连接蛋白突变与遗传性耳聋

1连接蛋白的结构与功能 连接蛋白是形成缝隙连接的蛋白单位,在脊椎动物细胞之间的通讯中起着关键作用,最近几年,越来越多的研究发现,连接蛋白与许多人类疾病关系密切,连接蛋白基因突变影响许多组织系统,并且同一基因突变与不同的疾病有关系,伴有β-连接蛋白突变的患者可以引起听觉系统,外周神经、皮肤以及眼部等病变[1-9].     

GJB2基因在遗传性聋中的检测

目的对遗传性聋家系进行GJB2基因突变检测,为该病的基因诊断提供依据。方法采用PCR直接测序法对20个非综合征型遗传性聋家系的先证者(均为耳聋患者)进行GJB2基因的突变检测。结果发现了三种碱基改变:109G>A、79G>A和341G>A。109G>A是已报道的具有争议的致病突变,本实验在两个隐性遗传性聋家系的先证者中检测到109G>A纯合突变,且与耳聋共分离。79G>A和341G>A是已报道的多态。结论本研究发现了具有争议的致病突变109G>A的纯合突变,极可能导致隐性遗传性聋。     

连接蛋白与遗传性耳聋

耳聋的遗传基础比较复杂，具有很强的遗传异质性。在人类约有15种连接蛋白，现证实有4种连接蛋白与遗传性耳聋有关，本从它们在组织和器官的表达、突变类型、引起耳聋的机制及临床应用等方面的研究进展进行综述。     

线粒体DNA突变与遗传性耳聋

由于线粒体ＤＮＡ研究方法的限制，线粒体ＤＮＡ突变与人类疾病关系的研究远远落后于核遗传病的认识。近年来由于ＰＣＲ及分子杂交等新技术的应用使线粒体疾病的研究取得了较大的进展，本简要综述了线粒体ＤＮＡ突变与多种遗传性耳聋症的最新分子遗传学研究进展。     

基因拷贝数变异在遗传性耳聋研究中的进展

耳聋是最常见的严重影响言语交流的残疾之一,每年至少有一半的新生聋儿是由于遗传缺陷引起的.引起耳聋的致病基因和突变种类众多,其中,基因拷贝数变异(Copy Number Variations,CNVs)被确认为是广泛存在于人类基因组DNA的重要变异形式,并且可以通过干扰基因表达来调控表型,是影响人类某些疾病的重要因素,其...     

EYA4突变致DFNA10型遗传性耳聋的研究进展

EYA4(Eye absent 4)(OMIM:603550)基因是Eya家族蛋白中的一员,编码转录激活因子相关蛋白,在胚胎发育过程中对组织特异性分化具有重要调控作用。EYA4蛋白含有高度保守的eyaHR和非保守的eyaVR两个功能域,eyaHR参与SIX/DACH蛋白之间的相互作用;eyaVR参与转录激活及机体固有免疫功能。EYA4致病突变会导致DFNA10型遗传性耳聋,临床表现为双侧对称的、迟发性、进展性感音神经性听力损失,发病初期常累及中频听力,呈谷型或平坦型听力曲线。目前已在世界范围内报道与DFNA10型耳聋相关EYA4的18种致病突变,多数突变引起EYA4翻译提前终止形成截短蛋白,单倍体剂量不足导致的失能效应是EYA4致聋的原因,EYA4的下调会引起内耳细胞的Na+/K+-ATP酶功能异常进而导致听觉障碍。构建合适的动物模型、鉴定更多的致病突变位点及深入的功能实验有助于进一步探索EYA4精确的致聋机制。     

连接蛋白基因与遗传性耳聋

遗传性耳聋是临床上一类常见疾病,近年来人们发现连接蛋白基因突变与其发病密切相关。通过对这类基因的鉴别和研究,为遗传性耳聋的诊断和治疗提供了新的思路和方法。本文就这一课题对相关文献进行了复习和回顾。     

耳聋基因检测在遗传性耳聋诊断及遗传咨询中的应用

目的从基因检测确定耳聋发病原因,同时为受检者的婚育遗传咨询与干预措施提供依据。方法对来自7个家庭共21例自愿受检者进行检测前咨询告知其检测的目的方法及意义后,使用耳聋基因芯片和直接测序法对其进行耳聋基因检测。结果在21例受检者中检出1例GJB2 235del C纯合突变、4例复合杂合突变。2例GJB2 235del C与SLC26A4 IVS7-2A>G双重杂合突变。12例杂合突变(2例GJB2基因杂合突变,10例SLC26A4基因杂合突变),2例SLC26A4基因与GJB2基因未检出。并对不同发病原因,不同就诊需求的耳聋家庭提供准确遗传咨询、指导和干预。结论临床上检测遗传性耳聋的基因对于遗传咨询、生育聋儿风险率评估、产前诊断以及开展治疗等均可提供重要的指导作用。     

MYO7A基因突变与遗传性耳聋

耳聋是临床上最常见的遗传性疾病之一。在所有耳聋患者中,遗传性聋约占50%,在所有先天性聋儿中,60%以上由遗传因素引起。与遗传性耳聋相关的基因大约有200多个,     

遗传性耳聋基因突变引起的听觉功能改变

遗传性耳聋依据临床表现可分为非综合征性耳聋(Nonsyndromic hearing impairment．NSHI)和综合征性耳聋(Syndromic hearing impairment，SHI)。各种遗传性耳聋的临床表现如下：     

特发性突聋患者GJB2 235delC突变分析

目的：分析GJB2 235delC突变在特发性突聋患者中的发生频率,探寻其与特发性突聋发病的可能相关性。方法：收集234例特发性突聋患者,以同期听力正常的80例体检人员为对照组。分别采外周静脉血,提取基因组DNA,经聚合酶链反应扩增GJB2基因编码区后,用限制性内切酶酶切的方法筛查235delC位点突变,同时对特发性突聋患者的临床资料汇总分析。结果：234例特发性突聋患者中,5例检测出GJB2 235delC杂合突变,突变检出率为2.1%,未检测出235delC纯合突变;对照组中未检出GJB2 235delC突变。2组人群235delC突变检出率差异无统计学意义（P〉0.05）。结论：特发性突聋患者中GJB2 235delC突变检出率低,提示该突变位点可能与特发性突聋的发病无相关性。     

Functional study of GJB2 in hereditary hearing loss

OBJECTIVES/HYPOTHESIS: The gene of the gap junction protein connexin 26 (Cx26) was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss (DFNB1). Although 35delG has been known as the major mutation in Western countries, 235delC was reported to be a specific form of mutation in Asian populations. The objective of the study was to identify how 235delC and E114G changes found in the Korean population affected the function of using molecular biological techniques. METHODS: Genes containing 235delC and E114G were cloned into the pcDNA3 vector, and HeLa cells were transfected with the recombinant DNA samples by the liposome complex method. The expression and subcellular localization of Cx26 were determined, using antibodies against amino acid sequences in the intracellular loop (IL) and N-terminal (NT) portions of Cx26. To analyze functions of the as a gap junction channel, we examined Lucifer yellow dye transfer between cells with a scrape-loaded technique. Wild-type (WT) with normal hearing was used as a positive control, and mock transfected cells were used as a negative control. RESULTS: Immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctate patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with anti-IL antibody but stained slightly just around the nucleus only with anti-NT antibody. In a functional study of, transfer of Lucifer yellow into contiguous cells was detected in both WT and E114G, but no transfer activity was observed in 235delC. CONCLUSIONS: The 235delC mutation showed a loss of targeting activity to the cell membrane and severe deterioration of gap junction activity. For the E114G, we did not find any difference from WT transfected cells.     

中国人群中GJB2基因变异与年龄相关性耳聋遗传易感性的关联性分析

目的 分析不同听力水平的中老年人GJB2基因的突变类型和基因型频率,探讨GJB2基因的各种突变和单核苷酸多态(SNPs)是否与年龄相关性耳聋的遗传易感性相关联.方法 通过普查共收集到648例中老年人的听力学资料和血样,根据听力学检查结果将其分为四组.提取基因组DNA,经聚合酶链反应(Polymerase chain reaction,PCR)扩增GJB2基因编码区,利用直接测序方法获得所有样本的基因型,区分致病突变和多态性改变,利用统计学分析方法研究各种突变和多态在各组的分布情况.结果 根据听力学结果将所有样本分为四组:正常对照组(157人)、轻度听力下降组(199人)、中度听力下降组(226人)、重度听力下降组(66人).通过直接测序的方法.发现4种移码突变,包括235delC杂合突变16例、299-300delAT杂合突变3例、176-191del16杂合突变1例、512insAACG杂合突变2例;1种错义突变109G>A;6种多态,包括79G>A、341A>G、608 T>C、457G>A、368C>A、571T>C;3种已报道但与疾病关系不确定的突变11G>A、187G>T、558G>A;5种没有报道过的突变,包括14C>T、253T>C、377T>C、478 G>A、594G>A.4种移码致病突变,1种错义突变和3种常见多态在各组间的分布未发现显著性统计学差异(P>0.05).结论 虽然未发现GJB2基因与年龄相关性聋具有显著的关联性,但伴随着各组间听阈的逐渐升高,致病突变所占的比率也逐渐升高,提示GJB2基因可能是年龄相关性聋遗传易感性的微效致病基因之一;可能因关联性比较微弱,因此在此研究样本量的基础上未能检出.     

Absence of GJB6 mutations in Indian patients with non-syndromic hearing loss

Objective

Hearing loss is the most frequent sensory defect in human being. Genetic factors account for at least half of all cases of profound congenital deafness. The 13q11-q12 region contains the GJB2 and GJB6 genes, which code connexin 26 (CX26) and connexin 30 (CX30) proteins, respectively. Mutations in the gene GJB2, encoding the gap junction protein connexin 26, are considered to be responsible for up to 50% of familial cases of autosomal recessive non-syndromic hearing loss and for up to 15-30% of the sporadic cases. It has also been reported that mutations in the GJB6 gene contribute to autosomal recessive and autosomal dominant hearing defects in many populations. The 342-kb deletion [del(GJB6-D13S1830)] of the Cx30 gene is the second most common connexin mutation after the CX26 mutations in some NSHL populations. The aim of this study was to screen GJB6 gene mutations in Asian Indian patients with autosomal non-syndromic hearing loss.

Methods

We screened 203 non-syndromic hearing loss patients, who were negative for homozygous mutations in GJB2 gene, for GJB6-D13S1830 deletion and mutations in coding regions of GJB6 using polymerase chain reaction, denaturing high performance liquid chromatography and direct sequencing.

Results

No deleterious mutation in GJB6 gene was detected in our study cohort.

Conclusion

The present data demonstrated that mutations in the GJB6 gene are unlikely to be a major cause of non-syndromic deafness in Asian Indians.     

新疆哈萨克族非综合征型聋GJB2基因突变的研究分析

目的：研究新疆哈萨克族非综合征型聋患者GJB2基因突变的情况。方法：调查对象为来自新疆地区的193例哈萨克族患者,采用直接测序法对非综合征型聋患者97例和健康对照96例进行GJB2基因突变的检测。结果：在编码区耳聋组共发现8种碱基改变：其中35delG纯和12例,79G〉A纯合5例,79G〉A杂合8例,79G〉A与608T〉C复合杂合1例,79G〉A与341A〉G复合杂合5例,235delC杂合4例,341A〉G杂合2例,439T〉G杂合1例,457G〉A杂合1例,521G〉A纯合2例。对照组发现4种已明确的常见多态性碱基改变。结论：本研究提示新疆哈萨克族非综合征型聋患者GJB2基因突变具有种族和地域性特点,该地区哈萨克族耳聋人群中GJB2有较高携带率,在本研究中35 delG为其常见突变方式。     

The deafness-causing mutation c.508_511dup in the GJB2 gene and a literature review

Conclusions: The mutation c.508_511dup in GJB2 gene has been incorrectly named as other mutations. It is essential to standardize mutation nomenclature to describe complex mutations. Objectives: This paper aimed to verify a series of patients with the frame-shift mutation c.508_511dup in the GJB2 gene and review the literature on related mutations. Methods: All the included patients with non-syndromic hearing loss (NSHL) carried the 504insAACG or c.508_511dup mutation of the GJB2 gene in the present study. Their parents were encouraged to participate. After written informed consent and clinic data had been obtained, genomic DNA was extracted from venous blood of participants. The target fragments were amplified by polymerase chain reaction (PCR) and subjected to bidirectional sequencing to identify sequence variations. Results: A total of 14 patients with prelingual NSHL and 6 normal parents were recruited. Genotyping revealed that one mutation, c.508_511dup (not 504insAACG), was homozygous in 1 patient, heterozygous in 2 patients and 3 parents, and compound heterozygous in 11 patients. Twelve patients had hearing loss caused by c.508_511dup in a homozygous or compound heterozygous form, and further study showed that it was wrongly named as 504insAACG. Additionally, according to the standard nomenclature, the previously reported mutations with distinct names from the literature review may be replaced by c.508_511dup.     

PCR-GeneScan法检测遗传性聋相关基因GJB2 235 delC和mtDNA A1555G突变

目的:检测GJB2 235delC杂合突变和mtDNA A1555G突变。方法:对120例样本进行诊断试验,其中测序GJB2 235delC杂合突变样本16例,mtDNA A1555G突变17例。用PCR方法对目标片段进行扩增,PCR产物在3100DNA sequencer(ABI)上聚丙烯酰胺胶毛细管电泳,GeneScan、GeneMarker软件数据分析。结果:120例样本均得到检测结果,检出GJB2 235delC杂合突变样本17例,mtDNA A1555G突变17例,1例正常样本误诊为235delC杂合突变而出现假阳性。结论:PCR-GeneScan技术可以同时检测2种不同基因的突变,单管多重PCR和GeneScan荧光标记法结合是同时检测多种突变一种新的思路,而且可能是一种有效的方法。     

高通量基因捕获测序技术在12个耳聋家庭中的应用

目的 分析12个耳聋家庭的临床特征,进行候选致病基因的突变检测。 方法 对12个耳聋家庭的患者进行病史采集、全身检查、听力学评估及颞骨CT检查;抽提家庭成员外周血基因组DNA;整理分析家系资料绘制系谱图;使用定向捕获联合二代测序技术进行耳聋基因检测;对可疑基因进行Sanger测序验证。 结果 12个耳聋家庭的13名耳聋患者表现为双侧不同程度的感音神经性耳聋。家庭1-7耳聋患者明确GJB2双等位基因突变致聋,分别是:c.235delC/ c.235delC,c.235delC/c.176del16,c.235delC/c.299delAT, c.235delC/c.511_512insAACG/c.235delC/c.605ins46,c.235delC/c.109G>A,c.109G>A /c.109G>A;家庭8-9先证者均为SLC26A4复合杂合突变致聋,分别是:c.589G>A/c.1975G>C, c.919-2A>G/ c.-2071_307+3801del7666; 其中,家庭10-12先证者,经过数据分析后分别得到8、5和3个可疑突变位点,在相应的家系中不与耳聋表型共分离,故未发现其致病基因。 结论 本研究明确了9个非综合征耳聋家庭的致病突变,同时证实高通量基因捕获测序技术是一种高效的耳聋基因检测工具。