Qatar Foundation Annual Research Forum Volume 2012 Issue 1

The cardiac muscle contraction is regulated by a set of proteins known as sarcomeric proteins, which are components of thick and thin filaments. Mutation in these proteins especially cardiac myosin binding protein-C, a multi-domain (C0-C10) protein, is one of the major causes of hypertrophic cardiomyopathy (HCM). However, structure-function relationship of this protein is unclear. Mutation E258K, which is located in the C-terminal of domain C1, has been shown to be associated with HCM in Egypt. Hence, the purpose of the study was to understand the molecular basis of this missense mutation. Molecular modelling study was performed using the available crystal structure of the domain C1. Here, we have carried out molecular dynamic simulations for both WT and E258K for 10 ns, and the structures that showed major changes were considered for further analyses. Our results suggest that the mutation can change the local structural stability through altering a series of intramolecular interactions. Moreover, as the mutation results in replacement of a negative amino acid by a positive one, thus, it affects the surface electrostatic properties of the domain (Figure). Hence, it might interfere with the binding to neighbouring domains and with the other sarcomeric proteins such as actin and myosin. E258K appears to affect the structural integrity of the domain C1 both directly and indirectly. It is hoped that these findings can help to understand the molecular mechanism of the disease.

Background and Objectives: Diabetic nephropathy (DN), a serious complication of diabetes, is characterized by hyperfiltration, hypertrophy, extracellular matrix accumulation, fibrosis and proteinuria leading to loss of renal function. In renal hypertrophy, tubules increase in size and cause accumulation of the extracellular matrix, and are also associated with alterations in renal sodium handling as well as hypertension; processes linked by involvement of the arachidonic acid (AA) metabolites 20-HETE and EETs. This study aims to determine the specific AA-metabolizing CYP450 isoforms present in proximal tubules (PT) that are altered by high glucose (HG) in cultured PTs, and in an animal model of diabetes. It intends to investigate the effects of alterations in CYP isoforms and/or AA-metabolite levels in DN. This work will investigate the mechanism of PT injury and the effect of inhibition of AA-metabolites in vitro and will also provide insight into the cross-talk between CYP450 isoforms and other sources of reactive oxygen species (ROS). Methods: Immunohistochemistry, hypertrophy, apoptosis, fibrosis, ROS generation, 20-HETE and EET formation, CYP4A and Nox protein expression, and mRNA levels were measured in vitro and in vivo. Results: Exposure of PT cells to HG resulted in apoptosis and hypertrophy. HG treatment increased ROS production and was associated with CYP4A and CYP2C upregulation, 20-HETE and EETs formation, and Nox oxidases upregulation. The effects of HG on Nox proteins and mRNA expression, matrix protein accumulation and apoptosis were blocked by HET0016, an inhibitor of CYP4A, and were mimicked by 20-HETE. Inhibition of EETs in vitro promoted the effects of HG on cultured proximal tubular cells. In parallel, the levels of CYPs 2B, 2C, and 4A were assessed in a rat model of streptozotocin-induced diabetes. There was significant induction of expression and activity over control of these CYPs associated with an increase in ROS production, Noxs expression, PTs injury, and this was prevented by insulin therapy. Conclusion: Our results indicate that hyperglycemia in diabetes has a significant effect on the expression of AA-metabolizing CYPs, manifested by increased AA metabolism, and might thus alter kidney function through alteration of type and amount of AA metabolites; this pathway is through an oxidative stress-dependant mechanism.

Background: Although the linkage between germline mutations of BRCA1 and hereditary breast/ovarian cancers is well established, recent evidence suggests that altered expression of wild type BRCA1 might contribute to the sporadic forms of breast cancer. The mechanism underlying the downregulation BRCA1 expression is not well understood. It might be dependent upon repressor activity that governs histone acetylation and DNA accessibility at the BRCA1 promoter. TNRC9 (TOX3) gene, highly associated with breast cancer susceptibility, encodes a nuclear protein of uncertain function but can modify chromatin structure. We hypothesized that constitutive expression of TNRC9 could be relevant to breast cancer biology through the modulation of BRCA1 activity. Methods: We assessed the associations of TNRC9 gene amplification with breast cancer onset and survival. The search for targets and effects of TNRC9 was performed using multifaceted molecular approaches. Results: The TNRC9 gene is often amplified and overexpressed in breast cancer, particularly in advanced breast cancer. TNRC9 gene amplification is associated with reduced disease-free and metastasis-free survival rates. TNRC9 significantly increased breast cancer cell proliferation, migration and survival of exposure to apoptotic stimuli, and tumor progression both in vitro and in mice models. Gene expression profiling, protein analysis and in silico assays of large datasets of breast and ovarian cancer samples suggested that TNRC9 and BRCA1 expression are inversely correlated. TNRC9 binds not only to BRCA1 promoter but also to the CREB complex, a BRCA1-transcriptional regulator. TNRC9 downregulates the expression of BRCA1 by altering the methylation status of its promoter. Conclusions: Our study unveils a molecular basis for a TNRC9 role in breast cancer and highlights a new paradigm in BRCA1 regulation.

Background: Ovarian cancer is the second leading cause of cancer-related death in women worldwide. Despite optimal cytoreduction and adequate adjuvant therapy, many patients will experience disease recurrence. Targeted therapies have been evaluated in ovarian cancer as a method to overcome resistant disease. Antiangiogenic therapy such as bevacizumab (Avastin; Genentech) has limited efficacy. Indeed, it increases hypoxia, drug resistance and, tumor rebound phenomenon observed after withdrawal. Hypothesis: We hypothesized that abnormalities in the tumor endothelium contribute to tumor growth which may be a direct source for chemotactic factors and might be responsible for residual microscopic disease and rebound effect following antiangiogenic treatment. Methods: Using a feeder-free Matrigel and spheroid models of ovarian cancer, we examined the effect of bevacizumab on residual disease. We used Akt-activated endothelial cells (EC) that replicates tumor endothelial biology and controls tumor growth, and human umbilical vein endothelial cells (HUVEC) to investigate the antiangiogenic activity of bevacizumab by angiogenesis and migration assays. We conducted an XTT assay to examine the effect of bevacizumab on proliferation of vascular endothelial growth factor (VEGF) producing human ovarian cancer cell lines. Finally, expression of FGF-2, phospho-Akt was assessed by Western blotting and flow cytometry. Results: We demonstrated the role of Akt-activated ECs in supporting expansion and self-renewal of ovarian cancer cells (OCC) in a residual disease context. We demonstrated that OCCs activate the endothelium, which displays resistance to bevacizumab. Bevacizumab had no effect on the proliferation of Akt-activated ECs, but significantly inhibited angiogenesis and delayed wound healing in HUVEC. We demonstrated that in this setting the cross-talk between cancer cells and ECs activates Pi3k/Akt inducing an autocrine loop through the pro-angiogenic factor fibroblast growth factor-2 (FGF-2) bypassing the VEGF-R pathway. We demonstrated that FGF-2 blocking would efficiently reverse the resistance to bevacizumab. Conclusion: Our data highlights the role of an activated endothelium in the constitution of the residual disease and resistance to bevacizumab. These results hint on the concept of using combination therapy to override drug resistance in ovarian cancer and to suppress or eradicate residual disease.

A consanguineous Arab family affected by a novel autosomal recessive disorder characterized by severe mental retardation and failure to thrive, was studied by Illumina 700K SNP genotyping, candidate gene mutation screening and whole exome sequencing for one affected member. Clinical findings include ptosis, bilateral epicanthic folds, striking midface hypoplasia, downturned mouth corners, thin upper vermillion, prominent ears, bilaterally short fourth metatarsal bone, bilateral fifth finger camptodactyly, mildly limited mobility in both knees and hypotonia. The gene was mapped by homozygosity mapping to 4 possible genome intervals, as the number of family members was insufficient for a significant LOD score. The positional candidate MAP2K1 was sequenced. No pathogenic mutations were identified. Whole exome target enrichment sequencing was performed on ABI SOLiD 4 System and Illumina HiSeq platforms for a single affected individual. Six non-synonymous variants were positioned within the homozygosity intervals. Two affecting evolutionary highly conserved amino acids with damaging effects according to PolyPhen and SIFT protein-modeling software, which were validated by Sanger sequencing to determine whether variants co-segregated with the disease phenotype. The variants were absent in 188 ethnically matched control chromosomes. One gene shows very limited expression in the brain while the other, a zinc finger protein, appears to be the disease causing mutation. Zinc finger proteins, a family of DNA and RNA binding proteins are transcriptional regulators controlling developmental cascades of gene expression especially during fetal brain development. Mutations in zinc finger domains interfere with normal brain development, are associated with non-syndromic X-linked mental retardation with impairments in adaptive behavior, and manifest during the developmental period causing severe mental retardation. The c.C5054G [p.S1685W] mutation affects 2 of the 3 ZNF407 isoforms, is located in the last third of the zinc finger domains and affects a serine residue in the alpha-helical part adjoining two zinc finger domains. This is thought to eliminate the functionality of downstream domains and interferes with expression of various genes under ZNF407 control during fetal brain development. Homozygosity mapping and whole exome sequencing of a single affected individual was the most effective, least labor intensive and most economical approach in identifying the mutation for this novel autosomal recessive zinc finger protein that causes a mental retardation syndrome.

Background & Objectives: Stein-Leventhal syndrome commonly known as polycystic ovary syndrome (PCOS) is the most common endocrinopathy among reproductive females. PCOS is a multisystem challenge causing not only gynecological issues such as irregular menstrual cycles, hyperandrogenism and infertility but also causing insulin resistance, type 2 diabetes and cardiovascular diseases. The objective of our study was to determine the incidence of PCOS among reproductive females at Qatar University using the National Institutes of Health (NIH), the Rotterdam consensus (Rott.) and the Androgen Excess Society (AES) criteria to determine the common phenotypes and hormonal parameters found in Qatar. Our aim was also to conduct a community-based study to make up for the lack of work done in female healthcare. Methods: A sample size of 121 females between the ages of 18-25 years were evaluated for symptoms of PCOS using family history of PCOS, body mass index and hormonal analysis. The sample was divided into the following four groups: control (n= 49), irregular menstrual cycles only (n= 13) , hyperandrogenism only (n= 27) and PCOS group (n= 31). Blood was drawn for measurement of TSH, progesterone, insulin, estradiol, SHBG, testosterone, DHEAS and prolactin. Modified Ferriman-Gallwey score was used for the evaluation of hirsutism. Results: The incidence of PCOS was found to be 25.83% (n= 31) using the Rotterdam criteria. Lower values were achieved using AES (20.83%, n= 25) and NIH (19.17%, n =23) definitions. Among our PCOS group, 16.13% (n= 5) were Qataris, 48.39% (n= 15) were Asians, 22.58% (n= 7) were Africans and 12.90% (n= 4) Europeans. Among the clinical features, family history of PCOS (p=0.037), hirsutism (p= 0) and irregular menstrual cycles (p= 0) were found to be significant factors in the PCOS group. Biochemical analysis showed elevated testosterone, elevated DHEAS and decreased SHBG hormones to be significant factors found in the PCOS group. Insulin and prolactin did not show any significant differences between the groups. Four individuals from the PCOS group were aware of their condition while 87% were unaware of their condition. Conclusions: Our study revealed a fairly significant prevalence of PCOS in the region and indicated a lack of awareness among people about their condition. There is need for government initiatives towards adequate investigations, management and treatment of PCOS.

Background: Apolipoprotein E (ApoE), a protein component of blood lipid particles, plays an important role in lipid transport and delivery. Single polymorphisms in residues 112 and 158 define the common E2, E3 and E4 alleles. In a study of Qataris, we observed that 17.4% of the African-derived genetic subgroup were heterozygotes for the rare Arg145Cys (R145C) variant that functions as a dominant trait with incomplete penetrance associated with dyslipidemia. Based on this, we hypothesized that the R145C polymorphism may be common in African-derived populations. Methods: The prevalence of the R145C variant worldwide was assessed in the 1000 Genomes Project (1000G) and then in 1012 Caucasians and 1226 African-Americans in New York City. Lipid profiles of the Qatari and New York R145C+ heterozygotes were compared to controls. Results: R145C+ Qatari heterozygotes had higher triglyceride levels compared to Qatari controls (p <0.007). The 1000Gs data demonstrated that the R145C polymorphism is rare in non-African derived populations, but present in 4.9-12.3% of Sub-Saharan African-derived populations. The R145C polymorphism was rare in New York City Caucasians (1/1012, 0.1%), but strikingly, 53 (4.3%) of 1226 New York City African-Americans were R145C+ heterozygotes, with an average of 52% higher fasting triglyceride levels compared to African-American R145C- controls (p <0.002). Conclusions: Based on these observations, there are likely to be millions worldwide derived from Sub-Saharan Africans that are ApoE R145C+. While larger epidemiological studies will be necessary to determine the long-term consequences of this polymorphism, the available evidence suggests it is a common cause of a mild triglyceride dyslipidemia.

Objectives: Stroke prevention therapy with oral anticoagulants (OAC) is reported to be under-utilized in patients with atrial fibrillation (AF) despite the overwhelming evidence supporting their use for stroke prevention. Rates and trends of the use of OAC from the developing world in this setting are lacking particularly in elderly patients. The aim of this study is to evaluate trends of utilization of OAC in elderly patients hospitalized with AF in a real-world population in a Middle-Eastern country over 20 years. Methods: Retrospective analysis of prospective registry of all patients aged above 70 years hospitalized with AF in Qatar from 1991 through 2010 was made. Rate and trends of OAC use was examined over the 20 years of the study. Results: During the 20-years period; 744 patients above the age of 70 years old were hospitalized for AF. Overall 26.1% of these patients received OAC with warfarin at hospital discharge while 60.2% received anti-platelets therapy with aspirin. The use of warfarin in this elderly group was significantly trending higher over the study period from 3.7% in the earlier years of the study to 39.5% in the latter years (P= 0.001). Anti-platelets therapy with aspirin also significantly increased from 47.4% in the earlier years of the study to 67.3% in the latter years (P= 0.001) [Table]. Conclusions: Although our study demonstrates that stroke prevention therapy with OAC and anti-platelets therapy are used with increasing frequency in elderly patients aged above 70 years over the 20-year study period, nonetheless they remain under-utilized. Further prospective studies are needed to investigate the reasons behind under-utilization of OAC in our region to help guide healthcare providers involved in the care of elderly people with AF to maximize potential benefit from OAC and anti-platelets while minimizing the hemorrhagic risk associated with these drugs.

Background: The incidence and prevalence of type 2 diabetes mellitus (T2DM) is on the rise in Qatar. The pathogenesis of T2DM is complex owing to molecular heterogeneity in the afflicted population. Current diagnostic methods rely on blood glucose measurements, which are non-informative with respect to progression of the disease to other associated pathologies. Thus predicting the risk and development of T2DM-related complications like cardiovascular disease remains a major challenge. Methods: We have used a combination of quantitative methods for characterization of circulating serum biomarkers of T2DM using a cohort of non-diabetic control subjects (n=76) and patients diagnosed with T2DM (n=106). In this case-control study, the samples were randomly divided as training and validation data sets. In the first step iTRAQ (isobaric tagging for relative and absolute quantification) based protein expression profiling was performed for identification of proteins displaying a significant differential expression in the two study groups. Five of these protein markers were selected for validation using multiple reaction-monitoring mass spectrometry (MRM-MS) and further confirmed with Western Blot and QRTPCR analysis. Results: We report the identification and verification of several biomarker candidates that can be potentially used for subset stratification of T2DM cohort. We identified a total of 227 high confidence proteins corresponding to 1393 peptides with greater than 95% confidence representing >40% sequence coverage. GO analysis predicted the differentially expressed proteins to be involved in a wide variety of cellular and metabolic processes. A total of 124 proteins showed a fold change of 2 or more between the control and T2DM serum samples. We report a set of five proteins viz. apolipoprotein-A-1, vitamin D binding protein, fibronectin, afamin and transthyretin as potent biomarkers, which are significantly up-regulated in diabetics. Conclusion: Our study revealed that many distinct molecular networks and metabolic pathways were activated in T2DM. It is hoped that future studies with larger cohorts as well those that focus on dissecting mechanistic roles will enhance our understanding of pathogenesis of T2DM and contribute to the development of targeted therapeutics in accordance with the personalized medicine paradigm.

Background and Objectives: Hematopoietic stem cells (HSC) are the most widely studied and characterized adult stem cells, which play an essential role in sustaining the formation of blood and immune system. The ease of their manipulation, the lack of serious ethical issues, and, in the autologous setting, the absence of their immunogenicity, have made them an attractive tool for developing stem cell-based therapies. Exploiting the properties of HSC by microRNA (miRNA) profiling offers an attractive approach to identify new regulators of stem cell fate. Although numerous miRNA have been screened from hematopoietic stem cells (HSC), the targets corresponding to many of these miRNA have not yet been fully elucidated. Therefore the objective of this study is to generate a miRNA profile from a subpopulation of adherent CD34+ HSC isolated from G- colony-stimulating factor mobilized peripheral blood aiming to understand the role of selected miRNA in regulating HSC stemness. Methods: CD34+ cells from patients' blood were isolated using a CD34+ isolation kit (Miltenyi Biotec) according to the manufacturer's protocol. miRNA profiling of adherent and nonadherent CD34+ cells was done using TaqMan Array MicroRNA Cards. Nanog expression levels was tested using a dual-luciferase reporter construct for miR-181a* or its mutant variant and Nanog 3′ UTR mRNA. Results: In this study, we have identified eight clusters of miRNA that were differentially expressed in an adherent subpopulation of CD34+ stem cells. Further analysis of one of the clusters by bioinformatics revealed that a miRNA, miR-181a*, which is highly expressed in the adherent CD34+ cells, affects the expression levels of Nanog, a stem cell surrogate marker. We show specifically by reporter assay and mutational analysis that miR-181a* targets a seedless 3′ compensatory site in the 3′UTR of Nanog and affects gene expression. We demonstrate that inhibiting miR-181a* upregulates the Nanog expression level, in addition to an increase in alkaline phosphatase activity. Conclusions: In conclusion, our results highlight a new stem cell-related target for the miR-181 family and show that miR-181a* directly targets Nanog in a subpopulation of CD34+ stem cells suggesting a possible role of miR-181a* in regulation of adherent CD34+ HSC cells stemness.