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.

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.

MicroRNA-183 Family in Inner Ear: Hair Cell Development and Deafness

miRNAs are essential factors of an extensively conserved post-transcriptional process controlling gene expression at mRNA level. Varoius biological processes such as growth and differentiation are regulated by miRNAs. Web of Science and PubMed databases were searched using the Endnote software for the publications about the role miRNA-183 family in inner ear: hair cell development and deafness published from 2000 to 2016. A triplet of these miRNAs particularly the miR-183 family is highly expressed in vertebrate hair cells, as with some of the peripheral neurosensory cells. Point mutations in one member of this family, miR-96, underlie DFNA50 autosomal deafness in humans and lead to abnormal hair cell development and survival in mice. In zebrafish, overexpression of the miR-183 family induces extra and ectopic hair cells, while knockdown decreases the number of hair cell. The miR-183 family (miR-183, miR-96 and miR-182) is expressed abundantly in some types of sensory cell in the eye, nose and inner ear. In the inner ear, mechanosensory hair cells have a robust expression level. Despite much similarity of these miRs sequences, small differences lead to distinct targeting of messenger RNAs targets. In the near future, miRNAs are likely to be explored as potential therapeutic agents to repair or regenerate hair cells, cell reprogramming and regenerative medicine applications in animal models because they can simultaneously down-regulate dozens or even hundreds of transcripts.

Effect of oxidized low density lipoprotein on the expression of Runx2 and SPARC genes in vascular smooth muscle cells

Vascular calcification is an important stage in atherosclerosis. During this stage, vascular smooth muscle cells [VSMC] synthesize many osteogenic factors such as osteonectin [encoded by SPARC]. Oxidative stress plays a critical role in atherosclerosis progression, and its accumulation in the vascular wall stimulates the development of atherosclerosis and vascular calcification. The osteonectin overexpression has been observed in the arterial wall during the course of atherosclerosis. However, the regulatory mechanism of oxidized low density lipoprotein [oxLDL]-mediated vascular calcification remains to be clarified. The aim of this study was to investigate the effect of oxLDL on the osteonectin gene expression through the Runx2 transcription factor. In this experimental study, VSMC were cultured in F-12K media and then treated with oxLDL. The expression of Runx2 and osteonectin genes was determined by real-time PCR method. Protein levels were investigated by the western blotting technique. The Runx2 gene was knocked down using siRNA in order to determine whether Runx2 regulates the osteonectin expression in VSMC induced by oxLDL. Then transfected cells were treated with oxLDL, and the expression levels of Runx2 and osteonectin were determined again. oxLDL was found to increase Runx2 and osteonectin gene expression [4.8 +/- 0.47- and 9.2 +/- 1.96-fold, respectively] after 48 h. Western blotting analysis confirmed the induced levels of Runx2 and osteonectin proteins. However, oxLDL-induced osteonectin expression was not observed to be blocked by Runx2 knockdown. The up-regulation of osteonectin by oxLDL is independent of Runx2, and it may be mediated by other transcription factors

Immunohistochemical analysis of mismatch repair proteins in iranian colorectal cancer patients at risk for Lynch syndrome

Hereditary non-polyposis colorectal cancer [HNPCC] is a common hereditary cancer predisposing syndrome has molecular and clinicopathological features still have remained ambiguous within Iranian populations. We discuss in this article some molecular and clinicopathological features of the condition. The study was a descriptive retrospective and designed on 1659 colorectal cancer [CRC] patients were screened based on early-onset disease and Amsterdam II criteria during 14 years [2000-2013]. Immunohistochemistry [IHC] staining was set up to detect expression of mismatch repair [MMR] genes on paraffin-embedded tissue sections of 31 HNPCC-CRC tumors. SPSS 19 software was used to analyze the data. IHC-MMR staining was absent in 7/31 individuals [22.6%] of which 4 cases showed IHC-Absent [IHC-A] in both MSH2 and MSH6 [57.1%], in 2 cases both MLH1 and PMS2 had negative staining [28.6%], and just in one case, MSH6 was defective [14.3%]. The frequency of CRC among IHC-A and IHC-Present [IHC-P] families was 67.5% and 27.9%, respectively. Also the most frequent extracolonic cancers in IHC-A group were: stomach [10%], small bowel [5%], and prostate [5%]; and in IHC-P group: stomach [18.4%], lung [10.9%], and breast [7.5%]. Average age of IHC-A individuals at diagnosis was 38.0 versus 45.3 years in IHC-P individuals. Overall, 20.8% and 57.1% of our index CRCs were localized proximal to the splenic flexure in IHC-P and IHC-A groups, respectively. Given the lack of enough information about molecular aspects of hereditary cancer syndromes like HNPCC in Iran, more evaluations are necessary on larger samples using complementary techniques such as MSI-testing and mutation analyses

[Population data on d7s2425 marker in five ethnic groups of the Iranian population: a highly informative marker for molecular diagnosis of ARNSHL]

SLC26A4 gene mutations are the second identifiable genetic cause of autosomal recessive nonsyndromic hearing loss [ARNSHL] after GJB2 mutations and are currently investigated in molecular diagnosis. In databases, several potential STR markers related to this region have been introduced. In this investigation, the characteristics and informativeness of D7S2425 CA repeat STR marker in SLC26A4 gene region was examined in five ethnic groups of the Iranian population. The locus was genotyped in 165 individuals of five different ethnic groups including Fars, Azari, Turkmen, Gilaki, and Arab using polymerase chain reaction [PCR] followed by polyacrylamide gel electrophoresis [PAGE] and fluorescent capillary electrophoresis. In this study, the results were analyzed by GeneMarker HID, Human STR Identity software, GenePop program, and Microsatellite Tools. Analysis of the allelic frequency revealed the presence of 8 alleles for D7S2425 marker in the Iranian population, of which the 246bp allele at the D7S2425 locus with 0.30% frequency was the most frequent. The 93.9% observed heterozygosity of the Gilak ethnic group was the highest among all ethnic groups. Analysis of deviations of the Hardy-Weinberg equilibrium demonstrated that all the ethnic groups were in equilibrium [P > 0.05] for D7S2425 locus. Finally, analysis of PIC value revealed that the D7S2425 marker could be considered as a highly informative marker in each ethnic group of the Iranian population [PIC value above 0.7]. Our data suggested that D7S2425 could be introduced as a highly informative marker in molecular diagnosis of SLC26A4 based ARNSHL by Linkage analysis

Compound Heterozygosity for Two Novel SLC26A4 Mutations in a Large Iranian Pedigree with Pendred Syndrome

OBJECTIVES: The aim of this study was to detect the genetic cause of deafness in a large Iranian family. Due to the importance of SLC26A4 in causing hearing loss, information about the gene mutations can be beneficial in molecular detection and management of deaf patients. METHODS: We investigated the genetic etiology in a large consanguineous family with 9 deaf patients from Fars province of Iran with no GJB2 mutations. Initially, linkage analysis was performed by four DFNB4 short tandem repeat markers. The result showed linkage to DFNB4 locus. Following that, DNA sequencing of all 21 exons, their adjacent intronic sequences and the promoter of SLC26A4 was carried out for mutation detection. RESULTS: Two novel mutations (c.863-864insT and c.881-882delAC) were identified in exon 7 of the gene, in both homozygous and compound heterozygous state in patients. CONCLUSION: Our results supported the importance of the SLC26A4 mutations in the etiology of hearing loss among the Iranian patients and therefore its mutation screening should be considered after GJB2 in the molecular diagnostics of hearing loss, especially when enlarged vestibular aqueduct or goiter is detected.

I405V and -629C/A polymorphisms of the cholesteryl ester transfer protein gene in patients with coronary artery disease

Cholesteryl ester transfer protein [CETP] plays a main role in high-density lipoprotein metabolism. CETP gene possesses several single nucleotide polymorphisms which have been associated with plasma high-density lipoprotein cholesterol [HDL-C] concentrations. The aim of this study was to determine the association of CETP -629C/A and I405V polymorphisms with coronary artery disease [CAD] in Iranian population. The presence of two CETP gene polymorphisms -629C/A and I405V were studied in 187 unrelated CAD cases and 136 controls. All the samples were clinically examined and lipid profile was estimated. Genotyping was performed using polymerase chain reaction/restriction fragment length polymorphism method. The frequency of-629C/A and I405V allelic variants were found to be 0.732 and 0.366 in cases and 0.658 and 0.348 in controls, respectively. The frequency of A allele of -629C/A polymorphism in cases was significantly higher than that of controls. HDL-C in AA genotype was higher than CA and CC genotypes in controls. No significant effect of II, IV and VV genotypes was found in lipid profiles. No significant association was found between CETP I405V polymorphism and increased risk of CAD in Azeri population studied. AA genotype of -629C/A increased HDL but the risk of CAD in this genotype might be higher than CC genotype

Contribution of GJB2 mutations and Four common DFNB loci in autosomal recessive non-syndromic hearing impairment in Markazi and Qom provinces of Iran

This study aimed to investigate the contribution of four common DFNB ["DFN" for deafness and "B" for autosomal resessive locus] loci and GJB2 gene mutations [exon 2] in hearing impairment in individuals living in Markazi and Qom provinces of Iran. Forty consanguineous Iranian families with at least three affected individuals in family or pedigree who suffer from an autosomal recessive non-syndromic congenital hearing impairment were the subjects of this study. Blood samples were taken from both hearing and non-hearing individuals, DNA was extracted and amplified by using specific primers for the coding region of GJB2 gene [exon 2]. The PCR product of GJB2 gene was then sequenced. Also short tandem repeat [STR] markers amplified by using specific primers for loci DFNB2, DFNB3, DFNB4 and DFNB21. At least 2 microsatellite markers [STR] for each DFNB locus exceeding to 4-6 markers for the linked families were used. The amplified markers were analyzed by conventional Polyacrylamide Gel Electrophoresis followed by silver staining. Six families were homozygous or compound heterozygous for GJB2 mutations and were excluded from further studies. Linkage analysis was carried out for the remaining 34 families by genotyping the flanked STR markers of DFNB2, DFNB3, DFNB4 and DFNB21 loci. Six families showed linkage; including one family to DFNB2, two families to DFNB3 and three families to DFNB4 locus while no family showed linkage to DFNB21 locus. Undoubtedly, the best understanding of the genetic basis of hearing loss in Iranian population will be achieved by performing similar experiments in other provinces and also by analyzing more loci

Mutation analysis of connexin 26 gene and del[GJB6-D13S1830] in patients with hereditary deafness from two provinces in Iran

Mutations in the connexin 26 [Cx26] gene at the DFNB1 locus on chromosome 13q12 are associated with autosomal recessive non-syndromic hearing loss [ARNSHL]. There are many known mutations in this gene that cause hearing loss. A single frameshift, at position 35 [35delG] accounts for 50% of mutations in the Caucasian population with carrier frequencies of 1.5-2.5%. In this study we investigated the prevalence of Cx26 gene mutations by directly sequencing the coding exon of this gene belonging to ARNSHL individuals from 53 families in Qom and Markazi provinces of Iran. Seven different Cx26 variants were identified. Five Cx26 mutations including 35delG, 233delC, 176del16, W24X, L90P were found in 10 of 53 families [18.87%]. One polymorphism V153I was also found. One variant A171T with unknown effects was also detected. Six of the 53 families were observed to have GJB2 mutations in both alleles [11.32%]. The most common mutation was 35delG. Three out of 10 families [30%] with GJB2 variants contained 35delG mutation in both alleles and the frequency of 35delG allele was 0.50 among 10 out of 53 families. Also screening for the 342-kb GJB6 deletion mutant did not reveal any large deletion among families studied. Thus, in the two provinces, contribution of GJB2 [Gap Junction Protein Beta 2] mutations to familial deafness appears to be less significant. This necessitates further assessment of the other known genes regions as well as a search for new genetic factors in hereditary deafness in the Iranian population