Genetics of Hearing Loss in North Iran Population: An Update of Spectrum and Frequency of GJB2 Mutations

Diagnosis of pre-lingual hearing loss (HL) is difficult owing to the high number of genes responsible. The most frequent cause of HL is DFNB1 due to mutations in the GJB2 gene. It represents up to 40% of HL cases in some populations. In Iran, it has previously been shown that DFNB1 accounts for 16-18% of cases but varies among different ethnic groups. Here, we reviewed results from our three previous publications and data from other published mutation reports to provide a comprehensive collection of data for GJB2 mutations and HL in northern Iran. In total, 903 unrelated families from six different provinces, viz., Gilan, Mazandaran, Golestan, Ghazvin, Semnan, and Tehran, were included and analyzed for the type and prevalence of GJB2 mutations. A total of 23 different genetic variants were detected from which 18 GJB2 mutations were identified. GJB2 mutations were 20.7% in the studied northern provinces, which was significantly higher than that reported in southern populations of Iran. Moreover, a gradient in the frequency of GJB2 mutations from north to south Iran was observed. c.35delG was the most common mutation, accounting for 58.4% of the cases studied. This study suggests that c.35delG mutation in GJB2 is the most important cause of HL in northern Iran.

Aspirin alleviates endothelial gap junction dysfunction through inhibition of NLRP3 inflammasome activation in LPS-induced vascular injury

The loss of endothelial connective integrity and endothelial barrier dysfunction can lead to increased vascular injury, which is related to the activation of endothelial inflammasomes. There are evidences that low concentrations of aspirin can effectively prevent cardiovascular diseases. We hypothesized that low-dose aspirin could ameliorate endothelial injury by inhibiting the activation of NLRP3 inflammasomes and ultimately prevent cardiovascular diseases. Microvascular endothelial cells were stimulated by lipopolysaccharide (2 μg/mL) and administrated by 0.1-2 mmol/L aspirin. The wild type mice were stimulated with LPS (100 μg/kg/day), and 1 h later treated with aspirin (12.5, 62.5, or 125 mg/kg/day) and dexamethasone (0.0182 mg/kg/day) for 7 days. Plasma and heart were harvested for measurement of ELISA and immunofluorescence analyses. We found that aspirin could inhibit NLRP3 inflammasome formation and activation in dose-dependent manner and has correlation between the NLRP3 inflammasome and the ROS/TXNIP pathway. We also found that low-concentration aspirin could inhibit the formation and activation of NLRP3 inflammasome and restore the expression of the endothelial tight junction protein zonula occludens-1/2 (ZO1/2). We assume that aspirin can ameliorate the endothelial layer dysfunction by suppressing the activation of NLRP3 inflammasome.

Mutations in GJB2 as Major Causes of Autosomal Recessive Non-Syndromic Hearing Loss: First Report of c.299-300delAT Mutation in Kurdish Population of Iran

BACKGROUND AND OBJECTIVES: Autosomal recessive non-syndromic hearing loss (ARNSHL) with genetic origin is common (1/2000 births). ARNSHL can be associated with mutations in gap junction protein beta 2 (GJB2). To this end, this cohort investigation aimed to find the contribution of GJB2 gene mutations with the genotype-phenotype correlations in 45 ARNSHL cases in the Kurdish population. SUBJECTS AND METHODS: Genomic DNA was extracted from a total of 45 ARNSHL families. The linkage analysis with 3 short tandem repeat markers linked to GJB2 was performed on 45 ARNSHL families. Only 9 of these families were linked to the DFNB1 locus. All the 45 families who took part were sequenced for confirmation linkage analysis (to perform a large project). RESULTS: A total of three different mutations were determined. Two of which [c.35delG and c.-23+1G>A (IVS1+1G>A)] were previously reported but (c.299-300delAT) mutation was novel in the Kurdish population. The homozygous pathogenic mutations of GJB2 gene was observed in nine out of the 45 families (20%), also heterozygous genotype (c.35delG/N)+(c.-23+1G>A/c.-23+1G>A) were observed in 4/45 families (8.8%). The degree of hearing loss (HL) in patients with other mutations was less severe than patients with c.35delG homozygous mutation (p < 0.001). CONCLUSIONS: Our data suggest that GJB2 mutations constitute 20% of the etiology of ARNSHL in Iran; moreover, the c.35delG mutation is the most common HL cause in the Kurdish population. Therefore, these mutations should be included in the molecular testing of HL in this population.

Effect of Gegensan on Expressions of NOS, α-SMA, Cx43, TGF-β1 mRNA and Protein in Penile Smooth Muscle Tissue of Alcoholic ED Rats / 中国实验方剂学杂志

Objective:To investigate the activity of nitric oxide synthase (NOS) and α-smooth actin (α-SMA) in rat penile smooth muscle tissue of rats with alcoholic erectile dysfunction (ED). The effects of protein gene 43 (connexin43, Cx43) and transforming growth factor β1 (TGF-β1) mRNA and protein expressions provide an experimental basis for the clinical application of Gegensan in the treatment of alcoholic ED. Method:SD rats were randomly divided into five groups:normal group, model group, and low,medium,high-dose Gegensan groups (5,10,20 g·kg-1). Except the normal group, the other groups were administered with drugs after alcohol intervention for 30 min at 15 mL·kg-1·d-1. Colorimetric assay was used to detect NOS activity in the penile smooth muscle tissue of alcoholic ED rats. Quantitative real-time fluorescence polymerase chain reaction(Real-time PCR) and Western blot were used to detect α-SMA, Cx43, TGF-β1 mRNA and protein expressions in smooth muscle tissue of alcoholic ED rats. Result:Compared with the normal group, the expressions of NOS, α-SMA and Cx43 mRNA and protein in the penile smooth muscle of the model group decreased significantly (Pβ1 mRNA and protein increased significantly (Pβ1 mRNA expression, and α-SMA mRNA and protein expressions in the penis tissue of rats with alcoholic ED were significantly up-regulated (PConclusion:Gegensan has an obvious protective effect on the structure of penile smooth muscle of alcoholic ED rats. The specific mechanism may be related to the regulation of NOS activity and a-SMA, Cx43 and TGF-β1 mRNA and protein expressions.

Effects of total flavonids of astragalus on arrhythmia,endoplasmic reticulum stress in mice with viral myocarditis / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of total flavonids of astragalus(TFA) on arrhythmia, endoplasmic reticulum stress and connexcin in mice with viral myocarditis and to clarify the mechanisms of TFA against viral myocarditis complicated with arrhythmia.@*METHODS@#Thirty-six male Balb/c mice were randomly divided into control group, viral myocarditis group and total flavonoids group (=12). The mice of viral myocarditis were intraperitonealy injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days. The mice of TFA group were intraperitoneal injected with 0.1 ml/day 10-950 TCID CVB3 for 3 days and treated with 0.1ml, 20 mg/L TFA by tail vein injection. At the end of the experiment, arrhythmia was detected by electrocardiogram, the heart of mice were stained by HE, the expressions of glucose-regulated protein 78(GRP78), endoplasmic reticulum stress signaling pathway factor activating transcription factor 4(ATF4) and connexcin 43(Cx43) were detected by Western blot.@*RESULTS@#The expressions of GRP78 and ATF4 were increased and the expression of Cx43 was decreased in viral myocarditis, while TFA inhibited these effect of viral myocarditis in heart of mice.@*CONCLUSIONS@#The antiarrhythmic effect of TFA may be related to the alleviation of endoplasmic reticulum stress and the increase of Cx43 expression.

Updates on gap junctions in central nervous system diseases / 国际儿科学杂志

Gap junctions play an important role in cell growth,development,differentiation,the transmission of nerve impulses and homeostasis.Increase or decrease in gap junctions may cause neurological dysfunctions,such as Alzheimer's disease,demyelinating disease,epilepsy,glioma,hypoxic ischemic encephalopathy.Therefore,research on gap junctions may provide important clues to understand demyelinating diseases,neural stem cell transplantation and post-iniury restoration.

Effect of Zuogui Pill on the Expression of Gap Junction Protein 43 in Ovarian Tissue of Mice with Chemo-therapy-induced Premature Ovarian Failure / 中国药房

OBJECTIVE:To study the effect of Zuogui pill on the expression of gap junction protein 43(Cx43)in ovarian tis-sue of mice with premature ovarian failure(POF)induced by intraperitoneal injection of cyclophosphamide(CTX),and explore its mechanism in the treatment of chemotherapy-induced POF. METHODS:72 mice were randomly divided into blank group,model group,Estradiol valerate tablet group (positive control,0.00013 g/kg),Zuogui pill low-dose,medium-dose,high-dose groups (13.65,40.95,122.85 g/kg)by body mass,10 in each group. Except for blank group,other groups were reduce POF model by ip CTX,once a day,for 20 d. Meanwhile,the mice were intragastrically administrated related medicines,once a day,for 30 d. After 2 h of last administration,reverse transcription-polymerase chain reaction method and Western blot method were used to detect the Cx43 mRNA and protein expressions in ovarian tissue respectively,and immunohistochemistry method was adopted to detect the distribution of Cx43 protein in ovarian tissue. RESULTS:Compared with blank group,Cx43 mRNA and protein expressions in ovarian tissue of mice in model group were obviously weakened,and the distribution of Cx43 protein in follicle and granulocytes were obviously reduced(P<0.01). Compared with model group,Cx43 mRNA and protein expressions in ovarian tissue of mice in Estradiol valerate tablet group and Zuogui pill medium-dose,high-dose groups were obviously strengthened,and the distribution of Cx43 protein in follicle and granulocytes were obviously increased(P<0.01). CONCLUSIONS:Zuogui pill can increase the Cx43 mRNA and protein expressions in ovarian tissue of CTX-induced POF mice,increase the distribution of Cx43 in follicle and granu-locytes and gap junction function,which may be one of the treatment mechanism of POF.

Immunohistochemical Study of Heart-regulating and Mind-calming Acupuncture for Adjusting Cx43 Expression in Myocardial Cells inaRabbit Model of Ventricular Arrhythmia / 上海针灸杂志

ObjectiveTo investigate the effect of heart-regulating and mind-calming acupuncture on the expression of gap junction protein connexin43 (Cx43) in myocardial cells in a rabbit model of ventricular arrhythmia.MethodA rabbit model of ventricular arrhythmia was made by injecting barium chloride into the marginal ear vein. Forty New Zealand rabbits were randomized into control, model, acupuncture and medication groups. The acupuncture group received heart-regulating and mind-calming acupuncture[at Lingtai(GV10), Shendao(GV11), bilateral Neiguan(PC6)and Baihui(GV20)]and the medication group, oral gavage of propafenone. Rabbit left ventricle was taken after the completion of the experiment. Cx43 expressions were compared and analyzed usingan immunohistochemical staining technique and a computer image processing system.ResultCx43 expression decreased significantly in the model group compared with the control group (P0.05).Conclusion Adjusting Cx43 expression in myocardial cells may be one of the target points for this acupuncture treatment of arrhythmia.

Screeing Using SNPscan Method for GJB2 Gene Mutation among Non-syndromic Deaf Patients of the Dominant Ethnic Minorities in Xinjiang / 听力学及言语疾病杂志

Objective To investigate the prevalence and characteristics of GJB2 mutations in Uygur,Hui, Kazak and Kirgiz ethnic patients with non-syndromic hearing loss(NSHL)from the Xinjiang Uygur Autonomous Region of China.Methods With the permission,we collected 565 patients with moderately severe to profound sen-sorineural hearing loss,including Uygur,Hui,Kazak and Kirgiz ethnic minorities from 14 cities of Xinjiang.Pe-ripheral blood samples were obtained to extract genomic DNA.The SNP classification technology was for common pathogenic GJB2 gene mutations.ResuIts The pathogenic allele frequency of GJB2 gene were 10.16%(87/856 ), 15.85%(13/82),10.16%(13/128),1.56%(1/64)in the NSHL patients of Uygur,Hui,Kazak and Kirgiz minori-ties,respectively.And these differences were statistically significant (χ2 =8.140,P=0.043).c.235delc was only found in the Uygur and Hui with the allele frequency of 5.14 %(44/856)and 13.41 %(11/82),respectively.And c.35delG was found in Uyhur,Hui,Kazak and Kirgiz with allele frequencies were 3.15% (27/856),1.21% (1/82),8.59%(11/128)and 1.56% (1/64),respectively.ConcIusion GJB2 gene mutations had a higher incidence in Xinjiang NSHL patients,GJB2 gene mutation spectrum had differences in Uygur,Hui,Kazak and Kirgiz,c. 235delC the hotspot mutation region in Uygur and Hui nationalities NSHL patients,while c.35delG is the hotspot mutation region in NSHL patients of Uygur,Kazak and Kirgiz ethnicities.

Advances in the study of arrhythmogenic mechanisms / 国际药学研究杂志

Arrhythmia is a common complication of cardiovascular diseases and a risk factor for human health. Especially, ventricular tachycardia and ventricular fibrillation may not only exacerbate original heart diseases, but also cause cardiac sudden death which has been an mportant death reason in China. However, anti-arrhythmic drugs nowadays cannot effectively treat these arrhythmias, with an efficiency of only 30%-60%, which indicates that our knowledge about arrhythmias is limited. Hence, to explore the potential mechanism, look for novel targets, and develop drugs with multiple-channel action are the focus of the research direction. Recent studies displayed that the atrial-specific potassium channels such as IKur and IKAch were involved in atrial fibrillation, which provided a prospective target for atrial fibrillation treatment. Calcium leak, gap junction protein and autoantibody against ICaL channel were shown to participate in arrhythmogenesis. These findings provided a theoretical basis for the development of more effective anti-arrhythmic drugs. Remarkably, as a kind of mportant RNA regulating gene expression, microRNA (miRNA) was shown to possess anti-arrhythmic activities which may prevent cardiac sudden death. miR-1, miR-133 and miR-590 regulated the arrhythmia in various types of animal models. Because of the multiple-gene regulation actions of miRNA, it has the potential to be developed as novel anti-arrhythmic target.

Connexin 43 expression in cultured interstitial cells of Cajal from guinea pigs / 第三军医大学学报

Objective To explore the expression of connexin 43 in the interstitial cells of Cajal (ICCs) from guinea pig bladder in vitro. Methods Bladder ICCs were primarily cultured from guinea pigs by collagenase digestion method. Primarily cultured bladder smooth muscle cells (SMCs) from guinea pig were taken as control. The expressions of c-kit and smooth muscle actin (SMA) were detected by immunofluorescent method. Immumofluorescent method and Western blotting were used to detect the expression of Connexin 43 in 2 groups of cells. Results The expression of c-kit was positive in ICCs but negative in the control group. On the contrary, the expression of SMA was negative in ICCs but positive in the control group. Connexin 43 expression was significantly higher in ICCs than that in the control group (P

A Functional Study of Gap Junction in GJB2 Mutations Associated with Hereditary Hearing Loss / 대한이비인후과학회지

BACKGROUND AND OBJECTIVES: GJB2 (Connexin 26), the gene of the gap-junction proteins, was found to be the main causative gene of autosomal recessive nonsyndromic hearing loss (DFNB1). Whereas 35delG was known as the major type mutation in the western countries, 235delC was reported as the specific form of mutation in Asian population. The objective of this study is to identify how two mutations (235 delC, E114G) found in the Korean population affect the function of GJB2 using the molecular biology techniques. MATERIALS AND METHODS: 235delC and E114G types of mutations were cloned in the pcDNA3 vector. HeLa cells were transfected with these cloned vectors by the liposome complex method. 1) 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. 2) To analyze functions of the GJB2, we examined the lucifer yellow dye transfer between cells with scrape-loaded technique. We used the wild-type (WT) Cx26 of normal hearing as a positive control, and mock cells as a negative control. RESULTS: The immunocytochemical analysis showed that cells transfected with E114G and WT gave characteristic punctuated patterns of reaction in the cell membrane with both antibodies. However, 235delC cells were not stained with the anti-IL antibody but only with the anti-NT antibody slightly around the nucleus regions. In the functional study of GJB2, transfer of lucifer yellow dye into contiguous cells was detected in E114G but not in 235delC. CONCLUSION: The 235delC type of mutation showed loss of their targeting activity on the cell membrane. As a result, the function of gap junction channels were severely deteriorated. With the E114G type mutation, we didn't find any difference when compared with the WT transfected cells. Above data indicate that types of GJB2 mutation are closely related to the status of hearing loss due to altered function of gap junction protein.