基于第三代测序技术和相对单倍型剂量分析的遗传性耳聋无创产前检测研究

Hereditary hearing loss is one of the most common monogenic genetic diseases, most of which is autosomal recessive. It is the most technically difficult type of disease in non-invasive prenatal testing of monogenic genetic diseases. The applicant applied non-invasive prenatal testing of common hereditary hearing loss with the use of circulating single-molecule amplification and resequencing technology (cSMART) previously and the accuracy rate was 91.3%,which means that NIPT for deafness genes still needs the further study. Our study intends to continue to target the two most common pathogenic genes of hereditary hearing loss: GJB2 and SLC26A4, obtain parental haplotype information through the third-generation sequencing technology, and calculate effective SNPs linked to disease sites. The cSMART was used to analyze the effective dose of these SNPs in the cfDNA of pregnant women to obtain the fetus’genotype. The stability, specificity and sensitivity of the method were verified in the GJB2/SLC26A4 deaf families. Our study is expected to significantly improve the scope and accuracy of non-invasive prenatal testing, in order to establish a technical platform for non-invasive prenatal testing of hereditary hearing loss, to achieve clinical application, and to improve the prevention of birth defects in China.

遗传性耳聋是最常见的单基因遗传病之一，绝大部分为常染色体隐性遗传，是单基因遗传病无创产前检测中最有技术难度的疾病类型。申请人先期应用环化单分子扩增和重测序技术(cSMART)进行了遗传性耳聋无创产前检测研究，准确率为91.3%，遗传性耳聋无创产前检测应用于临床还有待进一步研究。本课题拟继续针对中国遗传性耳聋最常见两个致病基因：GJB2与SLC26A4，通过第三代测序技术分析父母单倍型，计算得到有效的与致病变异连锁的SNP位点，再利用cSMART方法对孕妇外周血游离DNA中这些有效SNP位点进行突变剂量分析，获得胎儿的突变基因型；并在GJB2/SLC26A4耳聋家庭中验证此方法的稳定性、特异性及灵敏度。本课题将有望显著提高单基因病无创产前检测的适用范围及准确率，以期建立遗传性耳聋无创产前检测的技术平台，实现临床应用，完善我国耳聋出生缺陷的预防手段。

遗传性耳聋是最常见的单基因遗传病之一，绝大部分为常染色体隐性遗传，是单基因遗传病无创产前检测（NIPT）中最有技术难度的疾病类型。本课题针对中国遗传性耳聋最常见两个致病基因：GJB2与SLC26A4，收集40例耳聋家庭，突变类型包括SNV、小的 in/del(coding和intronic区域)和CNV。先利用PacBio 第三代测序技术，对10个耳聋家庭（GJB2与SLC26A4耳聋家庭各5个）的包含目标基因上下游50k区域进行捕获，构建出父母单倍体型，然后结合RHDO分析策略分析胎儿基因型。结果显示：10个胎儿经NIPT鉴定为1个GJB2单杂合，1个SLC26A4单杂合，1个SLC26A4野生型，此三个胎儿结果与有创产前诊断相符；4个仅明确了父源或母源基因型，所明确的基因型与有创产前诊断相符，剩余3个父源及母源突变均无法判断，总体来说与有创产前诊断符合率为30%。分析符合率不高的原因，考虑先期方案捕获的片段较短，无法获得足够SNP位点区分单体型，因此将捕获区域扩大至目标基因上下游1M区域。由于PacBio的三代测序大大超出预算，遂将测序方法调整为更为经济的10x Genomics的linked-read测序（此为模拟三代测序长片段读长的测序方式），对30个耳聋家庭（14个GJB2耳聋家庭，16个SLC26A4耳聋家庭）的包含目标基因上下游1M进行捕获，构建父母单倍体型，结合RHDO分析策略分析胎儿的基因型。结果显示：30个胎儿经NIPT鉴定为7个野生型，11个单杂合和12个纯合/复合杂合，与有创产前诊断结果一致，符合率为100%。本研究利用三代测序及linked-read测序的长片段读长特点，可提供足够的特异性SNPs构建单倍型，结合RHDO分析策略的NIPT，无需先证者，可检测包含CNV等各突变类型，适合普遍应用，具有替代有创产前诊断的临床潜力。

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