Biallelic variants in ZNF142 lead to a syndromic neurodevelopmental disorder.

Biallelic variants of the gene encoding for the zinc-finger protein 142 (ZNF142) have recently been associated with intellectual disability (ID), speech impairment, seizures, and movement disorders in nine individuals from five families. In this study, we obtained phenotype and genotype information of 26 further individuals from 16 families. Among the 27 different ZNF142 variants identified in the total of 35 individuals only four were missense. Missense variants may give a milder phenotype by changing the local structure of ZF motifs as suggested by protein modeling; but this correlation should be validated in larger cohorts and pathogenicity of the missense variants should be investigated with functional studies. Clinical features of the 35 individuals suggest that biallelic ZNF142 variants lead to a syndromic neurodevelopmental disorder with mild to moderate ID, varying degrees of delay in language and gross motor development, early onset seizures, hypotonia, behavioral features, movement disorders, and facial dysmorphism. The differences in symptom frequencies observed in the unpublished individuals compared to those of published, and recognition of previously underemphasized facial features are likely to be due to the small sizes of the previous cohorts, which underlines the importance of larger cohorts for the phenotype descriptions of rare genetic disorders.

Novel variants underlying autosomal recessive neurodevelopmental disorders with intellectual disability in Iranian consanguineous families.

BACKGROUND: Intellectual disability (ID) is a heterogeneous group of neurodevelopmental disorders that is characterized by significant impairment in intellectual and adaptive functioning with onset during the developmental period. Whole-exome sequencing (WES)-based studies in the consanguineous families with individuals affected with ID have shown a high burden of relevant variants. So far, over 700 genes have been reported in syndromic and non-syndromic ID. However, genetic causes in more than 50% of ID patients still remain unclear. METHODS: Whole-exome sequencing was applied for investigation of various variants of ID, then Sanger sequencing and in silico analysis in ten patients from five Iranian consanguineous families diagnosed with autosomal recessive neurodevelopmental disorders, intellectual disability, performed for confirming the causative mutation within the probands. The most patients presented moderate-to-severe intellectual disability, developmental delay, seizure, speech problem, high level of lactate, and onset before 10 years. RESULTS: Filtering the data identified by WES, two novel homozygous missense variants in FBXO31 and TIMM50 genes and one previously reported mutation in the CEP290 gene in the probands were found. Sanger sequencing confirmed the homozygote variant's presence of TIMM50 and FBXO31 genes in six patients and two affected siblings in their respective families. Our computational results predicted that the variants are located in the conserved regions across different species and have the impacts on the protein stability. CONCLUSION: Hence, we provide evidence for the pathogenicity of two novel variants in the patients which will expand our knowledge about potential mutation involved in the heterogeneous disease.

Hydro-alcoholic Extract of Achillea Wilhelmsii C. Koch Reduces the Expression of Cell Death-Associated Genes while Inducing DNA Damage in HeLa Cervical Cancer Cells.

BACKGROUND: Achillea wilhelmsii C. Koch hydroalcoholic extract (AWHE) is proven to induce cell death. Previous studies suggested that AWHE is an effective inhibitor against the proliferation of prostate cancer cells. The present study aimed to evaluate possible alterations of cell death-associated genes and determine the growth inhibitory activity of AWHE on HeLa cervical cancer cells. METHODS: The antiproliferative activity of AWHE was tested using the tetrazolium dye-based colorimetric assay (MTT assay). The mRNA levels of Vascular endothelial growth factor (VEGF), caspase-3, and Breast Cancer Susceptibility gene 1 (BRCA1) were measured using the real-time Polymerase Chain Reaction method. The in-cell levels of phosphorylated H2AX were determined using the in-cell ELISA method. The data were analyzed using the non-parametric ANOVA and Friedman tests. P<0.05 was considered statistically significant. RESULTS: Based on the MTT assay, The half-maximal inhibitory concentration and 81.99 µg/mL, respectively. The mRNA levels of BRCA1 increased after 12 and 24 hours of treatment (P<0.001), while the mRNA levels of VEGF significantly decreased after 12 hours (P=0.003) and 24 hours (P=0.001). Caspase-3 expression was increased in the HeLa cells after 6 and 12 hours (P<0.001) whereas γ-H2AX levels significantly increased after 24 and 48 hours of treatment (P<0.001). CONCLUSION: AWHE possesses growth inhibitory activity by altering the expression of cell death-associated genes. Using extracts from herbal plants may provide alternative strategies to be deployed in the fight against cancer.

Association of KIF26B and COL4A4 gene polymorphisms with the risk of keratoconus in a sample of Iranian population.

PURPOSE: Keratoconus (KTCN) is a congenital corneal eye disorder which correlates with abnormal distribution of the collagen fiber and causes loss of visual acuity. COLA4A gene has a substantive role in collagen synthesis, whereas KIF26B as a new candidate gene belonging to kinesin superfamily (KIFs) has been suggested to be associated with this disease. So, in this preliminary study, we simultaneously evaluated the effects of two single nucleotide polymorphisms, 222855rs7C/T and rs12407427C/T, on KTCN susceptibility in a sample of Iranian population. METHODS: The present case-control study consists of 144 patients confirmed with KTCN and 153 healthy controls. The variants are genotyped by using amplification refractory mutation system-polymerase chain reaction method. RESULTS: The findings disclosed that rs2228557C/T and rs12407427C/T polymorphisms significantly increased the risk of KTCN in measured (codominant1; p = 0.0001, codominant2; p = 0.0001, codominant3; p = 0.0006, dominant; p = 0.0001, over-dominant; p = 0.0005) and (codominant1; p = 0.0001, codominant3; p = 0.0005, recessive; p = 0.0001) inheritance patterns, respectively. CONCLUSION: Our results did prove a statistical association of both rs2228557 and rs12407427 genotypes (TT and CT + CC) and allele (T) with KTCN susceptibility in Iranian population. Further studies in other ethnicities are required to verify our results.

Association of TMPRSS2 Gene Polymorphisms with COVID-19 Severity and Mortality: a Case-Control Study with Computational Analyses.

Coronavirus disease 2019 (COVID-19) is a severe disease caused by a new variant of beta-coronavirus that first appeared in China. Human genetic factors, including polymorphisms, serve pivotal roles in the high transmission of SARS-CoV-2 and the stubbornly progressing sickness seen in a small but significant percentage of infected people; however, but these factors remain ill-defined. A total of 288 COVID-19 patients and 288 controls were genotyped for TMPRSS2 polymorphisms using both restriction fragment length polymorphism polymerase chain reaction (RFLP-PCR) and amplification refractory mutation system (ARMS)-PCR techniques. Different genotypes of TMPRSS2 polymorphisms were compared in terms of disease susceptibility and mortality. The statistical analysis showed that minor alleles of all studied variants statistically increased the risk of COVID-19, except for the rs75603675 C > A variant. The T allele of rs12329760 conferred an increased risk of COVID-19. Moreover, the AG/AC/TT/AG combination of genotypes significantly enhanced the risk of COVID-19 in our population. Different haplotypes of rs17854725/rs75603675/rs12329760/rs4303795 polymorphisms, including GACA, GACG, GATG, GATA, AATA, ACCG, ACTG, ACTA, GCCA, and GCTG, were found to be associated with increased risk of the disease (odds ratio > 1). Regarding the clinical and paraclinical characteristics, a statistically significant difference was found between non-severe and severe forms except for gender, platelet, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and underlying diseases. In addition, case genotypes of TMPRSS2 rs17854725 A > G, rs12329760 C > T, and rs4303795 A > G were significantly different regarding severe and non-severe forms of the disease (P-value < 0.001). Specifically, death was more frequent in carriers of the AG genotype of rs17854725 A > G (P-value = 0.022). Patients who carry the minor alleles of the four studied TMPRSS2 variants were rather vulnerable to COVID-19 infection. Our findings indicated that rs17854725 A > G (AA vs. AG and AA vs. GG), rs12329760 C > T (CC vs. CT and CC vs. TT), and rs4303795 A > G (AA vs. AG) genotypes of TMPRSS2 variations are associated with a more invasive disorder pattern. More studies on larger populations are needed to confirm our results.

Relationship between P2XR4 Gene Variants and the Risk of Schizophrenia in South-East of Iran: A Preliminary Case-Control Study and in Silico Analysis.

BACKGROUND: Schizophrenia (SZN) is a heterogeneous disorder. Recently, the role of purinergic receptor's signaling in mental disorders has implicated. There is no evidence regarding the association of P2XR4 single nucleotide polymorphisms (SNPs) and the risk of behavioral disorders. Therefore, this preliminary study, we determined the association of rs1169727A/G and rs25644A/G variants located in P2XR4 gene with the risk of SZN. METHODS: This case-control study was performed on 150 SZN patient referring to Baharan Hospital, Zahedan (Eastern of Iran) in 2018. Genotyping was done by tetra-amplification refractory mutation system polymerase chain reaction (Tetra ARMS-PCR). Different databases were used to determine the effects of the SNPs on the secondary structure of P2XR4 pre-mRNA and protein as well as binding of transcriptional regulators. RESULTS: The G allele of rs1169727 significantly increased the risk of SZN (OR=1.41, 95%CI=1.02-1.93, P=0.039), but there was no significant association was found between the other SNP and SZN. Moreover, GG model of rs1169727 (OR=2.46, 95%CI= 1.32-4.62, P=0.004) and rs25644 (OR=3.45, 95%CI= 1.12-5.10, P=0.013) increased the risk of SZN. The substitution of A and G alleles of rs1169727 significantly altered the secondary structure of pre-mRNA (P=0.1). In silico analysis revealed that rs25644A/G could act as an intronic cryptic donor site. Screening for flanking sequence of rs1169727A/G and rs25644A/G predicted a novel enhancer and silencer for both SNPs. CONCLUSION: rs1169727A/G and rs25644A/G are linked to SZN susceptibility in a sample of the Iranian population. In-silico analysis indicated that rs25644 have substantial roles in determining the pre-mRNA and protein structure of P2XR4 gene.

Association of a Novel KIF26B Gene Polymorphism with Susceptibility to Schizophrenia and Breast Cancer: A Case-Control Study.

BACKGROUND: KIF26B gene is found to play essential roles in regulating different aspects of cell proliferation and development of the nervous system. We aimed to determine if rs12407427 T/C polymorphism could affect susceptibility to schizophrenia (SZN) and breast cancer (BC), the two genetically correlated diseases. METHODS: The current case-control study was performed from Aug 2018 to Dec 2018. Briefly, 159 female pathologically confirmed BC cases referring to Alzahra Hospital, Isfahan, Iran, and 102 psychologically confirmed SZN patients (60 males and 42 females) admitted to Baharan Hospital, Zahedan, Iran, were enrolled. Using the salting-out method, genomic DNA was extracted, and variants were genotyped using allele-specific amplification refractory mutation system polymerase chain reaction (ARMS-PCR) method. RESULTS: The results revealed a significant association between the KIF26B rs12407427 codominant CT (P=0.001), CC (P=0.0001), dominant CT+CC, and recessive CC (P=0.001) genotypes with the risk of developing SZN. Significant correlations were also found regarding rs12407427 and BC susceptibility in different inheritance models, including over-dominant CT (P=0.026), dominant CT+CC (P=0.001), recessive CC (P=0.009), and codominant CT and CC (P=0.001) genotypes. The over-presence of the C allele was also correlated with an increased risk for SZN (P=0.0001) and BC (P=0.0001). Finally, computational analysis predicted that T/C variation in this polymorphism could change the binding sites in proteins involved in splicing. CONCLUSION: rs12407427 T/C as a de novo KIF26B variant might be a novel genetic biomarker for SZN and/or BC susceptibility in a sample of the Iranian population.

Silencing of Nucleostemin by siRNA Induces Apoptosis in MCF-7 and MDA-MB-468 Cell Lines.

One of the most important modulators involved in controlling apoptosis induction and viability of cancerous cells is nucleostemin (NS). Some studies revealed that NS is also needed to maintain the proliferation of embryonic neural stem cells and early embryogenesis. This study was designed to better elucidate the association between NS depletion status and apoptosis induction of both MCF-7 and MDA-MB-468 cell lines. We examined the effects of NS-targeting siRNAs on the expression of NS in MCF-7 and MDA-MB-468 human breast cancer cell lines by the Real-time polymerase chain reaction method. In addition, we investigated the correlation between knockdown of NS and viability rates and apoptosis induction in MCF-7 and MDA-MB-468 cell lines using the MTT assay and annexin V/PI staining, respectively. The NS-targeting siRNAs inhibited the viability of the cells in a dose- and time-dependent manner and induced apoptosis after 48 h in the cells. Thus, consistent with previous articles, this protein can be one of the regulators related to the inhibition of apoptosis and the increased viability of tumor-initiating cells in human breast cancer cell lines as well as other cancers.

Polymorphism in the 3'-UTR of LIF but Not in the ATF6B Gene Associates with Schizophrenia Susceptibility: a Case-Control Study and In Silico Analyses.

Schizophrenia (SCZ) is a multifactorial disorder caused by environmental and genetic factors. Studies have shown that various single-nucleotide polymorphisms (SNPs) in the binding sites of microRNAs contribute to the risk of developing SCZ. We aimed to investigate whether the variants located in the 3'-UTR region of LIF (rs929271T>G) and ATF6B (rs8283G>A) were associated with increased susceptibility to SCZ in a population from the south-east of Iran. In this case-control study, a total of 396 subjects were recruited. SNPs were genotyped via polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Genotyping results showed that the G allele of rs929271 significantly increased the risk of SCZ (OR = 1.58 95%CI = 1.19-2.10, p = 0.001). As for rs929271, the GG genotype of co-dominant (OR = 2.54 95%CI = 1.39-4.64, p = 0.002) and recessive (OR = 2.91 95%CI = 1.77-4.80, p < 0.001) models were strongly linked to SCZ. No significant differences were observed between rs8283 polymorphism and predisposition to SCZ. In silico analyses predicted that rs929271 might alter the binding sites of microRNAs, which was believed to have an unclear role in the development of SCZ. Moreover, rs929271 polymorphism changed the LIF-mRNA folding structure. These findings provide fine pieces of evidence regarding the possible effects of LIF polymorphism in the development of SCZ and regulation of the LIF gene targeted by microRNAs.

Relationship between Single Nucleotide Polymorphisms of GRHL3 and Schizophrenia Susceptibility: A Preliminary Case-Control Study and Bioinformatics Analysis.

Grainyhead-like (GRHL) transcription factors were recently linked to the etiology of neural tube defects (NTDs). Overlapping patterns in the variation of schizophrenia (SCZ) incidence with that of NTDs suggests the presence of common etiological risk factors. This preliminary study was designed to examine the relationship between two missense variants of GRHL3 gene (rs2486668C/G and rs545809A/T) and SCZ susceptibility among Iranians. Three hundred ninety subjects (192 patients confirmed with SCZ, and 198 healthy controls) were enrolled and genotyped. Statistical and bioinformatics analyzes were performed to determine the effects of the variants. In silico analyzes were performed to determine the effects of the variants on the secondary structure of GRHL3 protein and prediction of silencer motifs for each variation. Statistically significant differences were observed between the studied groups under codominant AA, dominant AT+AA, and recessive AA genetic contrast models for rs545809A/T. The presence of the A allele of rs545809A/T enhanced SCZ risk by 2.33 fold. In contrast, rs2486668C/G was not linked to SCZ susceptibility (P > 0.05). Bioinformatics analysis revealed that both missense SNPs caused substantial changes in the secondary structure of GRHL3-mRNA. Screening of the flanking sequences of rs545809A/T predicted silencer motifs for this SNP. Our results demonstrated that the rs545809A/T of GRHL3 gene could affect the risk of SCZ in Iranian populations. Replication studies are warranted to confirm these results.

Impact of Proliferator-Activated Receptor γ Gene Polymorphisms on Risk of Schizophrenia: A Case-Control Study and Computational Analyses.

Objective: Schizophrenia (SCZ) is a common psychiatric disorder characterized by a complex mode of inheritance. Peroxisome proliferator-activated receptor-γ (PPARG) mainly regulates lipid and glucose metabolisms while it is constitutively expressed in rat primary microglial cultures. This preliminary study was aimed to investigate the relationship of two polymorphisms in the PPARG gene, rs1801282 C/G, and rs3856806 C/T, to the risk of SCZ in the southeast Iranian population. Method : A total of 300 participants (150 patients with SCZ and 150 healthy controls) were enrolled. Genotyping was done using the amplification refractory mutation system polymerase chain reaction (ARMS-PCR) technique. Computational analyses were carried out to predict the potential effects of the studied polymorphisms. Results: A significant link was found between genotypes of rs1801282 and SCZ susceptibility. The G allele of rs1801282 in CG and GG form of the codominant model increased the risk of SCZ by 2.49 and 2.64 folds, respectively. With regards to rs3856806, enhanced risk of SCZ was also observed under different inheritance models except for the overdominant model. Also, the T allele of rs3856806 enhanced the risk of SCZ by 3.19 fold. Computational analyses predicted that rs1801282 polymorphism might alter the secondary structure of PPARG-mRNA and protein function. At the same time, the other variant created the binding sites for some enhancer and silencer motifs. Conclusion: Our findings showed that PPARG rs1821282 and rs3856806 polymorphisms associate with SCZ susceptibility. Replication studies in different ethnicities with a larger population are needed to validate our findings.

Achillea Wilhelmsii C. KochHydroalcoholic Extract Induces Apoptosis and Alters LIN28B and p53 Gene Expression in Hela Cervical Cancer Cells.

BACKGROUND: Inappropriate activation of the proto-oncogene LIN28B and inactivation of the p53 tumor suppressor, have been shown to have a critical role in tumorigenesis. Previous research has shown therapeutic potential for the use of herbal plants as an alternative strategy for cancer treatment. Achillae wilhelmsii C. Koch is a plant that has been traditionally used for its medicinal properties. The aim of this study was to investigate the cytotoxic and apoptosis-inducing effect of Achillea wilhelmsii C. Koch hydroalcoholic extract (AWHE) on HeLa cervical cancer cells and its effect on LIN28B and p53 expression. METHODS: The cytotoxic activity of AWHE was evaluated on HeLa cells using a trypan blue exclusion assay. The Annexin V/PI double staining assay was used to evaluate the apoptosis-inducing effect of the extract. The expression of LIN28B and p53 mRNA was measured using the real-time-PCR method. RESULTS: Treatment with AWHE was shown to induce cytotoxicity in both time and concentration-dependent manners (P<0.05). The proposition of HeLa cells undergoing apoptosis increased with increasing concentrations of AWHE (P<0.05). The mRNA levels of p53 increased following 12, 24, and 48 hours of AWHE treatment whereas the mRNA levels of LIN28B were significantly decreased after 4 to 12 hours of AWHE treatment (p<0.05). CONCLUSION: Our findings confirmed the pro-apoptotic function of AWHE on the cervical cancer HeLa cell line. This indicates that targeting the LIN28B signaling cascade may be a promising therapeutic strategy for cervical cancer. Further research is required to understand the therapeutic effects of AWHE in primary human cervical cancer cells and a pre-clinical cervical cancer model.

Novel Poly(Adenosine Diphosphate-Ribose) Polymerase (PARP) Inhibitor, AZD2461, Down-Regulates VEGF and Induces Apoptosis in Prostate Cancer Cells

Background: Prostate cancer (Pca) is a heterogeneous disease, and current treatments are not based on molecular stratification. Poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors have recently been found to be remarkably toxic to cells with defects in homologous recombination, particularly cells with BRCA-mutated backgrounds. Therefore, this preliminary study was designed to evaluate whether PTEN expression status could have an impact on the sensitivity of invasive Pca cells to the PARP inhibitor, AZD2461. Methods: MTT viability test, Annexin V­FITC/propidium iodide double staining, and caspase3 activity assay were used to evaluate the apoptosis and relative expression of PTEN and VEGF in PC-3 and DU145 cell lines using real-time PCR. Results: MTT results showed that the inhibitory effects of AZD2461 were higher in PC-3 than DU145 cells (with IC50 of 36.48 and 59.03 µM at 48 hours of treatment, respectively). Flow cytometric analysis also showed the same results. When exposed to 40 µM of AZD2461, PC-3 (38.8%) and DU145 (28%) cells underwent apoptosis (p < 0.05). Treatment of cells by AZD2461 also caused a significant increase in apoptosis through caspase3 activation in both cell lines. VEGF mRNA levels in PC-3 cells significantly decreased compared to adjusted untreated cells (p < 0.05) in all measured times while displaying different alteration patterns in DU145 cells (p < 0.05). Conclusion: AZD2461 suppresses the growth of prostate tumor cells since AZD2461 monotherapy could prove to be efficacious, especially against cells not expressing PTEN besides activating the possible apoptosis-independent cell death pathways.

Nitric Oxide Synthase 2 Polymorphisms (rs2779248T/C and rs1137933C/T) and the Risk of Type 2 Diabetes in Zahedan, Southeastern Iran.

BACKGROUND: Nitric oxide (NO) has been associated with insulin resistance and type 2 diabetes (T2D). NO is synthesized enzymatically from l-arginine (l-Arg) by three NO synthase (NOS) isoforms, Neuronal NOS (nNOS or NOS1), Inducible NOS (iNOS or NOS2), and Endothelial NOS (eNOS or NOS3). The impact of NOS2 gene polymorphism was investigated on the susceptibility of T2D in a sample of Iranian population (Southeastern of Iran). METHODS: In 2015, the present case-control study was conducted on 152 T2D patients and 157 healthy control subjects (HCs) referring to Bu-ali Hospital of Zahedan, eastern Iran. Genotyping of NOS2 rs2779248T/C and rs1137933C/T variants were done using the Tetra-Amplification Refractory Mutation System Polymerase Chain Reaction (Tetra-ARMS PCR) method. RESULTS: CT genotype of rs1137933C/T was significantly associated with increased risk of T2D (P<0.0001). The T allele of this single nucleotide polymorphism (SNP) was also strongly associated with T2D risk (P<0.0001). For rs2779248 T/C, TC genotype of this SNP decreased the risk of T2D (OR=0.25 95%CI= 0.15-0.42, P<0.0001); however, CC genotype of this SNP increased the risk of T2D (P<0.005). There was no significant association between clinical-demographic characteristics of T2D group with respect to both SNPS in dominant. CONCLUSION: CT genotype and C allele of NOS2 rs1137933 C/T polymorphism were associated with a higher risk of T2D, and no association was observed between T allele of NOS2 rs2779248 T/C polymorphism and T2D while TC genotype of this SNP decreased the risk of T2D in the study participants.

Antifungal effects of ethanolic and aqueous extracts of Vitexagnus-castus against vaginal isolates of Candida albicans.

BACKGROUND AND PURPOSE: Vulvovaginal candidiasis is one of the most common infections in female genital organs, which is caused by Candida species. Candida albicans is the causative agent of more than 80% of infections, and the role of non-Candida strains in the disease etiology is less prominent. The expansion of Azoles resistance among C. albicans strains is considered an important medical problem. According to previous studies, Vitex agnus-castus (vitex) has some antimicrobial effects. We aimed to evaluate the anti-fungal effects of aqueous and alcoholic extracts of vitex against clinical vaginal isolates of C. albicans in comparison with fluconazole. MATERIALS AND METHODS: Gas chromatography-mass spectrometry analysis was performed on vitex to identify its possible bioactive components. Forty C. albicans clinical isolates were identified by using germ tube, chlamydospore production, culture on CHROMagar, and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Finally, after the extraction of vitex, drug susceptibility test was carried out according to the clinical laboratory standards institute (CLSI) M27-S4 document guidelines. RESULTS: The major chemical components of vitex leaf as determined by gas chromatography included α-Pinene, isoterpinolene, caryophyllene, and azulene. The minimum inhibitory concentrations (MICs) of aqueous and alcoholic extracts of vitex, as well as fluconazole were within the ranges of 15.62-62.5, 7.81-15.62, and 0.25-8 µg/mL, respectively. CONCLUSION: Our findings showed that the alcoholic and aqueous extracts of vitex had antifungal activity against clinical isolates of C. albicans. Moreover, the alcoholic extract of vitex and fluconazole were more effective against clinical vaginal isolates of C. albicans compared to the aqueous extract of vitex.