Familial and sporadic GJB2-related deafness in iran: Review of gene mutations

Background: Mutations in the GJB2 gene encoding connexin 26 protein, are the main cause for autosomal recessive and sporadic non syndromic hearing loss in many populations. Here, we have taken together and reviewed results from our six previous publications, our unpublished data from ten Iranian provinces, as well as data from two previous mutation reports to provide a comprehensive collection of data for GJB2 mutations and deafness in Iran. Methods: In all, 1095 hearing impaired students and their deaf siblings from 890 families in 10 provinces of Iran were studied. The prevalence and type of the GJB2 gene mutations were investigated using nested PCR Ore screening strategy and direct sequencing of the coding exon of the gene. Results: Altogether 31 different genetic variants were detected from which 17 GJB2 mutations were identified. GJB2 mutations were found in 14.6% of deaf families (18.29% of familial and 12.7% of sporadic cases). We found GJB2 mutations in both alleles in 78% of GJB2 mutations chromosomes. However, 35deIG mutation was the most common GJB2 mutation accounting for 74.5% of the mutations in populations studied. Conclusion: Our data indicated that a specific combination of GJB2 mutations types and frequencies was presented in different populations of Iran. These results also highlight the importance of GJB2 mutations in development of hearing loss in familial and sporadic deaf families in different parts of the country and can be used as a basis of genetic counseling and clinical guideline in Iran

Oxidized Low-Density Lipoprotein and Upregulated Expression of Osteonectin and Bone Sialoprotein in Vascular Smooth Muscle Cells

Background: Oxidative stress has been associated with the progression of atherosclerosis and activation of genes that lead to increased deposition of proteins in the extracellular matrix. Bone sialoprotein (BSP) and osteonectin are proteins involved in the initiation and progression of vascular calcification. Objective: To investigate the effect of oxidized low-density lipoprotein on osteonectin and BSP expression in human aorta vascular smooth muscle cells (HA/VSMCs). Methods: We treated HA/VSMCs with oxidized low-density lipoprotein (oxLDL) and measured the relative expression of osteonectin and BSP genes using the real-time polymerase chain reaction (PCR) method. We investigated the protein levels produced by each gene using the western blotting technique. Results: oxLDL increased osteonectin and BSP levels (mean SD], 9.1 2.1]-fold and 4.2 0.75]-fold, respectively) after 48 hours. The western blotting results also confirmed the increased levels of osteonectin and BSP. Conclusion: oxLDL may enhance vascular calcification by promoting the expression of osteonectin and BSP

Expandable DNA repeat and human hereditary disorders

Background & Aims: Nearly 30 hereditary disorders in humans result from an increase in the number of copies of simple repeats in genomic DNA, including fragile X syndrome, myotonic dystrophy, Huntington’s disease, and Friedreich’s ataxia. One the most frequently occurring types of mutation is trinucleotide repeat expansion. The present study was conducted with the aim of investigating the cause and molecular mechanisms of repeat expansions DNA and their pathogenic mechanisms in diverse classes of genetic diseases. Methods: Scientific databases were searched using the keywords expandable DNA repeat fragile X, myotonic dystrophy, Huntington’s disease, and Friedreich’s ataxia. After primary screening, articles which were related to the studies topic were further considered and analyzed. Results: DNA repeats seem to be predisposed to such expansion due to their unusual structural features, which disrupt the cellular replication, repair, and recombination processes. The majority of these debilitating diseases are caused by repeat expansions in the noncoding regions of their resident genes. The pathogenic mechanism underling these disorders include loss of function in protein and gain of function in protein or ribonucleic acid (RNA). Conclusion: Although diseases caused by trinucleotide repeat expansion vary in their phenotypes, they are somewhat similar in their pathogenic mechanism and medical findings. It is likely that progress made in this field will be beneficial to patients who have other neurological diseases. © 2016, Kerman University of Medical Sciences. All rights reserved

Mutation analysis using the restriction site mutation (RSM) assay

The restriction site mutation RSM assay see Steingrimsdottir et al. H. Steingrimsdottir, D. Beare, J. Cole, J.F.M. Leal, Ž. Ž w T. Kostic, J. Lopez-Barea, G. Dorado, A.R. Lehmann, Development of new molecular procedures for the detection of genetic alteration in man, Mutat. Res. 353 1996 pp. 109–121 for a review has been developed as a genotypic mutation Ž . x . detection system capable of identifying mutations occurring in restriction enzyme sites of genomic DNA. Here we will report the steps taken to overcome some of the initial problems of the assay, namely the lack of quantitative data and limited sensitivity, the aim being to achieve a methodology suitable for the study of low dose chemical exposures. Quantitative data was achieved in the RSM assay by the inclusion of an internal standard molecule in the PCR amplification stage, thus allowing the calculation of both spontaneous and induced mutation frequencies. The sensitivity of the assay was increased through the discovery that intron sequences of genomic DNA accumulated more mutations in vivo compared to the exons, presumably due to differential selective pressure within genes G.J.S. Jenkins, I.deG. Mitchell, J.M. Parry, Enhanced w restriction site mutation RSM analysis of 1,2-dimethylhydrazine-induced mutations, using endogenous Ž . p53 intron sequences, Mutagenesis 12 1997 pp. 117–123 . This increased sensitivity was examined by applying the RSM assay to Ž . x analyse the persistence of N-ethyl-N-nitrosourea ENU -induced mutations in mice testes. Germ line mutations were sought Ž . in testes DNA 3, 10 and 100 days after ENU treatment. Mutations were detected in exons and especially intron regions, the intron mutations were more persistent, still being detected 100 days post-chemical treatment. Assignment of these mutations as ENU induced was complicated in some cases where the spontaneous mutation level was high. This theme of mutation persistence was further investigated by studying the presence of 4-nitroquinoline-1-oxide 4-NQO -induced DNA mutations Ž . in vitro. This study also analysed the relationship between DNA adduct formation and DNA mutation induction by the concurrent RSM analysis and 32P post-labelling analysis of 4-NQO treated human fibroblasts. The results demonstrated that early DNA mutations detected 4 days post-treatment by the RSM assay were probably ex vivo mutations induced by Taq polymerase misincorporation of 4-NQO adducted DNA, due to the maximum levels of 4-NQO adducts being present at this time point. A later mutational peak, after the adduct level had declined, was assumed to be due to DNA sequence changes produced in the fibroblasts by the in vivo processing of DNA adducts

Study of I405V polymorphism of cholesterol ester transfer protein gene in efficacy of statins on plasma level of high density lipoprotein cholesterol

زمینه و هدف: پروتئین انتقال دهنده کلسترول استر (CETP) در متابولیسم لیپوپروتئین با دانستیه بالا (HDL) و مسیر انتقال معکوس کلسترول نقش اساسی دارد. چند شکلی های ژن CETP مانند I405V (ایزولوسین به والین) که مستقیماً بر HDL کلسترول تاثیر می‌گذارد رونویسی از این ژن را تحت تاثیر قرار می‌دهد. هدف این مطالعه تعیین تاثیر پلی مورفیسم I405V ژن CETP بر سطح HDL کلسترول در پاسخ به درمان با استاتین‌ها می‌باشد. روش بررسی: در این مطالعه توصیفی - تحلیلی از بین بیمارانی که سطح لیپوپروتئین با دانسیته پایین کلسترول (LDL-C) بالاتر از 120 میلی گرم در دسی لیتر تحت درمان، 196 بیمار دریافت کننده لواستاتین و آتوراستاتین انتخاب شدند. در همه بیماران قبل و بعد از درمان پروفایل لیپیدی اندازه‌گیری شد. پلی مورفیسم I405V ژن CETP توسط تکنیک چند شکلی طول قطعه محدود (PCR- RFLP) تعیین گردید. سپس نتایج آزمایشات بیوشیمیایی در پلی مورفیسم‌های مختلف با استفاده از آزمون‌های t زوجی، ANOVA و توکی مورد مطالعه قرار گرفت. یافته‌ها: پس از درمان با لواستاتین در ژنوتیپ VV سطح کلسترول کاهش بیشتر و سطح HDL افزایش بیشتری نسبت به دو ژنوتیپ دیگر نشان داد (05/0

Investigation on two polymorphisms effective on HDL-C concentration in patients with coronary artery disease using restriction fragment length polymorphism

Background and aim: High density lipoprotein cholesterol (HDL-C) is a known inverse predictor of coronary heart disease (CHD). Cholesteryl ester transfer protein (CETP) and hepatic lipase (HL) are key proteins in HDL-C metabolism so that decreased CETP or HL activity is associated with high HDL-C. -629C/A polymorphism in promoter of CETP gene and-514C/T in promoter of HL gene were previously reported to reduce related protein level in plasma. In this study association of these polymorphisms with CHD related to HDL-C level were investigated. Methods: In this analytical-descriptive study 321 subjects underwent coronary angiography and divided in two groups base on angiogram (non CAD = 135 and CAD = 186). Serum lipids profile was measured by standard procedure and genotype was detected using PCR-RFLP method. Results: Overall the CETP genotype frequencies were in CAD patients: 58.8% (n=110), 28.9% (n=54) and 12.3% (n=23) and in non CAD patients: 45.2% (n=61), 41.5% (n=56) and 13.3% (n=18) for AA, CA and CC respectively. HL genotype frequencies were in CAD patients: 61.6% (n=114), 33.5% (n=62) and 4.9% (n=9) and in non CAD patients: 65.9% (n=89), 27.4% (n=37) and 6.7% (n=9) for CC, CT and TT respectively. In control group HDL-C concentration was higher for AA than CC genotype in -629C/A, and also for TT than CC genotype in -514C/T. Allele A in all subjects and T allele in woman were higher in CAD than non CAD group. A high increase in HDL-C level (10. mg/dl) was observed in individuals with CETP-AA/LIPC-TT and CETP-CA/LIPC-TT relative to CETP-CC/LIPC-CC across all subjects (P< 0.001) but there was no difference in CAD prevalence. Conclusion: Allele A from -629C/A, and T from -514C/T even with the increasing of HDL-C concentration had higher frequency in CAD than non CAD group. Therefore, it seems that HDL-C didn’t protect coronary artery when CETP or HL activity was reduced by these polymorphisms

Comparison of human dermal fibroblasts (HDFs) growth rate in culture media supplemented with or without basic fibroblast growth factor (bFGF)

Basic fibroblast growth factor (bFGF or FGF-2) is a member of the FGF family secreted by different kinds of cells like HDFs and it is an important nutritional factor for cell growth and differentiation. The HDFs release bFGF in culture media at very low. The present study aims to investigate the HDFs growth rate in culture media supplemented either with or without bFGF. In brief, HDFs were isolated from human foreskin sample and were cultured in vitro in media containing bFGF and lack of this factor. The cells growth rate was calculated by trypan blue. The karyotyping was performed using G-banding to investigate the chromosomal abnormality of HDFs in both groups. Total RNA of each groups were extracted and cDNA samples were synthesized then, real-time Q-PCR was used to measure the expression level of p27kip1 and cyclin D1 genes normalized to internal control gene (GAPDH). The karyotype analysis showed that HDFs cultured in media or without bFGF had normal karyotype (46 chromosomes, XY) and chromosomal abnormalities were not observed. The cell growth rates in both groups were normal with proliferated exponentially but the slope of growth curve in HDFs cultured in media containing bFGF was increased. Karyotyp test showed that bFGF does not affect on cytogenetic stability of cells. The survey of p27kip1 and cyclin D1 genes by real-time Q-PCR showed that the expression level of these genes were up-regulated when adding bFGF in culture media (p < 0.05). The findings of the present study demonstrate that appropriate supplementation of culture media with growth factor like bFGF could enhance the proliferation and differentiation capacity of cells and improve cells growth rate. Similarly, fibroblast growth factors did not induce any chromosomal abnormality in cells. Furthermore, in HDFs cultured in bFGF supplemented media, the p27kip1 and cyclin D1 genes were up-regulated and suggesting an important role for bFGF in cell-cycle regulation and progression and fibroblast division stimulation. It also suggests that the effects of bFGF on different cell types with/or without production of bFGF or other regulation factors be investigated in future

Study of -629C/A polymorphism of cholesteryl ester transfer protein gene in statin effects on plasma high density lipoprotein cholesterol level

Background and aim: Cholesteryl ester transfer protein (CETP) plays pivotal role in HDL metabolism and in reverse cholesterol transport (RCT) pathway. CETP gene variants such as -629C/A that affect HDL cholesterol directly modulates CETP gene tranh1ional activity. This study was aimed to determine influence of -629C/A polymorphism of CETP in statin effects with regard to plasma HDL cholesterol levels. Methods: In this deh1ive-analytical study 196 adult patients with LDL-C more than 120mg/dL were divided into two groups base on lovastatin and atorvastatin using. Lipid profile was measured in all subjects before and after treatment and -629C/A polymorphism of CETP promoter was studied using polymerase chain reaction/restriction fragment length polymorphism method. Data were compared with paired t-test and ANOVA in SPSS software. Results: Cholesterol was decreased and HDL was increased in AA genotype more than other genotypes by lovastatin but ApoA1 was increased in CC genotype. ApoA1 also was increased in CC genotype more than AA or AC genotypes by atorvastatin. Conclusion: In CC genotype lovastatin and specially atorvastatin increased ApoA1 in HDL particles more than other genotypes. Therefore treatment with lovastatin and atorvastatin is more effective in patients with CC genotype for raising HDL particles activity

Study of -629C/A polymorphism of cholesteryl ester transfer protein gene in statin effects on plasma high density lipoprotein cholesterol level

Background and aim: Cholesteryl ester transfer protein (CETP) plays in HDL metabolism and in reverse cholesterol transport (RCT) pivotal role pathway. CETP gene variants such as -629C/A that affect HDL cholesterol directly, modulates CETP gene transcriptional activity. This study was aimed to determine influence of -629C/A polymorphism of CETP in statin effects with regard to plasma HDL cholesterol levels. Methods: In this descriptive-analytical study, 196 adult patients with LDL-C more than 120mg/dL were divided into two groups base on lovastatin and atorvastatin using. Lipid profile was measured in all subjects before and after treatment and -629C/A polymorphism of CETP promoter was studied using polymerase chain reaction/restriction fragment length polymorphism method. Data were compared with paired t-test and ANOVA in SPSS software. Results: Cholesterol was decreased and HDL was increased in AA genotype more than other genotypes by lovastatin, but ApoA1 was increased in CC genotype. ApoA1 also was increased in CC genotype more than AA or AC genotypes by atorvastatin. Conclusion: In CC genotype, lovastatin and specially atorvastatin increased ApoA1 in HDL particles more than other genotypes. Therefore, treatment with lovastatin and atorvastatin is more effective in patients with CC genotype for raising HDL particles activity