Genetic Linkage Analysis of DFNB4, DFNB28, DFNB93 Loci in Autosomal Recessive Non-syndromic Hearing Loss: Evidence for Digenic Inheritance in GJB2 and GJB3 Mutations.

BACKGROUND: Autosomal recessive non-syndromic hearing loss (ARNSHL) a most frequent hereditary type of hearing impairment, exhibit tremendous genetic heterogeneity. We aimed to determine the contribution of three common DFNB loci (DFNB4, DFNB28, and DFNB93), and mutation analysis of Gap Junction Beta-2 gene (GJB2) and GJB3 genes in ARNSHL subjects in southern Iran. METHODS: Thirty-six large ARNSHL pedigrees (167 individuals) with at least two affected subjects (72 patients) were included in this descriptive study from Hormozgan Province of Iran, during 2014 - 2015. The variation of GJB2 and GJB3 genes were screened using direct sequencing method. The negative samples for GJB2 and GJB3 genes mutations were analyzed for the linkage to DFNB4, DFNB28, and DFNB93 loci by genotyping the corresponding short tandem repeat (STR) markers using polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) methods. RESULTS: DNA sequencing of GJB2 were identified heterozygous mutation (964 C/T) in 13.88% of the studied families. Three missense mutations (788G/A, 284C/T and 973G/C) were also detected in coding region of the GJB3 gene. The 284C/T mutation in the GJB3 occurs in compound heterozygosity along with the 964T/C mutation in the GJB2 in one family. Finally, we found no evidence of linkage to either of DFNB4, DFNB93 and DFNB28 loci. CONCLUSION: Highlighting the hypothesis that a genetic interaction between GJB2 and GJB3 genes could be lead to ARNSHL, however, no evidence of linkage to the DFNB loci was found. 284C/T variant in GJB3 gene might be pathogenic when accompanied by variant in GJB2 in a digenic pattern. However, further large-scale familial and functional studies are required to challenge this hypothesis.

Upregulated Expression of Circulating MicroRNAs in Kidney Transplant Recipients With Interstitial Fibrosis and Tubular Atrophy.

INTRODUCTION: The discovery of circulating microRNAs (miRNAs), as potential noninvasive diagnostic biomarkers, has opened new avenues of research for identifying transplant patients with chronic allograft dysfunction. The present study aimed to investigate the expression levels of 4 immune-related miRNAs, miR-21, miR-31, miR-142-3p, and miR-155, in plasma samples of kidney allograft recipients. MATERIALS AND METHODS: The plasma expression levels of the miRNAs were evaluated by quantitative real-time polymerase chain reaction in 53 kidney recipients with long-term stable allograft function (n = 27), biopsy-proven interstitial fibrosis and tubular atrophy (n = 26), and healthy controls (n = 15). The possible correlation between clinical parameters and the circulating miRNAs and the receiver-operating characteristic analysis were performed. RESULTS: Significantly upregulated expressions of miR-21 (P = .02), miR-142-3p (P = .048), and miR-155 (P = .005) were observed in plasma samples of recipients with interstitial fibrosis and tubular atrophy in comparison to the stable allograft function and healthy control groups. Expression level of the miR-21 in plasma was correlated with creatinine (r = -0.432, P = .03) and estimated glomerular filtration rate (r = 0.423, P = .031). Multivariable analysis indicated that miR-21, miR-142-3p, and miR-155 in plasma samples could discriminate almost most of the patients with interstitial fibrosis and tubular atrophy (area under curve, 0.802; sensitivity, 81%; specificity, 92%). CONCLUSIONS: Our data suggested that altered expression of miR-21, miR-142-3p, and miR-155 in plasma samples might be associated with kidney allograft dysfunction and could be used for graft monitoring in kidney transplantation.

Impaired expression of Drosha in breast cancer.

BACKGROUND: Impaired miRNAs processing pathway is one interesting scenario for global downregulation of the miRNAome in various types of malignancy. We previously reported that DGCR8 and Dicer genes dysregulated in patients with breast cancer. OBJECTIVE: To evaluate the expression pattern of Drosha in patients with breast cancer. METHODS: We evaluated the mRNA expression level of Drosha in 70 fresh breast carcinomas and adjacent non-neoplastic tissue using quantitative real-time PCR and assessed the possible correlation between its expression and clinicopathological parameters. RESULTS: Our results revealed that mRNA expression level of Drosha was decreased in tumors when compared to adjacent non-neoplastic tissue. However, this difference is not statistically significant (P > 0.05). Downregulation of Drosha is related to older age at diagnosis, higher histological grade, higher tumor size and metastasis. However, there was no significant correlation between Drosha expression level and clinicopathological parameters (P > 0.05). We found that Drosha expression negatively correlated with DGCR8 (P = 0.043), whereas dysregulated expression levels of Drosha and Dicer are positively correlated with to each other (P < 0.0001). CONCLUSION: This study provides evidence that the expression of Drosha is impaired in breast cancer. However, the molecular basis of observed expression pattern have remained inexplicable and should be further investigated.

Circulating miR-150, miR-192, miR-200b, and miR-423-3p as Non-invasive Biomarkers of Chronic Allograft Dysfunction.

BACKGROUND AND AIMS: Chronic allograft dysfunction (CAD) is the major cause of renal allograft loss and can only be diagnosed by invasive histological examinations. The current study aimed to determine whether or not the circulating miR-125a, miR-150, miR-192, miR-200b, miR-423-3p and miR-433 could serve as predictors of graft outcome in the renal transplant recipients with CAD. METHODS: To evaluate the expression levels of miRNAs, we used quantitative real-time PCR (qPCR) and analyzed the plasma samples of 53 renal transplant recipients, including: 27 recipients with stable graft function (SGF), 26 recipients with biopsy-proven interstitial fibrosis and tubular atrophy (IFTA) and 15 healthy controls. Possible correlation between the clinicopathological parameters and the studied circulating miRNAs was also evaluated. RESULTS: miR-150 (p <0.001), miR-192 (p = 0.003), miR-200b (p = 0.048) and miR-423-3p (p <0.001) were differentially expressed between IFTA and SGF plasma samples. Creatinine correlated with miR-192 (r = 0.414, p = 0.036) and miR-423-3p (r = -0.431, p = 0.028). Moreover, the estimated glomerular filtration rate (eGFR) significantly correlated with the circulating miR-192 (r = -0.390, p = 0.049) and miR-423 (r = 0.432, p = 0.028). Receiver operating characteristic (ROC) analysis indicated that four miRNAs possessed the best diagnostic value for discriminating IFTA from SGF recipients with the areas under the curve (AUC) of 0.87 and high sensitivity and specificity values of 78% and 91%, respectively. CONCLUSIONS: The results suggest that aberrant plasma levels of these miRNAs are associated with the renal allograft dysfunction. Therefore, they are proposed to be considered as potential diagnostic biomarkers for monitoring of renal graft function.

Differential expression of circulating miR-21, miR-142-3p and miR-155 in renal transplant recipients with impaired graft function.

BACKGROUND: The discovery of circulating microRNAs (miRNAs), as potential noninvasive diagnostic biomarkers, has opened new avenues of research for identifying patients with chronic failure in renal transplantation. The present study aimed to investigate the expression levels of four immune-related miRNAs (miR-21, miR-31, miR-142-3p and miR-155) in plasma samples of renal recipients. METHODS: The plasma expression levels of the miRNAs were evaluated by quantitative real-time PCR (qPCR) in 53 renal recipients with long-term stable allograft function, SGF (N = 27), and with biopsy-proven interstitial fibrosis and tubular atrophy (IFTA) (N = 26) and also healthy controls (N = 15). The possible correlation between clinical parameters and the circulating miRNAs and the receiver-operating characteristic (ROC) analysis were performed. RESULTS: Our results showed that expression of miR-21 (p = 0.023), miR-142-3p (p = 0.048) and miR-155 (p = 0.005) was significantly upregulated in plasma samples of recipients with IFTA in comparison with SGF and healthy control groups. Concentration of miR-21 (∆Ct value) in plasma was negatively correlated with creatinine (r = -0.432, p = 0.028) and positively correlated with eGFR (r = 0.423, p = 0.031). The multivariate ROC curve analysis indicated that miR-21, miR-142-3p and miR-155 in plasma samples could discriminate almost most of the IFTA patients (area under curve = 0.802, sensitivity = 81%, specificity = 92%). CONCLUSION: Our data suggested that altered expression of miR-21, miR-142-3p and miR-155 in plasma samples may be associated with renal dysfunction and can be used for graft monitoring.

Genetic Linkage Analysis of DFNB3, DFNB9 and DFNB21 Loci in GJB2 Negative Families with Autosomal Recessive Non-syndromic Hearing Loss.

BACKGROUND: Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common hereditary form of deafness, and exhibits a great deal of genetic heterogeneity. So far, more than seventy various DFNB loci have been mapped for ARNSHL by linkage analysis. The contribution of three common DFNB loci including DFNB3, DFNB9, DFNB21 and gap junction beta-2 (GJB2) gene mutations in ARNSHL was investigated in south of Iran for the first time. METHODS: In this descriptive study, we investigated sixteen large families with at least two affected individuals. After DNA extraction, GJB2 gene mutations were analyzed using direct sequencing method. Negative samples for GJB2 gene mutations were analyzed for the linkage to DFNB3, DFNB9 and DFNB21 loci by genotyping the corresponding short tandem repeat (STR) markers using polymerase chain reaction (PCR) and polyacrylamide gel electrophoresis (PAGE) methods. RESULTS: GJB2 mutations (283G>A and 29delT) were causes of hearing loss in 12.5% of families with ARNSHL and no evidence of linkage were found for any of DFNB3, DFNB9 and DFNB21 loci. CONCLUSION: GJB2 mutations are associated with ARNSHL. We failed to find linkage of the DFNB3, DFNB9 and DFNB21 loci among GJB2 negative families. Therefore, further studies on large-scale population and other loci will be needed to find conclusively linkage of DFNB loci and ARNSHL in the future.

Upregulation of the double-stranded RNA binding protein DGCR8 in invasive ductal breast carcinoma.

High-throughput experimental studies have indicated that the miRNAome is globally downregulated in various types of malignancy, and dysregulation of miRNAs processing component(s) is one possible mechanism for this phenomenon. Despite the progression in identifying cellular functions of Digeorge Syndrome Critical Region 8 (DGCR8) in miRNAs biogenesis, the role of altered expression of DGCR8 in the pathogenesis of invasive ductal breast carcinoma (IDC) has not yet been fully investigated. The objective of the present study was to evaluate DGCR8 mRNA expression in seventy fresh invasive ductal breast carcinomas and matched adjacent non-neoplastic tissues using quantitative real-time PCR and to assess the value of clinicopathological parameters on its expression. Our findings revealed that DGCR8 mRNA expression is upregulated in more than two-thirds of the cancerous specimens (68.66%) when compared to adjacent non-neoplastic tissue. This difference is statistically significant (P<0.05). We found that DGCR8 mRNA levels were increased in the high-grade and metastatic compared with those of both low-grade and non-metastatic. We demonstrated that there is not significant correlation between DGCR8 mRNA expression levels and clinicopathological parameters. In conclusion, our study suggested that upregulation of DGCR8 may be involved in tumorigenesis and aggressiveness of IDC and may serve as future therapeutic target.

Dysregulated expression of Dicer in invasive ductal breast carcinoma.

Several lines of evidence suggest that the global down-regulation of the microRNAome (miRNAome) involved in pathogenesis of various malignancies. Impaired microRNAs processing pathway is one possible mechanism for global down-regulation of the miRNAome. Dicer is a key enzyme in miRNA processing pathway, and dysregulation of its expression has been suggested as a possible cause of miRNAome alterations observed in various cancers. However, Dicer mRNA expression in invasive ductal breast carcinoma (IDC) has not been investigated in depth. Therefore, this study aimed to evaluate the mRNA expression of Dicer in IDC and also to assess the correlation of its expression with clinicopathological parameters including age, histological grade, tumor size and lymph node metastasis. We investigated the expression of the Dicer in seventy fresh invasive ductal breast carcinomas and matched adjacent non-neoplastic tissue by quantitative real-time PCR using validated reference genes. In addition, the possible impact of clinicopathological characteristics on Dicer expression levels was analyzed. Our results showed that Dicer mRNA expression is down-regulated in slightly more than half (51.43 %) of the tumor specimens when compared to adjacent non-neoplastic tissue. Comparison of the Dicer expression level between tumor and matched adjacent non-neoplastic tissue showed that there is no statistical significant differences between them (P = 0.425). We also found that Dicer mRNA expression in IDC samples was not correlated with clinicopathological features. In conclusion, our findings provide additional evidence to support the hypothesis that Dicer expression down-regulated in breast cancer. This study suggested that the decreased expression of Dicer may be potential underlying mechanism in pathogenesis of IDC.

Mitochondrial complex I gene variations; as a potential genetic risk factor in pathogenesis of multiple sclerosis.

BACKGROUND AND PURPOSE: Multiple sclerosis (MS) is an autoimmune-mediated inflammatory and debilitating disease of the central nervous system. Several investigations have suggested that the mitochondrial DNA encoded subunits of complex I gene variations are involved in the progression of MS. In this study, we investigated the possible association between mitochondrial complex I gene variations and MS in a Filipino population. MATERIAL AND METHODS: A total of 300 individuals were included in the present study, two-hundred patients with MS clinical symptoms, and one-hundred healthy subjects without MS clinical features. We amplified target genes of mtDNA using polymerase chain reaction technique (PCR), and sequenced these to evaluate mitochondrial complex I gene variations. RESULTS: We found nine variations (Nt 4216 T>C, Nt 5153 A>G, Nt 10142 C>T, Nt 11353 T>C, Nt 11935 T>C, Nt 12062 C>T, Nt 13042 G>A, Nt 13708 G>A and Nt 14179 G>A) in mtDNA-encoded complex I subunit genes. Our results showed that the prevalence of ND1, ND2, ND3, ND4 and ND5 gene variations was significantly higher in patients than in healthy controls (P<0.0001). Whereas, the frequency of Nt 14179 G>A variation in ND6 gene was significantly higher in the control group compared with the patients (P<0.0001). CONCLUSION: Taken together our data supports a strongly positive association between mitochondrial complex I gene variations and MS pathogenesis in a Filipino population.

Prevalence of thrombophilic gene polymorphisms in an azari population of iran.

There is several evidence suggests that thrombophilic gene polymorphisms may influence susceptibility to thromboembolic events. The prevalence of these polymorphisms is different in various races and ethnics. Accordingly, we studied the prevalence of Factor V (G1691A and A4070G), prothrombin G20210A and PAI-1 4G/5G in healthy northwest population of Iran. In this prospective study, 500 healthy individuals, who had no history of both personal and family history of thromboembolic disorders, were selected as a sample of healthy population in northwestern Iran. Genotyping of these polymorphisms was performed using the amplification refractory mutation system-polymerase chain reaction method. No significant differences were detected between the expected and observed frequencies of FV G1691A and A4070G, prothrombin G20210A polymorphisms (P>0.05), while the expected frequency of 4G allele was significantly more than observed frequency in the studied population (P<0.01). These findings were compared with other reports from various populations. In conclusion, the allele frequency for FV G1691A and PAI-1 4G/5G polymorphisms showed relative consistency compared to those of previous studies, while the incidence pattern of FV A4070G polymorphism in Northwestern population of Iran showed conflicting results regarding other studied population. The prothrombin G20210A polymorphism was observed at a higher frequency than other studied populations.

Apolipoprotein E genotyping in women with recurrent pregnancy loss: an in silico and experimental hybrid study.

The role of apolipoprotein E gene polymorphisms in the pathogenesis of recurrent pregnancy loss remains controversial. Therefore, our objective was to investigate the association between recurrent pregnancy loss and apolipoprotein E gene polymorphisms among northwest Iranian women, and also to predict the impact of these nonsynonymous single nucleotide polymorphisms on structure and function of apolipoprotein E protein. The subjects of our current study consisted of 100 women that have had two or more consecutive idiopathic first trimester miscarriages, and one hundred healthy women from the same geographical areas were used as a control group. After DNA extraction, we used a polymerase chain reaction-restriction fragment length polymorphism to genotype of the apolipoprotein E gene. In addition, we predicted the possible effects of amino acid substitutions at codons 112 and/or 158 on the structure and function of apolipoprotein E protein using Polymorphism Phenotyping online software v2. Our results showed that the rate of apolipoprotein E ε4 carriers and the frequency of the ε4 allele in the case group were statistically and significantly higher than those in the control group (P<0.05). Therefore, our data support the association of the Apo ε4 allele with RPL; however, in silico analysis predicted that the amino acid substitution at residue 112 (Apo ε4 allele) is a benign mutation. Accordingly, further studies are required to elucidate the mechanism(s) underlying the link between RPL pathogenesis and the Apo ε4 allele.

Polymorphisms in thrombophilic genes are associated with deep venous thromboembolism in an Iranian population.

It has been revealed that the inherited thrombophilia increases the risk of thrombosis in the venous system. To study the association of factor V G1691A, factor V HR2 (4070A/G), prothrombin G20210A, and PAI-1 (- 675 I/D, 5G/4G) polymorphisms with deep venous thromboembolism (DVT), these polymorphisms were investigated. A total of 193 patients who presented clinical symptoms of deep venous thromboembolism including 103 men and 90 women, and 500 healthy individuals without both personal and family histories of thromboembolic disorders including 275 men and 225 women were recruited into the study. Genotyping was carried out using the amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) technique. Our results showed that the genotype distribution for FV (G1691A and A4070G) and PAI-1 4G/5G polymorphisms in DVT patients were significantly higher than healthy control (P < 0.05). Also, the mutant allele frequencies for all studied polymorphisms differed significantly between the case and control groups (P < 0.05). We concluded that the prevalence of FV (G1691A and A4070G) and PAI-1 4G/5G polymorphisms increased the risk of DVT occurrence in subjects. These findings provide additional evidence to support the hypothesis that thrombophilic gene polymorphisms are involved in vascular thromboembolism.

The association between thrombophilic gene mutations and recurrent pregnancy loss.

PURPOSE: To determine whether the Factor V (1691G/A), Factor V HR2 (4070A/G), Prothrombin (20210G/A), PAI-1 (-675 I/D, 5G/4G), ACE (intron 16 I/D), Factor VII (Gln353Arg), Factor XIII (Val34Leu), ß-fibrinogen (-455G/A), Glycoprotein Ia (807C/T), tPA (intron 8 D/I) gene mutations could be risk factors for recurrent pregnancy loss (RPL). METHODS: Genotyping of thrombophilic gene mutations were carried out by amplification Refractory Mutation System-PCR (ARMS-PCR) method after DNA extraction. RESULTS: We found that the mutant allele frequencies of Factor V (1691G/A), Factor V HR2 (4070A/G), Prothrombin (20210G/A), PAI-1 (-675 I/D, 5G/4G), Factor XIII (Val34Leu) and ß-fibrinogen (-455G/A) were more seen in the case group compared with the healthy control; However, the difference between the two group is not statistically significant (p > 0.05). Whilst the mutant allele frequencies of other studied genes were lower in the case in comparison to the fertile control women (p > 0.05). CONCLUSION: Taken together, our data has shown that the prevalence of thrombophilic gene mutations was similar in women with RPL and healthy controls. Therefore, it appears that further studies on large-scale population and other genetic variants will be needed to conclusively find candidate genes for RPL unknown etiology in the future.

Methylenetetrahydrofolate Reductase C677T and A1298C Mutations in Women with Recurrent Spontaneous Abortions in the Northwest of Iran.

Introduction. Recurrent spontaneous abortion (RSA) is a significant obstetrical complication that may occur during pregnancy. Various studies in recent years have indicated that two common mutations (C677T and A1298C) of the methylenetetrahydrofolate reductase (MTHFR) gene are risk factor for RSA. This study was carried out to determine the influence of (C677T and A1298C) of the methylenetetrahydrofolate reductase (MTHFR) gene mutations with RSA. Materials and Methods. A total of 139 women were included in this study: 89 women with two or more consecutive miscarriages and 50 healthy controls. Total genomic DNA was isolated from blood leukocytes. To determine the frequency of the two common C677T and A1298C MTHFR gene mutations in the patients and controls, we used two methods, amplification refractory mutation system-PCR and PCR-restriction fragment length polymorphism. Results. There is no significant difference in the prevalence of 677T/T genotype among women with RSA and healthy controls (P = 0.285). Also no statistically significant difference in the frequency of A1298C MTHFR gene mutation was detected between the two groups (P = 0.175 ). Conclusion. In conclusion, the results indicate that the Amplification Refractory Mutation System-PCR method was in complete concordance with the results obtained by standard PCR-restriction fragment length polymorphism method. The results also show no significant difference in MTHFR C677T/A1298C genotype distribution among the two groups; therefore, further studies on larger population and other genetic variants to better understand the pathobiology of RSA are needed.