Expression pattern, prognostic value and potential microRNA silencing of FZD8 in breast cancer.

Breast cancer (BC) is one of the most widespread types of cancer affecting females, and therefore, early diagnosis is critical. BC is a complex heterogeneous disease affected by several key pathways. Among these, WNT proteins and their frizzled receptors (FZD) have been demonstrated to be crucial in regulating a number of cellular and molecular events in BC tumorigenesis. The role of the WNT receptor, FZD8, in BC has received minimal attention; for that reason, the present study examined the prognostic value of its protein expression pattern in a BC cohort. FZD8 cytoplasmic expression pattern analysis revealed that ~38% of the primary samples presented with a high expression profile, whereas ~63% of the samples had a low expression profile. Overall, ~46% of the malignant tissues in the lymph node-positive samples exhibited an increased FZD8 cytoplasmic expression, whereas 54% exhibited low expression levels. An increased expression of FZD8 was associated with several clinicopathological characteristics of the patients, including a low survival rate, tumor vascular invasion, tumor size and grade, and molecular subtypes. Affymetrix microarray triple-negative BC datasets were analyzed and compared with healthy breast tissues in order to predict the potential interfering microRNAs (miRNAs) in the WNT/FZD8 signaling pathway. A total of 29 miRNAs with the potential to interact with the WNT/FZD8 signaling pathway were identified, eight of which exhibited a significant prediction score. The target genes for each predicted miRNA were identified. On the whole, the findings of the present study suggest that FZD8 is a potential prognostic marker for BC, shedding some light onto the silencing mechanisms involved in the complex BC signaling.

Utility of Circulating Cell-Free DNA in Assessing Microsatellite Instability and Loss of Heterozygosity in Breast Cancer Using Human Identification Approach.

The diagnostic and prognostic utility of circulating cell-free DNA (cfDNA) in breast cancer (BC) patients was recently reported. Here, we investigated the use of cfDNA to examine microsatellite instability (MSI) and loss of heterozygosity (LOH) for early BC diagnosis. cfDNA and genomic DNA from 41 female BC patients and 40 healthy controls were quantified using NanoDrop spectrophotometry and real-time PCR. The stability of genomic and cfDNA was assessed using a high-resolution AmpFlSTR MiniFiler human identification kit. Significant increases in cfDNA plasma concentrations were observed in BC patients compared to controls. The genotype distribution of the eight autosomal short tandem repeat (STR) loci D7S820, D13S317, D21S11, D2S1338, D18S51, D16S539, FGA, and CSF1PO were in Hardy-Weinberg equilibrium. Significant differences in the allele frequencies of D7S820 allele-8, D21S11 allele-29, allele-30.2, allele-32.2, and CSF1PO allele-11 were seen between BC patients and controls. LOH and MSI were detected in 36.6% of the cfDNA of patients compared to genomic DNA. This study highlights the utility of plasma-derived cfDNA for earlier, less invasive, and cost-effective cancer diagnosis and molecular stratification. It also highlights the potential value of cfDNA in molecular profiling and biomarkers discovery in precision and forensic medicine.

The Prognostic Value of the Developmental Gene FZD6 in Young Saudi Breast Cancer Patients: A Biomarkers Discovery and Cancer Inducers OncoScreen Approach.

Wnt signalling receptors, Frizzleds (FZDs), play a pivotal role in many cellular events during embryonic development and cancer. Female breast cancer (BC) is currently the worldwide leading incident cancer type that cause 1 in 6 cancer-related death. FZD receptors expression in cancer was shown to be associated with tumour development and patient outcomes including recurrence and survival. FZD6 received little attention for its role in BC and hence we analysed its expression pattern in a Saudi BC cohort to assess its prognostic potential and unravel the impacted signalling pathway. Paraffin blocks from approximately 405 randomly selected BC patients aged between 25 and 70 years old were processed for tissue microarray using an automated tissue arrayer and then subjected to FZD6 immunohistochemistry staining using the Ventana platform. Besides, Ingenuity Pathway Analysis (IPA) knowledgebase was used to decipher the upstream and downstream regulators of FZD6 in BC. TargetScan and miRabel target-prediction databases were used to identify the potential microRNA to regulate FZD6 expression in BC. Results showed that 60% of the BC samples had a low expression pattern while 40% showed a higher expression level. FZD6 expression analysis showed a significant correlation with tumour invasion (p < 0.05), and borderline significance with tumour grade (p = 0.07). FZD6 expression showed a highly significant association with the BC patients' survival outcomes. This was mainly due to the overall patients' cohort where tumours with FZD6 elevated expression showed higher recurrence rates (DFS, p < 0.0001, log-rank) and shorter survival times (DSS, p < 0.02, log-rank). Interestingly, the FZD6 prognostic value was more potent in younger BC patients as compared to those with late onset of the disease. TargetScan microRNA target-prediction analysis and validated by miRabel showed that FZD6 is a potential target for a considerable number of microRNAs expressed in BC. The current study demonstrates a potential prognostic role of FZD6 expression in young BC female patients and provides a better understanding of the involved molecular silencing machinery of the Wnt/FZD6 signalling. Our results should provide a better understanding of FZD6 role in BC by adding more knowledge that should help in BC prevention and theranostics.

LRP6 Receptor Plays Essential Functions in Development and Human Diseases.

LRP6 is a member of the low-density lipoprotein receptor superfamily of cell-surface receptors. It is required for the activation of the Wnt/ß-catenin signalling pathway. LRP6 is detected in different tissue types and is involved in numerous biological activities such as cell proliferation, specification, metastatic cancer, and embryonic development. LRP6 is essential for the proper development of different organs in vertebrates, such as Xenopus laevis, chickens, and mice. In human, LRP6 overexpression and mutations have been reported in multiple complex diseases including hypertension, atherosclerosis, and cancers. Clinical studies have shown that LRP6 is involved in various kinds of cancer, such as bladder and breast cancer. Therefore, in this review, we focus on the structure of LRP6 and its interactions with Wnt inhibitors (DKK1, SOST). We also discuss the expression of LRP6 in different model systems, with emphasis on its function in development and human diseases.

Leptin Protein Expression and Promoter Methylation in Ovarian Cancer: A Strong Prognostic Value with Theranostic Promises.

Ovarian cancer (OC) is the deadliest among all gynecological cancers. Epidemiological studies showed that obesity might influence many cancers including OC. One of the key factors that may link obesity and OC is leptin (LEP), known as an adipokine with pleiotropic effects on body homeostasis. This study aims to investigate the expression pattern of LEP, assess the methylation profiles of LEP and their associations with clinicopathological features including survival outcomes of OC patients. The protein expression of LEP was evaluated in 208 samples using both tissue microarray and immunohistochemistry techniques. The methylation profiles of LEP were measured in 63 formalin-fixed, paraffin-embedded tumor tissues by quantitative polymerase chain reaction using a MethyLight assay. Our results showed a significant association of LEP protein overexpression with several clinicopathological variables, mainly tumor subtype, LVI, age of menarche, tumor size and stage (p < 0.04). Kaplan-Meier analysis (using low expression versus high expression as a discriminator) indicated that LEP protein overexpression is a powerful positive prognosticator of both OC recurrence (DFS) and disease-specific survival (DSS) in our OC cohort (log-rank p = 0.01 and p = 0.002, respectively). This implies that patients with high LEP expression profiles live longer with less recurrence rates. Methylation analysis results demonstrated a clear association between no/low LEP protein expression pattern (38%) and LEP promoter CpG island hypermethylation (43%). Results of this study suggest that LEP is a powerful prognosticator of OC recurrence and DSS. LEP expression in OC seems to be regulated by its promoter hypermethylation through gene partial/total silencing. Further multi-institutional studies using larger cohorts are required to demystify the intricate molecular functions of this leptin-driven effects in OC pathophysiology and to accurately assess its theranostic potential and validate its prognostic/predictive power in OC onset, progression towards more effective and personalized management of OC patients.

Investigation of Isocitrate Dehydrogenase 1 and 2 Mutations in Acute Leukemia Patients in Saudi Arabia.

Different forms of human cancer show mutations for isocitrate dehydrogenases 1 and 2 (IDH1/2). Mutation of these genes can cause aberrant methylation of the genome CpG islands (CGIs), which leads to an increase of suppressed oncogenes transcription or repression of active tumor suppressor gene transcription. This study aimed to identify the prevalence of IDH1/2 mutations in acute leukemia patients. The study cohort included 43 AML patients and 30 childhood ALL patients, from whom DNA bone marrow samples were taken. The alteration hotspots in codons IDH1 (R132) and IDH2 (R172 and R140) were examined via direct sequencing. Mutations in IDH1 were detected in 7 out of 43 (16.2%) AML patients; 5 of them occurred at codon R132. The other two mutations included a single-nucleotide polymorphism, which affected codon G105 in one patient. However, no mutation was detected in the IDH2 in any of the patients. Moreover, no mutations were detected in either IDH1 or IDH2 in ALL patients. The dominance of IDH1 mutations in AML, which was 16%, emphasizes the existence of the mutation in our population. On the other hand, IDH2 mutation was observed to be less frequent in both illnesses. Due to the limitation of using a small sample size, larger cohort screening is recommended to determine their usefulness as prognostic indicators.

Prognostic value of E-Cadherin and its tumor suppressor role in Saudi women with advanced epithelial ovarian cancer.

The extracellular matrix (ECM) disruption and cytoskeleton reorganization are crucial events in tumor proliferation and invasion. E-Cadherin (E-CAD) is a member of cell adhesion molecules involved in cell-cell junctions and ECM stability. The loss of E-CAD expression is associated with cancer progression and metastasis. This retrospective study aimed to assess E-CAD protein expression in ovarian cancer (OC) tissues and to evaluate its prognostic value. PATIENTS AND METHODS: 143 formalin-fixed and paraffin-embedded (FFPE) blocks of primary advanced stages OC were retrieved and used to construct Tissue microarrays. Automated immunohistochemistry technique was performed to evaluate E-CAD protein expression patterns in OC. RESULTS: E-CAD protein expression was significantly correlated with OC histological subtype (p < 0.0001), while borderline significant correlations were observed with both tumor grade (p = 0.06) and stage (p = 0.07). Interestingly, Kaplan-Meier survival analysis showed that OC patients with membranous E-CAD expression survived longer than those with no E-CAD expression mainly those at advanced stages (p < 0.009). Further in silico analysis confirms the key roles of E-CAD in OC molecular functions. CONCLUSION: we reported a prognosis value of membranous E-CAD in advanced stage OC patients. Further validation using larger cohorts is recommended to extract clinically relevant outcomes towards better OC management and individualized oncology.

Role of Oxidative Stress and the Identification of Biomarkers Associated With Thyroid Dysfunction in Schizophrenics.

Background: Schizophrenia is associated with a deficiency of dietary antioxidants like vitamin B6, B9, and B12 resulting in defective methylation leading to hyperhomocysteinemia. Hyperhomocysteinemia causes mitochondrial DNA damage, oxidative stress, vascular damage, and lipid peroxidation. Oxidative stress and increase in reactive oxygen species result in 8-oxodG production which induces apoptosis of both astrocytes and thyrocytes thus predisposing them to thyroid dysfunction and neurodegeneration. Furthermore, the presence of excessive free radicals increases thyroid thermogenesis causing hyperthyroidism or its excess may cause hypothyroidism by inhibiting iodide uptake. In the present study, we evaluated the various biomarkers associated with thyroid dysfunction in schizophrenics. Materials and Methods: 288 patients suffering from schizophrenia and 100 control subjects were screened for liver function tests (LFTs) such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and total bilirubin (TB). Also, the stress markers, namely malondialdehyde (MDA), homocysteine, cysteine, methionine, the thyroid profile including triiodothyronine (T3), thyroxine (T4), thyroid-stimulating hormone (TSH), thyroxine peroxide antibody (TPO-Ab); TSH receptor-Ab (TSHr-Ab), dietary antioxidants, lipids, cytokines, aminoacids and hormones, vitamins and trace elements, and other biochemical parameters. Results: The LFTs showed elevated levels of ALT (45.57 ± 4.87 Vs. 26.41 ± 3.76 U/L), AST (40.55 ± 1.34 Vs. 21.92 ± 3.65 U/L), ALP (121.54 ± 4.87 Vs. 83.76 ± 5.87 U/L), and total bilirubin (2.63 ± 0.987 Vs. 1.10 ± 0.056 mg/dl), in schizophrenics than controls. Increased levels of MDA (3.71 ± 0.967 Vs. 1.68 ± 0.099) and homocysteine (17.56 ± 2.612 Vs. 6.96 ± 1.987 µmol/L were observed in schizophrenics compared to the controls, indicating increased stress. Levels of cysteine and methionine were decreased in schizophrenics than the controls (1.08 ± 0.089 Vs. 4.87 ± .924 µmol/L and 17.87 ± 1.23 Vs. 99.20 ± 5.36 µmol/L). The levels of TPO-Ab (IU/ml), Tg-Ab (pmol/L), and TSHr-Ab (IU/L) were observed to be higher in the patients' group as compared to control subjects (9.84 ± 2.56 Vs. 5.81 ± 1.98, 55.50 ± 2.98 Vs. 32.95 ± 2.87 and 2.95 ± 0.0045 Vs. 1.44 ± 0.0023 respectively). Levels of Vitamin B6, B9, and B12 were also significantly decreased in the patients compared to the healthy controls. Conclusion: The schizophrenics, demonstrated altered liver function, increased stress markers, and decreased dietary antioxidants. Reduced primary and secondary antioxidant levels, may result in hyperhomocysteinemia and cause further DNA and mitochondrial damage. Therefore, homocysteine and/or prolactin levels may serve as candidate prognostic markers for schizophrenia. Also, both neurological symptoms and the susceptibility to thyroid disorders may be prevented in the initial stages of this debilitating disorder by appropriate dietary supplementation of antioxidants which can rectify a reduction in primary and secondary antioxidants, and disturbed prolactin-serotonin-dopamine interactions in schizophrenics.

Unraveling the Catha edulis Extract Effects on the Cellular and Molecular Signaling in SKOV3 Cells.

Khat (Catha edulis (Vahl) Endl.) is an evergreen flowering shrub used as a stimulant in many regions worldwide including East Africa, the Arabian Peninsula, Europe, and the United States. Chewing leaves of khat induces excitement and euphoria, which are primarily attributed to two major constituents, cathinone and cathine. Khat also contains other important constituents such as cathedulins. A considerable number of studies reported side effects induced by the khat extracts to both embryos and adults. These include teratogenicity and developmental retardation, oral cancer and ulcers, high blood pressure, and myocardial infarction. So far, little attention has been paid to the effects of khat extracts on the molecular signaling interactions. We aimed in this study to investigate this through evaluating the effects of khat extracts on SKOV3, a human ovarian adenocarcinoma cell line. We show, by in vitro assays, that khat induces several cellular defects including reduced cell size, cell membrane damage, and apoptosis. At high khat extract concentrations, the cell metabolic activity, cell cycle, and cellular proliferation were affected. RT-qPCR analysis showed an increase in the gene expression of the apoptotic marker BAX, the tumor suppressor p53, and the inflammatory cytokine IL-6. Protein expression analysis by immunostaining showed downregulation of ß-catenin, E-cadherin, and Ki-67 and upregulation of FZD8 and SPRY2, suggesting that Wnt and FGF signaling were implicated. SwissTargetPrediction in silico analysis showed that khat constituents cathine, cathinone, catheduline K2, and catheduline E5 bind to family A G-protein-coupled receptor, cause many neurological diseases and disorders such as Alzheimer's, schizophrenia, depression, and anxiety, and induce many ovarian cancer-related diseases. The analysis also showed that important signaling pathways such as CREB, Wnt, FGF, IL-6, and ERK/MAPK, and that of the endometrial cancer, and cell cycle were implicated. Upstream regulators of cathine and cathinone were found to potentially target several molecules including interleukin-8, MMP2, PLAU, and micro-RNAs. In conclusion, khat induces significant cellular and molecular changes that could potentially cause a wide range of serious diseases and syndromes. Such an impact could have a heavy burden on the health care system in the countries where khat is consumed.

Data mining analysis of human gut microbiota links Fusobacterium spp. with colorectal cancer onset.

Gut microbiota and their metabolites play a vital role in colon health and disease. Accumulating evidence suggests that the gut microbiota contributes to the risk of colorectal cancer (CRC). However, the role of a specific microbial community together with their metabolites contributing to the risk, initiation and progression of CRC is still unknown. Hence, we used a Bayesian Networks in combination with the IDA (Intervention calculus when the DAG is absent) to generate a graphical model that allows causal relationships to be inferred from observational data. Results from the analysis of publically available datasets showed that four species: Fusobacteium, Citrobacter, Microbacterium and Slaxkia have estimated non-null lower bounds of causal effects of CRC. These findings support the hypothesis that specific bacterial species (microbial markers) act in concert with locally modified microbiota to cause or influence CRC progression. Additional comprehensive studies are required to validate the potential use of F. nucleatum, Citrobacter as well as Slackia as microbial biomarkers in CRC for prevention, diagnosis, prognosis and/or therapeutics.

Catha edulis forsk mediates embryotoxic effects in rats: an experimental study / Catha edulis forsk modera los efectos embriotóxicos en ratas: un estudio experimental

Catha edulis Forsk leaves (Khat) is a flowering plant. A high proportion of the adult population in the Arabian Peninsula and the Horn of Africa chews it for its mild stimulant effect. The aim of the current study was to investigate the embryotoxic and teratogenic effects of the Khat extract using 60 female pregnant rats. These were divided to a Khat extract-treated group and a control group. Methanolic extract of Khat was orally given to the treated group 4 days before mating and up to day 16 of pregnancy with a dose of 100 mg/kg. Our results showed that significant number of embryos of the Khat-treated mothers were malformed and different in size and shape compared to embryos from the mothers of the control group. At day 8 of pregnancy, malformed embryos had ill developed primitive layers. By day 10 of pregnancy, neural tube and the somite were not formed compared to the control embryos. At later stages of pregnancy, embryos of the Khat-treated mothers appeared severely abnormal with opened neural groove and visceral pouches. Disrupted normal neural tube development, undifferentiated brain vesicles, incomplete closure of the brain flexures were also observed in these embryos. Highly significant increase in the number of the resorbed embryos of the Khat-treated mothers were observed (P < 0.01). The resorbed embryos appeared as a cellular collection in their placenta with some of their decidua had no visible embryonic tissues. In conclusions, Khat induced embryotoxic effects as well as severely affected the early normal embryonic development in rat.

Catha edulis (Khat) es una planta floreciente. Una alta proporción de la población adulta en la Península Arábiga y el Cuerno de África la mastica por su efecto estimulante. El objetivo del presente estudio fue investigar los efectos embriotóxicos y teratogénicos del extracto de Khat utilizando 60 ratas hembras preñadas. Estas se dividieron en un grupo tratado con extracto de Khat y un grupo control. El extracto metanólico de Khat se administró por vía oral al grupo tratado 4 días antes del apareamiento y hasta el día 16 de preñez con una dosis de 100 mg / kg. Los resultados mostraron que una cantidad significativa de embriones de las madres tratadas con Khat tenían malformaciones y eran diferentes en tamaño y forma en comparación con los embriones de las madres del grupo control. En el día 8 de preñez, los embriones malformados tenían capas primitivas mal desarrolladas. Para el día 10 de preñez, el tubo neural y el somito no se formaron en comparación con los embriones del grupo control. En etapas posteriores de la preñez, los embriones de las madres tratadas con Khat parecían severamente anormales con surcos neurales abiertos y bolsas viscerales. También se observaron alteraciones en el desarrollo normal del tubo neural, vesículas cerebrales indiferenciadas y el cierre incompleto de las flexiones cerebrales en estos embriones. Se observó un aumento altamente significativo en el número de embriones reabsorbidos de las madres tratadas con Khat (P <0,01). Los embriones reabsorbidos aparecieron como una colección celular en su placenta con algunas de sus deciduas sin tejidos embrionarios visibles. Khat indujo efectos embriotóxicos y afectó severamente el desarrollo embrionario normal temprano en la rata.

Frizzled-7 is required for Xenopus heart development.

Wnt signalling regulates cardiogenesis during specification of heart tissue and the morphogenetic movements necessary to form the linear heart. Wnt11-mediated non-canonical signalling promotes early cardiac development whilst Wnt11-R, which is expressed later, also signals through the non-canonical pathway to promote heart development. It is unclear which Frizzled proteins mediate these interactions. Frizzled-7 (fzd7) is expressed during gastrulation in the mesodermal cells fated to become heart, and then in the primary heart field. This expression is complementary to the expression of wnt11 and wnt11-R We further show co-localisation of fzd7 with other early- and late-heart-specific markers using double in situ hybridisation. We have used loss of function analysis to determine the role of fzd7 during heart development. Morpholino antisense oligonucleotide-mediated knockdown of Fzd7 results in effects on heart development, similar to that caused by Wnt11 loss of function. Surprisingly, overexpression of dominant-negative Fzd7 cysteine rich domain (Fzd7 CRD) results in a cardia bifida phenotype, similar to the loss of wnt11-R phenotype. Overexpression of Fzd7 and activation of non-canonical wnt signalling can rescue the effect of Fzd7 CRD. We propose that Fzd7 has an important role during Xenopus heart development.

Evaluation of the Effects of Airborne Particulate Matter on Bone Marrow-Mesenchymal Stem Cells (BM-MSCs): Cellular, Molecular and Systems Biological Approaches.

Particulate matter (PM) contains heavy metals that affect various cellular functions and gene expression associated with a range of acute and chronic diseases in humans. However, the specific effects they exert on the stem cells remain unclear. Here, we report the effects of PM collected from the city of Jeddah on proliferation, cell death, related gene expression and systems of biological analysis in bone marrow mesenchymal stem cells (BM-MSCs), with the aim of understanding the underlying mechanisms. PM2.5 and PM10 were tested in vitro at various concentrations (15 to 300 µg/mL) and durations (24 to 72 h). PMs induced cellular stress including membrane damage, shrinkage and death. Lower concentrations of PM2.5 increased proliferation of BM-MSCs, while higher concentrations served to decrease it. PM10 decreased BM-MSCs proliferation in a concentration-dependent manner. The X-ray fluorescence spectrometric analysis showed that PM contains high levels of heavy metals. Ingenuity Pathway Analysis (IPA) and hierarchical clustering analyses demonstrated that heavy metals were associated with signaling pathways involving cell stress/death, cancer and chronic diseases. qRT-PCR results showed differential expression of the apoptosis genes (BCL2, BAX); inflammation associated genes (TNF-α and IL-6) and the cell cycle regulation gene (p53). We conclude that PM causes inflammation and cell death, and thereby predisposes to chronic debilitating diseases.

Vascular endothelial growth factor (VEGFA) gene variation in polycystic ovary syndrome in a Tunisian women population.

BACKGROUND: Polycystic ovary syndrome (PCOS) is characterized by the growth of a number of small cysts on the ovaries which leads to sex hormonal imbalance. Women who are affected by this syndrome suffer from irregular menstrual cycles, decline in their fertility, excessive hair growth, obesity, acne and most importantly cardiac function problems. The vascular endothelial growth factor (VEGF) plays a pivotal role in tissue vascularization in general and in the pathogenesis of many diseases. The PCOS was found to be associated with high expression levels of VEGF. In women who undergo assisted reproductive procedures (ART), VEGF was found to be a key mediator of other factors to control ovary angiogenesis. Here, we set out to examine the association of VEGFA gene polymorphism with PCOS and its components in a population of Tunisia women to enhance our understanding of the genetic background leading angiogenesis and vascularization abnormalities in PCOS. METHODS: The association of VEGFA gene with PCOS and its components was examined in a cohort of 268 women from Tunisia involving 118 PCOS patients and 150 controls. VEGFA gene variations were assessed through the analysis of the following SNPs rs699947 (A/C), rs833061 (C/T), rs1570360 (G/A), rs833068 (G/A), rs3025020 (C/T), and rs3025039 (C/T). The linkage disequilibrium between SNPs was assessed using HAPLOVIEW software while combination of SNPs into haplotypes in the population and the reconstruction of the cladogram were carried-out by PHASE and ARLEQUIN programs, respectively. Genetic association and genotype-phenotype correlations were calculated by logistic regression and non-parametric tests (Kruskall-Wallis and Mann-Whitney tests), respectively, using StatView program. RESULTS: We observed 10 haplotypes in our studied cohort whereH1 (ACGG), H2 (ACAG), H7 (CTGG) and H8 (CTGA) were the most frequent. We observed the association of the genotype CT of the SNP rs30225039 with PCOS phenotype (P = 0.03; OR 95 % CI = 2.05 [1.07-3.90]) and a trend for correlation of the pair of haplotypes H2/H2 with prolactin levels in plasma (P = 0.077; 193.5 ± 94.3 vs 45.7 ± 7.2). These data are consistent with literature and highlight one more time the role of vascularization in the pathogeny of PCOS. CONCLUSIONS: LD pattern in VEGF locus showed a similar LD pattern between the Tunisian population and the CEU. More haplotypes in the Tunisian population than in CEU was observed (22 haplotypes vs 16 haplotypes) suggesting higher recombination rate in Tunisians. The study showed that there was any advantage of using haplotypes compared with SNPs taken alone.

Enhancement of Pathologist's Routine Practice: Reuse of DNA Extracted from Immunostained Formalin-fixed Paraffin-embedded (FFPE) Slides in Downstream Molecular Analysis of Cancer.

BACKGROUND/AIM: To date, the conventional formalin-fixed, paraffin-embedded (FFPE) technique is the gold-standard for preserving histomorphology. Once FFPE tissues are stained, slides are routinely archived along with their blocks at biobanks/hospitals. However, the reuse of fixed and stained biospecimens as DNA source is not a common routine practice worldwide and, thus, indicates the need of studies to investigate the feasibility of extracting DNA from already immunohistochemistry (IHC) FFPE-stained slides and its possible reuse in subsequent downstream molecular analyses. MATERIALS AND METHODS: FFPE IHC slides from colorectal cancer (CRC) patients were prepared and stored in the CEGMR Biobank. The workflow consists of digitalization of IHC stained slide's image, removing the slide cover-slip, crude dissection and DNA extraction. Following DNA quality assessment, mutation analysis of CTNNB1 and methylation profile of CDH1 were performed. RESULTS: High-quality DNA was obtained allowing 60% concordance between CDH1 methylation and membranous E-cadherin expression pattern. Clean CTNNB1 DNA chromatograms with evenly-spaced peaks were observed. CONCLUSION: This study is a proof of concept to recycle and reuse DNA from IHC stained slides with suitable concentration and integrity for further downstream molecular applications. These findings will enhance the pathologists' knowledge, attitudes and practices (KAP) towards the use of these biospecimens and support the implementation of this approach in clinical pathology practice. Therefore, the scientific community will benefit from the largest comprehensive database of human fully annotated FFPE biospecimens already available at their disposal in order to demystify the complexity and the heterogeneity of many challenging diseases and foster the transition towards precision medicine.

Frequent methylation of the KLOTHO gene and overexpression of the FGFR4 receptor in invasive ductal carcinoma of the breast.

Invasive ductal carcinoma of the breast is the most common cancer affecting women worldwide. The marked heterogeneity of breast cancer is matched only with the heterogeneity in its associated or causative factors. Breast cancer in Saudi Arabia is apparently an early onset with many of the affected females diagnosed before they reach the age of 50 years. One possible rationale underlying this observation is that consanguinity, which is widely spread in the Saudi community, is causing the accumulation of yet undetermined cancer susceptibility mutations. Another factor could be the accumulation of epigenetic aberrations caused by the shift toward a Western-like lifestyle in the past two decades. In order to shed some light into the molecular mechanisms underlying breast cancer in the Saudi community, we identified KLOTHO (KL) as a tumor-specific methylated gene using genome-wide methylation analysis of primary breast tumors utilizing the MBD-seq approach. KL methylation was frequent as it was detected in 55.3 % of breast cancer cases from Saudi Arabia (n = 179) using MethyLight assay. Furthermore, KL is downregulated in breast tumors with its expression induced following treatment with 5-azacytidine. The involvement of KL in breast cancer led us to investigate its relationship in the context of breast cancer, with one of the protagonists of its function, fibroblast growth factor receptor 4 (FGFR4). Overexpression of FGFR4 in breast cancer is frequent in our cohort and this overexpression is associated with poor overall survival. Interestingly, FGFR4 expression is higher in the absence of KL methylation and lower when KL is methylated and presumably silenced, which is suggestive of an intricate relationship between the two factors. In conclusion, our findings further implicate "metabolic" genes or pathways in breast cancer that are disrupted by epigenetic mechanisms and could provide new avenues for understanding this disease in a new context.

Individualized medicine enabled by genomics in Saudi Arabia.

The biomedical research sector in Saudi Arabia has recently received special attention from the government, which is currently supporting research aimed at improving the understanding and treatment of common diseases afflicting Saudi Arabian society. To build capacity for research and training, a number of centres of excellence were established in different areas of the country. Among these, is the Centre of Excellence in Genomic Medicine Research (CEGMR) at King Abdulaziz University, Jeddah, with its internationally ranked and highly productive team performing translational research in the area of individualized medicine. Here, we present a panorama of the recent trends in different areas of biomedical research in Saudi Arabia drawing from our vision of where genomics will have maximal impact in the Kingdom of Saudi Arabia. We describe advances in a number of research areas including; congenital malformations, infertility, consanguinity and pre-implantation genetic diagnosis, cancer and genomic classifications in Saudi Arabia, epigenetic explanations of idiopathic disease, and pharmacogenomics and personalized medicine. We conclude that CEGMR will continue to play a pivotal role in advances in the field of genomics and research in this area is facing a number of challenges including generating high quality control data from Saudi population and policies for using these data need to comply with the international set up.

Effects of increased paternal age on sperm quality, reproductive outcome and associated epigenetic risks to offspring.

Over the last decade, there has been a significant increase in average paternal age when the first child is conceived, either due to increased life expectancy, widespread use of contraception, late marriages and other factors. While the effect of maternal ageing on fertilization and reproduction is well known and several studies have shown that women over 35 years have a higher risk of infertility, pregnancy complications, spontaneous abortion, congenital anomalies, and perinatal complications. The effect of paternal age on semen quality and reproductive function is controversial for several reasons. First, there is no universal definition for advanced paternal ageing. Secondly, the literature is full of studies with conflicting results, especially for the most common parameters tested. Advancing paternal age also has been associated with increased risk of genetic disease. Our exhaustive literature review has demonstrated negative effects on sperm quality and testicular functions with increasing paternal age. Epigenetics changes, DNA mutations along with chromosomal aneuploidies have been associated with increasing paternal age. In addition to increased risk of male infertility, paternal age has also been demonstrated to impact reproductive and fertility outcomes including a decrease in IVF/ICSI success rate and increasing rate of preterm birth. Increasing paternal age has shown to increase the incidence of different types of disorders like autism, schizophrenia, bipolar disorders, and childhood leukemia in the progeny. It is thereby essential to educate the infertile couples on the disturbing links between increased paternal age and rising disorders in their offspring, to better counsel them during their reproductive years.

Whole-exome analysis of foetal autopsy tissue reveals a frameshift mutation in OBSL1, consistent with a diagnosis of 3-M Syndrome.

BACKGROUND: We report a consanguineous couple that has experienced three consecutive pregnancy losses following the foetal ultrasound finding of short limbs. Post-termination examination revealed no skeletal dysplasia, but some subtle proximal limb shortening in two foetuses, and a spectrum of mildly dysmorphic features. Karyotype was normal in all three foetuses (46, XX) and comparative genomic hybridization microarray analysis detected no pathogenic copy number variants. RESULTS: Whole-exome sequencing and genome-wide homozygosity mapping revealed a previously reported frameshift mutation in the OBSL1 gene (c.1273insA p.T425nfsX40), consistent with a diagnosis of 3-M Syndrome 2 (OMIM #612921), which had not been anticipated from the clinical findings. CONCLUSIONS: Our study provides novel insight into the early clinical manifestations of this form of 3-M syndrome, and demonstrates the utility of whole exome sequencing as a tool for prenatal diagnosis in particular when there is a family history suggestive of a recurrent set of clinical symptoms.

pecific nutrient combination effects on tax, NF- κB and MMP-9 in human T-cell lymphotropic virus -1 positive malignant T-lymphocytes.

BACKGROUND: Adult T-cell Leukemia (ATL) is a disease with no known cure. The disease manifests itself as an aggressive proliferation of CD4+ cells with the human T-cell Lymphotropic virus type 1 (HTLV-1). The leukemogenesis of the virus is mainly attributed to the viral oncoprotein. Tax activates the Nuclear Factor kappa B (NF-κB) which stimulates the activity and expression of the matrix metalloproteinase-9 (MMP-9). The objective of this study was to investigate the efficacy of a specific nutrient synergy (SNS) on proliferation, Tax expression, NF-κB levels as well as on MMP-9 activity and expression both at the transcriptional and translational levels in two HTLV-1 positive cell lines, HuT-102 and C91-PL at 48h and 96h of incubation. Cytotoxicity of Epigallocatechin-3-gallate (EGCG) was assayed using CytoTox 96 Non-radioactive and proliferation was measured using Cell Titer96TM Nonradioactive Cell Proliferation kit (MTT- based assay). Enzyme linked immunosorbant assay (ELISA) and electrophoretic mobility shift assay (EMSA) were used to assess the effect of SNS on NF-κB mobility. Zymography was used to determine the effects of SNS on the activity and secretion of MMP-9. The expression of MMP-9 was done using RT-PCR at the translational level and Immunoblotting at the transcriptional level. RESULTS: A significant inhibition of proliferation was seen in both cell lines starting at a concentration of 200µg/ml and in a dose dependent manner. SNS induced a dose dependent decrease in Tax expression, which was paralleled by a down-regulation of the nuclearization of NF-κB. This culminated in the inhibition of the activity of MMP-9 and their expression both at the transcriptional and translational levels. CONCLUSIONS: The results of this study indicate that a specific nutrient synergy targeted multiple levels pertinent to the progression of ATL. Its activity was mediated through the NF-κB pathway, and hence has the potential to be integrated in the treatment of this disease as a natural potent anticancer agent.