摘要
最早的新生儿听力筛查工作起始于1964年，其在我国的开展也已经超过10年，迄今已在早期发现聋病方面取得了卓越成效。随着新生儿听力筛查工作的广泛开展和临床经验的积累，逐渐发现在新生儿听力筛查中存在б些局限，即并不是所有的听力损失均会在出生后立即表现。那么，对于迟发型听力损失，则新生儿的听力筛查似乎就更显得无能为力了。遗传学的研究和分子流行病学的数据使我们认识到遗传基因在维护听力健康和发现听力异常中的重要性。检出这些已经发病或潜在的听力损失者的最佳方法是对所有新生儿的血斑进行遗传学分子筛查，以便发现他们是否具有引起迟发性听力损失的高危病因。2007年，王秋菊教授提出了在广泛开展的新生儿听力筛查的基础上，融入聋病易感基因分子水平筛查的理念，在新生儿出生时或出生后3天内，除了进行常规的听力筛查外，同时进行新生儿聋病常见易感基因的筛查，策略上包括普遍人群筛查和目标人群筛查。本研究正是基于此理念展开，共分为三个部分：
第一部分新生儿听力及基因联合筛查106,513例研究报告
本课题组应用专有的含有听力筛查信息和血样信息的新生儿遗传疾病筛查采样卡，可以方便快捷准确地进行了新生儿听力及基因的同步筛查，发现了具有重要临床意义的研究结果。本研究发现，在106,513例新生儿中，总计有4,192例（4个位点3,055例+16个位点1,137例）存在常见耳聋基因的突变，其中205例为未通过，3,987例为携带者。而更为重要的是在这205例未通过者中有169例为MTRNR1基因m.1555A>G或m.1494C>T突变者，意味着这169名新生儿将对氨基糖苷类药物极其敏感，如果接触药物将产生不可逆的耳聋情况。另外，令人兴奋的是发现了多达3987例耳聋基因的致病突变的携带者，这些携带者在未来的婚配中，如果与同样基因型的携带者婚配将有25％的概率产生耳聋后代。结合GJB2、GJB3、SLC26A4和MTRNR1四个基因的筛查，发现本研究中的新生儿总体的三个耳聋基因的4个致病突变的携带率达到了2.87%（3,055/106,513），天津地区四个耳聋基因20个致病突变的携带率达到了5.65%（3,298/58,397），本研究中总共发现致病基因携带率为3.94％(4,192/106,513)。这个数字比通过听力筛查得到的重度聋的0.1％高出了39倍，而更为重要的是其中大部分难以通过目前的听力筛查手段得以发现。本结果为新生儿听力和基因联合筛查的开展和推广提供了理论基础和依据。
第二部分新生儿聋病易感基因快速筛查试剂盒的研发与临床验证
第б章新生儿聋病易感基因快速筛查试剂盒的研发
本研究以四引物扩增受阻PCR（ARMS-PCR）为技术原理，建立了针对人线粒体MTRNR1基因m.1555A>G, m.1494C>T突变、SLC26A4基因c.919-2A>G突变、GJB2基因c.235delC突变四个位点进行基因型检测的技术方法；本研究最终获得了分别针对四个位点的多重PCR反应体系，包括核心制剂配制方法、PCR反应程序、基因型分析方法；本研究所获得的反应体系对四个位点的基因型分析具有特异性，对DNA样品的敏感度达到了104个拷贝数。
第二章新生儿聋病易感基因快速筛查试剂盒的临床验证
本课题组应用自主设计并拥有专利的耳聋基因快速筛查试剂盒，可以方便快捷地进行新生儿四个常见耳聋突变（GJB2c.235delC; SLC26A4c.919-2A>G; MTRNR1mt.1555A>G and mt.1494C>T）筛查，根据12,224例新生儿的临床验证，发现筛查结果稳定、准确，阳性结果经直接测序验证未见假阳性。
本研究发现，在参与筛查的共计12,224例新生儿中，有258例存在常见耳聋基因的突变，其中有22例为线粒体MTRNR1基因m.1555A>G或m.1494C>T突变者，意味着这些新生儿将对氨基糖苷类药物极其敏感，如果接触微量耳毒性药物将产生不可逆的耳聋。本筛查方案还筛查出3例GJB2基因c.235delC纯合突变者，这3例新生儿的听力筛查皆未通过，做到了早期确诊耳聋及其病因，为下б步早期干预或接受电子耳蜗手术提供依据。另外，本研究还发现了233例GJB2c.235delC和SLC26A4c.919-2A>G杂合突变携带者，这些新生儿中的大部分都通过了常规新生儿听力筛查，这些携带者在未来的婚配中，如果与同样基因型的携带者婚配将有25％的概率产生耳聋后代。
第三部分新生儿听力与基因联合筛查结果解读与筛查模式探讨
对耳聋基因筛查的结果，必须结合听力筛查的结果来联合进行，新生儿基因筛查的结果报告形式也是以通过和未通过来表示。本研究结果使我们发现了那些耳聋基因的携带者和致病突变患儿，其中很大б部分用目前的听力筛查方法均显示为通过，然而家长没有意识到这些婴幼儿却存在着潜在的致聋危机。本研究发现的MTRNR1m.1555A>G或m.1494C>T突变的新生儿，在获知基因筛查的结果之后，就可以做到有效地避免接触药物而保持б个很好的听力。由于线粒体突变属于母系遗传，还可以避免其耳聋的传递。
本研究通过对已开展的多中心新生儿听力和基因联合筛查结果进行分析，探讨联合筛查的意义，分析了对联合筛查结果的解读方式，根据临床实践中的经验教训，建立了比较切实可行的听力和基因联合筛查模式。在进行新生儿聋病易感基因的筛查时，策略方面最为重要的是强调在新生儿听力筛查的基础上进行的聋病易感基因的普遍筛查。根据本课题组在13个省市启动的新生儿听力筛查为基础的新生儿聋病易感基因筛查的多中心合作研究，建立了新生儿听力和基因联合筛查的模板。
Congenital hearing impairment is a distressing disorder which occurs inabout1-3in1000live births. According to the correlated data reported by theWHO,278million people worldwide have moderate to profound hearing loss inbilateral ears. Most people who have hearing impairment live in developingcountries. In China, there are approximately21million people with hearingimpairment out of the60million people classified as disabled，and of those21million with hearing loss, approximately0.8million are younger than7years old.This number has continued to increase by more than30,000newborns withcongenital deafness annually.
To date,64nonsyndromic deafness genes and more than1000discretedeafness causing mutations have been described (http://hereditaryhearingloss.org).Despite the genetic heterogeneity, there are still a limited number of mutationalhot spots of hereditary deafness. Among Chinese children, epidemiologicalstudies indicated that at least36%cases of congenital hearing impairment were resulted from GJB2, SLC26A4or mitochondrial12S rRNA (MTRNR1) mutations(GJB2,18.31%; SLC26A4,13.73%; MTRNR1,3.96%).
Therefore, for early disclosure of genetic causes leading to onset inchildhood or aminoglycoside-sensitive hearing loss and identification of carrierswith those pathogenic mutations, integration of genetic tests into the currenthearing screening programs is essential for maintaining hearing health of childrenwho are most vulnerable to both genetic and environmental risk exposures. In thisreport, we first thoroughly analyzed the universal newborn hearing and the newlydeveloped genetic screening conducted106,513neonates in13provinces ofChina. Then, we develop a new Tetra-primer Amplification Refractory MutationSystem-PCR (ARMS-PCR) Kit to detect the four most commonly or specificpoint mutations of the three genes (GJB2c.235delC; SLC26A4c.919-2A>G;MTRNR1mt.1555A>G and mt.1494C>T). A simple and economical genotypingmethod involving a single PCR reaction followed by gel electrophoresis. Inlarge-scale newborn screening this rapid, method for point mutation genotypingwill provide both cost and time benefits compared to current methods. At last, weproposed several strategies for improving the concurrent audiological and genetictesting based on the practical evidence from the largest population in the world.These studies were eomposed of three parts below:
PART1: A Report of Newborn Hearing Concurrent GeneticScreening in106,513Neonates in China
This study was undertaken in13provinces of China.106,513newbornbabies received hearing concurrent genetic screening. The hearing screening wasperformed in two step protocol with OAE or AABR. Blood sample wascollected with a universal newborn genetic screening card. And three commongene, MTRNR1, GJB2and SLC26A4were screened with standard protocol.Among all the106,513neonates,91.48%(97,433/106,513）individuals passed thefirst-step hearing screening,3.41%(3638/106,513) babies passed only one side,and the other5.11%(5,442/106,513) were bilaterally referred. Gene screeningfound3,055individuals had one or two mutant alleles, the carrier rate is2.87%(3,055/106,513) among the entire newborn population. The risk for hearing losswas100%(33/33) for those newborns carrying causative GJB2or SLC26A4mutations (homozygotes or compound heterozygotes). Total169newborns withMTRNR1mt.1555A>G or mt.1494C>T pathogenic mutation, who would sufferfrom sudden hearing loss once applying aminoglycoside drugs.
PART2: Operate a Newborn Genetic Screening Procedure forHigh Risk Deafness-associated Mutations with a NewTetra-primer ARMS PCR Kit
The new ARMS-PCR screening kit was designed to detect four high riskdeafness-associated mutations (GJB2c.235delC, SLC26A4c.919-2A>G, mtDNA12S rRNA mt.1555A>G and mt.1494C>T). The kit was able to amplify bothwild-type and mutant alleles with a control fragment. The proposed method wasconducted to genotype the above four deafness gene mutations in four PCRreactions. Each mutation was genotyped by a set of four primers, twoallele-specific inner primers, and two common outer primers. A mismatch at thepenultimate or antepenult nucleotide of the3’ terminus was introduced in order tomaximize specificity. The16primers were used for the amplification of genomicDNA as a template. Amplified fragments were separated by electrophoresis. Wedesigned and validated the kit with wild and mutant type DNA samples that hadbeen previously been confirmed by Sanger sequencing. Then12,224newbornswere enrolled, and those samples with mutations were further validated withsequencing too. Among12,224newborns,258individuals had one or two mutantalleles, with the carrier rate being2.11%(258/12,224). For GJB2c.235delCmutation,3cases was homozygote and129cases were heterozygote carriers;For SLC26A4c.919-2A>G mutation,104cases were heterozygotes carriers, andno homozygotes were found; for MTRNR1mt.1555A>G mutation,16cases wasidentified; six cases of MTRNR1mt.1494C>T mutation were detected. All mutations were detected with high specificity. Mutation samples were confirmedvia Sanger sequencing. No false positive was found. We developed a user-friendlyscreening kit for deafness-associated mutations. It provided rapid, reproducible,and cost-effective detection of deafness gene mutation without special equipment.The kit allowed the detection of the four high risk deafness-associated mutationswith only4single tube PCR reactions. In the future, the kit could be applied tolarge population-based epidemiological studies for newborn hearing defectsscreening.
PART3: Analyze Different Results of a Newborn HearingConcurrent Genetic Screening Procedure and toDevelop a Spread-Model
This study indicated that the conventional newborn hearing screeningprogram for newborns could be improved by adding the genetic component. Thegenetic tests could immediately confirm hearing impaired babies referred byconventional hearing screening test and identified the cause of deafness withinone month. And genetic tests have the advantage of immediately determining themost important cause for preventable deafness, in particular resulting fromMTRNR1m.1555A> G or m.1494C>T, which can be efficiently eliminated byaverting the use of risky aminoglycoside antibiotics. The genetic tests aid in theidentification of newborn carriers of causative alleles, who tended to have a higher incidence of hearing loss and should be given more attention in future. To avoidthese causative alleles to be transmitted to next generation and maintain thecarriers’ normal hearing. So, genetic tests complements the current newbornhearing screening program and provides additional insights beyond whatconventional audiological tests reveal, thus significantly advancing the currentpractice to improve the newborn hearing screening as a means of early discoveryof genetic risk factors.
引文
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[1] Mahboubi H, Dwabe S, Fradkin M, Kimonis V, Djalilian HR. Genetics ofhearing loss: where are we standing now. Eur Arch Otorhinolaryngol.2012.269(7):1733-45.
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[4] Schrijver I. Hereditary non-syndromic sensorineural hearing loss:transforming silence to sound. J Mol Diagn.2004.6(4):275-84.
[5] Cohen M, Phillips JA3rd. Genetic approach to evaluation of hearing loss.Otolaryngol Clin North Am.2012.45(1):25-39.
[6] Hilgert N, Smith RJ, Van Camp G. Forty-six genes causing nonsyndromichearing impairment: which ones should be analyzed in DNA diagnostics.Mutat Res.2009.681(2-3):189-96.
[7] Smith RJ, Hone S. Genetic screening for deafness. Pediatr Clin North Am.2003.50(2):315-29.
[8] Lenz DR, Avraham KB. Hereditary hearing loss: from human mutation tomechanism. Hear Res.2011.281(1-2):3-10.
[9] Dai P, Yu F, Han B, et al. GJB2mutation spectrum in2,063Chinesepatients with nonsyndromic hearing impairment. J Transl Med.2009.7:26.
[10] Martinez AD, Acuna R, Figueroa V, Maripillan J, Nicholson B.Gap-junction channels dysfunction in deafness and hearing loss. AntioxidRedox Signal.2009.11(2):309-22.
[11] Liu XZ, Xia XJ, Ke XM, et al. The prevalence of connexin26(GJB2)mutations in the Chinese population. Hum Genet.2002.111(4-5):394-7.
[12] Dai P, Yu F, Han B, et al. The prevalence of the235delC GJB2mutationin a Chinese deaf population. Genet Med.2007.9(5):283-9.
[13] Kokotas H, Petersen MB, Willems PJ. Mitochondrial deafness. Clin Genet.2007.71(5):379-91.
[14] Masindova I, Varga L, Stanik J, et al. Molecular and hereditarymechanisms of sensorineural hearing loss with focus on selectedendocrinopathies. Endocr Regul.2012.46(3):167-86.
[15] Lu J, Li Z, Zhu Y, et al. Mitochondrial12S rRNA variants in1642HanChinese pediatric subjects with aminoglycoside-induced andnonsyndromic hearing loss. Mitochondrion.2010.10(4):380-90.
[16] Chen G, Wang X, Fu S. Prevalence of A1555G mitochondrial mutation inChinese newborns and the correlation with neonatal hearing screening. IntJ Pediatr Otorhinolaryngol.2011.75(4):532-4.
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