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非综合征性前庭水管扩大患者拷贝数变异、KCNJ10基因的筛查及基因表达的研究

发布时间:2018-09-08 16:44
【摘要】: 非综合征性前庭水管扩大(non-syndromic Enlarged Vestibular Aqueduct,EVA)是一种最常见的内耳畸形,其在感音神经性聋的患者中约占10%左右。临床表现为学龄前发病的感音神经性听力损失,部分患者存在有低频骨气导差,表现为混合性听力损失;其听力损失从轻度到极重度不等,听力常呈现波动性或进行性下降,最终达重度或极重度,从而影响语言的学习或正常的交流。前庭水管扩大的患者在听力下降前常有感冒、发热、轻微头部外伤或其它使颅内压增高的诱发因素,因此对于该病的早期发现、早期诊断、早期干预成为预防或推迟其发病的有效措施。前庭水管扩大是一种常染色体隐性遗传性疾病,其与染色体7q31上的SLC26A4基因突变有密切关系。在前庭水管扩大的患者中,约有65-92%的患者可以检测到该基因的突变。SLC26A4基因属于阴离子转运家族,有12个跨膜区,N末端和C末端位于细胞内,表达于甲状腺、肾脏和内耳。介导硫酸根、碳酸氢根、甲酸根、草酸根、氢氧根、氯、碘及果糖等多种单价或二价的离子的转运,在机体离子成分平衡的维持中发挥重要作用。当SLC26A4基因发生突变后,突变体的蛋白表达及功能均发生变化,从而导致疾病的发生。随着对SLC26A4基因研究的逐渐深入,发现该基因突变具有很大的异质性:包括基因突变的形式、基因突变发生的位点及不同种族该基因突变图谱等均存在有很大的异质性。近年来的研究表明虽然非综合征性前庭水管扩大和SLC26A4基因突变有密切的关系,但国内外的研究均发现在相当一部分前庭水管扩大的病人中,未找到SLC26A4基因突变或仅找到一个单等位基因突变。为了明确这部分病人的致病机理;探讨其它遗传因素在这部分病人的耳聋发病中的作用,本课题研究了拷贝数变异和参与内淋巴钾离子调节的基因-KCNJ10基因突变在非综合征性前庭水管扩大的发病中的作用。为完善耳聋基因诊断的程序和方法提供依据。同时针对中国人群所特有的SLC26A4突变图谱,选取了几种常见的热点突变,进行了SLC26A4基因突变后的表达研究,以期为进一步探索SLC26A4基因突变的致病分子机制奠定基础。 第一部分应用MLPA方法对非双等位基因突变的SLC26A4基因拷贝数变异的筛查 拷贝数变异(CNVs)是指在人类基因组中广泛存在的,从1000bp到数百万bp范围内的缺失、插入、重复和复杂多位点的变异。是近年来发现的在人类基因组中除SNPs之外,另一类丰富的多态性来源,CNVs除广泛存在于正常个体的基因组中,也与染色体重组和一些遗传性疾病的发生有密切关系。常规的一些检测技术无法检测到CNVs的存在。 为了解拷贝数变异是否在非综合征性前庭水管扩大的发病机制中起作用,本部分研究应用多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLP A)技术对解放军总医院聋病分子诊断中心所收集到的非综合征性前庭水管扩大的患者中没有检测到SLC26A4基因任何突变及仅检测到单个等位基因突变的患者进行SLC26A4基因拷贝数变异的筛查,寻找可能存在的致病性的拷贝数变异。在39例未找到SLC26A4基因突变及68例仅找到SLC26A4单等位基因突变的非综合征性前庭水管扩大的病人中;均未发现有明显的拷贝数变异的存在。基于试验结果我们推断有以下几种可能:①SLC26A4基因的外显子中确实不存在有拷贝数变异;因此拷贝数变异不构成非综合征性大前庭水管扩大的致病机制。②SLC26A4基因中存在有拷贝数的变异,但这些拷贝数变异没有存在于开放阅读框架区,而是存在于内含子区或上、下游的调控区等部位,用现有的MLPA检测试剂盒无法检测到。③在SLC26A4基因周围或其他对SLC26A4起调控作用的基因中,存在有拷贝数变异,这些拷贝数变异通过改变这些基因的表达或功能,间接地影响SLC26A4基因,从而导致非综合性前庭水管扩大的出现。 本研究首次将CNVs应用于非综合征性前庭水管扩大病人的基因筛查,为非综合征性前庭水管扩大病人的基因诊断提供了一个新的视角,从而完善该病的基因诊断。 第二部分非综合征性前庭水管扩大患者KCNJ10基因突变的筛查 KCNJ10基因位于人类染色体1q23,编码一种内向整流性钾离子通道,主要表达在大脑,内耳和肾脏。在内耳中,KCNJ10基因编码的钾离子通道主要表达在血管纹,对于耳蜗电位(EP)的形成起重要作用,血管纹产生耳蜗电位(EP)并向耳蜗内淋巴分泌钾离子。耳蜗电位是耳蜗转导电流的主要驱动力量;钾离子电流是最主要的耳蜗转导电流。耳蜗转导电流产生了听力。KCNJ10突变可以导致耳蜗电位的消失,从而导致听力的损失。近年来的研究表明在Pendred综合征和非综合征性的前庭水管扩大中有KCNJ10基因突变的存在,由此推测KCNJ10基因突变在非综合征性的前庭水管扩大的发病机制中起作用。 为了解KCNJ10基因突变是否在非综合征性前庭水管扩大的发病机制中起作用,本部分研究对解放军总医院聋病分子诊断中心所收集到的非综合征性前庭水管扩大的患者进行了KCNJ10基因突变的筛查。在107例携带SLC26A4单杂合突变及未检测到突变的病例中,共检测到3例KCNJ10基因突变,其中有812 GA杂合突变2例,1042 CT杂合突变1例。在311例已筛查到SLC26A4基因双等位基因突变(纯合突变和复合杂合突变)患者中共筛查到12例KCNJ10突变;其中812GA杂合突变11例;1042CT杂合突变1例。在229例正常人中共筛查到14例KCNJ10突变;其中812GA杂合突变11例,1042CT杂合突变2例,811CT杂合突变1例。对那些带有SLC26A4双等位基因突变,同时又带有KCNJ10基因突变患者的父母进行上述两个基因的筛查,结果表明其父母中必有一方为同时带有SLC26A4和KCNJ10单杂合的携带者,但是临床症状却表现正常,既没有听力的下降,也没有前庭水管的扩大。这同国外有关研究的观点截然不同。根据以上实验结果我们初步得出以下几种推论:①KCNJ10的单杂合突变在中国人群中是一种普遍存在的现象;②中国人群中KCNJ10最常见的突变类型是812GA;③KCNJ10虽然在耳蜗电位及听觉形成过程中起重要作用,但KCNJ10的单杂合突变和Pendred综合征及非综合征性前庭水管扩大之间并没有必然的联系;④KCNJ10和SLC26A4的复合杂合突变不一定导致Pendred综合征或非综合征性前庭水管扩大;对于同时有SLC26A4和KCNJ10基因单杂合突变的非综合征性前庭水管扩大患者,其致病原因有可能并不是由KCNI10突变引起的,而有可能是由于其他原因,如另一种目前我们尚未知的基因突变或环境与基因的相互作用引起的。 本研究初步分析了KCNJ10与非综合征性前庭水管扩大之间的关系,表明了KCNJ10基因突变和非综合征性前庭水管扩大的发生没有必然的联系,为完善非综合征性前庭水管扩大的基因诊断提供了依据。 第三部分中国人群中非综合征性前庭水管扩大患者常见的SLC26A4基因突变的表达研究 SLC26A4基因突变可引起Pendred综合征或非综合征性前庭水管扩大。SLC26A4基因突变具有很大的异质性。基因突变的形式,不同种族该基因突变图谱等均存在有很大的异质性。这些异质性提示SLC26A4突变后其致病的分子机制有可能也存在一定的异质性。中国人群中SLC26A4突变图谱和世界上其他人群的突变图谱有着明显的不同。在中国人群中常见的几种热点突变类型是IVS7-2AG; 2168AG (H723R);1174AT (N392Y); 1229CT (T410M);2027TA,而这些突变引起的分子功能改变的具体机制目前为止尚未见报道。因此,本研究即以中国人群中最常见的突变类型为研究对象,构建上述几种常见的SLC26A4突变质粒,并将其转染至HEK293细胞中,以观察其突变发生后在细胞内的表达情况,结果显示在本实验中所选取的四种SLC26A4基因突变发生后,其编码的Pendrin蛋白均滞留在细胞质内,而野生型Pendrin蛋白则表达于细胞膜上,由此推测SLC26A4突变发生后,其编码的Pendrin蛋白无法定位于胞膜上,而滞留于细胞质内,无法完成正常的阴离子交换功能,从而导致疾病的发生。本研究明确了中国人群中常见SLC26A4突变类型的蛋白表达,为下一步研究突变体的离子转运功能的变化奠定了基础,为采取干预措施,挽救病人的听力提供可能。
[Abstract]:Non-syndromic Enlarged Vestibular Aqueduct (EVA) is one of the most common inner ear malformations, accounting for about 10% of patients with sensorineural deafness. Patients with enlarged vestibular aqueduct often have a cold, fever, minor head trauma or other predisposing factors that increase intracranial pressure before hearing loss. Vestibular aqueduct enlargement is an autosomal recessive inherited disease closely related to SLC26A4 gene mutation on chromosome 7q31. About 65-92% of patients with vestibular aqueduct enlargement can detect the gene. SLC26A4 gene belongs to the anion transporter family. It has 12 transmembrane domains. The N-terminal and C-terminal domains are located in cells and express in thyroid, kidney and inner ear. As the SLC26A4 gene mutation occurs, the protein expression and function of the mutant change, leading to the occurrence of disease. With the deepening of the study of the SLC26A4 gene, it is found that the mutation has great heterogeneity: including the form of gene mutation, the site of gene mutation and the mutation of the gene in different races. Recent studies have shown that although there is a close relationship between non-syndromic vestibular aqueduct enlargement and SLC26A4 gene mutation, domestic and foreign studies have found that in a considerable number of patients with vestibular aqueduct enlargement, no SLC26A4 gene mutation or only a single allele mutation was found. To clarify the pathogenesis of these patients and to explore the role of other genetic factors in the pathogenesis of deafness in these patients, we studied the role of copy number variation and KCNJ10 gene mutation involved in endolymphatic potassium ion regulation in the pathogenesis of non-syndromic vestibular aqueduct enlargement. At the same time, the expression of SLC26A4 gene mutation was studied by selecting several common hot spot mutations according to the specific mutation map of SLC26A4 gene in Chinese population, in order to lay a foundation for further exploring the molecular mechanism of SLC26A4 gene mutation.
The first part is to screen copy number variation of SLC26A4 gene with non allelic mutation by MLPA.
Copy number variations (CNVs) refer to deletions, insertions, repetitions, and complex multilocus variations that are widespread in the human genome, ranging from 1000bp to millions of bp. CNVs are a rich source of polymorphisms found in recent years in the human genome, in addition to SNPs. CNVs are also widely present in the genome of normal individuals, but also stained. There is a close relationship between body weight and the occurrence of some genetic diseases. Conventional detection techniques can not detect the presence of CNVs.
In order to understand whether copy number variation plays a role in the pathogenesis of non-syndromic vestibular aqueduct enlargement, this part studied the application of multiplex ligation-dependent probe amplification (MLP A) technology to non-syndromic vestibular aqueduct enlargement collected by the Center for Molecular Diagnosis of Deafness in PLA General Hospital. No mutation in the SLC26A4 gene was detected in large patients and only one allele mutation was detected in patients with SLC26A4 gene copy number mutation screening for possible pathogenic copy number mutations. There were no significant copy number variations in patients with enlarged vestibular aqueduct. Based on the results, we concluded that there were several possibilities: (1) There was no copy number variation in the exon of SLC26A4 gene; therefore, copy number variations did not constitute the pathogenesis of non-syndromic enlarged vestibular aqueduct. (2) SLC26A4 gene Copy number variations exist, but these variations do not exist in the open reading frame region, but in the intron region, upstream and downstream regulatory regions, which can not be detected by the existing MLPA detection kits. These copy number variations indirectly affect the SLC26A4 gene by altering the expression or function of these genes, resulting in non-comprehensive vestibular aqueduct enlargement.
In this study, CNVs was used for the first time in gene screening of patients with non-syndromic vestibular aqueduct enlargement, providing a new perspective for gene diagnosis of non-syndromic vestibular aqueduct enlargement, thus improving the genetic diagnosis of the disease.
The second part is the screening of KCNJ10 gene mutation in patients with non syndromic vestibular aqueduct enlargement.
KCNJ10 gene is located on human chromosome 1q23 and encodes an inward rectifier potassium channel, which is mainly expressed in the brain, inner ear and kidney. In the inner ear, KCNJ10 gene encodes potassium channels which are mainly expressed in stria vascularis and play an important role in the formation of cochlear potential (EP). The stria vascularis produces cochlear potential (EP) and secretes to endolymph nodes of the cochlea. Cochlear potential is the main driving force of cochlear conduction current; potassium current is the main conduction current. Cochlear conduction current produces hearing. KCNJ10 mutation can lead to loss of cochlear potential and lead to hearing loss. The mutation of KCNJ10 gene may play a role in the pathogenesis of non-syndromic vestibular aqueduct enlargement.
To understand whether KCNJ10 gene mutation plays a role in the pathogenesis of non-syndromic vestibular aqueduct enlargement, this study screened non-syndromic vestibular aqueduct enlargement patients collected from the Molecular Diagnostic Center for Deafness in PLA General Hospital for mutations in the KCNJ10 gene. Three mutations of KCNJ10 gene were detected, including 812 GA heterozygous mutation in 2 cases and 1 1042 CT heterozygous mutation in 1 case.Twelve KCNJ10 mutations were detected in 311 patients with SLC26A4 gene double allele mutation (homozygous mutation and compound heterozygous mutation); 11 were 812 GA heterozygous mutation; 1 was 1042 CT heterozygous mutation. A total of 14 KCNJ10 mutations were screened in 229 normal subjects, including 11 812GA heterozygous mutations, 2 1042CT heterozygous mutations, and 1 811CT heterozygous mutation. However, the clinical symptoms were normal. There was no hearing loss or vestibular aqueduct enlargement. This is quite different from the viewpoint of foreign studies. Based on the above results, we can draw the following conclusions: 1. The single heterozygous mutation of KCNJ10 is one in Chinese population. (2) The most common mutation type of KCNJ10 in Chinese population is 812GA; (3) Although KCNJ10 plays an important role in cochlear potential and auditory development, there is no necessary association between single heterozygous mutation of KCNJ10 and endred syndrome and non-syndromic vestibular aqueduct enlargement; (4) The combination of KCNJ10 and SLC26A4. Heterozygous mutations do not necessarily lead to Pendred syndrome or non-syndromic vestibular aqueduct enlargement; for non-syndromic vestibular aqueduct enlargement patients with simultaneous single heterozygous mutations in SLC26A4 and KCNJ10 genes, the cause may not be caused by mutations in KCNI10, but by other causes, such as another one that we have not yet identified. The mutation of the known gene or the interaction between the environment and the gene.
This study preliminarily analyzed the relationship between KCNJ10 and non-syndromic vestibular aqueduct enlargement, which indicated that there was no necessary relationship between KCNJ10 gene mutation and non-syndromic vestibular aqueduct enlargement, and provided a basis for improving gene diagnosis of non-syndromic vestibular aqueduct enlargement.
Part III Expression of SLC26A4 gene mutations in non-syndromic vestibular aqueduct enlargement in Chinese population
SLC26A4 gene mutation can cause Pendred syndrome or non-syndromic vestibular aqueduct enlargement. SLC26A4 gene mutation has great heterogeneity. There are great heterogeneity in the form of gene mutation and the mutation map of this gene in different races. These heterogeneity suggest that the molecular mechanism of pathogenesis of SLC26A4 mutation may also exist. The mutation profiles of SLC26A4 in the Chinese population are significantly different from those in other populations in the world. Several hotspot mutation types commonly seen in the Chinese population are IVS7-2AG; 2168AG (H723R); 1174AT (N392Y); 1229CT (T410M); 2027TA, and the specific mechanism of these mutations leading to molecular functional changes is still unknown. Therefore, we constructed the SLC26A4 mutant plasmids and transfected them into HEK293 cells to observe the intracellular expression of the four SLC26A4 mutants selected in this study. It is speculated that the pendrin protein encoded by SLC26A4 can not be localized on the cell membrane after mutation, but can not be retained in the cytoplasm to perform normal anion exchange function, leading to the occurrence of disease. The expression of SLC26A4 mutant protein in Chinese population lays a foundation for the further study of the changes of ion transport function of the mutant, and provides the possibility for intervening measures to save the patient's hearing.
【学位授予单位】:中国人民解放军军医进修学院
【学位级别】:博士
【学位授予年份】:2010
【分类号】:R764.43

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