耳聋是一种常见的遗传病，遗传异质性高、人群携带率高，诊断价值高，普筛需求高。目前已发现有100多个基因与耳聋发生相关，中国遗传性耳聋的发病率约为1-3/1000，每年大约新增6万聋儿，包括3.5万新生听力障碍儿童和2.5万迟发性和药物性耳聋。耳聋的高发病率归结于中国人群约4-5%的突变基因携带率，基因突变检测具有很高的诊断价值。因此，本研究基于微液滴数字PCR（Droplet digital PCR, ddPCR）以口腔拭子为检测样本建立了耳聋常见突变位点无创检测方法，尤其是首次实现了药物性耳聋基因突变异质率的精准定量检测。此外，以孕妇血浆游离DNA（Cell-free DNA, cfDNA）为检材进行了耳聋无创产前检测的（Noninvasive prenatal testing, NIPT）初步研究。研究结果显示基于ddPCR的检测方法简便快速、灵敏度高、精准可靠是一种有前景的无创耳聋基因突变检测方法。
  首先，本研究基于ddPCR以口腔拭子为检测样本建立了超高灵敏度的耳聋无创检测方法，该方法极大降低了检测模板量且不会对被检测者造成创伤。目前耳聋突变检测方法主要有微阵列基因芯片、质谱、测序技术等，这些方法通量高、准确性高，但不足之处在于检测样本来源主要以成人静脉血、新生儿足跟血为主，均需要针刺取样，属于侵入性取样方式，基于这些技术开发无创检测方法的报道较少。口腔拭子是一种无创检测样本，采样简便且成本低，但样本含量波动大量化不可控且纯度较血液低。为实现无创检测的目的，本研究基于灵敏度高、准确性高的ddPCR和口腔拭子样本建立了耳聋常见8位点无创检测方法并进行系统研究，结果显示该方法特异性可以达到100%，最低检测限为5拷贝，在10⁵-10拷贝范围内检测线性好且R²>0.9976，在10⁵-10拷贝范围内定量的准确性和重复性好。本方法对-20℃冻存和常温保存90天的口腔拭子所提取的核酸均可准确检出，进一步对23例干血斑和42例口腔拭子临床样本进行检测，检出率为100%，与Sanger测序结果一致率为100%。
  进一步基于ddPCR的超高灵敏度和绝对定量的优势，研究首次实现药物性耳聋基因突变异质率的精准定量检测。在某种程度上异质率水平是决定线粒体功能、疾病严重程度及复杂临床表型的最重要的影响因素之一。目前，药物性耳聋基因异质性突变的检测尚无金标准方法，对异质率定量检测的报道较少，尤其是较低水平异质率检测。因此，本研究首次基于ddPCR对具有重大筛查意义的药物性耳聋m.1494C>T和m.1555A>G位点突变异质率进行定量检测，结果表明该方法在0%-100%的异质率范围内检测线性好且R²>0.9990。本方法可检测低至1%的异质率。进一步对14例干血斑进行定量及分型检测，ddPCR结果与芯片和Sanger测序结果比对，从1例野生型样本中检测出低水平的异质突变，7例均质突变样本均检测出不同水平的异质突变。最后，对2个药物性耳聋家系的5位母系成员口腔拭子进行检测，异质突变样本均精准定量，均质突变样本均检测到不同水平的异质突变且异质率在每代间传递逐渐递增。
  最后，基于ddPCR的快速简便、超高灵敏度和绝对定量的优势，开展了耳聋NIPT的初步研究。目前，耳聋无创产前诊断的常用方法有新一代测序技术和ddPCR技术。新一代测序技术检测价格高、操作复杂且结果准确性影响因素较多。因此本研究基于更为简便灵活的ddPCR技术建立遗传性耳聋无创产前诊断方法。首先通过ddPCR对不同基因型样本和不同浓度样本进行特异性和定量准确性验证，结果显示ddPCR可对不同浓度样本进行定量检测且具有较高的灵敏度和特异性。然后，通过片段化DNA对临床常见两种孕妇cfDNA进行模拟，ddPCR检测结果显示当胎儿cfDNA≥5%时定量准确性和重复性较好。最后，对3例耳聋家系进行定量检测，检测结果与侵入性产前诊断结果一致，检测结果精准可靠。
  本研究以ddPCR技术平台为基础，以口腔拭子为检材，建立了适合大规模开展的耳聋无创筛查方法。该方法不仅能对常见位点进行分型检测还能对药物性耳聋突变异质率进行精准定量，快速简便、灵敏度高、准确性高，有望成为新一代耳聋基因突变筛查方法。基于方法的灵活性和简便性，也可用于线粒体其它相关疾病检测，为线粒体疾病的分子机制研究、临床诊断、基因治疗提供了精准的分析方法。本研究进一步以cfDNA为检材完成了耳聋无创产前诊断的初步研究，该方法在cfDNA检测方面具有较好的灵敏度和准确性，是一种可靠的耳聋无创产前检测方法。

  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°C. 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>0.9990, 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.