Identification of ITPR1 gene as a novel target for hsa-miR-34b-5p in non-obstructive azoospermia: a Ca2+/apoptosis pathway cross-talk.

MiR-34b-5p has been reported as a non-invasive diagnostic biomarker for infertility. However, no gene targets regulating the mechanism of cation of this miRNA are known. In this study, using gene set enrichment analysis the Inositol 1,4,5-Trisphosphate Receptor Type 1 (ITPR1) gene was identified as the sole target for hsa-miR-34b-5p, and found significantly overexpressed in non-obstructive azoospermia (NOA) patients. This finding was confirmed by qRT-PCR on fresh testicular tissues from NOA patients. Then, pathway enrichment analysis as well as the diagnostic value analysis of hsa-miR-34b-5p/ITPR1 indicated ITPR1 as a hub gene in the calcium (Ca2+)-apoptosis pathway, and a valuable predictive biomarker for NOA. Moreover, gene expression and histological assays showed the association of the effects of ITPR1's increased expression on spermatogenesis failure through induction of apoptosis in NOA patients. These data suggested that the hsa-miR-34b-5p/ITPR1 axis could serve as a potential regulatory predictive biomarker for human spermatogenesis through the Ca2+-apoptosis pathway cross-talk.

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.

The LncRNA MIAT is identified as a regulator of stemness-associated transcript in glioma.

BACKGROUND: Myocardial infarction-associated transcript (MIAT) is a long non-coding RNA (lncRNA) with altered expression in different diseases and malignancies. In this study, the potential expression and function of lncRNA MIAT in intuition and progression of brain cancer was investigated. METHODS AND RESULTS: At first, TCGA data analysis demonstrated that lncRNA MIAT is significantly upregulated in various malignancies, especially its expression is dramatically elevated in brain tumors. In line with the data, we further evaluated the expression of MIAT in a series of brain tumor tissue, and our results revealed that the expression of MIAT was noticeably overexpressed in glioblastoma (p = < 0.0001). We further found that the expression of MIAT was markedly upregulated in low-grade brain tumors rather than high-grade ones. To further investigate the biological function of MIAT in brain cancer cells, its expression was suppressed by si-RNA-mediated knocking down. Inhibition of MIAT resulted in reduced proliferation of brain tumor cells followed by cell cycle arrest at the G1 phase, and significant induction of apoptosis, and senescence, but limited the migration ability and epithelial-mesenchymal-transition (EMT). Moreover, knocking-down of MIAT reduced the expression of stemness factors, followed by upregulation of their downstream miRNAs (micro RNAs), let-7a-5p, and miR-29b-3p. CONCLUSIONS: Altogether, our data demonstrated that lncRNA MIAT could control proliferation, migration, and metastasis of brain cancer cells via regulating the Nanog/ Sox2 / let-7a-5p / miR-29b-3p axis. This data could introduce lncRNA MIAT as a novel oncogene in brain cancer pathogenesis.

AOPEP variants as a novel cause of recessive dystonia: Generalized dystonia and dystonia-parkinsonism.

INTRODUCTION: The genetic basis of autosomal-recessive dystonia remains poorly understood. Our objective was to report identification of additional individuals with variants in AOPEP, a recently described gene for recessively inherited dystonic disorders (OMIM:619565). METHODS: Ongoing analysis on a high-throughput genetic platform and international case-recruitment efforts were undertaken. RESULTS: Novel biallelic, likely pathogenic loss-of-function alleles were identified in two pedigrees of different ethnic background. Two members of a consanguineous Iranian family shared a homozygous c.1917-1G>A essential splice-site variant and featured presentations of adolescence-onset generalized dystonia. An individual of Chinese descent, homozygous for the nonsense variant c.1909G>T (p.Glu637*), displayed childhood-onset generalized dystonia combined with later-manifesting parkinsonism. One additional Iranian patient with adolescence-onset generalized dystonia carried an ultrarare, likely protein-damaging homozygous missense variant (c.1201C>T [p.Arg401Trp]). CONCLUSIONS: These findings support the implication of AOPEP in recessive forms of generalized dystonia and dystonia-parkinsonism. Biallelic AOPEP variants represent a worldwide cause of dystonic movement-disorder phenotypes and should be considered in dystonia molecular testing approaches.

LncRNA LOC100507144 acts as a novel regulator of CD44/Nanog/Sox2/miR-302/miR-21 axis in colorectal cancer.

Long noncoding RNAs (lncRNAs) appear as vital regulators and biomarkers in many human cancers. LOC100507144 is a validated lncRNA located in the neighborhood of CD44 in a head-to-head configuration, and its expression and function in cancer cells are still unknown. This research aimed to find out more about the expression and function of this lncRNA in colorectal cancer (CRC). Our expression data represented that the expression of LOC100507144 transcript was substantially higher in tumors with advanced stages, lymph node metastasis, and vascular invasion. Loss-of-function examinations demonstrated that LOC100507144 contributed to CRC cell proliferation by restricting apoptosis, cellular senescence, and promoting cell cycle. Gain-of-function experiments also confirmed these results. Our data illustrated that LOC100507144 enhanced the migration and the epithelial to mesenchymal transition (EMT) of CRC cells, accompanied by the generation of cells with stemness characteristics. Our findings revealed that the knocking-down of LOC100507144 inhibited the expression of crucial stemness factors, including CD44, Nanog, and Sox2, and accordingly resulted in suppressing their targets, miR-302 and miR-21. Overall, the current study's findings for the first time reveal that LOC100507144 could enhance CRC progression and metastasis through regulation of the CD44/Nanog/Sox2/miR-302/miR-21 axis.

Meta-analysis of gene signatures and key pathways indicates suppression of JNK pathway as a regulator of chemo-resistance in AML.

The pathways and robust deregulated gene signatures involved in AML chemo-resistance are not fully understood. Multiple subgroups of AMLs which are under treatment of various regimens seem to have similar regulatory gene(s) or pathway(s) related to their chemo-resistance phenotype. In this study using gene set enrichment approach, deregulated genes and pathways associated with relapse after chemotherapy were investigated in AML samples. Five AML libraries compiled from GEO and ArrayExpress repositories were used to identify significantly differentially expressed genes between chemo-resistance and chemo-sensitive groups. Functional and pathway enrichment analysis of differentially expressed genes was performed to assess molecular mechanisms related to AML chemotherapeutic resistance. A total of 34 genes selected to be differentially expressed in the chemo-resistance compared to the chemo-sensitive group. Among the genes selected, c-Jun, AKT3, ARAP3, GABBR1, PELI2 and SORT1 are involved in neurotrophin, estrogen, cAMP and Toll-like receptor signaling pathways. All these pathways are located upstream and regulate JNK signaling pathway which functions as a key regulator of cellular apoptosis. Our expression data are in favor of suppression of JNK pathway, which could induce pro-apoptotic gene expression as well as down regulation of survival factors, introducing this pathway as a key regulator of drug-resistance development in AML.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

hsa-miR-423 rs6505162 Is Associated with The Increased Risk of Breast Cancer in Isfahan Central Province of Iran.

OBJECTIVE: Thirteen million cancer deaths and 21.7 million new cancer cases are expected in the world by 2030. Breast cancer is considered as the main cause of cancer mortality in women aged 20-59 years. microRNAs (miRNAs) regulate gene expression at the post-transcriptional level and they are highly expressed in malignancies, including breast cancer. The role of miRNAs in the pathogenesis of breast cancer is not fully understood. In the present study, for the first time, the impact of hsa-miR-423 rs6505162 on breast cancer risk was investigated in the central province of Iran, Isfahan. MATERIALS AND METHODS: This case-control study was conducted on 153 clinicopathological proven breast cancer patients and 153 sex-matched healthy women with no history of any cancer type and relative patients. The patients and controls were genotyped and association of their clinical characteristics with hsa-miR-423 rs6505162 genotype was analyzed. RESULTS: The findings indicated that CC genotype of hsa-miR-423 rs6505162 was associated with the increased risk of breast cancer [odds ratio (OR)=2.37, 95% confidence interval (CI)=1.29-4.35 and P=0.0023, CC vs. AA]. CONCLUSION: The data suggested that hsa-miR-423 rs6505162 could be considered as a novel risk factor in breast cancer pathogenesis in Isfahan province of Iran.

CG/CA genotypes represent novel markers in the NPHS2 gene region associated with nephrotic syndrome.

Nephrotic syndrome (NS) is considered as a primary disease of the kidney that represents a heterogeneous group of glomerular disorders occurring mainly in children. It is generally divided into steroid-sensitive and steroid-resistant forms, depending upon the patient's response to steroid therapy. Among the genes involved, the NPHS2 gene has been reported as the causative gene in steroid resistant form of nephrotic syndrome. In the present study, heterozygosity rate, allelic frequency and linkage of rs2274625 and rs3829795 markers were investigated in the NPHS2 gene region. To determine the SNP alleles, tetra-primer ARMS PCR was used. After genotyping rs2274625 and rs3829795 polymorphic markers in 120 unrelated individuals and nine trios families, the data were analysed using various computer programs such as UCSC Genome Browser, dbSNP and SNPper. Based on the statistical analysis of the results, for rs2274625 marker, allele frequency for C and T alleles was 97% and 3%, respectively. For rs3829795 marker allele frequency for G and A alleles was 55% and 45%, respectively. The values of heterozygosity index for the examined markers were 5% for rs2274625 and 45/8% for rs3829795. Consequently, two informative haplotypes, CG/CA, were identified in the NPHS2 gene region through combination of these two markers. These haplotypes can serve as appropriate tools for the identification of heterozygous carriers and linkage analysis of nephrotic syndrome disease in the Iranian families with an affected child.

Significant Association of miR-605 rs2043556 with Susceptibility to Breast Cancer.

BACKGROUND: MicroRNAs (miRNAs) are noncoding RNA molecules, which directly regulate gene expression. It has been documented that single nucleotide polymorphisms in miRNA genes could alter the regulation of miRNA expression and function. OBJECTIVE: In this study, the allele and genotype frequency of miR-605 rs2043556 and its association with breast cancer were investigated in the Iranian population. METHODS: Genotyping was performed in 162 females affected with breast cancer and 180 healthy individuals. Genotyping was performed using Restriction Fragment Length Polymorphism (RFLP) followed by Sanger sequencing. RESULTS: The data showed the presence of Hardy Weinberg equilibrium (HWE) for this marker in the Iranian population. Allelic frequency for A and G allele was 0.75 and 0.25, respectively. Odd ratios for the association between miR-605 rs2043556 AG/GG genotypes was 3.86 with p-value= 0. CONCLUSION: The results indicated an increased risk for breast cancer susceptibility for miR-605 rs2043556 in the Iranian population.

A comprehensive study of phenylalanine hydroxylase gene mutations in the Iranian phenylketonuria patients.

Phenylketonuria (PKU) is a metabolic disorder caused by mutations in the phenylalanine hydroxylase (PAH) gene. After thalassemia, PKU is considered as the most common autosomal recessive diseases in the Iranian population. Therefore, an efficient diagnostic strategy is required to identify disease-causing mutations in this population. Following our first report in 2003, here we presented a comprehensive study on the mutation spectrum of the PAH gene in the Iranian population. This study was performed on 280 unrelated chromosomes from 140 Iranian patients with classic PKU. All 13 exons as well as exon-intron boundaries of the PAH gene were analyzed by direct DNA sequencing. Thirty four different mutations were identified by a mutation detection rate of 100%. IVS10-11G > A, p.P281L, R261Q, p.F39del and IVS11+1G > C were the most prevalent mutations with frequencies of 26.07%, 19.3%, 12.86%, 6.07 and 3.93%, respectively. All other mutations represented a relative frequency less than 3.5%. The data from this study provided a comprehensive spectrum of the PAH gene mutations which can facilitate carrier detection and prenatal diagnosis of PKU disease in the Iranian population.

A novel compound-primed multiplex ARMS-PCR (CPMAP) for simultaneous detection of common PAH gene mutations.

In this study, we introduce a novel compound-primed multiplex ARMS PCR (CPMAP) for simultaneous detection of common PAH gene mutations. This approach was used successfully for simultaneous identification of six most common PAH gene mutations in 137 phenylketonuria patients in the Iranian population. A total of six normal and six mutant allele-specific primers and 4 common primers containing a tag sequence of 12 base pair at the 5'-end were designed and used in two separate optimized multiplex ARMS reactions followed by hot-start PCR. The products were separated and visualized on 3% agarose gel. The CPMAP genotyping data were completely in accordance with the direct sequencing results. The CPMAP suggests a reliable, economical and rapid method for simultaneous detection of PAH point mutations using conventional PCR, which could be applied for diagnosis of other gene mutations.

UGT1A1 gene linkage analysis: application of polymorphic markers rs4148326/rs4124874 in the Iranian population.

OBJECTIVES: Mutations in the UGT1A1 gene are responsible for hyperbilirubinemia syndromes including Crigler-Najjar type 1 and 2 and Gilbert syndrome. In view of the genetic heterogeneity and involvement of large numbers of the disease causing mutations, the application of polymorphic markers in the UGTA1 gene could be useful in molecular diagnosis of the disease. MATERIALS AND METHODS: In the present study, two polymorphic markers including rs4148326 and rs4124874 in the UGT1A1 gene region were characterized. The markers were selected using bioinformatics analysis of the UGT1A1 gene region and genotyped in 212 unrelated healthy individuals and 13 family trios in the Iranian population using Tetra-Primer ARMS PCR technique. The allele frequency and population status of the alleles were estimated using GENEPOP, FBAT, PowerMarker and Arlequin software. RESULTS: The results indicated that in the case of rs4148326 marker, allele frequency for T and C allele was 66.04% and 33.96%, respectively. For rs4124874 marker, allele frequency for G and T alleles was 39.4% and 60.6%, respectively. The values of heterozygosity index for the markers examined were 64.1 for rs4148326 and 72.1 for rs4124874, respectively. The haplotype estimation analysis of the markers resulted in three informative haplotypes with frequencies ≥0.05. Moreover, the results suggested the presence of linkage disequilibrium between two markers. CONCLUSION: Altogether, the data suggested that rs4148326 and rs4124874 could be introduced as informative markers for molecular diagnosis of Crigler-Najjar type 1 and 2 and Gilbert syndrome in the Iranian population.

F1174V mutation alters the ALK active conformation in response to Crizotinib in NSCLC: Insight from molecular simulations.

Crizotinib is an efficient antineoplastic drug for treatment of non-small cell lung carcinoma (NSCLC), which is identified as an anaplastic lymphoma kinase (ALK) inhibitor. F1174V is a recently identified acquired point mutation relating to the Crizotinib resistance in NSCLC patients. The mechanism of Crizotinib resistance relating to F1174V mutation as a non-active site mutation remains unclear. In this study, the molecular dynamic simulation was used to investigate the possible mechanisms by which F1174V mutation may affect the structure and activity of ALK kinase domain. The results suggested that F1174V mutation could cause two important secondary structure alterations, which led to the local conformational change in ALK kinase domain. This causes more positive free energy in the mutant complex in comparison with the wild-type one. In addition, our structural analyses illustrated that F1174V mutation could result in some important interactions, which represent the key characteristics of the ALK active conformation. This study provided a molecular mechanism for ALK Crizotinib resistance caused by F1174V mutation,which could facilitate designing more efficient drugs.

Comparative genomics of human stem cell factor (SCF).

Stem cell factor (SCF) is a critical protein with key roles in the cell such as hematopoiesis, gametogenesis and melanogenesis. In the present study a comparative analysis on nucleotide sequences of SCF was performed in Humanoids using bioinformatics tools including NCBI-BLAST, MEGA6, and JBrowse. Our analysis of nucleotide sequences to find closely evolved organisms with high similarity by NCBI-BLAST tools and MEGA6 showed that human and Chimpanzee (Pan troglodytes) were placed into the same cluster. By using JBrowse, we found that SCF in Neanderthal had a single copy number similar to modern human and partly conserved nucleotide sequences. Together, the results approved the gene flow and genetics similarity of SCF among human and P. troglodytes. This may suggest that during evolution, SCF gene transferred partly intact either on the basis of sequence or function from the same ancestors to P. troglodytes, the ancient human like Neanderthal, and then to the modern human.

Association of rs1738074 polymorphism of TAGAP gene with susceptibility to multiple sclerosis in the Iranian population.

Multiple sclerosis (MS) is one of the most common diseases of the central nervous system (CNS) in the Iranian population. To date, association of many genes with the prevalence and progression of the disease have been investigated. In the present study, the impact of rs1738074 single nucleotide polymorphism (SNP) in the TAGAP gene (TAGAP rs1738074) on the risk of MS was evaluated in a sample of the Iranian population. In a case control study, genotyping was performed on 300 patients and normal individuals. The data were analyzed using Pearson's chi-square test. The results showed a significant difference in the SNP frequency between case and control groups (p-value=0.049). The genotype frequencies of TT, TC and CC in patients were 10.67%, 51.33% and 38%, respectively, and in normal individuals were 20.66%, 42.67% and 36.67%, respectively. The results showed a significant difference in the genotype frequency of T/T between the patient and control groups (p<0.05). Interestingly, individuals with T/T genotype were estimated to be less susceptible to MS ((p-value=0.025), Fisher's exact test), odd ratio was 2.18 (controls versus MS patients) with 95% CI: 1.137-4.187. The results suggested that TAGAP rs1738074 polymorphism could be considered as a risk factor in the prevalence of MS in the Iranian population.

The pros and cons of the fourth revision of thalassaemia screening programme in Iran.

Objective To evaluate the repercussions of recent changes to the cut-offs used in the first screening step of the pre-marital screening programme for thalassaemia prevention in Iran. Methods The profiles of 984 subjects referred to a genetic laboratory, and the tests of 242 parents of children with thalassaemia major were assessed for red blood cell (RBC) indices, haemoglobin (Hb) A2 levels and results of Hb electrophoresis. Results Of 407 suspected thalassaemia minor (STM) cases, 18 proved positive for thalassaemia minor on molecular analysis (18/407, confidence interval 2.6-6.9%). If the revised screening cut-offs had been used to determine who would undergo molecular analysis, two of these cases would not have been identified. Only 4.4% of suspected cases with lower than normal RBC indices (mean corpuscular volume <80 fl and mean corpuscular Hb <27 pg) and HbA2 (<3.5%) were diagnosed with thalassaemia minor. Conclusion The thalassaemia major prevention programme is performed in two separate steps. One step involves the screening of subjects and identification of ß-thalassaemia minor, suspected cases for thalassaemia minor (STM), and normal subject groups. The other step concerns the identification of thalassaemia minor in the STM group. Changing the cut-offs at the first screening step does not result in significant improvement from an economic view, and is associated with significant risk at the second screening step.

Studies on Non-synonymous Polymorphisms Altering Human DNA Topoisomerase II-Alpha Interaction with Amsacrine and Mitoxantrone: An In Silico Approach.

BACKGROUND: DNA topoisomerase II-α (Top2-α), an essential enzyme for the management of DNA during replication, transcription, recombination, and chromatin remodeling, is one of the most important anticancer targets. Numerous molecules have been designed as Top2-α inhibitors. However, several studies have shown that polymorphisms and mutations in Top2 have conferred resistance to most of these anticancer drugs. The aim of this study was to computationally examine the mechanisms by which genomic variations in Top2-α could affect its resistance to Amsacrine and Mitoxantrone as important inhibitors of the enzyme. RESULTS: The results showed that variants K529E, R568H, R568G and T530M could affect Top2-α inhibition by Amsacrine causing possible drug-resistant. Moreover, R487K, and Y481C variants could change the response of the enzyme to Mitoxantrone. CONCLUSION: These results could facilitate the prediction and development of more effective drugs for Top2-α variants, making the cancer chemotherapy more effectiv.

D5S351 and D5S1414 located at the spinal muscular atrophy critical region represent novel informative markers in the Iranian population.

Spinal muscular atrophy (SMA) is a degenerative neuromuscular disease associated with progressive symmetric weakness and atrophy of the limb muscles. In view of the involvement of numerous point mutations and deletions associated with the disease, the application of polymorphic markers flanking the SMA critical region could be valuable in molecular diagnosis of the disease. In the present study, D5S351 and D5S1414 polymorphic markers located at the SMA critical region in the Iranian populations were characterized. Genotyping of the markers indicated the presence of six and nine different alleles for D5S351 and D5S1414, respectively. Haplotype frequency estimation in 25 trios families and 75 unrelated individuals indicated the presence of six informative haplotypes with frequency higher than 0.05 in the studied population. Furthermore, the D' coefficient and the χ(2) value for D5S351 and D5S1414 markers revealed the presence of linkage disequilibrium between the two markers in the Iranians. These data suggested that D5S351 and D5S1414 could be suggested as informative markers for linkage analysis and molecular diagnosis of SMA in the Iranian population.