中国听神经病谱系障碍患者PJVK与OTOF基因临床遗传特征分析

发布时间：2018-04-05 13:51

本文选题：听神经病　切入点：听神经病谱系障碍　出处：《中国人民解放军医学院》2012年硕士论文

【摘要】：听神经病(auditory neuropathy, AN)又称为听神经病谱系障碍(auditory neuropathy spectrum disorder, ANSD),描述了一种内毛细胞和听神经突触和/或听神经本身功能不良所致的听力障碍,不同于典型的感音神经性听力损失。典型的临床表现是言语理解力受损,而言语觉察阈和纯音听阈可以正常,也可以严重受损；听力学检查特点是耳声发射(otoacoustic emissions,OAE)和耳蜗微音电位(cochlear microphonics,CM)正常引出,听性脑干反应(ABR)无明显分化的波形或严重异常。目前听神经病的病因、病变部位和发病机制尚不十分明确,导致临床(?)预的预测和评估上存在难度。2008年,Starr等提议按病损部位对AN分型：如只是听神经受累,内毛细胞和突触正常,称为“听神经病变”(auditory nerve disorder),如果内毛细胞突触病变而听神经正常,称为“听突触病变”(auditory synaptic disorder)。按病变部位分类,有助于精确定义本病,为临床干预提供指导。然而,目前尚无临床测试方法可以精确地确定病变部位。近年来,一系列听神经病相关基因的发现,从分子水平揭示了听神经病的病理机制,使得根据基因导致的不同病变部位对听神经病进行分型成为可能,在非综合征型聋病例中尤为重要。目前研究发现与常染色体隐性遗传非综合征性听神经病相关的基因有PJVK基因和OTOF基因。本研究应用候选基因突变筛查方法,选取2003-2011年收集和保存的明确诊断的150例听神经病患者,开展中国听神经病人群PJVK基因的突变特征研究,并在本课题组前期完成76例听神经病患者OTOF基因筛查的基础上进一步针对17例听神经病患者开展OTOF基因筛查，，进而结合临床表型与基因型探讨温度敏感性听神经病患者的临床遗传学特征。从遗传学角度阐述听神经病的病因学机制及其病变部位,探讨不同类型听神经病患者的临床特点和遗传学特征。本研究共分三个部分：第一部分中国听神经病谱系障碍患者PJVK基因临床遗传学特征分析选取2003年一2011年确诊的听神经病患者150例(分别来自北京、天津、河北、河南、山东、山西等20个省市,男女比例为1：1.05),针对PJVK基因的7个外显子设计引物,采取聚合酶链式反应(PCR)扩增目的片段,应用PCR产物直接测序法进行PJVK基因突变检测；并针对发现的突变位点,在100名听力正常人中进行验证。分析中国听神经病患者该基因的遗传特征及相关听神经病的临床特点。研究发现：在150例听神经病患者中共检测到PJVK基因位于外显子区域的突变5种,4种为国际上未见报道的突变：1种为无义突变c.886CT,3种为错义突变c.437GA,c.740GT和c.887GA；还发现1种已报道的致病突变c.548GA。在150例听神经病患者中PJVK基因突变携带率为3.3%。携带c.886CT突变的患者临床表现为温度敏感性听神经病,该突变使第296位氨基酸位点处形成终止密码子,导致编码的蛋白质提前终止,在150例病人中仅1例携带该突变(0.67%),99例正常人中均未发现该突变,提示此为致聋突变。c.437GA,c.740GT, c.548GA和c.887GA均引起氨基酸的改变,突变氨基酸位点在不同物种间高度保守,且在正常对照者中未检测到这些突变,考虑与听神经病的发病密切相关。携带c.437GA突变患者23岁发病,听力曲线为覆盆形,低频中度、中重度听力损失,中频听力正常,高频轻度听力损失,言语识别率左耳68%,右耳64%；携带c.548GA突变患者21岁发病,听力曲线为低频上升型,低频中度、中重度听力损失,高频听力正常,言语识别率左耳32%,右耳18%；携带c.740GT突变患者16岁发病,波动性听力损失,听力损失在中度到轻度之间,言语识别率左耳88%,右耳86%；携带c.887GA突变的患者表现为婴幼儿时期发病,听力曲线为中频“U”型,高、低频为轻度听力损失,中频为中重度听力损失,言语识别率左耳80%,右耳76%。第二部分中国听神经病谱系障碍患者OTOF基因临床遗传学特征分析本研究在本课题组前期完成76例听神经病患者OTOF基因筛查的基础上进一步针对17例听神经病患者开展OTOF基因筛查。收集患者临床听力学资料,针对OTOF基因的47个外显子设计44对引物,采取聚合酶链式反应(PCR)扩增,应用PCR产物直接测序法进行OTOF基因突变检测;针对发现的突变位点,在100名听力正常人中进行验证。以了解这些听神经病患者OTOF基因的遗传学特征及临床特点。研究发现：本研究共检测到OTOF基因位于外显子区域的变异29种,其中可能的致病突变有11种,多态性改变有18种。在可能致病的11种突变中,有8种是本研究发现的新的突变,包括4种移码突变(p.G558A fsX21,p.L795SfsX5, p.I1108H fsX69, c.5833delG),1种无义突变(p.Y1133X),和3种错义突变(p.Q265L,p.G541S和p.R1928C)；此外本研究还发现了三种已报道的致病突变(p.N727S, p.Q994V fsX6, c. ivs4023+1GA)。这11种可能的致病突变,目前仅在中国人群中发现,未见其他地域人群中相关报道。本研究在1名温度敏感性听神经病患者中检测到p.G541S和p.L795SfsX5复合杂合突变,在其他听神经病患者及正常人中均未发现该突变,这是与温度敏感性听神经病相关的新的突变。在3名婴幼儿听神经病患儿中检测到其中1人携带OTOF基因p.G558AfsX21杂合突变、p.Q994VfsX6杂合突变和p.Q265L杂合突变；另1名患儿携带OTOF基因c.4023+1GA纯合突变；其余1名患儿携带OTOF基因c.5833delG和p.Y1133X复合杂合突变；三名患儿都表现为婴幼儿时期发病,双耳对称的极重度聋。此外,本研究还在3例听神经病患者中还检测到3种单杂合突变：携带p.11108H fsX69突变患者表现为双耳对称的重度耳聋,平坦型听力曲线；携带p.N727S突变患者表现为双耳中重度聋,平坦型听力曲线；携带p.R1928C突变的患者表现为双耳极重度聋。第三部分温度敏感性听神经病谱系障碍患者临床遗传学特征分析本研究针对3名温度敏感性听神经病患者开展听神经病相关基因OTOF和PJVK检测,并回顾分析其临床特征。研究发现：在3名温度敏感性听神经病患者中,1名患者携带OTOF基因p.G541S和p.L795S fsX5复合杂合突变,1名患者携带OTOF基因p.Q994V fsX6杂合突变和PJVK基因p.R296X杂合突变,1名患者未检测到PJVK基因或OTOF基因致病突变。携带OTOF基因p.Q994V fsX6杂合突变和PJVK基因p.R296X杂合突变的患者听力状况在一天之内随不同时间体温的变化而有所变化,听力好时对日常谈话的声音有反应,能够执行指令性动作,可以交流并正确回答问题;听力不好时对大的声音没有反应,不能完成指令性动作,对周围事物漠然。携带OTOF基因p.G541S和p.L795S fsX5复合杂合突变患儿主诉为发热时听力明显下降交流困难,体温正常时听力好转可交流。
[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.

【学位授予单位】：中国人民解放军医学院
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R764.43

【参考文献】