【作者】 王剑；

【导师】 邱建华；

【作者基本信息】 第四军医大学 ， 耳鼻咽喉科学， 2011， 博士

【摘要】 听神经病是一种临床表现较为特殊的疾病,主要的听力学特征包括听性脑干反应缺失或严重异常,耳声发射正常,镫骨肌反射消失或阈值升高,纯音听力图多以低频听阈损失为主。患者的主要主诉是言语分辨率差而无法与人正常交流。听神经病与一般的感音神经性聋的显著差异,正引起越来越多的关注。但是对于此病的病因、发病机制及疾病的转归仍不明确。因此分析认识疾病的发展与转归,有利于深入了解该病并指导患者生活。另外,我们希望通过基因水平的研究以获得该病的致病基因,从而明确该病的发病机理,寻找可能的治疗手段。试验方法和结果1听神经病患者随访研究通过对2001年1月至2009年7月间就诊于我科的听神经病患者进行随访调查,分析听神经病患者的纯音听阈测试、声导抗测试、ABR、DPOAE、ENG及VEMP等的变化特征。随访者均为非综合征型听神经病患者,共74人,男性32人,女性42人,发病年龄3~33岁,平均16.69±4.42岁。患者纯音听阈损失以低频损失为主(70.1%),左右耳间无差别。随访1~9年,平均3.74±1.96年,患者纯音听阈水平在随访期内无明显差异,无向高频发展的趋势。随着患者病程和随访期的延长,言语识别率为0的患者比例有显著的增加。随访期内镫骨肌反射阈值与引出率无明显变化。随访期间患者DPOAE无变化,并无继发性耳声发射消失的表现。随访期间患者ENG异常耳数量、VEMP阈值及引出率均无明显变化,并无加重趋势。总之,随访期内听神经病患者的听力学表现基本无变化,未观察到明显加重或减轻。对于该病的发展与转归,仍需扩大随访人群并延长随访期,以全面了解该病的自然病程。2听神经病患者OOOOTTTOOF基因与PPPPJJJVVK基因突变筛查OTOF基因与PJVK基因是隐性遗传性耳聋的致病基因,目前有证据显示其突变与听神经病相关。为了明确散发听神经病患者上述两种基因的突变情况,我们进行了全外显子测序筛查。70名受检者中,OTOF基因共检出10种影响氨基酸编码的突变,其中4种未见报道,包括4023+1G>A,G368R,N727S,A809D。PJVK基因共检出3种影响氨基酸编码的突变,其中2种未见报道,包括1059+2A>C,R183Q。但是检出的突变均为单杂合突变,尚不能确定其突变形式是该患者的致病原因。3听神经病遗传家系的收集与整理临床中我们注意总结和收集耳聋家系以供基因研究。我们共收集到7个听神经病家系。其中5个为常染色体显性遗传的非综合症性听神经病家系,2个是伴有外周神经病的X染色连锁的隐性遗传性听神经病家系。为我们后期的研究奠定了坚实的基础。4X染色体连锁的隐性遗传性听神经病家系的致病基因筛查我们对一个临床资料较为完整的X染色体连锁的隐性遗传性听神经病家系进行了全外显子捕获测序,逐步缩小检测范围,最终通过家系连锁确定了该家系的致病基因为AIF(apoptosisinducefactor,凋亡诱导因子)基因,突变为错义突变S314N。5AAAIIF基因在听神经病患者中的突变筛查我们对70名散发的听神经病患者进行了AIF基因的全外显子测序筛查,在男患者中共检出9种错义突变,包括L191P、I287T、Q332H、G360R、R422W、A440V、P475L、E558V、D559G,及一个位于5‘UTR区的纯合突变,均未见报道。我们认为AIF基因在听神经病患者中的高突变率可能提示其在听神经病致病过程中扮演着重要的角色。6AAAIIF基因的定位与表达研究。我们通过RT-PCR及组织化学染色的方法,对AIF基因在大鼠内耳的表达做了初步研究,发现AIF广泛分布在Corti器、内外毛细胞、基底膜、螺旋神经节细胞及血管纹等处。同时新生大鼠与成年大鼠耳蜗基底膜AIFmRNA的表达量无明显差异。结果初步提示我们,AIF在内耳中广泛表达,其功能异常可能影响声音信号的转化与传导。我们仍需进一步进行功能研究以明确其致病机理。总之,我们对听神经病的了解还比较少,通过本研究,我们初步探明了听神经病的自然转归,了解了隐性遗传性听神经病相关基因OTOF与PJVK基因在散发听神经病患者中的致病性,并通过对一个X染色体连锁的隐性遗传听神经病家系进行研究,定位了一个新的听神经病致病基因AIF,并在散发的听神经病患者中检出9种错义突变,明确了AIF基因在听神经病患者中的重要性。通过实验手段对AIF基因在大鼠耳蜗做了明确定位,为进一步研究其功能奠定了基础。更多还原

【Abstract】 Auditory neuropathy (AN) is a specific hearing disorder. The main clinicalcharacters include auditory brainstem responses (ABR) absent or abnormalseverely and evoked otoacoustic emissions (EOAE) normal, which reflects thefunction of auditory nerve with inner hair cells and outer hair cells, respectively.AN was first named by Starr in 1996, and interested a lot of researchers studyingthis special disease. Auditory tests results in AN patients distinguished fromother sensorineural hearing loss also include acoustic reflex (AR) absence orthreshold elevation, pure tone auditory threshold elevation mainly at lowerfrequencies and vestibular affection as vestibular evoked myogenic potentials(VEMP) and/or electronystagram (ENG) abnormal. The core complain of ANpatients is speech discrimination disability and difficulty of verbalcommunication especially in noises. However, there is no effective treatment forAN patients, and we still have no idea of the causes and the changes in thecourse of this disease. Therefore we follow up a bunch of adult AN patients andanalyse the changes in auditory tests, hoping to understand the natural processand the prognosis of the disease. Neonatal hyperbilirubinemia, anoxianeonatorum, premature birth and ototoxic drug history can explain the causes in some congenital AN, but most posteriority AN patients has no clear history ofsuch risk factors at all. Some AN patients had been detected genes mutations ascause of their disease, but we do not know if genes variations are the cause ofsporadic adult patients. Thus, we designed this study to explore whether ourpatients carry variations in related genes.MethodMethods and results1. Follow-up study of auditory neuropathy patientsThe main body of this study is 74 out patients diagnosed as AN during 2001January till 2009 July. All the patients test pure tone audiometry, acousticimpedance, ABR, DPOAE, ENG, VEMP and speech discrimination during thefollow-up period. The average ages of AN onset are 16.69±4.42 years and thecourse of follow up are 3.74±1.96 years. There is no difference in the pure toneaudiometry between left and right ears of the patients, and both ears affect lowfrequencies mostly (70.1%). During the course of follow up, the pure tonethreshold has no significant changes and has no trend affecting higherfrequencies. Word discrimination score has no changes during follow up, but thenumber of ears with 0% word discrimination rate increased during the followingcourse. AR, DPOAE, ENG and VEMP have no statistic differences between thefirst and last visits, but the recordable VEMP threshold and latency areapparently different from control. In conclusion, during following course, thereis no big difference in almost all the auditory tests. We need to visit more ANpatients and follow up a longer period to understand better about the specialdisease.2. OTOF gene and PJVK gene variations sequencing in late onset ANpatientsOTOF and PJVK are the pathogenetic genes detected in some AN patients.However, almost all the mutations are detected in early onset patients, who hadlittle ability to hear and speak. There is no variation map in adult AN patients,and we wonder if OTOF and/or PJVK are the pathogenetic genes of late onset AN patients. We tested 70 sporadic acquired AN patients, and sequenced all theexons as well as introns near exons of OTOF and PJVK. We have detected 10variations that altered amino acid coding, 4 out of which had not reported before,including 4023+1 G>A, G368R, N727S and A809D. We have detected 2 novelmissense mutations in PJVK gene. However, all of the new variations are thesingle heterozygous in single patients, so we can not say that these variations arethe only causes of these patients. We still need to sequence more patients anddetect more related genes.3. Hereditary AN families clinical and genetic features analyzingSome AN patients has clear family history. In clinical study, we collectedsome precious hereditary AN families as follow. We collected 7 AN familiesappearing autosomal dominant inheritance phenotype and 2 AN families withperipheral neuropathy showing X-linked recessive inheritance phenotype. Weevaluated the phenotype and genetic characters of these families in detail.4. Pathogenetic gene sequencing of an X-linked recessive AN familyWe focused on an X-linked recessive AN family searching for theirpathogenetic gene, which had not detected yet. We used the exome capture chip(Nimblgen) and sequencing chip (Solexa) in the propositus for variations in theentire chromosome. Then we narrow the scope of candidate genes step by step,and finally confirmed gene AIF as the pathogenetic gene of the family, whosepathogenetic mutation is S314N.5. AIF gene sequencing in late onset AN patientsWe sequenced the 70 AN patients for all the AIF exons and introns near exons.Interestingly, we deteceed 9 missense mutations in 9 male patients as follow:L191P, I287T, Q332H, G360R, R422W, A440V, P475L, E558V and D559G, aswell as a variation in the 5’UTR of the upper stream in a boy. All the abovevariations are novel and did not detected in control. We believe that AIF gene iscritical in AN pathogenesis, and the mutations of AIF gene could be the causesof the patients. 6. Fluorescence dye and mRNA express of AIF in normal rat inner earIn order to make sure the location of AIF staining in normal cochlea, we usedimmunofluorescent and RT-PCR techniques. We observed that AIF wasdistributed broadly in the inner ear cells, especially in hair cells. There is novisible difference among the expression of AIF mRNA among neonatal andadult cochlea basilar membranes and neonatal spiral ganglion cells. Thus, weknow that AIF is a important molecule in mitochondria and expressed stably inthe inner ear, and we should do more work to fully understand the function ofAIF in hearing loss. AIF could be the very key to the AN.ConclusionWe are still at the beginning of AN studying. In this study, we get the generalknowledge of the natural out come of AN, we realized the variations carryingstates of OTOF gene and PJVK gene in late onset AN patients, we found a newAN related pathogenetic gene in an X-linked AN family with peripheralneuropathy, we confirmed the importance of AIF in AN pathogenesis as detected10 novel pathogenetic mutations, and we make clear the expression of AIF inneonatal and adult rat inner ear. All our work urged to reveal the pathogenesis ofAN, so that to find some curable methods to make the patients recover.更多还原