)and gross deletion(n=4).Besides,15 missense mutations and 2 non-frameshifting deletions were classified to be pathogenic according to the Standards and Guidelines for the Interpretation of Sequence Variants proposed by ACMG.Using Cohen’s Kappa coefficient to compare the W index and genetic analysis as predictors of subtypes of WS.There was a poor concordance between the W index>1.95 and the mutation analysis.The ROC curves indicated that the W index had a less discriminatory power for WS1.De novo mutations were more frequently seen for SOX10(15/20)than PAX3 mutations(3/13,P=0.005).Asymmetrical hearing loss were more frequently seen for PAX3 mutations(4/13)than SOX10(0/20,P=0.017).Excessive freckle was more frequent in probands with MITF mutations(9/22)than those with SOX10 mutations(0/20,P=0.001)and PAX3 mutations(0/13,P=0.013).The most frequently aberrations were malformed vestibule and semicircular canals(49%,27/55).And comblined cochlea hypoplasia Ⅲ was frequent in probands with SOX10 mutations(15/20)but absent in those with PAX3(0/13,P=0.000)or MITF mutations(0/22,P=0.000).Besides,we present a WS family including three WS patients with no mutations were identified through NGS.A SINE-VNTR-Alu(SVA)retrotransposition insertion into intron 2 of SOXIO gene was detected by gennome sequencing by long-read Nanopore sequencer.This retrotransposon insertion was identified by targeted long-read Pacbio sequencing.And gel electrophoresis of the PCR products showed bands of different size in the three patients and control samples in the family.Experimental validation such as transcriptome analyses still need to be finished.In addition,we report a sporadic WS case which was initially identified as de novo mutation of SOXIO.Analysis of tissue of diflerent embryonic origin in the father confirmed mosaicism in both somatic cells and reproductive cells by PCR and deep sequencing of the target mutation.Part Ⅱ Analysis of Genotype-Phenotype Correlations in Treacher Collins SyndromeTreacher Collins syndrome is a clinically and genetically heterogeneous disorder characterized by craniofacial dysostosis and hearing loss mainly caused by mutations in TCOFI,POLRID,and POLRIC.In our study,detailed phenotypic and genetic analyses were performed on five Chinese sporadic or familial TCS cases.A novel gross deletion,a novel small deletion and two known deletions within TCOF1 as well as a known mutation in POLRID were identified.To our knowledge,this is the first report of Chinese TCS cases caused by a gross deletion within TCOF1.These findings expand the mutation spectrum of TCS in the Chinese population.We propose that analyses such as NGS or MLPA capable of detecting large deletions be considered for TCS molecular diagnosis.Furthermore,we discuss the correlation between the genotype and phenotype in TCS and conclude that no relationship of phenotype severity with the size of the deletion or the type or location of the mutation.Part Ⅲ Exploration of Early Non-invasive Prenatal Test Based on Endocervical Fetal TrophoblastThe retrieval of trophoblasts from the endocervical canal of pregnant women is another non-invasive approach for prenatal diagnosis,and its feasibility has been long discussed.This study demonstrated the presence of fetal cells in transcervical samples based on the detection of chromosome Y signal by ordinary PCR and STR analysis.Cells expressing HLA-G were identified among transcervical cells.The percentage of transcervical samples that contained HLA-G+ cells was lower than previously reported.While about 70%transcervical samples contained HLA-G+cells in our study.Our detection rate for fetal cells was about 41%.However,this study was still hampered by a number of technical factors.Further optimization of the protocol is required before transcervical trophoblasts can be retrieved in a reliable way.