| Hearing loss,a common clinical neurosensory disorder in humans,is characterized by high genetic heterogeneity,high carrier rate of mutation,high diagnostic value,and high demand for universal screening.To date,more than 100 genes associated with hearing loss have been identified.In China,the incidence of congenital hearing loss is approximately 1-3/1,000.The number of deafness children increased about 60,000 per year including 35,000 newborn deafness infants,and 25,000 age-related and aminoglycoside-induced hearing loss.The high incidence of hearing loss is due to the 4-5%carrier rates of deafness-associated mutations among Chinese population.The detection of hearing loss gene mutations has high diagnostic value.In this study,a noninvasive and accurate detection method for hotspot deafness-associated mutations based on buccal swabs and droplet digital PCR(ddPCR)was developed,especially accurately quantified the heteroplasmy rate of aminoglycoside-induced hearing loss mutations.In addition,a preliminary study of noninvasive prenatal testing(NIPT)method based on ddPCR and cell-free DNA(cfDNA)was carried out.All of the results showed that the ddPCR-based method is a promising detection method for deafness-associated mutations due to its high sensitivity and accuracy.First,a noninvasive detection method based on buccal swab and ddPCR was developed,this method greatly reduce the input of template DNA and won't bring trauma and pain for participants.Currently,various techniques including micro-array,mass spectrometry and next generation sequencing have been applied to screen deafness-associated mutations and showed good performance.However,these techniques are invasive since they mainly isolated sample DNA from peripheral blood and heel blood of newborns.In addition,noninvasive detection methods based on these techniques for deafness-associated mutation screening are seldom reported.Buccal swab is a kind of noninvasive sample which is easy to collect and low in cost.However,the purity of buccal swab DNA is lower than that of blood and the total yield of DNA is variable.With the aim to achieve noninvasive detection,a noninvasive and accurate detection method for eight hotspot mutations was developed,the results showed that the specificity was 100%and the detection limit was 5 copies.The assay was linear from 105 to 10 copies with R2>0.9976 and showed a good reproducibility from 105 to 10 copies.We also verified buccal swab is a reliable sample since the DNA extracted from a single buccal swab could still be accurately quantified after stored for 90 days at either room temperature or-20?.Furthermore,all clinical samples(23 DBS and 42 buccal swabs)were accurately quantified and genotyped,and the results were verified by the results of Sanger sequencing.Furthermore,the heteroplasmy rate of mitochondrial gene was accurately quantified for the first time due to the highly sensitivity and accuracy of ddPCR.Several study results showed that heteroplasmy rate may be one of the factors that affect complicated clinical phenotypes of mitochondrial-associated disease.Up to now,there is no "golden standard" for the detection of mitochondrial gene heteroplasmy rate.Few reports addressed the detection of heteroplasmy rate especially low level heteroplasmy rate for aminoglycoside-induced hearing loss.Therefore,an accurate quantification method was developed for heteroplasmy rate detection of m.1494C>T and m.1555A>G sites,which have important effect on medication guidance,the results showed that the assay was linear from 0%-100%with R2>0.99 90,heteroplasmy rate was accurately quantified as low as 1%for both sites.Moreover,14 DBSs and 5 buccal swabs from 2 aminoglycoside-induced hearing loss families were tested by ddPCR,and the results were verified by results of Sanger sequencing.Low level heteroplasmy rate was detected from 1 wildtype DBS,7 mutant DBSs,and 3 buccal swab from 3 members of aminoglycoside-induced hearing loss families.The average heteroplasmy rates showed a trend of gradual increase over generations.Last,a preliminary study of NIPT for hereditary hearing loss based on ddPCR and cfDNA was carried out.Next generation sequencing technology and ddPCR have been applied to NIPT.However,the next generation sequencing technology has high detection cost,complicated operation and the results could be affected by many factors.Therefore,we selected ddPCR which is more simple and flexible to develop NIPT for hereditary hearing loss.In this study,samples of different genotype and different concentrations were genotyped and quantified,the results showed that ddPCR had a good performance for specificity and accuracy.In addition,two kinds of cfDNA of pregnant women were simulated by fragment DNA.The results showed that ddPCR showed a good performance for simulated samples as the ration of fetal cfDNA ?5%.Moreover,3 hereditary hearing loss families were tested,and the results were in accordance with the results of invasive prenatal testing.In this study,a noninvasive detection method suitable for universal screening was developed based on ddPCR platform and buccal swab.This method is not only used to genotype hotspot mutations but also to quantify heteroplasmy rate of aminoglycoside-induced hearing loss mutations.This method is expected to be the second generation screening method due to its simplicity,high sensitivity and accuracy.This method is also applicable to other mitochondrial related disease due to its flexibility and simplicity,and may provide more scientific data for the analysis of complicated phenotypes and gene treatment of mitochondrial diseasse.Furthermore,a preliminary study of noninvasive prenatal testing method was carried out based on ddPCR and cfDNA,the results showed that the ddPCR-based methos had high sensitivity and specificity for cfDNA detection,and it was a reliable noninvasive prenatal testing method for hereditary hearing loss. |
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