Exome sequencing identifies novel variants associated with non-syndromic hearing loss in the Iranian population.

Autosomal recessive non-syndromic hearing loss (ARNSHL) is a public health concern in the Iranian population, with an incidence of 1 in 166 live births. In the present study, the whole exome sequencing (WES) method was applied to identify the mutation spectrum of NSHL patients negative for GJB2 gene mutations. First, using ARMS PCR followed by Sanger sequencing of the GJB2 gene, 63.15% of mutations in patients with NSHL were identified. Among the identified mutations in GJB2:p.Val43Met and p.Gly21Arg were novel. The remaining patients were subjected to WES, which identified novel mutations including MYO15A:p.Gly39LeufsTer188, ADGRV1:p.Ser5918ValfsTer23, MYO7A: c.5856+2T>c (splicing mutation), FGF3:p.Ser156Cys. The present study emphasized the application of WES as an effective method for molecular diagnosis of NSHL patients negative for GJB2 gene mutations in the Iranian population.

Association of DROSHA rs6877842, rs642321 and rs10719 polymorphisms with increased susceptibility to breast cancer: A case-control study with genotype and haplotype analysis.

BACKGROUND: Multiple lines of evidence suggest that single nucleotide polymorphisms (SNPs) in genes encoding components of the microRNA processing machinery may underlie susceptibility to various human diseases, including cancer. OBJECTIVE: The present study aimed to investigate whether rs6877842, rs642321 and rs10719 SNPs of DROSHA, a key component of the miRNA biogenesis pathway, are associated with increased risk of breast cancer. METHODS: A total of 100 patients diagnosed with breast cancer and 100 healthy women were included. Following extraction of DNA, genotyping was performed by tetra primer- amplification refractory mutation system-PCR (T-ARMS-PCR) technique. Under the co-dominant, dominant and recessive inheritance models, the association between DROSHA SNPs and breast cancer risk was determined by logistic regression analysis. The association of DROSHA SNPs with patients' clinicopathological parameters was assessed. Also, haplotype analysis was performed to evaluate the combined effect of DROSHA SNPs on breast cancer risk. RESULTS: We observed a statistically significant association between DROSHA rs642321 polymorphism and breast cancer susceptibility (P < 0.05). Under the dominant inheritance model, DROSHA rs642321 polymorphism was significantly associated with increased risk of breast cancer (OR: 6.091; 95% CI: 3.291-11.26; P = 0.0001). Our findings demonstrated that DROSHA rs642321 T allele can contribute to the development of breast cancer (OR: 3.125; 95% CI: 1.984-4.923; P = 0.0001). We also found that GTC and GTT haplotypes conferred significant risk for breast cancer (OR: 2.367; 95% CI: 1.453-3.856; P = 0.0001 and OR: 7.944; 95% CI: 2.073-30.43; P = 0.0001, respectively). CONCLUSIONS: These results provide the first evidence that DROSHA rs642321 polymorphism is associated with increased risk of breast cancer. However, further studies are needed to firmly validate these findings.

A rapid and simple method for simultaneous determination of three breast cancer related microRNAs based on magnetic nanoparticles modified with S9.6 antibody.

Cancer progression is typically associated with the simultaneous changes of multiple microRNA (miR) levels. Therefore, simultaneous determination of multiple miR biomarkers exhibits great promise in early diagnosis of cancers. This research seeks to discuss a simple biosensing method for the ultrasensitive and specific detection of the three miRs related to the breast cancer based on S9.6 antibody coated magnetic beads, titanium phosphate nanospheres, and screen-printed carbon electrode. To prepare signaling probes, three hairpin DNAs (hDNAs) were labeled with three encoding titanium phosphate nanospheres with large quantities of different heavy metal ions (zinc, cadmium, lead), which have been utilized to discriminate the signals of three microRNA targets in relation with the corresponding heavy metal ions. After that, these hairpin structures hybridize with miR-21, miR-155 and miR-10b to form miR-21/hDNA1, miR-155/hDNA2 and miR-10b/hDNA3 complexes, which were captured by S9.6 antibodies (one anti-DNA/RNA antibody) pre-modified on magnetic bead surface. Therefore, the specific preconcentration of targets from complex matrixes can be carried out using magnetic actuation, increasing the sensitivity and specificity of the detection. The biosensor was suitably applied for direct and rapid detection of multiple microRNAs in real sample. It was observed that there were no significant differences between the results obtained by the suggested method and qRT-PCR as a reference method. So, this method makes an ultrasensitive novel platform for miRNAs expression profiling in clinical diagnosis and biomedical research.

A novel signal amplification tag to develop rapid and sensitive aptamer-based biosensors.

Determination of microRNAs (miRNAs) as valuable blood-borne biomarkers has attracted many scientific attentions. However, analytical methods are still restricted by miRNAs intrinsic characteristics. In this study, for the first time, novel blackberry-like magnetic DNA/FMMA nanospheres were synthesized and mounted on a gold stir-bar as signal amplification probes. To produce this strong electrochemical signal label, double strand DNAs were immobilized on gold coated magnetic nanospheres through a hybridization chain reaction followed by reversible addition-fragmentation chain-transfer polymerization, which brought a great quantity of the electroactive tags (FMMA) on the nanosphere surface. These nanospheres were then fixed on the gold stir-bar as signal probes. The magnetic DNA/FMMA nanosphere probes can be released by substituting with the newly emerging DNA fragments of catalyzed hairpin assembly products. Eventually, these signal probes were magnetically enriched on the electrode surface to produce electrochemical signal and finally, the biosensor was developed to detect miRNA-106a (model target). The suggested aptamer-based biosensor demonstrated considerable selectivity, acceptable storage stability, high specificity, and excellent performance in real sample analysis without any pretreatments. As a result, current study reveals that the developed strategy has a great potential for the early diagnosis of gastric cancer and additionally the clinical monitoring of any miRNA sequences.

Identification of Novel miRNAs in the F8 Gene Via Bioinformatics Tools.

BACKGROUND: Hemophilia A is an X-linked bleeding disorder resulting in a deficiency of plasma clotting factor VIII and caused by mutations in the FVIII gene (F8 gene). MicroRNAs (miRNAs) in body fluids are promising biomarker candidates for Hemophilia A, due to their stability in body fluids and accessibility by non- or minimally-invasive procedures. Therefore; Advances in miRNA analysis methods resulted in a wide range of publications on miRNAs as putative biomarkers. OBJECTIVE: Here we tried to scan the F8 gene region to predict a novel miRNA and identify it as a regulator of the F8 gene. MATERIALS AND METHODS: To this aim, the ability to express novel miRNAs in F8 locus was assessed via reliable bioinformatics databases such as SSCprofiler, RNAfold, miREval, FOMmiR, MaturBayes, miRFIND, UCSC genome browser, Deep Sequencing, and miRBase. RESULTS: Data analysis from the relevant databases offers one stem-loop structure that is predicted to express a novel miRNA. CONCLUSIONS: The diagnosis of Hemophilia A with the help of these types of biomarkers is a non-invasive procedure that has been demonstrated to have a significant role in the early diagnosis of the disease. Hopefully, the proposed candidate sequence will be confirmed in vitro and become a non-invasive biomarker in the near future.

Experimental validation of a predicted microRNA within human FVIII gene.

Hemophilia A is an X-linked bleeding disorder that occurs due to the deficiency of Factor VIII (FVIII) protein clotting activity. The mutations in the F8 gene, which encodes FVIII coagulating protein have been widely reviewed. However, there is a wide range of criteria that in addition to F8 gene mutations, different molecular mechanisms may be associated with hemophilia A. Various functions of FVIII could be related to the hypothetical small non-coding RNAs, located within the F8 gene sequence. Therefore, miRNAs that can post-transcriptionally regulate gene expression might confer susceptibility to developing hemophilia A. Here, we have selected a bioinformatically predicted hairpin structure sequence in the first intron of the F8 gene that has the potential to produce a real miRNA (named put-miR1). We tried to experimentally detect the predicted miRNA via RT-PCR following its precursor overexpression in HEK 293 cell lines. Despite the accuracy of miRNA prediction, according to the reliable bioinformatics studies, we couldn't confirm the existence of considered mature miRNA in transfected cells. We hope that through changing experimental conditions, designing new primers, or altering cell lines and expression vectors, the exogenous and endogenous expression of the predicted miRNA will be confirmed.

MicroRNA and Hemophilia-A Disease: Bioinformatics Prediction and Experimental Analysis.

OBJECTIVE: Hemophilia-A is a common genetic abnormality resulted from decreased or lack of factor VIII (FVIII) pro-coagulant protein function caused by mutations in the F8 gene. Majority of molecular studies consider screening of mutations and their relevant impacts on the quality and expression levels of FVIII. Interestingly, some of the functions in FVIII suggest a probable involvement of small non-coding RNAs embedded within the sequence of F8 gene. Therefore, microRNAs which are encoded within the F8 gene might have a role in hemophilia development. In this study, miRNAs production in the F8 gene was investigated by bioinformatics prediction and experimental validation. MATERIALS AND METHODS: In this experimental study, bioinformatics tools have been utilized to seek the novel microRNAs inserted within human F8 gene. The ability to express new microRNAs in F8 locus was studied through reliable bioinformatics databases such as SSCProfiler, RNA fold, miREval, miR-FIND, UCSC genome browser and miRBase. Then, expression and processing of the predicted microRNAs were examined based on bioinformatics methods, in the HEK293 cell lines. RESULTS: We are unable to confirm existence of the considered mature microRNAs in the transfected cells. CONCLUSION: We hope that through changing experimental conditions, designing new primers or altering cell lines as well as the expression of vectors, exogenous and endogenous expressions of the predicted miRNA will be confirmed.

Multiplex PCR based screening for micro/partial deletions in the AZF region of Y-chromosome in severe oligozoospermic and azoospermic infertile men in Iran.

BACKGROUND: Infertility is a health problem which affects about 10-20% of married couples. Male factor infertility is involved approximately 50% of infertile couples. Most of male infertility is regarding to deletions in the male-specific region of the Y chromosome. OBJECTIVE: In this study, the occurrence of deletions in the AZF region and association between infertility and paternal age were investigated in Iranian men population. MATERIALS AND METHODS: To assess the occurrence of Y chromosomal microdeletions and partial deletions of the AZF region, 100 infertile men and 100 controls with normal spermatogenesis were analyzed. AZFa, AZFb, AZFc and partial deletions within the AZFc region were analyzed using multiplex PCR method. Finally, the association between paternal age and male infertility was evaluated. RESULTS: No AZFa, AZFb or AZFc deletions were found in the control group. Seven infertile men had deletions as the following: one AZFb, five AZFc, and one AZFab. Partial deletions of AZFc (gr/gr) in 9 of the 100 infertile men (9/100, 9%) and 1 partial AZFc deletions (gr/gr) in the control group (1/100, 1%) were observed. In addition, five b2/b3 deletions in five azoospermic subjects (5/100, 5%) and 2 partial AZFc deletions (b2/b3) in the control group (2/100, 2%) were identified. Moreover, the risk of male infertility was influenced by the paternal age. CONCLUSION: The results of this study suggested that the frequency of Y chromosome AZF microdeletions increased in subjects with severe spermatogenic failure and gr/gr deletion associated with spermatogenic failure.

Association between XPD (Lys751G1n) Polymorphism and Lung Cancer Risk: A Population-Based Study in Iran.

OBJECTIVE: People are usually susceptible to carcinogenic aromatic amines, present in cigarrette smoke and polluted environment, which can cause DNA damage. Therefore, maintenance of genomic DNA integrity is a direct result of proper function of DNA repair enzymes. Polymorphic diversity could affect the function of repair enzymes and thus augment the risk of different cancers. Xeroderma pigmentosum group D (XPD) gene encodes one of the most prominent repair enzymes and the polymorphisms of this gene are thought to be of importance in lung cancer risk. This gene encodes the helicase, which is a component of transcription factor IIH and an important part of the nucleotide excision repair system. Studies reveal that individuals with Lys751Gln polymorphism of XPD gene have a low repairing capacity to delete the damages of ultraviolet light among other XPD polymorphisms. MATERIALS AND METHODS: In this case-control study, first Lys751Gln polymorphism was genotyped, then its association with lung cancer risk was analyzed. Genomic DNA was extracted from the whole blood sample of 640 individuals from Iran (352 healthy individuals and 288 patients). Allele frequencies and heterozygosity of Lys751Gln polymorphism were determined using polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: According to statistical analyses, lung cancer risk in individuals with Lys751Gln polymorphism (Odd Ratio=1.8, 95% Confidence Interval 0.848-3.819) is approximately twice as high as that of Lys/Lys genotype, however 751Gln/Gln genotype did not relate to lung cancer risk (Odd Ratio=0.7, 95% Confidence Interval 0/307-1/595). CONCLUSION: This study suggests that heterozygous polymorphism (Lys/Gln) increases the sensitivity of lung cancer risk, while homozygous polymorphism (Lys/Lys) probably decreases its risk and C allele frequency shows no remarkable increase in the patients.

BanI/D13S141/D13S175 represents a novel informative haplotype at the GJB2 gene region in the Iranian population.

Non-syndromic sensorineural hearing loss (NSHL) represents the most common cause of hearing loss in the Iranian patients. In view of the large numbers of mutations identified in GJB2, mutations analysis of the gene has been time-consuming and cost-ineffective. Alternatively, molecular markers that are highly linked to the GJB2 gene have proven to be useful in carrier detection and prenatal diagnosis of NSHL families. These markers usually show a population-dependent-based haplotype frequency. However, to date, no information on the genotyping and frequency of the markers is present for the Iranian population. In this study, genotyping and analysis of the haplotype frequency of three markers, including BanI, D13S141, and D13S175, at the GJB2 region were investigated. The haplotype frequency was estimated using PHASE program. The input data contained two alleles (+ and -) for BanI, four alleles for D13S141, and seven alleles for D13S175. Among the 42 possible haplotypes examined, four haplotypes showed relatively high frequencies (≥5%). Therefore, a combination of BanI/D13S141/D13S175 could be suggested as an informative haplotype for possible carrier detection and prenatal diagnosis of NSHL in the Iranian population.