新生儿及婴儿的听觉特性与山东地区耳聋热点基因突变及其听力学表现的研究
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摘要
研究背景及目的：
新生儿听力损失是较常见的出生缺陷。我国听力残疾人约有2057万,每年出生1950万的新生儿,其中约有0.1368%患有致残性听力损失,每年约新增2～3万个听障儿童。由于3岁以前是言语-语言发育最重要和最关键的时期,先天性听力损失将极其严重地影响儿童言语、认知和情感的发育,以及未来接受教育和工作的机遇。因此,“早发现,早诊断,早治疗”是避免因聋致哑、促进患儿发育的较好方式。通过新生儿听力普遍性筛查项目的实施,能够对先天性听力损失及早发现、及早进行干预,从而在一定程度上减少了听力残疾和语言障碍造成的损失和社会负担。
但就目前新生儿听力筛查发展状况而言,听力损失的确诊、干预和康复方面还面临许多挑战。在听阈评估的技术方面,过去的几十年中临床广泛应用的听性脑干反应(ABR)是对婴幼儿听力损失做出诊断的最为重要的手段之一。但是不同的刺激声所诱发的脑干听性反应是极其不同的。短声诱发的听性脑干反应(ABR)与2000至4000Hz频阈范围的听敏度呈高度相关,但对中低频听力损失可能会产生假阴性结果(即500至2000Hz以下)。听觉频率特异性测试是近年来广泛用于临床的比较成熟的听力学检测技术,包括短纯音听性脑干反应(tone-burst ABR),短音听性脑干反应(tone-pip ABR),听觉稳态诱发电位(ASSR)和行为听力测试等。它们可以对不同频率的最小可听到的刺激声强度进行检测,可以明确在不同频率的听力下降状况,使诊断更加明确具体；更为重要的是,它可以为6月龄以下不会进行言语表达的较小婴儿的助听器选配提供较为可靠的听力曲线,使其助听器选配更加个体化。否则将大大影响助听器参数的设置,使助听效果不能达到最优化,从而直接影响患儿的聆听效果和言语康复水平。美国婴幼儿听力联合委员会2007年发表的形势报告,就听力损失的早期发现及早期干预亦提出了指导方针,支持对新生儿和较小婴儿进行普遍听力筛查、评估和干预。其指南中提出,听力损失的诊断性测试中应包括频率特异性电生理检查,如听觉稳态反应(ASSR)、短纯音听性脑干反应(tone-burst ABR)等。因此,开展新生儿和／或婴幼儿听觉频率特异性测试技术,既是听力学发展的大势所趋,又是小儿临床听力学领域所面临的挑战和亟需解决的问题。
目前,国外在频率特异性听力测试方面的研究尚缺乏针对小婴儿和新生儿的临床标准。我国在新生儿和婴幼儿频率特异性电生理检测的应用还刚刚起步,对于新生儿听力损失的频率特异性特点还不甚了解,对于快速的脑发育阶段中,听力损失的频率特异性变化还不清晰,从而无法为听力损失儿童的早期干预提供有力科学依据。本课题组前期研究表明,听力损失婴幼儿的听阈(ABR)水平随年龄增长存在波动。但是对于听力损失新生儿和婴幼儿,神经的可塑性对各频率影响还未见报道。只有准确地把握听力损失患儿听觉神经系统的发育特点和规律,明确其随年龄演变的规律,才能有效确定助听器各项参数及调试和监测周期,不断调整康复方案；把握可塑性调节的关键点并及时给予合适的外在干预,使早期干预更加有效,这无疑会对其康复产生强有力的推动作用。
近十年来,遗传学和分子生物学的发展极大地推动了耳聋发生的分子机制的研究。研究表明,基因突变具有显著的种族特异性,即不同地区及种族的患者,同一基因的突变类型不完全相同,每个种族有其特异性热点突变。GJB2,SLC26A4和mtDNA A1555G,是中国人非综合征型聋常见的热点突变。遗传学研究表明,先天性重度～极重度听力损失中50%以上与遗传因素有关,其中70%的患儿属于非综合征型听力损失(NSHI)。通过对耳聋大家系的连锁分析和基因定位克隆等技术,目前已经定位了104个非综合征型耳聋基因座位。遗传流行病学研究认为,通过对GJB2, SLC26A4和mtDNA A1555G三种基因的检测,可以发现26.65%的中国北方地区语前聋患者；通过对GJB2的235delC等位基因突变位点的检测,可有高达15%患者得到明确诊断。SLC26A4突变导致的前庭导水管扩大是内耳最常见的畸形,在先天性聋中占5%～10%,其突变阳性率为可达97%。本研究通过对GJB2、SLC26A4和·mtDNA A1555G三种基因的检测,可以初步掌握山东地区听力听力损失婴幼儿及儿童耳聋基因的热点突变位点和突变率,明确基因型与听力损失临床表型之间的关系,不仅对估计疾病的预后和康复效果起着非常重要的作用,而且为今后耳聋基因筛查和诊断提供依据,为优生优育提供决策基础,对降低听力损失患儿的出生率,对提高出生人口质量,减轻社会压力起着不可估量的作用。
综上所述,明确听觉发育的频率特异性,将直接指导早期干预的具体实施和个体化康复计划的制定；了解了听力损失的病因尤其是基因突变位点,对遗传咨询、减少病残儿的出生具有极其深远的社会意义。
研究方法：
1.研究对象及分组：选取0～6月龄听力正常婴儿80例(160耳),按纠正月龄分为四组：新生儿组、42 d组、3月龄组和6月龄组,每组20例(40耳),男女例数均等。听力正常婴儿的入选标准为：38～41孕周足月娩出,出生体重＞2500 g,生后Apgar评分1 min＞8分,无新生儿重症监护史,无耳毒性药物应用史及耳聋家族史,各项体检指标均为正常；听力检查：瞬态诱发耳声发射(TEOAE)测试能正常引出,短声听性脑干反应(click ABR) V波反应阈正常听力级≤30 dB,声导抗测试0～3月龄以1 kHz鼓室图为单峰型,6月龄以226 Hz鼓室图为A型,峰压值在-100daPa～+100daPa之间为正常。
基因检测：选取2007年1月～2009年7月在济南市妇幼保健院新生儿听力筛查康复中心就诊、经至少6个月随访确诊为单耳和／或双耳轻度～极重度非综合征型听力损失的婴幼儿及儿童146例,其中男77例,女69例。初诊年龄6w～10岁,年龄中位数为18 m。所有患儿均来自山东省内14个地市,146例中144例均接受过新生儿听力普遍筛查,2例漏筛；接受过听力筛查的患儿中,133例患儿因复筛“未通过”而转诊至我院进一步确诊；11例初筛“通过”,因“语言发育迟缓”或“头部外伤后听力下降”等原因就诊。
2.测试技术：
①病史采集听力检测前,由患儿家长填写“听力异常儿童登记表”,内容包括：患儿家庭基本信息、出生史、个人史(生后患病及耳毒性药物应用情况、头部外伤史等)、体检情况、母亲妊娠史、分娩史、耳聋家族史等,重要信息(如家族史等)需与不同家庭成员单独沟通,以确保内容的真实性和准确性。
②声导抗和耳声发射测试：在进行电耳镜检查并清除外耳道耵聍后,使用美国GSI tympernometry中耳分析仪进行探测音为226 Hz和1 kHz鼓室声导抗测试。测试起始压力为+200 daPa,终止压力为-400 daPa,压力变化速度为50daPa／s,方向由正向负。采用丹麦Medsen耳声发射仪进行TEOAE测试，仪器自动完成探头自检和结果判读。以上测试在一安静的房间内进行,环境噪声小于40dB(A)。
③click ABR和tone-pip ABR测试：采用丹麦Medsen ICS听觉诱发电位仪,记录电极放置于前额正中近发际处,参考电极置于双侧乳突,地极置于鼻根部,极间电阻≤3 kΩ。click ABR测试的刺激信号为短声,刺激速率为20.1次／s,最大输出强度为正常听力级(下同)98 dB,叠加次数2000次,带通滤波100～3000 Hz,以反应阈≤30 dB为正常。tone-pip ABR采用上升、下降时间均为2ms,平台时间为0 ms的短音。带通滤波同click ABR,增益为105。通过ER-3A插入式耳机给声,将0.25、0.5、1、2、4、8 kHz处能够引出ABRV波的最小强度定为其反应阈。临床上以通过瞬态诱发性耳声发射(TEOAE),鼓室图(226Hz和／或1000Hz探测音)正常,短声听性脑干反应(click ABR)波V反应阈≤30dBnHL,500Hz短音听性脑干反应(tone-pip ABR)反应阈≤40dBnHL为听力正常的诊断标准。
④ASSR测试：采用丹麦Medsen ICS听觉诱发电位仪,记录电极放置于前额正中近发际处,参考电极置于双侧乳突,地极置于鼻根部,极间电阻≤3 kΩ。ASSR刺激声信号载波频率为0.25、0.5、1、2、4、8 kHz,左耳调制频率分别为79、92、77、84、85、100 Hz,右耳为86、97、81、95、88、102 Hz,放大增益为200 k。带通滤波为65～105 Hz,每个强度最多扫描480次；调幅深度为100%,调频深度为25%,ER-3A插入式耳机双耳12个频率同时给声测试,测试步距为5 dB,系统自动记录反应并判定各频率所能引起反应的最小正常听力级。所有电生理测试均在隔声屏蔽室内进行,环境噪声小于17 dB(A)。
⑤医学评估和影像学检查：对所有听力损失患儿均进行医学评估和颞骨高分辨率CT扫描。医学评估的内容包括：详细的体格检查,包括眼科、耳鼻咽喉科、口腔、甲状腺功能、心脏、肾脏以及神经系统检查等,从而鉴别综合征型与非综合征型听力损失。前庭导水管扩大(enlarged vestibular aqueduct,EVA)的诊断标准：高分辨率颞骨CT轴位,从半规管总脚到前庭水管外口1／2处直径＞1.5mm；MRI检查发现双侧小脑半球表面有条弧形或椭圆形囊状物高信号影。
⑥基因组DNA的提取：经本院伦理委员会认可,在知情同意的前提下,对各组听力损失患儿抽取静脉血5～10ml,用于基因组DNA的提取和保存。采用北京天根公司生产的基因组DNA提取试剂盒在全血中提取基因组DNA,琼脂糖电泳和紫外分光光度计定量和纯度分析,-20°保存。
⑦引物序列：应用在线引物设计软件Primer3设计引物序列,引物由上海英骏公司合成(中国)GJB2引物：上游引物CX26-F:TTGGTGTTTGCTCAGGAAGA下游引物CX26-R:GGCCTACAGGGGTTTCAAAT扩增产物长度：960bpSLA26A4引物：上游引物P8-F:AAGTTCAGCATTATTTGGTTGACA下游引物P8-R:TGGTTGTTTCTTCCAGATCACA扩增产物长度：305bpmtDNA A1555G引物：上游引物Mito-F:TCAACCTCACCACCTCCT下游引物Mito-R:TTTGTCGCCTCTACCTAT扩增产物长度：767bp
⑧PCR反应体系：10×Buffer2.5ul,2.5Mm／ulDNTP 2ul,5U／ulrTaq酶0.5ul(Takara公司),20Mm／ul上下游引物0.3ul,100ng／ulDNA 1ul,ddH2O补齐至25ul。
⑨PCR扩增在ABI公司(美国)9700热循环仪上完成,具体程序如下：
GJB2：94℃预变性5min；94℃变性30sec,58℃退火30sec,72℃延伸30sec,30个循环后72℃延伸7min。反应结束后,取2ul进行1%琼脂糖电泳检测,PCR产物4℃保存待直接测序。
SLC26A4 IVA7-2：94℃预变性5min；94℃变性30sec,53℃退火45sec,72℃延伸60sec,35个循环后72℃延伸7min。反应结束后,取2ul进行1%琼脂糖电泳检测,PCR产物4℃保存待直接测序。
mtDNA12SrRNA A1555G:95℃预变性5min,95℃变性30sec,62℃退火30sec,72℃延伸70sec,30个循环后72℃延伸7min。酶切反应体系：10×Tango Buffer2ul,Alw26I限制性内切酶0.2ul,PCR产物5ul,ddH20补齐至20ul,在37℃水浴箱内消化3小时,取10ul进行2%琼脂糖电泳检测酶切结果。PCR扩增片段没有A1555G突变,可被AlW26I酶切割成326bp和441bp二条带,相反扩增片段有A1555G突变,该酶切位点消失,由AlW26I酶切经琼脂糖电泳后仍为一条带,进行PCR产物测序。
⑩PCR产物测序：应用ABI公司(美国)3730型DNA测序仪进行直接测序。应用DNAStar软件包中的Seqman (Lasergene公司,美国)软件与NCBI网站公布的标准DNA序列进行序列对比分析,检测待查个体的碱基序列改变,查明其在氨基酸序列中的位置以及相应氨基酸改变。
3.统计学方法：采用SPSS10.0统计软件,分别计算各组听力正常婴儿70 dB短声刺激下ABR的潜伏期和波间期、0.25～8 kHz tone-pip ABR和ASSR反应阈的均值和标准差,并进行独立样本t检验和均数的方差分析；组间率和频数的比较采用卡方(x2)检验,以P=0.05为统计学意义界值。
研究结果：
1.70 dB正常听力级短声刺激下,短声ABRⅠ、Ⅲ、Ⅴ波潜伏期、Ⅰ～Ⅲ、Ⅲ～Ⅴ、Ⅰ～Ⅴ波间期随月龄增加逐渐缩短,波Ⅰ于42 d前、波Ⅲ于3个月前发育变化显著(P值＜0.05)
2.短音(tone-pip) ABR波形与短声ABR波形相似,Ⅰ、Ⅲ、Ⅴ波潜伏期随频率增加逐渐缩短,波形分化逐渐清晰。
3.不同频率、不同月龄tone-pip ABR和ASSR反应阈水平差异虽有统计学意义(P＜0.05),但是均无生理学意义(相差＜10dB)。除0.25 kHz外,其余频率tone-pip ABR反应阈均低于ASSR。
4.不同月龄tone-pip ABR和ASSR听力曲线形状相似。
5.GJB2基因突变检测：146例患儿中,共检出GJB2基因突变44例(男23例,女21例),占30.14%(44／146),发现GJB2基因的6种突变类型,其中良性多态突变3种：79G＞A(41例)、341A＞G(36例)和608T＞C(3例),等位基因频率分别是16.44%(48／292)、14.73%(43／292)和1.03%(3／292)；致病突变3种：235delC／235delC(18例)、235delC／176-191del16(4例)和235delC／299delAT(3例),其等位基因频率为21.23%(62／292)、1.37%(4／292)和1.03%(3／292)。其中,以235delC纯合突变最为常见,占72%(18／25)其次为235delC／176-191del16和235delC／299delAT。所有146例患儿中,235delC纯合突变18例,占12.33%；复合杂合突变7例,占4.79%；235delC杂合突变19例,占13.01%,总突变率为30.14%；其中纯合突变和复合杂合突变被认为是致病突变,因此25例患儿可以明确为GJB2基因突变致聋,占17.12%。
6.SLC26A4基因IVS7-2位点突变检测：146例患儿中,共检出SLC26A4基因IVS7-2位点A＞G突变23例(男14例,女9例),占15.75%(23／146)；其中纯合突变10例,杂合突变13例,等位频率为11.30%(33／292)。纯合突变和复合杂合突变被认为是致病突变,因此10例患儿可以明确为SLC26A4基因突变致聋,占6.85%。
7.mtDNA 1555位点A＞G突变检测：146例患儿中,检出1例mtDNA 1555位点A>G突变,系杂合突变,突变频率为0.68%(1／146),等位基因频率为0.34%(1／292)。
8.医学评估：146例患儿中,除1例GJB2基因突变(235delC／299delAT)患儿伴有眼外直肌发育异常,1例SLC26A4基因杂合突变患儿伴有外耳畸形外,其余患儿均未见异常。
9.影像学检查：146例听力损失患儿中,128例接受了高分辨率颞骨CT扫描,占87.67%；CT结果显示：前庭导水管扩大(EVA)20例(其中1例合并听小骨畸形),Mondini畸形6例。其中4例存在GJB2突变,占所有GJB2突变患儿的11.36%(4／44),13例存在SLC26A4基因突变,占所有SLC26A4基因突变患儿的56.52%(13／22),mtDNA A1555G基因突变的患儿仅1例,颞骨CT未见异常。SLC26A4基因突变患儿中内耳畸形检出率明显高于GJB2基因突变组和未检出突变组(p＜0.05)
10.GJB2基因突变的听力学表现：听力损失程度从轻度～极重度均可见,以中度及中度以上为主,占95.5%(42／44)；既有对称性听力损失(38例),又有非对称性听力损失(5例)；除1例单侧(左耳)重度听力损失外,其余43例均为双耳受损；有耳聋家族史的多见,占40.91%(18／44)；随访中发现听力损失程度加重1例(重度→极重度)。
11.SLC26A4基因IVS7-2位点A＞G突变的听力学表现：听力损失程度轻度～极重度均可见,90%以上(20／22)表现为先天性听力损失,以波动性、非对称性听力损失居多,占45.45%(10／22)；非对称性听力损失11例,占47.83%(11／23)。随访中发现,轻、中度听力损失患儿容易出现听力波动,诱因多为上呼吸道感染和头部外伤,大多经及时的激素治疗后可恢复到波动前水平,个别也可自行恢复。
12.mtDNA 1555突变的听力学表现：患儿系第二胎,男性,因3岁“自幼不会说话”就诊,未接受过新生儿听力筛查,体格检查无异常发现,家长否认氨基糖甙类类药物应用史及耳聋家族史,听力学表现为先天性极重度感音神经性听力损失。
13.初筛“通过”的听力损失患儿基因检测：146例听力损失患儿中,11例初筛时“通过”的新生儿听力筛查,占7.53%(11／146)；11例中,3例存在GJB2基因突变,检出率为27.3%(3／11),1例SLC26A4基因IVS7-2位点A＞G突变,7例基因检测阴性。
14.基因型与听力表型之间的关系：GJB2基因突变的患儿中,不同听力损失程度的患儿所占比例分别为：轻度4.55%(2／44),中度11.36%(5／44),重度22.73%(10／44),极重度61.36%(27／44)；SLC26A4基因突变的患儿中,不同听力损失程度的患儿所占比例分别为：轻度17.39%(4／23),中度21.74%(5／23),重度43.48%(10／23),极重度17.39%(4／23)。对两组中不同程度听力损失的构成比采用2×4行列表的卡方(x2)检验进行比较,结果显示：两组中不同程度听力损失的构成比差异有统计学意义(x2=12.3637,p=0.0062,p＜0.05),即GJB2基因突变患儿以重度～极重度听力损失为主,而SLC26A4基因突变的患儿则以中度～重度听力损失居多。
研究结论：
1.0-6月正常婴儿tone-pip ABR的潜伏期和波间期随月龄增加逐渐缩短,而反应阈水平无明显变化。
2.tone-pip ABR和ASSR均有稳定的频率特异性,tone-pip ABR反应阈低于ASSR,可能更接近婴幼儿的实际听阈。
3.听力损失儿童中GJB2、SLC26A4和mtDNA A1555G突变的检出率分别为30.14%、15.07%和0.68%,其中GJB2突变的检出率最高,主要致病突变的基因型为235delC／235delC,235delC／176-191del16和235delC／299delAT。mtDNAA1555G突变的检出率较低,除地域和种族差异外,可能与检测对象年龄尚小、无氨基糖甙类药物应用史有关。通过GJB2、SLC26A4和mtDNA 12SrRNA基因的检测,可为23.97%的患儿明确病因(纯合或复合杂合突变35例)
4.基因型与听力表型之间的关系：不同基因型所致听力损失的临床表现多种多样,且GJB2和SLC26A4基因突变患儿都可伴有内耳解剖异常；但是GJB2基因突变以重度～极重度、对称性听力损失、内耳解剖正常者为主,而SLC26A4基因突变患儿中非对称性听力损失多见,程度以中度、重度居多,一半患儿可以合并内耳畸形。尽管不同基因突变所致的听力表型呈现一定特点,但是不能从听力表型推测其基因型,反之亦然。此结果提示：临床上进行基因诊断时,应将单侧及双侧、包括轻度在内的所有听力损失患儿均纳入检测对象。
5.有必要在新生儿听力普遍筛查的基础上,联合开展新生儿耳聋基因筛查。GJB2、SLC26A4和mtDNA12SrRNA A1555G基因是主要的候选基因。
创新和意义：
1.首次建立了国内0～6月龄婴儿频率特异性听力测试的正常值,为探索正常大脑听觉特性随月龄发育演变的规律、研究听觉可塑性提供了依据；同时,比较了短音听性脑干反应(tone-pip ABR)和听觉稳态诱发反应(ASSR)反应阈的频率特性,并制定了临床实用的测试方案。
2.首次在国内较大样本地报道山东地区听力损失小儿中耳聋基因GJB2、SLC26A4和ntDNA12SrRNA A1555G的常见的突变位点和突变率；在新生儿听力普遍筛查的基础上,研究了GJB2和SLC26A4基因型与听力表型之间的关系。而且,在国内首次报道了4例GJB2基因突变患儿存在内耳解剖异常。
Background and objectives:
Hearing loss is the common birth defect in newborns.There are about 20.57 million people with hearing loss and 0.1368 of newborns have hearing loss annually in china,which means that there are 20～30 thousand hearing loss children increased every year.As we know,it is a very important stage for speech & languagr development before three-year old.The congenital hearing loss will affect the speech,recognition,emotion,education and work opportunities.Therefore,the early diagnosis and early intervention is a better way to avoid dumb and promote children's development.
In universal newborn hearing screening programe,there are many chanlenges on the aspects of diagnosis,intervention and rehabilitation presently. In the past decades,auditory brainstem response(ABR) was widely used in clinic as the main diagnosed mathod for infants with hearing loss.But the evoked potentials were very different in different stimulus.The click ABR was highly correlated with the threshold between 2000 to 4000Hz frequency range,while it might cause false nagative result in middle-low frenquency hearing loss.Recently,the frequency specific hearing tests were widely used clinically,including tone-burst ABR,tone-pip ABR,auditory steady state response(ASSR) and behavior hearing test.They could test infants with hearing loss in each frequency and provide the detailed information for diagnosis.More importantly,the frequency specific tests could provide the reliable profile of hearing loss and then make the hearing aid fitting more individually.
The Joint Committee on Infant Hearing (JCIH) Year 2007 Position Statement further proposed that the frequency-specific assessment of ABR testing is needed to determine the degree and configuration of hearing loss in each ear for fitting of amplification devices. It also suggested the frequency-specific assessment can be applicated in the diagnosis tests, such as ASSR, tone-burst ABR and so on.
In the prsent study, the frequency specific hearing tests were still lack of the clinical criteria for newborns and infants. In addition, the present data of tone-pip ABR and ASSR are mostly from the adults and children, lacking of the reports on newborns and infants. So we know little about the auditory development in the early stage of life,especially on the aspect of the frequency change.The previous study of our team revealed that the threshold of ABR in newborns and infants changed with the increaseing of the month.In this experiment, we analyzed the features of development of tone-pip ABR and ASSR, and established the data for normal infants within the first 6 months of life. This work will provide the theory basis and the application reference for early diagnosis and intervention of congenital hearing loss.
The aim of this study is to evaluate the gene mutational analysis of the GJB2, SLC26A4, mtDNA A1555G on children with SNHL, and to identify the cause of SNHL in Shandong province and to explore the relationships between the gene mutations and their adiological characters.
Methods:
1.80 infants with normal hearing participated in the present study. These subjects were assigned to four groups including 2-day newborns as group 1,6-week old as group 2,3-month old as group 3 and 6-month old infants as group 4 for analysis. Every group had 20 cases (10male and 10 female) with 40 ears. The criteria for normal hearing of infants was:full-term infants with 38 to 41gestational weeks, the birth weight was not less than 2500g, the Apgar score after birth was not less than 8 within one minute, no history of newborn intensive care unite(NICU), no family history of hearing loss and not using any ototoxic drugs. Their physical examinations were within normal range.
From Jan 2008 to July 2009,peripheral blood was collected from 146 children with SNHL confirmed by otoacoustic emissions (OAE),typernormetry,auditory brainstem response(ABR),tone-pip ABR,auditory steady state response(ASSR) and clinical physical examination,who aged 6 wks to 68 months.The degree of hearing loss was divided into mild(31～50dB),moderate(51～70dB),sever(71～90dB) and profound (≥91 dB)n HL according to WHO (1997) criteria.
2. Measurement Method:
①Case History:Before the hearing tests,the information about the history of diliverary,the disease history of the infants,physical examination,the history of pregnant mother and the family history of hearing loss etc.
②GSI 901 tympernometry was used to measure the middle ear function. The pressure was produced from+200 daPa to-400 daPa at the speed of 50 daPa per second. The probe tone was produced at 1000 Hz and 226 Hz for the infants less than 6 months old and just 6 months old respectively.
③TEOAEs were measured for each subject, using the Danmark Medsen diagnostic equipment, which could show PASS or REFER automatically.
④The ABR was performed by the auditory evoked potential system(Medsen, ICS). The recording electrode was positioned on the foread, the ground electrode was positioned at the nasal placement, and the reference electrodes were positioned at the mastoid bilaterally. The impendence was not more than 3kΩacross any two of them. The stimulus was delivered through an ER-3A insert earphone at the rate of 20.1 per second. The output limit was 98 dBnHL. The superposition was 2000 times at each intensity.The band-pass filter was from 100 to 3000 Hz. The test intensity was given at 70 dB nHL firstly and decreased by 20 dB nHL untill the minimal level of the response threshold of wave V appeared.The tone-pip stimuli was produced in 2-0-2 blackman envelope, and the responses were recorded at the minimal level in nHL at the octave frequencies from 250 to 8000 Hz. The stimuli was delivered through an ER-3A insert earphone at the rate of 21.1 per second. The band-pass filter was from 100 to 1500 Hz.
⑤The ASSR measurements were presented by the ASSR system(Medsen, ICS) with the carrier frequencies from 250 to 8000Hz and the modulation rates from 77 to 102 Hz. The recording was lasting 8 minutes in the 100%amplitude-modulated (AM) and 25%frequency-modulated (FM) through an ER-3A insert earphone. The amplification gain was 200k, the band-pass filter was from 65 to 105 Hz. The stimulus was produced synchronously at 12 frequencies bilaterally with 5 dB steps. The output limit was 120 dB nHL.The minimal level of responses were detected in nHL automatically. Every electro-physilogial hearing assements above were conducted in the sound-proof room, and the ambient noise was not more than 17dB(A).
⑥The high resolution CT scan was used for infants with hearing loss.
⑦The polymerase chain reaction(PCR) and sequencing technique were used to analyze the coding region of GJB2,SLC26A4,mtDNA A1555G gene.The sequences were analysed with DNAStar.
3.Statistical Analysis
All data were analyzed by using SPSS 10.0 software packages, T test and one-way analysis were employed. The differences between groups were recognized statistically significant as the p value not more than 0.05. If there were statistical differences amongs the groups, the SNK (Student-Newman-Keuls) test were used for comparing every two groups. The difference between two groups was recognized statistically significant as the q value not more than 0.05.
Results:
1. For click ABR at 70dB nHL, the absolute and interpeak wave latencies ofⅠ,Ⅲ,Ⅴ,Ⅰ-Ⅲ,Ⅲ-ⅤandⅠ-Ⅴdecreased as the age increased (p<0.05). The developmental changes were obvious in waveⅠandⅢbefore 6 wk and 3-month respectively (p<0.05).
2.The tone-pip ABR had similar waveforms as the click ABR, its wave latencies decreased and the waveforms got better with the age and frequency increased.
3.The thresholds of tone-pip ABR and ASSR had different thresholds at 250 to 8000 Hz in 0～6-month infants, but no physiological differences of them. The tone-pip ABR thresholds were significantly lower than those of ASSR from 0.5 to 8 kHz (p<0.05).
4. Both ASSR and ABR had satable and similar audiograms between different age groups in infants.
5. 44 of 146 (30.14%)cases with SNHL were found to carry GJB2 mutations,18 cases were identified as 235delC/235delC homozygotes,4 of 44 were identified as 235delC/176-191dell6 compound heterozygotes,3 were identified as 235delC/299～300delAT compound heterozygotes,19 were identified as 235delC heterozygotes.
6. 23 of 146 (15.75%) cases were found to carry SLA26A4 mutations,including 10 IVA7-2 A>G homozygotes and 13 heterozygotes.The incidence of hearing loss caused by SLA26A4 gene mutations was 6.85%.
7. Only 1 of 146(0.68%) was found to carry mtDNA A1555G mutations, who was heterozygotes.
8. Totally,128 of 146 (87.67%) cases were received high resilution CT scan and 27 cases of them had positive results.56.52%of cases with SLC26A4 mutations had inner ear malformation, which was higher than GJB2 and no-mutation groups.4 cases with GJB2 mutations were diagnosed with enlarged vestibular aqueduct (EVA), a novel reported in Shandong Province for the first time.
9. The audiological characters of GJB2 and SLA26A4 mutations were diversity. The degree of the hearing loss were mostly sever to profound for GJB2 mutations while moderate to sever for SLC26A4 mutations.
Conclusions:
1. The wave latency ofⅠ,Ⅲ,Ⅴand inter-wave latency ofⅠ-Ⅲ,Ⅲ-ⅤandⅠ-Ⅴof tone-pip ABR were decreased as the age increase, while the thresholds had no physiology changes within 0～6 month after birth.
2.Both tone-pip ABR and ASSR had stable frequency specificity. Compared with the ASSR, tone-pip ABR had lower response threshold and maybe nearer to the hearing level of the infant.
3. The incidence of GJB2, SLC26A4 and mtDNA A1555G mutations were 30.14%,15.75%and 0.68%respectively for children with NSHI. The main mutations of GJB2 gene were 235delC/235delC,235delC/176-191del16 and 235delC/299delAT. 23.97%(35/146) of all with NSHI were diagnosed with heredity though the conbine tests of Molecular genetic method. Screening and diagnosis for these three mutations are nesseory and effective to the children with SNHL found by universal newborn hearing screening programe.
4. The audiological characteristics of GJB2 and SLA26A4 mutations were diversity.
5. Molecular genetic screening and diagnosis for GJB2, SLA26A4 and mtDNA A1555G mutations were nesseory and effective on the basis of universal newborn hearing screening programe.
Innovations and Meanings:
1.This study first established the normal data of tone-pip ABR and ASSR, and investigated the frequency specificity between them in infants aged 0～6 months,which provided the referenced evidence for the, development of human auditory system and effective policy of frequency specific hearing tests in clinical practice.
2. This study first reported the incidence of GJB2, SLA26A4 and mtDNA A1555G mutations in a large sample in Shandong province of China, on the basis of universal newborn hearing screening program. This study also investigated the relationship between gene mutations and clinical denonstrations,and reported that 4 cases with inner ear malformation carried GJB2 but no SLA26A4 mutations.
新生儿听力损失是较常见的出生缺陷。我国听力残疾人约有2057万,每年出生1950万的新生儿,其中约有0.1368%患有致残性听力损失,每年约新增2～3万个听障儿童。由于3岁以前是言语-语言发育最重要和最关键的时期,先天性听力损失将极其严重地影响儿童言语、认知和情感的发育,以及未来接受教育和工作的机遇。因此,“早发现,早诊断,早治疗”是避免因聋致哑、促进患儿发育的较好方式。通过新生儿听力普遍性筛查项目的实施,能够对先天性听力损失及早发现、及早进行干预,从而在一定程度上减少了听力残疾和语言障碍造成的损失和社会负担。
但就目前新生儿听力筛查发展状况而言,听力损失的确诊、干预和康复方面还面临许多挑战。在听阈评估的技术方面,过去的几十年中临床广泛应用的听性脑干反应(ABR)是对婴幼儿听力损失做出诊断的最为重要的手段之一。但是不同的刺激声所诱发的脑干听性反应是极其不同的。短声诱发的听性脑干反应(ABR)与2000至4000Hz频阈范围的听敏度呈高度相关,但对中低频听力损失可能会产生假阴性结果(即500至2000Hz以下)。听觉频率特异性测试是近年来广泛用于临床的比较成熟的听力学检测技术,包括短纯音听性脑干反应(tone-burst ABR),短音听性脑干反应(tone-pip ABR),听觉稳态诱发电位(ASSR)和行为听力测试等。它们可以对不同频率的最小可听到的刺激声强度进行检测,可以明确在不同频率的听力下降状况,使诊断更加明确具体；更为重要的是,它可以为6月龄以下不会进行言语表达的较小婴儿的助听器选配提供较为可靠的听力曲线,使其助听器选配更加个体化。否则将大大影响助听器参数的设置,使助听效果不能达到最优化,从而直接影响患儿的聆听效果和言语康复水平。美国婴幼儿听力联合委员会2007年发表的形势报告,就听力损失的早期发现及早期干预亦提出了指导方针,支持对新生儿和较小婴儿进行普遍听力筛查、评估和干预。其指南中提出,听力损失的诊断性测试中应包括频率特异性电生理检查,如听觉稳态反应(ASSR)、短纯音听性脑干反应(tone-burst ABR)等。因此,开展新生儿和／或婴幼儿听觉频率特异性测试技术,既是听力学发展的大势所趋,又是小儿临床听力学领域所面临的挑战和亟需解决的问题。
目前,国外在频率特异性听力测试方面的研究尚缺乏针对小婴儿和新生儿的临床标准。我国在新生儿和婴幼儿频率特异性电生理检测的应用还刚刚起步,对于新生儿听力损失的频率特异性特点还不甚了解,对于快速的脑发育阶段中,听力损失的频率特异性变化还不清晰,从而无法为听力损失儿童的早期干预提供有力科学依据。本课题组前期研究表明,听力损失婴幼儿的听阈(ABR)水平随年龄增长存在波动。但是对于听力损失新生儿和婴幼儿,神经的可塑性对各频率影响还未见报道。只有准确地把握听力损失患儿听觉神经系统的发育特点和规律,明确其随年龄演变的规律,才能有效确定助听器各项参数及调试和监测周期,不断调整康复方案；把握可塑性调节的关键点并及时给予合适的外在干预,使早期干预更加有效,这无疑会对其康复产生强有力的推动作用。
近十年来,遗传学和分子生物学的发展极大地推动了耳聋发生的分子机制的研究。研究表明,基因突变具有显著的种族特异性,即不同地区及种族的患者,同一基因的突变类型不完全相同,每个种族有其特异性热点突变。GJB2,SLC26A4和mtDNA A1555G,是中国人非综合征型聋常见的热点突变。遗传学研究表明,先天性重度～极重度听力损失中50%以上与遗传因素有关,其中70%的患儿属于非综合征型听力损失(NSHI)。通过对耳聋大家系的连锁分析和基因定位克隆等技术,目前已经定位了104个非综合征型耳聋基因座位。遗传流行病学研究认为,通过对GJB2, SLC26A4和mtDNA A1555G三种基因的检测,可以发现26.65%的中国北方地区语前聋患者；通过对GJB2的235delC等位基因突变位点的检测,可有高达15%患者得到明确诊断。SLC26A4突变导致的前庭导水管扩大是内耳最常见的畸形,在先天性聋中占5%～10%,其突变阳性率为可达97%。本研究通过对GJB2、SLC26A4和·mtDNA A1555G三种基因的检测,可以初步掌握山东地区听力听力损失婴幼儿及儿童耳聋基因的热点突变位点和突变率,明确基因型与听力损失临床表型之间的关系,不仅对估计疾病的预后和康复效果起着非常重要的作用,而且为今后耳聋基因筛查和诊断提供依据,为优生优育提供决策基础,对降低听力损失患儿的出生率,对提高出生人口质量,减轻社会压力起着不可估量的作用。
综上所述,明确听觉发育的频率特异性,将直接指导早期干预的具体实施和个体化康复计划的制定；了解了听力损失的病因尤其是基因突变位点,对遗传咨询、减少病残儿的出生具有极其深远的社会意义。
研究方法：
1.研究对象及分组：选取0～6月龄听力正常婴儿80例(160耳),按纠正月龄分为四组：新生儿组、42 d组、3月龄组和6月龄组,每组20例(40耳),男女例数均等。听力正常婴儿的入选标准为：38～41孕周足月娩出,出生体重＞2500 g,生后Apgar评分1 min＞8分,无新生儿重症监护史,无耳毒性药物应用史及耳聋家族史,各项体检指标均为正常；听力检查：瞬态诱发耳声发射(TEOAE)测试能正常引出,短声听性脑干反应(click ABR) V波反应阈正常听力级≤30 dB,声导抗测试0～3月龄以1 kHz鼓室图为单峰型,6月龄以226 Hz鼓室图为A型,峰压值在-100daPa～+100daPa之间为正常。
基因检测：选取2007年1月～2009年7月在济南市妇幼保健院新生儿听力筛查康复中心就诊、经至少6个月随访确诊为单耳和／或双耳轻度～极重度非综合征型听力损失的婴幼儿及儿童146例,其中男77例,女69例。初诊年龄6w～10岁,年龄中位数为18 m。所有患儿均来自山东省内14个地市,146例中144例均接受过新生儿听力普遍筛查,2例漏筛；接受过听力筛查的患儿中,133例患儿因复筛“未通过”而转诊至我院进一步确诊；11例初筛“通过”,因“语言发育迟缓”或“头部外伤后听力下降”等原因就诊。
2.测试技术：
①病史采集听力检测前,由患儿家长填写“听力异常儿童登记表”,内容包括：患儿家庭基本信息、出生史、个人史(生后患病及耳毒性药物应用情况、头部外伤史等)、体检情况、母亲妊娠史、分娩史、耳聋家族史等,重要信息(如家族史等)需与不同家庭成员单独沟通,以确保内容的真实性和准确性。
②声导抗和耳声发射测试：在进行电耳镜检查并清除外耳道耵聍后,使用美国GSI tympernometry中耳分析仪进行探测音为226 Hz和1 kHz鼓室声导抗测试。测试起始压力为+200 daPa,终止压力为-400 daPa,压力变化速度为50daPa／s,方向由正向负。采用丹麦Medsen耳声发射仪进行TEOAE测试，仪器自动完成探头自检和结果判读。以上测试在一安静的房间内进行,环境噪声小于40dB(A)。
③click ABR和tone-pip ABR测试：采用丹麦Medsen ICS听觉诱发电位仪,记录电极放置于前额正中近发际处,参考电极置于双侧乳突,地极置于鼻根部,极间电阻≤3 kΩ。click ABR测试的刺激信号为短声,刺激速率为20.1次／s,最大输出强度为正常听力级(下同)98 dB,叠加次数2000次,带通滤波100～3000 Hz,以反应阈≤30 dB为正常。tone-pip ABR采用上升、下降时间均为2ms,平台时间为0 ms的短音。带通滤波同click ABR,增益为105。通过ER-3A插入式耳机给声,将0.25、0.5、1、2、4、8 kHz处能够引出ABRV波的最小强度定为其反应阈。临床上以通过瞬态诱发性耳声发射(TEOAE),鼓室图(226Hz和／或1000Hz探测音)正常,短声听性脑干反应(click ABR)波V反应阈≤30dBnHL,500Hz短音听性脑干反应(tone-pip ABR)反应阈≤40dBnHL为听力正常的诊断标准。
④ASSR测试：采用丹麦Medsen ICS听觉诱发电位仪,记录电极放置于前额正中近发际处,参考电极置于双侧乳突,地极置于鼻根部,极间电阻≤3 kΩ。ASSR刺激声信号载波频率为0.25、0.5、1、2、4、8 kHz,左耳调制频率分别为79、92、77、84、85、100 Hz,右耳为86、97、81、95、88、102 Hz,放大增益为200 k。带通滤波为65～105 Hz,每个强度最多扫描480次；调幅深度为100%,调频深度为25%,ER-3A插入式耳机双耳12个频率同时给声测试,测试步距为5 dB,系统自动记录反应并判定各频率所能引起反应的最小正常听力级。所有电生理测试均在隔声屏蔽室内进行,环境噪声小于17 dB(A)。
⑤医学评估和影像学检查：对所有听力损失患儿均进行医学评估和颞骨高分辨率CT扫描。医学评估的内容包括：详细的体格检查,包括眼科、耳鼻咽喉科、口腔、甲状腺功能、心脏、肾脏以及神经系统检查等,从而鉴别综合征型与非综合征型听力损失。前庭导水管扩大(enlarged vestibular aqueduct,EVA)的诊断标准：高分辨率颞骨CT轴位,从半规管总脚到前庭水管外口1／2处直径＞1.5mm；MRI检查发现双侧小脑半球表面有条弧形或椭圆形囊状物高信号影。
⑥基因组DNA的提取：经本院伦理委员会认可,在知情同意的前提下,对各组听力损失患儿抽取静脉血5～10ml,用于基因组DNA的提取和保存。采用北京天根公司生产的基因组DNA提取试剂盒在全血中提取基因组DNA,琼脂糖电泳和紫外分光光度计定量和纯度分析,-20°保存。
⑦引物序列：应用在线引物设计软件Primer3设计引物序列,引物由上海英骏公司合成(中国)GJB2引物：上游引物CX26-F:TTGGTGTTTGCTCAGGAAGA下游引物CX26-R:GGCCTACAGGGGTTTCAAAT扩增产物长度：960bpSLA26A4引物：上游引物P8-F:AAGTTCAGCATTATTTGGTTGACA下游引物P8-R:TGGTTGTTTCTTCCAGATCACA扩增产物长度：305bpmtDNA A1555G引物：上游引物Mito-F:TCAACCTCACCACCTCCT下游引物Mito-R:TTTGTCGCCTCTACCTAT扩增产物长度：767bp
⑧PCR反应体系：10×Buffer2.5ul,2.5Mm／ulDNTP 2ul,5U／ulrTaq酶0.5ul(Takara公司),20Mm／ul上下游引物0.3ul,100ng／ulDNA 1ul,ddH2O补齐至25ul。
⑨PCR扩增在ABI公司(美国)9700热循环仪上完成,具体程序如下：
GJB2：94℃预变性5min；94℃变性30sec,58℃退火30sec,72℃延伸30sec,30个循环后72℃延伸7min。反应结束后,取2ul进行1%琼脂糖电泳检测,PCR产物4℃保存待直接测序。
SLC26A4 IVA7-2：94℃预变性5min；94℃变性30sec,53℃退火45sec,72℃延伸60sec,35个循环后72℃延伸7min。反应结束后,取2ul进行1%琼脂糖电泳检测,PCR产物4℃保存待直接测序。
mtDNA12SrRNA A1555G:95℃预变性5min,95℃变性30sec,62℃退火30sec,72℃延伸70sec,30个循环后72℃延伸7min。酶切反应体系：10×Tango Buffer2ul,Alw26I限制性内切酶0.2ul,PCR产物5ul,ddH20补齐至20ul,在37℃水浴箱内消化3小时,取10ul进行2%琼脂糖电泳检测酶切结果。PCR扩增片段没有A1555G突变,可被AlW26I酶切割成326bp和441bp二条带,相反扩增片段有A1555G突变,该酶切位点消失,由AlW26I酶切经琼脂糖电泳后仍为一条带,进行PCR产物测序。
⑩PCR产物测序：应用ABI公司(美国)3730型DNA测序仪进行直接测序。应用DNAStar软件包中的Seqman (Lasergene公司,美国)软件与NCBI网站公布的标准DNA序列进行序列对比分析,检测待查个体的碱基序列改变,查明其在氨基酸序列中的位置以及相应氨基酸改变。
3.统计学方法：采用SPSS10.0统计软件,分别计算各组听力正常婴儿70 dB短声刺激下ABR的潜伏期和波间期、0.25～8 kHz tone-pip ABR和ASSR反应阈的均值和标准差,并进行独立样本t检验和均数的方差分析；组间率和频数的比较采用卡方(x2)检验,以P=0.05为统计学意义界值。
研究结果：
1.70 dB正常听力级短声刺激下,短声ABRⅠ、Ⅲ、Ⅴ波潜伏期、Ⅰ～Ⅲ、Ⅲ～Ⅴ、Ⅰ～Ⅴ波间期随月龄增加逐渐缩短,波Ⅰ于42 d前、波Ⅲ于3个月前发育变化显著(P值＜0.05)
2.短音(tone-pip) ABR波形与短声ABR波形相似,Ⅰ、Ⅲ、Ⅴ波潜伏期随频率增加逐渐缩短,波形分化逐渐清晰。
3.不同频率、不同月龄tone-pip ABR和ASSR反应阈水平差异虽有统计学意义(P＜0.05),但是均无生理学意义(相差＜10dB)。除0.25 kHz外,其余频率tone-pip ABR反应阈均低于ASSR。
4.不同月龄tone-pip ABR和ASSR听力曲线形状相似。
5.GJB2基因突变检测：146例患儿中,共检出GJB2基因突变44例(男23例,女21例),占30.14%(44／146),发现GJB2基因的6种突变类型,其中良性多态突变3种：79G＞A(41例)、341A＞G(36例)和608T＞C(3例),等位基因频率分别是16.44%(48／292)、14.73%(43／292)和1.03%(3／292)；致病突变3种：235delC／235delC(18例)、235delC／176-191del16(4例)和235delC／299delAT(3例),其等位基因频率为21.23%(62／292)、1.37%(4／292)和1.03%(3／292)。其中,以235delC纯合突变最为常见,占72%(18／25)其次为235delC／176-191del16和235delC／299delAT。所有146例患儿中,235delC纯合突变18例,占12.33%；复合杂合突变7例,占4.79%；235delC杂合突变19例,占13.01%,总突变率为30.14%；其中纯合突变和复合杂合突变被认为是致病突变,因此25例患儿可以明确为GJB2基因突变致聋,占17.12%。
6.SLC26A4基因IVS7-2位点突变检测：146例患儿中,共检出SLC26A4基因IVS7-2位点A＞G突变23例(男14例,女9例),占15.75%(23／146)；其中纯合突变10例,杂合突变13例,等位频率为11.30%(33／292)。纯合突变和复合杂合突变被认为是致病突变,因此10例患儿可以明确为SLC26A4基因突变致聋,占6.85%。
7.mtDNA 1555位点A＞G突变检测：146例患儿中,检出1例mtDNA 1555位点A>G突变,系杂合突变,突变频率为0.68%(1／146),等位基因频率为0.34%(1／292)。
8.医学评估：146例患儿中,除1例GJB2基因突变(235delC／299delAT)患儿伴有眼外直肌发育异常,1例SLC26A4基因杂合突变患儿伴有外耳畸形外,其余患儿均未见异常。
9.影像学检查：146例听力损失患儿中,128例接受了高分辨率颞骨CT扫描,占87.67%；CT结果显示：前庭导水管扩大(EVA)20例(其中1例合并听小骨畸形),Mondini畸形6例。其中4例存在GJB2突变,占所有GJB2突变患儿的11.36%(4／44),13例存在SLC26A4基因突变,占所有SLC26A4基因突变患儿的56.52%(13／22),mtDNA A1555G基因突变的患儿仅1例,颞骨CT未见异常。SLC26A4基因突变患儿中内耳畸形检出率明显高于GJB2基因突变组和未检出突变组(p＜0.05)
10.GJB2基因突变的听力学表现：听力损失程度从轻度～极重度均可见,以中度及中度以上为主,占95.5%(42／44)；既有对称性听力损失(38例),又有非对称性听力损失(5例)；除1例单侧(左耳)重度听力损失外,其余43例均为双耳受损；有耳聋家族史的多见,占40.91%(18／44)；随访中发现听力损失程度加重1例(重度→极重度)。
11.SLC26A4基因IVS7-2位点A＞G突变的听力学表现：听力损失程度轻度～极重度均可见,90%以上(20／22)表现为先天性听力损失,以波动性、非对称性听力损失居多,占45.45%(10／22)；非对称性听力损失11例,占47.83%(11／23)。随访中发现,轻、中度听力损失患儿容易出现听力波动,诱因多为上呼吸道感染和头部外伤,大多经及时的激素治疗后可恢复到波动前水平,个别也可自行恢复。
12.mtDNA 1555突变的听力学表现：患儿系第二胎,男性,因3岁“自幼不会说话”就诊,未接受过新生儿听力筛查,体格检查无异常发现,家长否认氨基糖甙类类药物应用史及耳聋家族史,听力学表现为先天性极重度感音神经性听力损失。
13.初筛“通过”的听力损失患儿基因检测：146例听力损失患儿中,11例初筛时“通过”的新生儿听力筛查,占7.53%(11／146)；11例中,3例存在GJB2基因突变,检出率为27.3%(3／11),1例SLC26A4基因IVS7-2位点A＞G突变,7例基因检测阴性。
14.基因型与听力表型之间的关系：GJB2基因突变的患儿中,不同听力损失程度的患儿所占比例分别为：轻度4.55%(2／44),中度11.36%(5／44),重度22.73%(10／44),极重度61.36%(27／44)；SLC26A4基因突变的患儿中,不同听力损失程度的患儿所占比例分别为：轻度17.39%(4／23),中度21.74%(5／23),重度43.48%(10／23),极重度17.39%(4／23)。对两组中不同程度听力损失的构成比采用2×4行列表的卡方(x2)检验进行比较,结果显示：两组中不同程度听力损失的构成比差异有统计学意义(x2=12.3637,p=0.0062,p＜0.05),即GJB2基因突变患儿以重度～极重度听力损失为主,而SLC26A4基因突变的患儿则以中度～重度听力损失居多。
研究结论：
1.0-6月正常婴儿tone-pip ABR的潜伏期和波间期随月龄增加逐渐缩短,而反应阈水平无明显变化。
2.tone-pip ABR和ASSR均有稳定的频率特异性,tone-pip ABR反应阈低于ASSR,可能更接近婴幼儿的实际听阈。
3.听力损失儿童中GJB2、SLC26A4和mtDNA A1555G突变的检出率分别为30.14%、15.07%和0.68%,其中GJB2突变的检出率最高,主要致病突变的基因型为235delC／235delC,235delC／176-191del16和235delC／299delAT。mtDNAA1555G突变的检出率较低,除地域和种族差异外,可能与检测对象年龄尚小、无氨基糖甙类药物应用史有关。通过GJB2、SLC26A4和mtDNA 12SrRNA基因的检测,可为23.97%的患儿明确病因(纯合或复合杂合突变35例)
4.基因型与听力表型之间的关系：不同基因型所致听力损失的临床表现多种多样,且GJB2和SLC26A4基因突变患儿都可伴有内耳解剖异常；但是GJB2基因突变以重度～极重度、对称性听力损失、内耳解剖正常者为主,而SLC26A4基因突变患儿中非对称性听力损失多见,程度以中度、重度居多,一半患儿可以合并内耳畸形。尽管不同基因突变所致的听力表型呈现一定特点,但是不能从听力表型推测其基因型,反之亦然。此结果提示：临床上进行基因诊断时,应将单侧及双侧、包括轻度在内的所有听力损失患儿均纳入检测对象。
5.有必要在新生儿听力普遍筛查的基础上,联合开展新生儿耳聋基因筛查。GJB2、SLC26A4和mtDNA12SrRNA A1555G基因是主要的候选基因。
创新和意义：
1.首次建立了国内0～6月龄婴儿频率特异性听力测试的正常值,为探索正常大脑听觉特性随月龄发育演变的规律、研究听觉可塑性提供了依据；同时,比较了短音听性脑干反应(tone-pip ABR)和听觉稳态诱发反应(ASSR)反应阈的频率特性,并制定了临床实用的测试方案。
2.首次在国内较大样本地报道山东地区听力损失小儿中耳聋基因GJB2、SLC26A4和ntDNA12SrRNA A1555G的常见的突变位点和突变率；在新生儿听力普遍筛查的基础上,研究了GJB2和SLC26A4基因型与听力表型之间的关系。而且,在国内首次报道了4例GJB2基因突变患儿存在内耳解剖异常。
Background and objectives:
Hearing loss is the common birth defect in newborns.There are about 20.57 million people with hearing loss and 0.1368 of newborns have hearing loss annually in china,which means that there are 20～30 thousand hearing loss children increased every year.As we know,it is a very important stage for speech & languagr development before three-year old.The congenital hearing loss will affect the speech,recognition,emotion,education and work opportunities.Therefore,the early diagnosis and early intervention is a better way to avoid dumb and promote children's development.
In universal newborn hearing screening programe,there are many chanlenges on the aspects of diagnosis,intervention and rehabilitation presently. In the past decades,auditory brainstem response(ABR) was widely used in clinic as the main diagnosed mathod for infants with hearing loss.But the evoked potentials were very different in different stimulus.The click ABR was highly correlated with the threshold between 2000 to 4000Hz frequency range,while it might cause false nagative result in middle-low frenquency hearing loss.Recently,the frequency specific hearing tests were widely used clinically,including tone-burst ABR,tone-pip ABR,auditory steady state response(ASSR) and behavior hearing test.They could test infants with hearing loss in each frequency and provide the detailed information for diagnosis.More importantly,the frequency specific tests could provide the reliable profile of hearing loss and then make the hearing aid fitting more individually.
The Joint Committee on Infant Hearing (JCIH) Year 2007 Position Statement further proposed that the frequency-specific assessment of ABR testing is needed to determine the degree and configuration of hearing loss in each ear for fitting of amplification devices. It also suggested the frequency-specific assessment can be applicated in the diagnosis tests, such as ASSR, tone-burst ABR and so on.
In the prsent study, the frequency specific hearing tests were still lack of the clinical criteria for newborns and infants. In addition, the present data of tone-pip ABR and ASSR are mostly from the adults and children, lacking of the reports on newborns and infants. So we know little about the auditory development in the early stage of life,especially on the aspect of the frequency change.The previous study of our team revealed that the threshold of ABR in newborns and infants changed with the increaseing of the month.In this experiment, we analyzed the features of development of tone-pip ABR and ASSR, and established the data for normal infants within the first 6 months of life. This work will provide the theory basis and the application reference for early diagnosis and intervention of congenital hearing loss.
The aim of this study is to evaluate the gene mutational analysis of the GJB2, SLC26A4, mtDNA A1555G on children with SNHL, and to identify the cause of SNHL in Shandong province and to explore the relationships between the gene mutations and their adiological characters.
Methods:
1.80 infants with normal hearing participated in the present study. These subjects were assigned to four groups including 2-day newborns as group 1,6-week old as group 2,3-month old as group 3 and 6-month old infants as group 4 for analysis. Every group had 20 cases (10male and 10 female) with 40 ears. The criteria for normal hearing of infants was:full-term infants with 38 to 41gestational weeks, the birth weight was not less than 2500g, the Apgar score after birth was not less than 8 within one minute, no history of newborn intensive care unite(NICU), no family history of hearing loss and not using any ototoxic drugs. Their physical examinations were within normal range.
From Jan 2008 to July 2009,peripheral blood was collected from 146 children with SNHL confirmed by otoacoustic emissions (OAE),typernormetry,auditory brainstem response(ABR),tone-pip ABR,auditory steady state response(ASSR) and clinical physical examination,who aged 6 wks to 68 months.The degree of hearing loss was divided into mild(31～50dB),moderate(51～70dB),sever(71～90dB) and profound (≥91 dB)n HL according to WHO (1997) criteria.
2. Measurement Method:
①Case History:Before the hearing tests,the information about the history of diliverary,the disease history of the infants,physical examination,the history of pregnant mother and the family history of hearing loss etc.
②GSI 901 tympernometry was used to measure the middle ear function. The pressure was produced from+200 daPa to-400 daPa at the speed of 50 daPa per second. The probe tone was produced at 1000 Hz and 226 Hz for the infants less than 6 months old and just 6 months old respectively.
③TEOAEs were measured for each subject, using the Danmark Medsen diagnostic equipment, which could show PASS or REFER automatically.
④The ABR was performed by the auditory evoked potential system(Medsen, ICS). The recording electrode was positioned on the foread, the ground electrode was positioned at the nasal placement, and the reference electrodes were positioned at the mastoid bilaterally. The impendence was not more than 3kΩacross any two of them. The stimulus was delivered through an ER-3A insert earphone at the rate of 20.1 per second. The output limit was 98 dBnHL. The superposition was 2000 times at each intensity.The band-pass filter was from 100 to 3000 Hz. The test intensity was given at 70 dB nHL firstly and decreased by 20 dB nHL untill the minimal level of the response threshold of wave V appeared.The tone-pip stimuli was produced in 2-0-2 blackman envelope, and the responses were recorded at the minimal level in nHL at the octave frequencies from 250 to 8000 Hz. The stimuli was delivered through an ER-3A insert earphone at the rate of 21.1 per second. The band-pass filter was from 100 to 1500 Hz.
⑤The ASSR measurements were presented by the ASSR system(Medsen, ICS) with the carrier frequencies from 250 to 8000Hz and the modulation rates from 77 to 102 Hz. The recording was lasting 8 minutes in the 100%amplitude-modulated (AM) and 25%frequency-modulated (FM) through an ER-3A insert earphone. The amplification gain was 200k, the band-pass filter was from 65 to 105 Hz. The stimulus was produced synchronously at 12 frequencies bilaterally with 5 dB steps. The output limit was 120 dB nHL.The minimal level of responses were detected in nHL automatically. Every electro-physilogial hearing assements above were conducted in the sound-proof room, and the ambient noise was not more than 17dB(A).
⑥The high resolution CT scan was used for infants with hearing loss.
⑦The polymerase chain reaction(PCR) and sequencing technique were used to analyze the coding region of GJB2,SLC26A4,mtDNA A1555G gene.The sequences were analysed with DNAStar.
3.Statistical Analysis
All data were analyzed by using SPSS 10.0 software packages, T test and one-way analysis were employed. The differences between groups were recognized statistically significant as the p value not more than 0.05. If there were statistical differences amongs the groups, the SNK (Student-Newman-Keuls) test were used for comparing every two groups. The difference between two groups was recognized statistically significant as the q value not more than 0.05.
Results:
1. For click ABR at 70dB nHL, the absolute and interpeak wave latencies ofⅠ,Ⅲ,Ⅴ,Ⅰ-Ⅲ,Ⅲ-ⅤandⅠ-Ⅴdecreased as the age increased (p<0.05). The developmental changes were obvious in waveⅠandⅢbefore 6 wk and 3-month respectively (p<0.05).
2.The tone-pip ABR had similar waveforms as the click ABR, its wave latencies decreased and the waveforms got better with the age and frequency increased.
3.The thresholds of tone-pip ABR and ASSR had different thresholds at 250 to 8000 Hz in 0～6-month infants, but no physiological differences of them. The tone-pip ABR thresholds were significantly lower than those of ASSR from 0.5 to 8 kHz (p<0.05).
4. Both ASSR and ABR had satable and similar audiograms between different age groups in infants.
5. 44 of 146 (30.14%)cases with SNHL were found to carry GJB2 mutations,18 cases were identified as 235delC/235delC homozygotes,4 of 44 were identified as 235delC/176-191dell6 compound heterozygotes,3 were identified as 235delC/299～300delAT compound heterozygotes,19 were identified as 235delC heterozygotes.
6. 23 of 146 (15.75%) cases were found to carry SLA26A4 mutations,including 10 IVA7-2 A>G homozygotes and 13 heterozygotes.The incidence of hearing loss caused by SLA26A4 gene mutations was 6.85%.
7. Only 1 of 146(0.68%) was found to carry mtDNA A1555G mutations, who was heterozygotes.
8. Totally,128 of 146 (87.67%) cases were received high resilution CT scan and 27 cases of them had positive results.56.52%of cases with SLC26A4 mutations had inner ear malformation, which was higher than GJB2 and no-mutation groups.4 cases with GJB2 mutations were diagnosed with enlarged vestibular aqueduct (EVA), a novel reported in Shandong Province for the first time.
9. The audiological characters of GJB2 and SLA26A4 mutations were diversity. The degree of the hearing loss were mostly sever to profound for GJB2 mutations while moderate to sever for SLC26A4 mutations.
Conclusions:
1. The wave latency ofⅠ,Ⅲ,Ⅴand inter-wave latency ofⅠ-Ⅲ,Ⅲ-ⅤandⅠ-Ⅴof tone-pip ABR were decreased as the age increase, while the thresholds had no physiology changes within 0～6 month after birth.
2.Both tone-pip ABR and ASSR had stable frequency specificity. Compared with the ASSR, tone-pip ABR had lower response threshold and maybe nearer to the hearing level of the infant.
3. The incidence of GJB2, SLC26A4 and mtDNA A1555G mutations were 30.14%,15.75%and 0.68%respectively for children with NSHI. The main mutations of GJB2 gene were 235delC/235delC,235delC/176-191del16 and 235delC/299delAT. 23.97%(35/146) of all with NSHI were diagnosed with heredity though the conbine tests of Molecular genetic method. Screening and diagnosis for these three mutations are nesseory and effective to the children with SNHL found by universal newborn hearing screening programe.
4. The audiological characteristics of GJB2 and SLA26A4 mutations were diversity.
5. Molecular genetic screening and diagnosis for GJB2, SLA26A4 and mtDNA A1555G mutations were nesseory and effective on the basis of universal newborn hearing screening programe.
Innovations and Meanings:
1.This study first established the normal data of tone-pip ABR and ASSR, and investigated the frequency specificity between them in infants aged 0～6 months,which provided the referenced evidence for the, development of human auditory system and effective policy of frequency specific hearing tests in clinical practice.
2. This study first reported the incidence of GJB2, SLA26A4 and mtDNA A1555G mutations in a large sample in Shandong province of China, on the basis of universal newborn hearing screening program. This study also investigated the relationship between gene mutations and clinical denonstrations,and reported that 4 cases with inner ear malformation carried GJB2 but no SLA26A4 mutations.
引文
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