本文選題：聽(tīng)神經(jīng)病　切入點(diǎn)：聽(tīng)神經(jīng)病譜系障礙　出處：《中國(guó)人民解放軍醫(yī)學(xué)院》2012年碩士論文

【摘要】：聽(tīng)神經(jīng)病(auditory neuropathy, AN)又稱為聽(tīng)神經(jīng)病譜系障礙(auditory neuropathy spectrum disorder, ANSD),描述了一種內(nèi)毛細(xì)胞和聽(tīng)神經(jīng)突觸和/或聽(tīng)神經(jīng)本身功能不良所致的聽(tīng)力障礙,不同于典型的感音神經(jīng)性聽(tīng)力損失。典型的臨床表現(xiàn)是言語(yǔ)理解力受損,而言語(yǔ)覺(jué)察閾和純音聽(tīng)閾可以正常,也可以嚴(yán)重受損；聽(tīng)力學(xué)檢查特點(diǎn)是耳聲發(fā)射(otoacoustic emissions,OAE)和耳蝸微音電位(cochlear microphonics,CM)正常引出,聽(tīng)性腦干反應(yīng)(ABR)無(wú)明顯分化的波形或嚴(yán)重異常。 目前聽(tīng)神經(jīng)病的病因、病變部位和發(fā)病機(jī)制尚不十分明確,導(dǎo)致臨床(?)預(yù)的預(yù)測(cè)和評(píng)估上存在難度。2008年,Starr等提議按病損部位對(duì)AN分型：如只是聽(tīng)神經(jīng)受累,內(nèi)毛細(xì)胞和突觸正常,稱為“聽(tīng)神經(jīng)病變”(auditory nerve disorder),如果內(nèi)毛細(xì)胞突觸病變而聽(tīng)神經(jīng)正常,稱為“聽(tīng)突觸病變”(auditory synaptic disorder)。按病變部位分類,有助于精確定義本病,為臨床干預(yù)提供指導(dǎo)。然而,目前尚無(wú)臨床測(cè)試方法可以精確地確定病變部位。近年來(lái),一系列聽(tīng)神經(jīng)病相關(guān)基因的發(fā)現(xiàn),從分子水平揭示了聽(tīng)神經(jīng)病的病理機(jī)制,使得根據(jù)基因?qū)е碌牟煌∽儾课粚?duì)聽(tīng)神經(jīng)病進(jìn)行分型成為可能,在非綜合征型聾病例中尤為重要。目前研究發(fā)現(xiàn)與常染色體隱性遺傳非綜合征性聽(tīng)神經(jīng)病相關(guān)的基因有PJVK基因和OTOF基因。 本研究應(yīng)用候選基因突變篩查方法,選取2003-2011年收集和保存的明確診斷的150例聽(tīng)神經(jīng)病患者,開(kāi)展中國(guó)聽(tīng)神經(jīng)病人群PJVK基因的突變特征研究,并在本課題組前期完成76例聽(tīng)神經(jīng)病患者OTOF基因篩查的基礎(chǔ)上進(jìn)一步針對(duì)17例聽(tīng)神經(jīng)病患者開(kāi)展OTOF基因篩查，，進(jìn)而結(jié)合臨床表型與基因型探討溫度敏感性聽(tīng)神經(jīng)病患者的臨床遺傳學(xué)特征。從遺傳學(xué)角度闡述聽(tīng)神經(jīng)病的病因?qū)W機(jī)制及其病變部位,探討不同類型聽(tīng)神經(jīng)病患者的臨床特點(diǎn)和遺傳學(xué)特征。 本研究共分三個(gè)部分： 第一部分中國(guó)聽(tīng)神經(jīng)病譜系障礙患者PJVK基因臨床遺傳學(xué)特征分析 選取2003年一2011年確診的聽(tīng)神經(jīng)病患者150例(分別來(lái)自北京、天津、河北、河南、山東、山西等20個(gè)省市,男女比例為1：1.05),針對(duì)PJVK基因的7個(gè)外顯子設(shè)計(jì)引物,采取聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增目的片段,應(yīng)用PCR產(chǎn)物直接測(cè)序法進(jìn)行PJVK基因突變檢測(cè)；并針對(duì)發(fā)現(xiàn)的突變位點(diǎn),在100名聽(tīng)力正常人中進(jìn)行驗(yàn)證。分析中國(guó)聽(tīng)神經(jīng)病患者該基因的遺傳特征及相關(guān)聽(tīng)神經(jīng)病的臨床特點(diǎn)。 研究發(fā)現(xiàn)：在150例聽(tīng)神經(jīng)病患者中共檢測(cè)到PJVK基因位于外顯子區(qū)域的突變5種,4種為國(guó)際上未見(jiàn)報(bào)道的突變：1種為無(wú)義突變c.886CT,3種為錯(cuò)義突變c.437GA,c.740GT和c.887GA；還發(fā)現(xiàn)1種已報(bào)道的致病突變c.548GA。在150例聽(tīng)神經(jīng)病患者中PJVK基因突變攜帶率為3.3%。 攜帶c.886CT突變的患者臨床表現(xiàn)為溫度敏感性聽(tīng)神經(jīng)病,該突變使第296位氨基酸位點(diǎn)處形成終止密碼子,導(dǎo)致編碼的蛋白質(zhì)提前終止,在150例病人中僅1例攜帶該突變(0.67%),99例正常人中均未發(fā)現(xiàn)該突變,提示此為致聾突變。c.437GA,c.740GT, c.548GA和c.887GA均引起氨基酸的改變,突變氨基酸位點(diǎn)在不同物種間高度保守,且在正常對(duì)照者中未檢測(cè)到這些突變,考慮與聽(tīng)神經(jīng)病的發(fā)病密切相關(guān)。攜帶c.437GA突變患者23歲發(fā)病,聽(tīng)力曲線為覆盆形,低頻中度、中重度聽(tīng)力損失,中頻聽(tīng)力正常,高頻輕度聽(tīng)力損失,言語(yǔ)識(shí)別率左耳68%,右耳64%；攜帶c.548GA突變患者21歲發(fā)病,聽(tīng)力曲線為低頻上升型,低頻中度、中重度聽(tīng)力損失,高頻聽(tīng)力正常,言語(yǔ)識(shí)別率左耳32%,右耳18%；攜帶c.740GT突變患者16歲發(fā)病,波動(dòng)性聽(tīng)力損失,聽(tīng)力損失在中度到輕度之間,言語(yǔ)識(shí)別率左耳88%,右耳86%；攜帶c.887GA突變的患者表現(xiàn)為嬰幼兒時(shí)期發(fā)病,聽(tīng)力曲線為中頻“U”型,高、低頻為輕度聽(tīng)力損失,中頻為中重度聽(tīng)力損失,言語(yǔ)識(shí)別率左耳80%,右耳76%。 第二部分中國(guó)聽(tīng)神經(jīng)病譜系障礙患者OTOF基因臨床遺傳學(xué)特征分析 本研究在本課題組前期完成76例聽(tīng)神經(jīng)病患者OTOF基因篩查的基礎(chǔ)上進(jìn)一步針對(duì)17例聽(tīng)神經(jīng)病患者開(kāi)展OTOF基因篩查。收集患者臨床聽(tīng)力學(xué)資料,針對(duì)OTOF基因的47個(gè)外顯子設(shè)計(jì)44對(duì)引物,采取聚合酶鏈?zhǔn)椒磻?yīng)(PCR)擴(kuò)增,應(yīng)用PCR產(chǎn)物直接測(cè)序法進(jìn)行OTOF基因突變檢測(cè);針對(duì)發(fā)現(xiàn)的突變位點(diǎn),在100名聽(tīng)力正常人中進(jìn)行驗(yàn)證。以了解這些聽(tīng)神經(jīng)病患者OTOF基因的遺傳學(xué)特征及臨床特點(diǎn)。 研究發(fā)現(xiàn)：本研究共檢測(cè)到OTOF基因位于外顯子區(qū)域的變異29種,其中可能的致病突變有11種,多態(tài)性改變有18種。在可能致病的11種突變中,有8種是本研究發(fā)現(xiàn)的新的突變,包括4種移碼突變(p.G558A fsX21,p.L795SfsX5, p.I1108H fsX69, c.5833delG),1種無(wú)義突變(p.Y1133X),和3種錯(cuò)義突變(p.Q265L,p.G541S和p.R1928C)；此外本研究還發(fā)現(xiàn)了三種已報(bào)道的致病突變(p.N727S, p.Q994V fsX6, c. ivs4023+1GA)。這11種可能的致病突變,目前僅在中國(guó)人群中發(fā)現(xiàn),未見(jiàn)其他地域人群中相關(guān)報(bào)道。 本研究在1名溫度敏感性聽(tīng)神經(jīng)病患者中檢測(cè)到p.G541S和p.L795SfsX5復(fù)合雜合突變,在其他聽(tīng)神經(jīng)病患者及正常人中均未發(fā)現(xiàn)該突變,這是與溫度敏感性聽(tīng)神經(jīng)病相關(guān)的新的突變。在3名嬰幼兒聽(tīng)神經(jīng)病患兒中檢測(cè)到其中1人攜帶OTOF基因p.G558AfsX21雜合突變、p.Q994VfsX6雜合突變和p.Q265L雜合突變；另1名患兒攜帶OTOF基因c.4023+1GA純合突變；其余1名患兒攜帶OTOF基因c.5833delG和p.Y1133X復(fù)合雜合突變；三名患兒都表現(xiàn)為嬰幼兒時(shí)期發(fā)病,雙耳對(duì)稱的極重度聾。此外,本研究還在3例聽(tīng)神經(jīng)病患者中還檢測(cè)到3種單雜合突變：攜帶p.11108H fsX69突變患者表現(xiàn)為雙耳對(duì)稱的重度耳聾,平坦型聽(tīng)力曲線；攜帶p.N727S突變患者表現(xiàn)為雙耳中重度聾,平坦型聽(tīng)力曲線；攜帶p.R1928C突變的患者表現(xiàn)為雙耳極重度聾。 第三部分溫度敏感性聽(tīng)神經(jīng)病譜系障礙患者臨床遺傳學(xué)特征分析 本研究針對(duì)3名溫度敏感性聽(tīng)神經(jīng)病患者開(kāi)展聽(tīng)神經(jīng)病相關(guān)基因OTOF和PJVK檢測(cè),并回顧分析其臨床特征。 研究發(fā)現(xiàn)：在3名溫度敏感性聽(tīng)神經(jīng)病患者中,1名患者攜帶OTOF基因p.G541S和p.L795S fsX5復(fù)合雜合突變,1名患者攜帶OTOF基因p.Q994V fsX6雜合突變和PJVK基因p.R296X雜合突變,1名患者未檢測(cè)到PJVK基因或OTOF基因致病突變。 攜帶OTOF基因p.Q994V fsX6雜合突變和PJVK基因p.R296X雜合突變的患者聽(tīng)力狀況在一天之內(nèi)隨不同時(shí)間體溫的變化而有所變化,聽(tīng)力好時(shí)對(duì)日常談話的聲音有反應(yīng),能夠執(zhí)行指令性動(dòng)作,可以交流并正確回答問(wèn)題;聽(tīng)力不好時(shí)對(duì)大的聲音沒(méi)有反應(yīng),不能完成指令性動(dòng)作,對(duì)周圍事物漠然。攜帶OTOF基因p.G541S和p.L795S fsX5復(fù)合雜合突變患兒主訴為發(fā)熱時(shí)聽(tīng)力明顯下降交流困難,體溫正常時(shí)聽(tīng)力好轉(zhuǎn)可交流。
[Abstract]:Auditory neuropathy (auditory neuropathy, AN) also known as auditory neuropathy spectrum disorder (auditory neuropathy spectrum disorder, ANSD), describes a kind of inner hair cells and auditory nerve synapses and / or listen to the hearing nerve itself due to dysfunction, unlike typical sensorineural hearing loss. The typical clinical manifestation is verbal comprehension impaired speech detection threshold and pure tone threshold can be normal, can also be severely damaged; audiological examination is characterized otoacoustic emission (otoacoustic emissions, OAE) and cochlear microphonics (cochlear microphonics, CM) normal leads, auditory brainstem response (ABR) no obvious differentiation of the waveform or severely abnormal.
At present, the etiology of auditory neuropathy, the lesion and the pathogenesis is not very clear, leading to clinical prediction and evaluation of pre (?) on the difficulty of.2008, Starr suggested that the type of AN according to the site of lesion: just as auditory nerve involvement, inner hair cells and synaptic normal, called auditory neuropathy ("auditory nerve disorder), if the inner hair cell synapses and normal auditory nerve lesions, called" listen to synaptic lesions (auditory synaptic disorder). According to the classification of the lesion, contribute to the precise definition of the disease, and provide guidance for clinical intervention. However, at present no clinical test method can accurately determine the lesion in recent years. Here, a series of auditory neuropathy related genes were revealed to the pathological mechanism of an at the molecular level, which can be classified according to the different location of auditory neuropathy caused by gene in nonsyndromic deafness. It is particularly important in the case that the genes associated with the autosomal recessive nonsyndromic acoustic neuropathy are found to be PJVK and OTOF genes.
In this study, candidate gene screening method for diagnosis, selection of 2003-2011 years of collection and preservation of the 150 patients with auditory neuropathy, carry out China listen to mutation of the PJVK gene in patients with nerve group, and completed in the previous 76 cases of auditory neuropathy screening based on OTOF gene in 17 patients with auditory neuropathy for further development screening of OTOF gene, and then combined with the clinical phenotype and genotype of temperature sensitive auditory neuropathy patients. Clinical genetics characteristics from the perspective of genetics to etiology of neuropathy and lesion location, to explore the different types of clinical characteristics and genetic characteristics of patients with neuropathy.
This study is divided into three parts:
Analysis of the clinical genetic characteristics of PJVK gene in patients with acoustic neuropathic disorders in China
From 2003 2011 diagnosed with auditory neuropathy (150 cases were from Beijing, Tianjin, Hebei, Henan, Shandong, Shanxi and other 20 provinces and cities, the ratio of male to female was 1:1.05), to design primers for PJVK gene exon 7, by polymerase chain reaction (PCR) amplification, using direct sequencing of PCR products method for detection of PJVK gene mutation; and for mutations that were verified in 100 normal subjects. Chinese to clinical features of genetic characteristics analysis of the gene and the related neuropathy patients with auditory neuropathy.
The study found: in the 150 patients with auditory neuropathy were detected in the PJVK gene mutation in exon region 5, 4 reports of international no mutation: 1 nonsense mutation c.886CT, 3 missense mutations c.437GA, c.740GT and c.887GA; also found that 1 reported pathogenic mutations in c.548GA. 150 cases of auditory neuropathy patients PJVK gene mutation rate was 3.3%.
Carrying the clinical manifestations of patients with c.886CT mutations for temperature sensitive auditory neuropathy, the mutation of 296th amino acid sites are formed at the stop codon, resulting in early termination of encoding protein, in the 150 cases, only 1 cases have the mutation (0.67%), 99 cases of normal people were not found the mutation, suggesting that this is caused by deaf mutant.C.437GA, c.740GT, c.548GA and c.887GA were caused by the change of amino acid, amino acid mutation sites are highly conserved in different species, and in normal controls was not detected in these mutations, considered closely related with auditory neuropathy pathogenesis. Carrying the c.437GA mutation in patients 23 years of age onset, audiogram raspberry shaped, low to moderate, in severe hearing loss, if normal hearing, high-frequency mild hearing loss, speech recognition rate is 68% the left ear, right ear 64%; carrying c.548GA mutation patients 21 years of age of onset, hearing curve into low rise, low to moderate, in Severe hearing loss, high frequency hearing, speech recognition rate is 32% the left ear, right ear 18%; carrying c.740GT mutation patients 16 years of age of onset, fluctuating hearing loss, hearing loss in moderate to mild, speech recognition rate is 88% the left ear, right ear 86%; the patients carrying the c.887GA mutation is childhood disease, audiogram if the "U" type, high frequency, mild hearing loss, if moderate to severe hearing loss, speech recognition rate is 80% the left ear, right ear 76%.
Analysis of the clinical genetic characteristics of OTOF gene in the second part of Chinese auditory neuropathic disorder
The study on our early completion of 76 cases of OTOF patients with neuropathy to genetic screening further for 17 patients with auditory neuropathy in OTOF gene screening. Clinical audiology data were collected, 47 exons and 44 pairs of primers were designed according to OTOF gene, by polymerase chain reaction (PCR) amplification, application of PCR products direct sequencing to detect mutations in the OTOF gene for mutations; that was validated in 100 normal hearing people. In order to understand the genetic characteristics and clinical features of these auditory neuropathy patients with OTOF gene.
The findings of this study were detected in OTOF gene exon 29 mutation region, which may be the pathogenic mutation of 11 kinds of polymorphism change 18. In May 11 pathogenic mutations, 8 are found in the research of new mutations, including 4 frameshift mutation (p.G558A fsX21, p.L795SfsX5 p.I1108H, fsX69, c.5833delG), 1 (p.Y1133X), nonsense mutations and 3 missense mutations (p.Q265L, p.G541S and p.R1928C); this study also found three mutations have been reported (p.N727S, p.Q994V fsX6, pathogenic C. ivs4023+1GA). The 11 possible pathogenic mutations currently only found in China population, while there are no relevant reports in other areas of the crowd.
In this study, 1 temperature sensitive auditory neuropathy were detected in p.G541S and p.L795SfsX5 compound heterozygous mutations in other auditory neuropathy patients and normal people were not found in the mutant, which is associated with temperature sensitive auditory neuropathy associated with new mutations. In 3 infants to detection 1 of them carrying OTOF gene heterozygous p.G558AfsX21 the sudden neuropathy in children with p.Q994VfsX6 heterozygous mutation and p.Q265L heterozygous mutation; another 1 patients carrying OTOF gene homozygous c.4023+1GA mutation; the remaining 1 patients carrying OTOF gene c.5833delG and p.Y1133X compound heterozygous mutations; three patients had infantile onset, bilateral symmetric extremely severe hearing loss. In addition, this research in 3 cases with auditory neuropathy were detected in 3 heterozygous mutations: carrying the p.11108H fsX69 mutation patients showed bilateral symmetric severe deafness, flat audiogram articles; Patients with p.N727S mutations were characterized by moderate to severe deafness and a flat type of hearing curve, and patients with p.R1928C mutation showed bipolar deafness.
Analysis of the clinical genetic characteristics of third patients with temperature sensitive acoustic neuropathy
In this study, the OTOF and PJVK detection of auditory neuropathy related genes were carried out in 3 patients with temperature sensitive acoustic neuropathy, and the clinical features were reviewed and analyzed.
The study found: in the 3 temperature sensitive auditory neuropathy patients, 1 patients with OTOF gene p.G541S and p.L795S fsX5 compound heterozygous mutations, 1 patients with OTOF gene p.Q994V fsX6 heterozygous mutation and PJVK gene p.R296X heterozygous mutation, 1 patients did not detect the PJVK gene or OTOF gene mutations.
Change the state of hearing in the patients carrying OTOF gene p.Q994V fsX6 heterozygous mutation and PJVK gene p.R296X heterozygous mutation in a day with different time temperature and the change of hearing good conversation voice response, can carry out mandatory action, can communicate and answer the questions correctly; listening to loud sounds bad no response, not mandatory action, indifferent to their surroundings. Carrying the OTOF gene p.G541S and p.L795S fsX5 compound heterozygous mutation with fever in children with hearing loss was found in the normal body temperature when listening communication difficulties, improved communication.

【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R764.43

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