Genetics Laboratories

FUNCTIONAL ANALYSIS OF NOVEL HEART SPECIFIC GENES

 -Functional Analysis Of Novel Heart Specific Genes in mice and man: A subtractive hybridization cDNA library specific to BALB/c mouse heart tissue was previously constructed in our laboratory. The purpose of this preliminary study was to find novel and rarely expressed genes that have important roles in cardiac formation and function.

The aim of the studies is to analyze the unknown transcripts in detail. Therefore, detailed bioinformatics analyses, Northern Blotting, and in situ hybridization techniques are exerted. As the final step, functional analysis based on cell culture systems is being planned. Moreover, the cell culture experiments will be designed considering the nature of the studied gene products.

-Deciphering ROR-a target genes in monocyte and endothelial cells and understanding their roles in atherosclerosis: Atherosclerosis is caused by inflammatory response initiated by endothelial cells and macrophages in the intima. ROR-alpha is a hypoxia-sensitive transcription factor which regulates systemic inflammatory response and cholesterol metabolism by its target genes expressed in the liver.

The aim of this project is to retrieve new data on the role of ROR-alpha atherosclerosis development. The project is expected to provide potential molecular targets that may be used for the prevention or the treatment of atherosclerosis in the future. Expressions of which are supposed to be changed depending on intracellular cholesterol level and circadian rhythm, will be tested in cell culture unit of the Immunology Department to elucidate relationship of atherosclerosis with the conditions in question. In the end of this project, novel genes involving atherosclerosis development and alterations of their expression profiles upon certain inducers will be indentified.  

 Molecular Cardivascular Epidemiology

 -Cardiovascular Clinical Epidemiology: Coronary Heart Disease is one of the major health problems causing death. As a study that reflects the profile for cardiovascular risk factors in the Turkish population, TEKHARF (TARF) Project has been running since 1990. In this long-term project, broad spectrums of conventional cardiovascular and metabolic risk factors are being investigated. The study has examined the hypothesis that functional variants of certain genes that are associated with cardiovascular events, and has identified that genes that play role in lipid, energy and glucose metabolism are important in risk determination.

Genetic Risk Factors for Cardiovascular Disease and Metabolic Syndrome in Turkish People followed for 15 years (TEKHARF-Genetics), TUBITAK 2005-2008: Our aim is to study the polymorphisms and haplotypes of the selected genes, and their association with cardiovascular and metabolic events. Thus, genetic risk factors that are of equal value to conventional risk factors will be identified. These genes that are found to be associated will be statistically analysed and identified as genetic risk factors for the Turkish population. Among these risk factors lipocalin family is one of the most important interest areas.

Association of the APOD gene with cardiovascular diseases and metabolic syndrome: ApoD belongs to lipocalin family, which plays role in the transport of hydrophobic molecules. Our previous findings show that ApoD is present among genes that are expressed mainly in the heart and its expression is significantly decreased on atherosclerotic plaques.

Genotype distribution of selected polymorphic points (“SNP”) are being identified in the TEKHARF-DNA bank of 2235 people specific for Turkish Population in association with cardiovascular diseases and/or metabolic syndrome. In addition to this, ApoD-protein analyses in peripheral blood of patients are planned for determination of the correlation between the discovered ApoD variances and protein amounts. Integration of study findings will contribute to the construction of the highly predictive “genetic risk panel” specific for the Turkish population.

Neuro-genetics

-Parkinson and Alzheimer Diseases Research:

Our study group works on the genetic basis of neurological disorders and holds about 1.085 DNA samples from patients with various movement disorders concerning  Parkinson’s Disease, Alzheimer Disease, dystonia, ataxia, Restless Leg Syndrome and healthy controls in the laboratory. This research is performed using a variety of methodologies. The goal of our research is to identify genetic variability that causes or contributes to neurological diseases in order to facilitate understanding of the molecular processes underlying disease. With this perspective, the ongoing projects are detailed as below:

Autosomal-recessive gene mutation frequencies in Turkish population: The objective is to evaluate the phenotype and frequencies of mutations in genes associated to Parkinson’s (PD) and Alzheimer’s Diseases.

PRKN Gene: The project team has finalized experiments on PRKN, DJ1 and Pink1 genes so far by MLPA (Multiplex Ligation-dependent Probe Amplification). Our data suggest that the PRKN gene mutations belong to the most frequent form associated with PD in Turkey. The prevalence rate with regard to the age of onset in our population is comparable to those previously described.

LRRK2 Gene: A total of 255 patients with parkinson from 223 families originating from Turkey were recruited to the study. Our results suggest that mutations in these regions of LRRK2 gene are not the main causes of PD in the Turkish population. In future, the rest of the genomic regions of the LRRK2 gene are going to be analysed in further detail.

DJ-1 and Pink1 Genes: Our study including exclusion of mutations in genes already known to be involved autosomal-recessive (AR) PD (Parkin, DJ-1 and Pink1), identification of new loci for AR parkinsonism by homozygosity mapping using SNP microarrays (in collaboration with the team from INSERM UMR/S 679, Prof Alexis Brice, Paris, France).

PSEN1, PSEN2 and APP Genes: The main goal of this research is to identify mutations in three genes namely, presenilin 1 (PSEN1), presenilin 2 (PSEN2) and amyloid precursor protein (APP), which are associated with familial as well as sporadic forms of Alzheimer disease.

 
 

Molecular Hematology Group

Intracellular interactions and functional analysis of WNT target genes

WNT pathway plays role in normal and malign hematopoietic cells. WNT proteins regulate “self renewal capacity” of hematopoietic stem cells and T and B-lymphocytes. We determined a multistep deregulation in the T-cell and B-cell acute lymphoblastic leukemia patients. Our goal is to determine the functional organization of molecules in different lineage leukemia. To do this we are using different approaches including shRNA interference, proximity ligation assay, reporter assay etc. Our findings so far, indicate that abnormal WNT signaling exists in T-ALL.

Genomic analysis of Childhood Acute Lymphoblastic Leukemia

Childhood acute lymphoblastic leukemia (ALL) is the most common malignancy in children and mechanisms of leukemic transformation are not well defined. In this project, we aimed to use of high resolution, genome-wide approaches to identify genomic aberrations contributing to leukemogenesis.

 T-cell ALL (T-ALL), results from the malignant transformation of normal developing T cells in the thymus, the so-called thymocytes. Aberrant regulations of signaling pathways that control normal T-cell developmentin the thymus are importantfor T-ALL leukemogenesis, where as chromosomalaberrations leading to abnormal fusion proteins, as often found as causative factor in precursor B-ALL, are not commonly found inT-ALL. In our first study, we performed whole genome expression arrays where we defined a new subset of T-ALL with deregulated WNT signaling and a novel molecular marker is found regulated in this T-ALL subgroup. Novel oncogenic proteins and miRNA deregulations are still in validation process.

Relapsed childhood ALL carries a poor prognosis, despite intensive retreatment, owing to intrinsic drug resistance. Hence, in the second study, we aimed to understand the genetic events that contribute to relapse and chemo resistance, and identify novel targets of therapy. Three high-throughput assays were used to identify genetic and epigenetic changes at relapse. Using matched diagnosis/relapse bone marrow samples from children with relapsed B- ALL and T-ALL we evaluated gene expression, copy number abnormalities (CNA), and DNA methylation. There were many genetic alterations that evolved from diagnosis to relapse. We identified a distinct signature of differentially expressed genes from diagnosis to relapse associated with early relapse, and DNA methylation analysis found increased promoter methylation at relapse.

Targeted amplicon deep sequencing in high-risk ALL patients.

We are currently conducting a targeted amplicon deep sequencing screening of candidate genes in high-risk acute leukemia. We are a member of ELN (European Leukemia Net) and a collaborator of IRON II study, which is an international project under this organization. IRON II “The Interlaboratory RObustness of Next-generation sequencing (IRON) study” conducted for the deep analysis of the candidate risk genes in hematological malignancies. Targeted amplicon deep sequencing is a valuable tool to determine the background complexity (clonal variety) of the individual patients.

This study has started with the aim of sequencing high priority genes in a large cohort by using GS FLX sequencing instruments. We are currently conducting a targeted amplicon deep sequencing screening of candidate genes in high-risk acute leukemia. Istanbul University, Institute of Experimental Medicine (DETAE)-Genetics Department is participating the project in ALL and CML sub-teams. As a part of IRON II Study, we are aim to analyze candidate genes to characterizing molecular markers for disease classification, patient subgroup identification or new markers for treatment in ALL and CML patients.

Epilepsy and Rare Neurological Disorders Research Group:

Our main research interest mainly focuses on identification of genetic factors contributing to epilepsy. Epilepsy is a chronic heterogeneous disease manifested by recurrent, unprovoked seizures at any age. This is the second most common condition after headache with a prevalence of 0.5-1%. Among all epilepsies, idiopathic generalized epilepsy (IGE) is of great interest since it accounts for almost 30 per cent of all epilepsies and it tends to be genetically determined. The clinical features are characterized by age-related occurrence of generalized seizures in the absence of any detectable brain lesions or metabolic abnormalities. Childhood and juvenile absence epilepsy, juvenile myoclonic epilepsy and epilepsies with generalized tonic-clonic seizures represent the most common IGE syndromes.

–          Molecular genetic dissection of the heritable risk of IGEs by genome-wide linkage and association studies and candidate gene sequence analyses

–          Genome-wide array-based methylation profiling of parent-offspring trios displaying vertical inheritance of the IGE trait, i.e. one of the parents is affected along with the child.

–          Differential expression analysis of apoptosis and inflammation related genes in Mesial Temporal Lobe Epilepsy using human brain tissue.

–          Whole genome expression analysis using rat models of febrile seizure in order to investigate latent period of epileptogenesis.

We are also interested in studying rare inherited disorders, which are quite frequent in Turkey, mostly due to an increased rate of consanguineous marriages and presence of families having multiple children traditionally. Identification of new genes from families with rare disorders will eventually serve as models for complex disorders. We are currently working with such families displaying rare neurological disorders including disorders of brain development that are associated with epilepsy and ataxia. We would like to extend this research to other rare disorders.

–          Gene mapping pipeline: consanguineous families with rare recessive disorders with more than two affected individuals are recruited. In this regard, whole genome genotyping is performed in both affected and unaffected individuals from each family, followed by statistical analysis to map the gene region and to detect any pathogenic copy number changes in affected individuals. Candidate gene search is then employed for prioritized genes lying in this region in order to find disease-associated variants. If the gene region is extensive or if more than one gene region is detected, exome sequencing is used to study all exons in the genome. Genes in the linkage regions are primarily analyzed among the whole exome data. The variants identified are validated by conventional sequencing and scanning methods and then subjected to segregation analysis and frequency detection in a matched control group. In relatively small families where linkage analysis fails to map a single strong gene region, high-throughput screening is possible for all genes in candidate regions via exome sequencing.

Finally we have extended our research to SNP-array based ‘molecular karyotyping’ where we aim to detect copy number variations associated with children born with multiple congenital and mental retardation.

Immunogenetics

 Many disease states, including autoimmune diseases and cancer, are characterized by the presence of antibodies directed against self-antigens (autoantigens). These circulating antibodies or their antigen targets serve as potential biomarkers for understanding disease pathogenesis and defining its clinical outcomes, further with developing new therapeutic approaches.

Protein arrays are poised to become a central proteomics technology both important in basic research and biotechnological developments. Protein arrays are used in for in vitro functional interaction screens and particularly to detect antibodies or autoantigens in individual patient during disease or to monitor immune responses. Moreover, since most proteins are made by recombinant methods, there is direct connectivity between results on protein arrays and DNA sequence information.

We are mainly investigating new autoantigens in Behçet’s Disease (BD) which is a chronic inflammatory disorder of unknown pathogenesis, characterized by recurrent uveitis, skin lesions, oral aphthous and genital ulcers. Central nervous system involvement (Neuro-Behçet’s disease, NBD) is one of the most serious complications of BD and is encountered in 5-15% of the patients. Our approaches are based on protein array methods for the search of disease specific proteins that may have medical or diagnostic potentials as disease markers or drug targets and their corresponding gene sequences with respect to amino acid sequences of these antigens and their epitopes as well.

In our recent projects, we identified new autoantigens (PINK1, STIP1 and SWAP70) in BD or NBD by SEREX (Serological analysis of recombinant expression of cDNA expression libraries) and protein arrays (ImaGenes). New identified autoantigens are studied further for validation by ELISA and Western Blot.

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