中国人常见GJB2基因突变表达载体的构建及鉴定

目的:构建中国人常见GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp与增强型绿色荧光蛋白(EGFP)的融合蛋白表达载体,寻找体外研究GJB2基因缺失突变致聋机制的有效途径.方法:用体外定点突变法构建235 de1C、299-300 delAT和176 de1 16 bp突变全序列与EGFP表达载体,以此为模板PCR扩增突变有效表达序列,将PCR产物连接到pMD19-T载体中,EcoRI/BamHI双酶切克隆载体,测序鉴定序列正确性后,将酶切产物插入pEGFP-N1载体中,脂质体转染HEK293细胞,荧光显微镜观察表达的融合蛋白.结果:GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp在HEK293细胞中高效表达,表达主要位于细胞质中.结论:成功构建了中国人常见GJB2基因突变235 de1C、299-300 delAT和176 de1 16 bp与EGFP的融合蛋白表达载体,为进一步研究其致聋机制奠定了基础.     

中国人常见GJB2基因突变片段TA克隆载体构建

目的应用TA克隆技术构建含中国人常见GJB2基因突变片段的载体,为构建突变绿色荧光蛋白融合载体提供基础。方法首先利用定点诱变法在体外构建235delC．299-300delAT和176del16bp绿色荧光蛋白非融合载体,以此为模板利用PCR扩增突变基因片段,然后将PCR扩增产物克隆到TA载体上,用限制性内切酶法和测序法鉴定重组质粒序列正确性。结果用限制性内切酶法和测序方法证实扩增片段为目的片段。结论成功地构建了235delC、299-300delAT和176del16bp突变基因片段TA克隆载体,为构建突变绿色荧光蛋白融合载体、进一步研究突变致聋机制奠定了基础。     

人GJB6基因表达载体的构建及鉴定

目的 构建人野生型Cx30与红色荧光蛋白DsRed的融合蛋白表达载体,为揭示Cx30突变患者发病机制提供实验依据.方法 用PCR法扩增GJB6基因,将PCR产物与T载体连接,用双切酶酶切pEASY-GJB6与载体DsRed-N1,连接回收后的片断,构建野生型Cx30编码序列与PDsRed2表达载体,测序鉴定序列正确性.将GJB6-DsRed用脂质体转染HEK293细胞,荧光显微镜观察表达的融合蛋白.结果 GJB6-DsRed在HEK293细胞中高效表达,表达主要位于细胞膜中.结论 成功构建了人野生型Cx30与红色荧光蛋白DsRed的融合蛋白表达载体,为进一步研究非综合征性聋的致聋机制奠定了基础.     

靶向人Trb3基因shRNA真核表达载体的构建及鉴定

目的 构建并鉴定靶向果蝇同源蛋白3(tribbles-related protein 3,Trb3)基因的短发夹状RNA(short hairpin RNA,shRNA)真核表达载体,并检测其对靶基因的沉默效应.方法 根据Genbank数据库中Trb3序列,设计、合成3对互补并特异性编码其shRNA序列的寡核苷酸,克隆...     

人Bcl-2基因重组腺病毒的构建及其在螺旋神经节细胞中的表达

目的构建表达人Bcl-2基因的重组腺病毒，并研究其在原代培养螺旋神经节细胞(spiral ganglion cells，SGC)的表达特性。方法采用细菌内同源重组法，从pUC-CAGGS,／Bcl-2质粒中获得人Bcl-2 cDNA，克隆到腺病毒穿梭质粒pAdTrack-CMV上，后者和腺病毒骨架质粒pAdEasy-1在细菌内同源重组，在HEK293细胞中包装成为重组Ad增强型绿色荧光蛋白／Bcl-2腺病毒，电镜观察病毒颗粒，用病毒感染原代培养的大鼠螺旋神经节细胞，在荧光显微镜下观察感染效率，用逆转录聚合酶链反应方法检测Bcl-2基因在螺旋神经节细胞的转录表达，用Western Blot检测其蛋白的表达。结果经酶切鉴定证实重组腺病毒质粒构建成功，电镜显示包装细胞中有大量病毒颗粒存在，呈规则六边形。荧光显微镜下观测到感染病毒的SGC细胞发出明亮的绿色荧光。分别在mRNA水平及蛋白水平证实有外源性Bcl-2基因的表达。结论细菌内同源重组法是一种简便、高效的制备方法。构建的重组Ad增强型绿色荧光蛋白／Bcl-2腺病毒能有效的感染SGC，并可在SGC中正确地转录和翻译，为进一步研究腺病毒介导的Bcl-2基因对螺旋神经节损伤的保护作用奠定基础。     

国人conneXin 2 6基因突变与先天性感音神经性耳聋的相关性研究

目的探讨国人耳聋人群中connexm26基因的突变频率和位点.方法收集15例有遗传性耳聋家族史的病例和252例散发性先天性耳聋病例血液样本,使用PCR-SSCP方法分析connexin26基因编码区突变.同时采用PSDM和BsBsiYI酶切的方法,直接检测异常connexin26基因35delG的突变.结果检出突变样本46例,其中散发耳聋患者中38例,突变率为15.1%;有家族史的聋儿15例中8例,突变率为53.3%.46例中5份有相似的异常电泳带,PCR产物直接测序,其形式为79位G→A的突变;另外在散发耳聋患者中还发现2例251delT和233delC,PDSM分析未发现有35delG的突变.结论国人先天性耳聋患者中存在着connexin26基因的高突变率,但突变热点与国外报道的不同,推测connexin26基因突变有明显的种族特异性.     

Identification of 605ins46, a novel GJB2 mutation in a Japanese family

Connexin 26 gene (GJB2) mutations are known to be responsible for a significant portion (30–80%) of autosomal recessive congenital severe to profound deafness. More than 60 recessive mutations in GJB2 have been reported and most consist of point mutations of a nucleotide. We report here a novel insertional GJB2 mutation consisting of a long repetitive nucleotide sequence. As compound heterozygotes of this mutation with 235delC express sensorineural hearing loss of variable severity, further analysis of the phenotype–genotype relationship is required.     

Association of GJB2 gene mutation with cochlear implant performance in genetic non-syndromic hearing loss

Objective

To analyze the association of GJB2 gene mutations with cochlear implant performance in children.

Methods

Sixty-five consecutive children who underwent cochlear implantation due to congenital profound senseurineural hearing between 2006 and 2008 were included in the study. In children, GJB2 gene mutation analysis was performed. Their auditory performance was assessed using MAIS, MUSS and LittlEARS tests.

Results

Twenty-two of sixty-five patients GJB2 mutations, and 35delG was the most frequent mutation. No significant difference was found between the auditory performance of mutation positive and negative children after one year follow up (p > 0.05).

Conclusion

GJB2 gene mutations do not impact on the outcome of cochlear implantation.     

GJB2 mutations in hearing impairment: identification of a broad clinical spectrum for improved genetic counseling

OBJECTIVES/HYPOTHESIS: Hearing impairment has a high prevalence affecting approximately 1 in 1000 newborn children. Alterations in the gap junction protein beta 2 (GJB2) and gap junction protein beta 6 (GJB6) are associated with nonsyndromic hearing impairment and should have a significant impact on genetic counseling. STUDY DESIGN: Various cases of nonsyndromic hearing impairment were screened for alterations in GJB2 and GJB6 in this clinical study. METHODS: The prevalence of mutations in GJB2 encoding for connexin 26 in a patient group with nonsyndromic hearing impairment comprising 45 families and 57 sporadic cases was initially determined by sequencing. The role of GJB2 was then assessed in individuals with hearing impairment (3 families and 20 sporadic cases) who are usually excluded from analysis because of the presence of additional symptoms or in cases in which a role for nongenetic factors cannot be eliminated. In hearing-impaired individuals with heterozygous GJB2 mutations the recently identified 342-kb deletion truncating GJB6 called del(GJB6-D13S1830) as a digenetic component in hearing impairment was excluded by polymerase chain reaction. RESULTS: Autosomal recessively inherited GJB2 mutations induced hearing impairment in 25.5% of individuals in the nonsyndromic hearing impairment group. GJB2 alterations were also seen in 17.4% of individuals in whom additional symptoms or a role for nongenetic involvement could not be excluded. In all, 15 different alterations in GJB2 were detected, including the previously unknown 154G>C, 557C>T, and 682C>T mutations, and these were correlated to clinical parameters. CONCLUSION: Improved genetic counseling can be performed by screening for GJB2 alterations in patients with nonsyndromic hearing impairment including patients within groups for which a role for exogenetic factors cannot be excluded. Specific genetic counseling for GJB2-linked hearing impairment in heterozygotes will depend on future research.     

人工耳蜗植入患者的缝隙连接蛋白相关基因（GJB2）突变分析

目的探讨人工耳蜗植入患者的缝隙连接蛋白相关基因（gap junction protein beta 2，GJB2）突变情况，分析耳聋的遗传学发病机制。方法对接受人工耳蜗植入的241例患者进行聋病基因GJB2基因突变筛查。结果241例人工耳蜗植入患者中检测出65例GJB2基因突变，其中1例为新发现突变GJB2 235delC／598G〉A。结论人工耳蜗植入患者中GJB2基因突变的发生率为27．0％，GJB2基因突变是人工耳蜗植入人群中耳聋的主要致病因素之一。     

Correlation between GJB2 mutations and audiological deficits: personal experience

Mutations in GJB2 gene are the most common cause of genetic deafness. More than 100 mutations have been described. The aim of this work is to describe the personal experience in genetic hearing loss, investigating the audiological and genetical characteristics of Cx26 deafness and correlating genotype and phenotype. We performed audiological and genetical evaluation in 154 patients affected by non-syndromic deafness of different degree. All patients showed a bilateral symmetrical sensorineural hearing loss. From the genetical analysis 127 probands resulted as negatives while 27 as positives (51.8% homozygous for 35 delG, 14.8% compound heterozygosis and 33.3% single mutation); 7.5% of patients had a mild deafness, 37% moderate, 33.3% severe and 22.2% profound. The c.35 delG mutation was detected in 66.6% of patients. Three mutations were found in compound heterozygosis with 35 delG, six different single mutations already described, and a new mutation S138G were also found. Correlation between genotype and phenotype confirmed the high variability of hearing loss.     

High carrier frequency of the GJB2 mutation (35delG) in the north of Iran

OBJECTIVE: Mutations in the GJB2 gene are a major cause of autosomal recessive and sporadic non-syndromic hearing loss in many populations. A single mutation of this gene (35delG) accounts for approximately 70% of mutations in Caucasians with a carrier frequency of 2-4% in Europe. This study aims to determine the rate of 35delG carrier frequency in Iran. METHODS: Genomic DNA was extracted from a total of 550 unaffected unrelated subjects from 4 provinces of Iran following the standard phenol chloroform procedure. The one base pair deletion (35delG) was analysed using a nested PCR procedure; 35delG mutation carriers were subsequently confirmed by sequence analysis. Moreover, using the Binomial probability distribution, we compared the 35delG carrier frequency of Iranian population with the various Middle Eastern and overall European populations. RESULTS: Of the four populations studied, we found a high carrier frequency of 2.8% in Gilan province in the north of Iran. The overall 35delG carrier frequency was found to be 1.25% in the populations studied (our present and previous data) which is similar to the overall 35delG carrier frequency detected in Middle Eastern populations, but Significantly lower than that identified in European populations.     

Prevalence of the c.35delG and p.W24X mutations in the GJB2 gene in patients with nonsyndromic hearing loss from North-West Romania

Objective

In Central and South-Eastern European countries, the most frequent mutation types responsible for congenital nonsyndromic sensorineural hearing loss (NSHL) are c.35delG and p.W24X (15-55.8% and 2.5-4.3%, respectively). The aim of the study was to determine for the first time in Romania the prevalence of c.35delG and p.W24X mutations in patients with NSHL.

Material

75 unrelated children with NSHL from Transylvania (North-West Romania).

Methods

a. Audiological examination (otoscopy, tympanogram, acoustic otoemission and tonal audiogram or auditory evoked potentials); b. detection of the c.35delG (semi-nested-PCR, RFLP and ARMS-PCR analysis) and p.W24X (ARMS-PCR analysis) mutations.

Results

Audiological examination allowed the diagnosis of hearing loss of various degrees: moderate in 8 patients (10.7%), severe in 14 cases (18.7%), profound in 53 patients (70.6%). The number of reported mutation cases as against the number of alleles indicates a 33.3% frequency rate for c.35delG mutation and respectively 5.3% for p.W24X mutation. All 22 patients with 35delG/c.35delG genotype (19 patients), c.35delG/p.W24X genotype (2 patients) or p.W24X/p.W24X genotype (1 patient) presented profound/severe hearing loss.

Conclusion

Our study confirms that the frequency rate of the two mutations analyzed in patients with NSHL from North-West Romania is comparable to that seen in other Central and South-Eastern European countries. The homozygote or compound heterozygote states represent a major risk factor for profound or severe deafness. Audiological screening in newborns and genetic testing in confirmed congenital hypoacusis cases are compulsory for early therapeutic intervention (hearing prosthesis or cochlear implant) and genetic counselling.