Ph.D. Theses

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Absence epilepsy in Turkish patients: a novel gene at susceptibility locus 2q36 and the role of GABRG2
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2009., 2009.) Çapan, Özlem Yalçın.; Çağlayan, S. Hande.
Idiopathic absence epilepsies (IAE) are complex disorders mainly caused by genetic factors. Two major whole-genome linkage studies performed on many IAE samples pointed to chromosome 2q36 as a susceptibility locus for absence epilepsies. Whole genome linkage study on absence epilepsy model Wag/Rij rats, also showed the syntenic 2q33-37 region to be linked to the quantative trait of absence seizures. Candidate ion channel genes at 2q36 were screened but causative mutation could not be identified. On the other hand, mutations have been found in the subunits of GABA receptors and Ca channels in a few patients. At present, therefore, the complete picture of pathogenesis of the absence seizures is not known. In this study, to assess the possible role of 2q36 region in absence epilepsy, 205 Turkish absence patients and 219 healthy controls were used in an association analysis. Based on the haplotype block structure of the Turkish population in this region 10 tagSNPs were selected to cover the 160kb region at 2q36. The patients were subgrouped according to the syndrome and seizure types. The results revealed a significant association of two neighboring SNPs (rs7588807 and rs2840128) with JAE syndrome and even higher significant association with GTCS with the same SNP, rs7588807 that resided in the INHA gene. The point mutation and qPCR analysis of the INHA gene revealed mutations/variations in several patients and a large deletion that covered 30-50 kb in at least seven JAE patients supporting the association of INHA with the epilepsy phenotype and its establishment as a novel gene involved in the pathogenesis of JAE. The presence of other candidate genes in the deleted region paves the way for further molecular genetic analysis to reveal the role of each candidate gene in the pathogenesis of epilepsies, if any. The study further supports the role of GABRG2 in the pathogenesis of absence seizures by the identification of novel variations/mutations especially affecting the splice sites in CAE patients.
Amyotrphic lateral sclerosis in Turkey : Studies on Familial and sporadic ALS using high-throughput genomic technologies
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Özoğuz, Aslıhan.; Başak, A. Nazlı.
Amyotrophic Lateral Sclerosis (ALS) is a late-onset neurodegenerative disease, characterized by death of motor neurons in cortex, brainstem and spinal cord. It is a multifactorial disease with interacting pathogenic mechanisms. Most incidences are sporadic (SALS), while 10 per cent of cases have a family history (FALS). The genetics of ALS is complex; eight genes and six loci with autosomal dominant (AD), autosomal recessive and X-linked patterns of inheritance have been identified thus far. In the framework of this study, 198 Turkish ALS cases were investigated for possible mutations in the SOD1 gene. Five FALS cases were shown to carry disease-causing SOD1 mutations, while six were carriers of a rare polymorphism. In the next step, AD, nonconsanguineous FALS and juvenile cases were analyzed for the TDP-43, FUS and ANG genes via DNA sequencing. One homozygous D90A case, represented as recessive, was investigated by haplotype analysis and was compared to 21 Scandinavian ALS cases in search of a common ancestry. Additionally, 15 FALS and 13 juvenile cases, who were negative for the tested genes, were analyzed by whole genome genotyping for identification of new ALS genes. While no significant regions were obtained, a recessive family was preselected for the identification of homozygosity regions. Five candidate genes located within homozosity regions were examined in one of the family member; no mutations were identified. The same individual was also assessed by whole exome resequencing. Furthermore, this study also contributed to a collaborative genome-wide association study in SALS, where 14 month-survival advantage was shown for homozygous CC allele at rs1541160 SNP in the gene coding KIFAP3. This is the most comprehensive study performed in Turkey on the molecular genetics of ALS. The high-throughput methodologies used and the findings presented in this thesis are expected to shed light to the complex pathogenesis of amyotrophic lateral sclerosis.
Beta thalassemia in Turkey: |distribution, diversity, evolution and phenotype-genotype correlations
(Thesis (Ph.D.)- Bogazici University. Institute for Graduate Studies in Science and Engineering, 1999., 1999.) Tadmouri, Ghazi Omar.; Başak, A. Nazlı.
The present study illustrates the results of years of research on different aspects of beta-thalassemia in Turkey. Methods to detect the C-T change at position -158 upstream of the Gy-globin gene and the (AT)xTy motif 5' to the beta-globin gene were established and implemented. Analysis of these polymorphisms explained the reason behind the increased levels of fetal hemoglobin in nine out of 31 beta-thalassemia patients analyzed and demonstrated a dominant effect exerted by the XmnI Gy-globin polymorphism. Molecular screening of beta-globin genes in 19 beta-thalassemia individuals by genomic DNA sequencing uncovered the presence of 14 mutations; three of these are seen for the first time in Turkey. Another achievement made during this study is the compilation of beta-globin gene data collected since 1988 in a single repository. This allowed an easy mean to investigate the distribution of beta-globin gene mutations in various regions and towns of Turkey. This also demonstrated that the distribution of beta-thalassemia mutant alleles differed within each geographical area with a decreased gradient of mutation numbers from the East to the West of Anatolia. Analysis of nine polymorphic nucleotides and the (AT)xTy motif 5' to the beta-globin gene in 204 non-related beta-globin genes from Turkey exhibited 12 sequence haplotypes. Samples from the Black Sea region demonstrated a remarkable level of genetic heterogeneity in contrast to the homogeneity in Central Anatolian samples. Of the 22 beta-globin mutations analyzed, 18 were related with single sequence haplotypes and each of the other four were associated with a minimum of two sequence haplotypes. Our results demonstrate that the heterozygote advantage against malaria in Anatolia may have occurred at 6500-2000 BC by the oldest beta-thalassemia allele (i.e.,IVS-I-110 G-A). From that date on, most of the common beta-thalassemia mutations in Turkey were established and by the 13th century AD most of them were brought to frequencies close to what is observed at present.
Cellular and molecular analysis of regenerative neurogenesis in the zebrafish (Danio rerio) olfactory epithelium
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2021., 2021.) Kocagöz, Yiğit.; Fuss, Stefan H.
The olfactory epithelium (OE) has a high regenerative capacity that ensures the lifelong replacement of olfactory sensory neurons (OSNs). Different modes of neurogen esis originate from the selective activity of two distinct stem cell populations in the OE. In mammals, maintenance and regenerative neurogenesis are attributed to the selective activity of globose basal (GBC) and horizontal basal cells (HBC), respectively. How ever, the identity and function of similar progenitor cells in the OE of non-mammalian vertebrates remain elusive. This study demonstrates that a dual progenitor cell sys tem also exists in the zebrafish OE but with distinctive features when compared to mammalian models. HBC-like cells reside within the basal layer along the sensory OE, while GBC-like cells are confined to two isolated regions flanking the sensory OE. The positional and functional differences between these progenitor populations establish a balance between constitutive and regenerative modes of neurogenesis in the OE. Here, OE regeneration in response to global and OSN-specific injuries is examined in detail. The OE launches a rapid regenerative response upon chemical lesion, which results in HBC-like cell activation and OSN regeneration within the sensory OE. However, in response to selective OSN ablation, accelerated OSN regeneration is characteristically seen around the OE margins similar to the neurogenic activity in the intact OE. This crucial finding suggests that different injury conditions may affect HBC- and GBC-like cells selectively. RNA-sequencing analysis of the regenerating OE suggests that the diffusible signaling factor HB-EGF may have a critical role at the onset of regenerative neurogenesis. Exogenous HB-EGF administration induces OSN regeneration, while pharmacological inhibition of HB-EGF activity suppresses the regenerative response.
Characterization of GPR139 and investigating its possible role in brain development
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2022., 2022) Aslan, Tolga.; İyison, Necla Birgül.
G protein coupled receptors are a large family of cell surface receptors and are involved in a plethora of physiological functions from vision to various neuronal func tions. They have been a major target in the pharmaceutical industry and more than one third of currently available drugs target a GPCR. Although extensively studied, there are still 140 orphan GPCRs. GPR139 is an orphan GPCR in the rhodopsin fam ily and is primarily expressed in medial habenula. In addition to its cognate ligand, its function is still not known. At the time of the initiation of our study, the literature only suggested a possible role of GPR139 in neurological disorders such as ADHD and Alzheimer’s disease. Objective of this study is to investigate possible the role(s) of GPR139 in brain development via employing a Gpr139 knock-out mice model, and via in vivo ablation of Gpr139 in adult mice, and subjecting the animals to a set of behavioral experiments to measure the impact of deletion and ablation, respectively. In addition to animal experiments, various bioinformatic analyses were also performed to further characterize Gpr139. Our findings showed that Gpr139 is expressed differen tially in the brain tissue during embryonic development and its expression is most likely controlled by histone modifications and not DNA methylation. We also showed that ablation of GPR139 does not have any significant effects on spatial learning, locomotor activity, recognition memory and anxiety.
Characterization of novel Wnt/β-catenin pathway targets
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2016., 2016.) Akiva, İzzet.; İyison, Necla Birgül.
The Wnt/β-catenin signaling pathway is an evolutionary conserved pathway which has important functions in vertebrate early development such as axis formation, cellular proliferation and morphogenesis. The activation of this pathway leads to translocation of the transcriptional activator β-catenin into the nucleus where it activates T-cell factor/Lymphoid enhancer factor (Tcf/Lef) family of transcription factors, which regulate expression of developmental and cell cycle-related genes. Apart from its roles in various cellular processes, Wnt/β-catenin signaling pathway is also one of the most important intracellular pathways implicated cancer progression. A significant number of identified target molecules of this pathway, are known to have tumorigenic characters when mutated. Previous studies confirmed BRI3 (Brain protein I3) gene to be one of the transcriptional target genes of Wnt/β-catenin pathway. We used various approaches for the functional characterization of its novel targets. As a first step in our study, IFITM3 and MGAT1 proteins were confirmed as interaction partners for BRI3 by Yeast-two Hybrid and Co-IP techniques. BRI3 was found to be upregulated in response to TNF-α treatment and overexpression of BRI3 resulted in an increase in NFkB promoter activity. On the other hand, MGAT1 is a putative novel target of Wnt/β-catenin signaling pathway and determined to be upregulated in response to β-catenin activation. Cell proliferation and migration assays showed that, Huh7 cells stably expressing each of the BRI3 and MGAT1 genes have greater proliferative and invasive capabilities compared to control Huh7 cells. Furthermore, in vivo xenograft experiments were performed and it was determined that the stable overexpression of both of these genes in Huh7 cell lines lead to increased rate of tumor growth in NUDE/SCID mice. The resulting tumors were subjected to transcriptomic analyses using RNA-Sequencing technique in order to determine which pathways and biological processes take part in the cancer initiation process.
Characterization of thermophilic gene expression enzymes
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Çağlayan, Melike.; Bilgin, Neşe.
The DNA polymerase I genes of seven newly identified Geobacillus species within the family Bacillaceae, were cloned, sequenced and overexpressed in Escherichia coli. The polA gene of these species encodes DNA polymerase I of 878 amino acid residue protein with a predicted molecular weight of 99.3 kDa. Similarity analyses suggested that DNA polymerases belong to family A polymerases and lack 3'-5' exonuclease activity. The complete coding sequences of the genes were submitted into the GenBank. The recombinant (His)6-tagged DNA polymerases were purified by Ni2+-affinity chromatography and the homogeneous proteins were obtained after TEV protease digestion. DNA polymerases from Geobacillus anatolicus (Gana DNApolI) and Geobacillus kaue strain NB (Gkaue DNApolI) were further characterized in vitro and optimum conditions with respect to temperature, pH, monovalent and divalent ions were determined. Geobacillus DNA polymerase I fragment (GF DNApolI) was cloned and purified after homology modeling using Bacillus DNA polymerase I fragment (BF) as the model protein structure. The accuracy of GF DNApolI was measured by two M13 based fidelity assays which score errors produced during in vitro DNA synthesis of the lacZα complementation gene in M13mp2 DNA at 37°C, 50°C and 72°C. Base substitution errors increase three-fold when temperature is raised from 37°C to 72°C. DNA sequencing of the phage mutants showed that some of the base substitutions are more temperature sensitive than others. The most common base substitution error is the misincorporation of dGMP opposite to template G. Single nucleotide incorporations for both correct and for incorrect nucleotides were also studied under single-turnover conditions at 22°C, 37°C and 50°C. For both correct and incorrect dNTP insertions, the rate of polymerization, kpol, increased (seven- and four-fold, respectively) when temperature is raised from 22°C to 50°C, whereas only a slight change in Kd was observed. As a result, kinetic efficiency of the enzyme (kpol/Kd) shows five-fold increase over this temperature range.
Defining novel cancer genes, sage tags and co-regulated regions of the human genome evolving from the search and identification of novel Wnt/TCF/B-catenin targets
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2009., 2009.) Kavak, Erşen.; Koman, Ahmet.; İyison, Necla Birgül.
The Wnt signaling pathway is involved in many differentiation events during embryonic development and can lead to tumor formation after aberrant activation of its components. β-catenin, a cytoplasmic component, plays a major role in the transduction of canonical Wnt signaling. The first part of this study aimed at identifying novel genes that are regulated by active β-catenin/TCF signaling in hepatocellular carcinoma-derived Huh7 cells with high (transfected) and low β-catenin/TCF activities. High TCF activity Huh7 cells led to earlier and larger tumor formation when xenografted into nude mice. SAGE (Serial Analysis of Gene Expression), genome-wide microarray and in silico promoter analysis were performed in parallel, to compare gene expression between low and high β-catenin/TCF activity clones, and also those that had been rescued from the xenograft tumors. SAGE and genome-wide microarray data were compared and contrasted. BRI3 and HSF2 were identified as novel targets of Wnt/β-catenin signaling after combined analysis and confirming experiments including qRT-PCR, ChIP, luciferase assay and lithium treatment. In the course of analyzing SAGE and microarray data of Huh7 cells, we felt the need to overlay this data with different data sources. Starting with this motivation in the second part of the study, we performed the first meta-analysis of SAGE and microarray data together, which we termed common gene expression patterning (COGENT). By using COGENT data, we introduced the “being multi-cancer” concept for genes being differentially expressed in several types of tumors. There are not only multi-cancer genes but also multi-cancer co-regulated regions (RIDGEs) on the genome. “Multi-cancerness” of a gene correlates well with the number publications stating that gene in a cancer context, its rank within a single study and the number of its orthologs. There are many multi-cancer genes which have not been related to tumorigenesis as yet. In addition, there are 81 tumor associated RIDGEs (TA-RIDGEs) on the human genome which change similarly in multiple types of tumors.
Development of single cell genetic testing strategies : assessment of MHC compatibility, meiotic recombinations and beta-thalassemia in human embryos
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2011., 2011.) Taylan, Fulya.; Altıok, Ender.; Bilgin, Neşe.
Single cell molecular biology, a relatively new scientific branch, is promising to study unique questions and leading to novel applications in biology and medicine. Single cell studies have been challenged by difficulties in selection and isolation of appropriate cells, low amplification efficiencies, allele drop outs, PCR contaminations and inefficiency of conventional analysis strategies. This study has explored the possibilities of analyzing multiple genetic conditions particularly concerning the beta globin and the HLA regions in human embryos. The HLA genes, beta-globin gene, and the associated microsatellites have been amplified simultaneously by multiplex PCR. DNA sequencing has been optimized for high resolution genotyping. The real-time PCR and melting curve analysis have been adapted for the first time for rapid and reliable analysis of the HLA compatibility. Use of microsatellites of the extended HLA locus has enabled more accurate and efficient detection of the allele drop outs, contaminations and recombinations. Amplification and informative detection have been obtained for 1012 blastomeres out of 1180 human embryos used in this study, giving a detection rate of 86%. A total of 122 (13%) embryos were found unaffected from beta thalassemia and had identical genotype at ten HLA regions. Transfer of 94 embryos that have resulted into 16 pregnancies with 14 healthy offsprings indicates the feasibility of the single cell applications for preventive medical approaches. Microsatellite typing of the extended HLA locus has enabled to study the characteristics of the meiotic recombinations in human embryos. The recombination rate was determined as 0.44 cM/Mb, 2.1 fold less compared to the general genomic recombination rate of 0.92 cM/Mb. It was 3.83 fold higher in the maternal MHC regions compared to the paternal MHC regions. Upto 14 fold difference was observed among individuals. Breakpoints of recombinations in the class II region were clustered between the DRB1 and DPB1 genes covering the TAP1 and TAP2 genes.
Disease gene discovery by linkage mapping and exome analysis
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) Bölükbaşı, Esra Yıldız.; Özören, Nesrin.; Tolun, Aslı.
Disease gene identification is an important area in genetics. Consanguineous marriages are very common in some populations and lead to emergence of rare diseases. To uncover the functions of our genes and the molecular bases of diseases, novel disease genes need to be identified. Later the effect of the mutation on the protein could be investigated by various analyses. Also, discovery of a new disease gene can be a glimmer of hope for the patients who are hoping for definite diagnosis and development of therapies. The steps of the disease gene identification is mapping the locus by linkage analysis or homozygosity mapping, identifying the causative mutation by exome sequence analysis and relating the mutation to the disease pathology, thus uncovering the molecular pathogenesis. Possible or known effect of the mutation can be investigated via computational algorithms and in the light of the information in published studies and databases. In this thesis study, causative genes were searched in eight consanguineous families afflicted with six different recessive diseases. The two identified genes and the identified mutations are PDIA3 (p.Cys57Tyr) in Syndromic Intellectual Disability family and CEP19 (p.Tyr65*) in Bardet-Biedl Syndrome family. Besides, in BBS family possible modifiers GLI1 p.Gly274Arg, CCDC28B p.Phe110Phe, MKKS/BBS6 p.Ile339Val, C8ORF37 p.Ala178Val and TMEM67 p.Asp799Asp were identified/detected. In Isolated Intellectual Disability family, missense PTRHD1 p.Cys52Tyr mutation confirmed that the gene is responsible for ID. Gene expression assay revealed wide expression in brain. In Intellectual Disability and Hypothyroidism family missense TPO p.Asp240Gly was identified. Intronic splicing c.6375-1G>C in SPTBN2 in Spinocerebellar Ataxia family unraveled the basis of dominant vs recessive effects of the variants in this gene. Synonymous p.Ile382Ile in TNKS2 in one of the Pleuroparenchymal Fibroelastosis families was identified as the strongest candidate. No candidate variant could be identified in the other two families, indicating genetic heterogeneity for this disease.
Divergent functions of NLRP7 in embryogenesis, inflammation and oncogenesis
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2019., 2019.) Garipcan, Aybüke.; Özören, Nesrin.
NLRP7 is a novel protein about which we have limited information. To date, mutations in NLRP7 gene have been associated with recurrent Complete Hydatidiform Mole (RCHM) and NLRP7 is accepted to be the first causative gene for RHM. CHM is a gestational disease characterized by hyper trophoblast proliferation with no embryo formation. Furthermore, NLRP7 expression was found to be elevated in testicular seminoma, endometrium cancer and embryonal carcinoma. Yet, the possible mechanisms of action of NLRP7 in these biological conditions or pathways have not been enlightened. In this thesis, we generated patient derived induced Pluripotent Stem Cells (iPSC) to address the role of NLRP7 in HM. We revealed that inadequate NLRP7 levels expedited the di↵erentiation of iPSCs towards the trophoblasts through BMP4. Recovery of NLRP7 expression or BMP pathway inhibition decelerated excessive di↵erentiation of patient iPSCs to trophoblasts. Also, as NLRP7 is an NOD Like Receptor Family member, it is expected to play a role in innate immunity. We have found that infection of human monocytic cells with Pseudomonas aeruginosa activated the NLRP7 inflammasome followed by increased IL-1! secretion. Clearly, stable overexpression of NLRP7 is correlated with increased secretion of pro-inflammatory cytokines such as; TNF-alpha, IL-6, i-309. In addition, to elucidate the possible proto-oncogenic role of NLRP7 in tumorogenesis, we performed xenograft experiments in vivo. We found that stable expression of NLRP7 in the endometrial cancer cell line (Hec1a) resulted in increased tumor growth. Furthermore, potential interaction partners of NLRP7 were identified after co-immunoprecipitation followed by mass spectrometry. Our results shed further light to the overlapping and diverging molecular pathways regulated by NLRP7 in embryogenesis, inflammation and oncogenesis.
Effects of extremely low frequency electromagnetic fields on caspase activities
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Ozansoy, Mehmet.; Başak, A. Nazlı.; Denizhan, Yağmur.
All life on Earth is bathed in a sea of natural low-frequency electromagnetic fields from conception to death. Since the World Health Organization (WHO) launched its international electromagnetic fields (EMF) project in 1996, it has conducted international reviews of the evidence on whether exposure to static and extremely low frequency (ELF) fields might be harmful to health. ELF fields for WHO’s EMF project are defined as those having frequencies above zero and below 300 Hz. In the framework of this study, the activation of seven different caspases will be investigated systematically, when extremely low frequency electromagnetic fields, which are thought to be an environmental hazard according to WHO, are applied to the HEK 293 cell line. The selected frequency will be 50 Hz, which is the power transmission line frequency in most parts of the world. Two different magnetic field strengths will be applied to HEK 293 cells, and two different exposure durations will be chosen. Caspase activity levels are to be measured at different time points after exposure. The common pattern seen in all of them was the oscillation of enzyme activities from the beginning. At 100 μT, caspases gave four peaks at four, eight, 16 and 34-hour incubation periods. This oscillatory behavior can also be seen when 25 μT magnetic field was applied, but the behaviors of the enzymes were different in a certain extent. The location and the number of the peaks at 25 μT exposure were quite variable, but the activity periods of all caspases seemed to be shorter than those exposed to 100 μT. The data presented here indicate that when ELF-EMF is applied to the HEK 293 cells, all seven caspases investigated are found to be activated, but this activation shows an oscillatory pattern, and in the long run it seems to be damped by some intracellular mechanisms.
Elucidating the role of unzipped in the visual system of drosophila
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Terzioğlu Kara, Ece.; Çelik, Arzu.
The Drosophila eye is composed of 800 ommatidial units, each containing eight photoreceptors (PR). The outer PRs R1-R6 are responsible for motion detection, while the inner PRs R7 and R8 and are responsible for colour vision. The outer PRs express Rhodopsin1 (Rh), while the inner PRs express Rh3-Rh6. The pale subtype is formed when the R7 cells express Rh3 and the coupled R8 cells express Rh5, while in the yellow subtype R7 cells express Rh4 and the coupled R8 cells express Rh6. This final state of differentiation is reached after a sequential recruitment process of PRs at the 3rd instar larval stage. At the initial stage, the PR cells start differentiating and their axons target to their temporal layers. At the terminal stage the inner and outer PRs are distinguished from each other, each PR expresses a specicific Rh molecule, and the axons target to their final layers in the optic lobe. The aim of this study was to identify new molecules involved in inner PR differentiation, mainly focusing on three genes expressed in R8 cells that have been identified in a previously performed enhancer-trap screen. One of these genes, unzipped was chosen for further analysis because of its function as cell adhesion molecule (CAM). By immunostainings the expression of Uzip was revealed to be in R8 cells and a subgroup of glial cells at the 3rd instar larval stage, R8 cells and glial cells at the 48 hour pupa stage, and glial cells in the adult stage where some of them project to the medulla. Immunostainings showed that Uzip does not have a direct effect on PR formation and glia expression at the 3rd instar larvae and pupae stages; or on subtype specification at the adult stage. For axonal targeting both the downregulation of Uzip in the target area and the misexpression in neighboring PRs and glia, which are not in the target region, lead to an increase in axonal misprojection phenotype. According to our suggested model, Uzip expressing R8 cells follow the adhesive signal from the Uzip expressing glial cells at the target region of projection. This study identifies Unzipped and describes its expression and involvement in axonal targeting the visual system.
From genome scan to disease gene identification
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2008., 2008.) Uğur, Sibel Aylin.; Tolun, Aslı.
In the framework of this study, genome wide linkage scans of the following five inherited disorders were evaluated with parametric programs and suggestive loci were subsequently investigated with fine-mapping studies and candidate gene approach. Split-Hand/Foot Malformation (SHFM) affects the central rays of the autopod. We identified a novel SHFM locus at 12q13.11-q13 and a homozygous WNT10b mutation (p.R332W) in all affected individuals plus in an asymptomatic female. We propose that either a second locus contributes to the manifestation of SHFM phenotype or a suppressor locus prevented trait manifestation in the non-penetrant female. This is the first reported WNT10b mutation on the pathogenesis of limb development and recessive mutation in SHFM. Hypomyelination and congenital cataract is a recessive white matter disorder caused by mutations in gene DRCTNNB1A. Here we report a large intragenic deletion that does not lead to congenital cataract in all of the patients in an afflicted family. A novel form of recessive cone rod dystrophy was mapped to chromosome 17p13.2-p13.1, and the disease gene was identified as GUCY2D encoding the retinal guanylyl cyclase gene. The mutation (p.I949T) resided in the catalytic domain of the protein where other mutations had previously been associated with Leber congenital amaurosis, a common cause of childhood blindness. The milder phenotype observed in our patients implicate that either the mutation does not disturb the catalytic activity completely or modifier locus/loci interfere with the phenotype. Lastly, a recessive form of mental retardation and a dominant arthrogryposis syndrome were mapped to chromosomes 7q21.3-q31.1 and 13q31.3-q32.1, respectively.
From mutations to disease mechanism in Rett Syndrome, breast cancer, and congenital hypothyroidism
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2008., 2008.) Barış, İbrahim.; Battaloğlu, Esra.
Epidemiological studies provide the correlative data to understand the etiology of human inherited diseases and develop efficient genetic testing assays. Additionally, the accumulated data of genetic and epigenetic findings, expression profiling, and proteomics allows disease diagnosis, to understand the molecular mechanisms leading to the disease pathogenesis, and to develop efficient therapeutic approaches. In the framework of this thesis, we have investigated genetic and epigenetic changes and performed genotypephenotype correlations to unravel the molecular mechanisms that lead to three different diseases, Rett Syndrome, breast cancer, and congenital hypothyroidism. The genetic basis of Rett Syndrome (RTT) was investigated in a total of 71 RTT patients. A heterogeneous spectrum of disease-causing MECP2 mutations was identified in 68.2 per cent of a clinically well defined group of cases whereas in only 12.5 per cent of the patients referred for differential diagnosis suggesting that this gene does not represent a major cause of the disease among patients with Rett-like features. For the first time, we have identified gene duplications as causative mutations in female atypical RTT cases. Consistent with the animal models, our results support the possibility that duplication of MECP2 that leads to increased expression might underlie some cases of X-linked delayedonset neurobehavioral disorders including Rett Syndrome. Our results showed that exon rearrangements that could not be detected by standard techniques contribute to 19.3 per cent of these MECP2 mutations, and should be considered in especially RTT variants in order to determine the actual significance of the gene in the etiology of RTT. Genotype/phenotype correlation was performed based on comparison of severity score of patients with the type and location of the mutation and the XCI pattern. The results did not reveal a statistically significant correlation, but, the patients with exon deletions were found to be more severely affected than patients with all other types of mutations and patients with exon duplications to present with severe eye contact problems. Additionally, we have developed and validate a novel multiplexed amplification refractory mutation system (ARMS) assay for identification of seven common mutations that accounts for almost 65 per cent of all MECP2 gene mutations. The validation studies revealed that our novel assay is an efficient, reliable, and cost-effective screen for molecular genetic testing of patients with RTT. Furthermore, we tested the effect of DNA concentration on reliablity and reproducibility of SYBR green dye-based Real Time PCR analysis to detect the MECP2 exon rearrangments. The results suggested that Real Time PCR analysis is reliable for determination of the exon copy number if the DNA amount is in the range of 1-50 ng. To our knowledge, there are no known reports investigating the role of methylation of hHR23A and hHR23B genes in the tumor tissues. We have characterized the 5' flanking region of the hHR23A and hHR23B genes using web-based analysis and investigated the involvement of methylation status of putative promoter region of hHR23A and hHR23B genes in breast carcinogenesis. The observations of the hypermethylation of hHR23A gene and the presence of methylated conserved motifs and transcription binding sites in hHR23B gene among the analyzed tumor tissues suggested the involvement of methylation of hHR23 genes in the breast carcinogenesis. Investigation of epigenetic changes in tumor samples of breast cancer patients was a pioneering work since available literature implicates its presence only in cell lines. Since our CH patient was the first case with Bamforth Syndrome and suffered the plasma cholinesterase deficiency, the genetic mechanisms leading to congenital hypothyroidism and prolonged paralysis after mivacurium were investigated. In contrast to other reported two patients with TTF2 gene mutation, the presence of thyroid tissue in our patient suggested further phenotypic heterogeneity associated with human TTF-2 mutations. The functional study with a collaborative work also helped to understand the genetic mechanisms and provided original evidence that implicated differential effects of TTF-2 mutations on downstream target genes required for normal human thyroid organogenesis.
Functional and structural insights into a novel insect G protein-coupled receptor, allatostatin receptor type C of pine processionary moth
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 2020., 2020.) Shahraki, Aida.; İyison, Necla Birgül.
Insect neuropeptides regulate di erent aspects of insect physiology. They exert their function via binding to their cognate receptor belonging to Class A G protein coupled receptors (GPCRs). Neuropeptide receptors are potential targets for nextgeneration pesticides. Allatostatin (AST) neuropeptides regulate the development of insects through the inhibition of juvenile hormone (JH) secretion. Here, Allatostatin receptor type C (AstR-C) of pine processionary moth, a pest in Mediterranean countries, was extracted from the whole genome sequencing (WGS) data. The receptor was cloned and characterized combining via di erent approaches. Using resonance energy transfer (RET)-based techniques, kinetics of G protein coupling and -arrestin recruitment were investigated. Homology modeling, docking and molecular dynamics (MD) simulation approaches were conducted to predict the orthosteric pocket of the receptor which was validated by in silico and in vitro methods. The binding pocket was subjected to virtual screening studies to nd agonists. As a result, it was found that binding of the native ligand at sab-nanomolar and nanomolar ranges to the receptor induces Gi/o protein coupling and -arrestin recruitment, respectively. Kinetics studies revealed that a brief stimulation of the receptor at nanomolar range is enough to obtain a long-lasting response. The accuracy of the predicted orthosteric pocket was validated via G protein activation assay. Q2716:55 (Ballesteros-Weinstein numbering) was found to be critical for G protein-dependent activation of AstR-C. Virtual screening studies resulted in obtaining a small molecule capable of activating the receptor.
Functional enrichment methodology for analyzing omics data to study the aetiology of rare diseases
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2018., 2018.) Saygı, Ceren.; Özören, Nesrin.; Sezerman, Uğur.
Rare diseases (RDs) are a large and diverse group of disorders and defined by low prevalence, in other words, it is any disease that affects a small percentage of the population. According to OMIM and Orphanet, ~7000 different RDs have been estimated, but the number of phenotypes that remain to be defined could be considerably higher. The difficulty in obtaining the correct diagnosis is the most dramatic problem to be solved for the patients, about 30% still lack a diagnostic definition. The patients living with rare diseases visit an average of 7.3 physicians before receiving an accurate diagnosis and the mean length of time from symptom onset to accurate diagnosis is 4.8 years. Late diagnoses delay specific treatments and may have severe and life-threatening consequences. Molecular diagnosis is the most prominent way to facilitate earlier and accurate diagnosis, and hence an effective treatment for rare undiagnosed cases. In this dissertation project, a novel bioinformatics workflow is constructed for whole-exome/genome sequencing data analysis, variant prioritization and pathogenicity prediction from a cascade of different tools shading light into different aspects of the diagnostic process. The pathogenicity mechanisms of mutations are elucidated via molecular dynamics (MD) simulations. The newly developed pipeline is planned to be used for diagnosis of undiagnosed patients with a suspected genetic disorder, where other testing modalities have been inconclusive or noninformative. The workflow was tested on several undiagnosed clinical cases with their family members and achieved high success rates by identifying the causative variant. For two of these families, the pathogenicity mechanisms of mutations were described via MD simulations, and these findings have been submitted to two different SCI journals and passed the editorial approval. The diagnosis of one of these families was Periventricular Nodular Heterotopia, while the other was Nail Dysplasia-10. Both of the diseases are extremely rare that is seen in one in a million cases. In conclusion, we developed a unique workflow for molecular diagnosis of rare undiagnosed diseases. Our pipeline contributes to the already existing knowledge through the combination of population frequency, pathogenicity prediction tools, gene intolerance scores, and MD simulations for the first time.
Gene hunt in four inherited diseases
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Çetinkaya, Murat.; Tolun, Aslı.
Genetic linkage analysis is applied to identify loci that harbor gene or genes associated with a disease. It is possible to map disease loci by observing alleles the segregation of microsatellite or SNP markers with the disease in families, it is possible to map disease loci. Availability of densely spaced microsatellite marker maps and genomewide SNP scans have made such studies feasible. In the framework of this thesis, genomewide linkage data were evaluated with parametric linkage analysis software and haplotype segregation studies for four inherited disorders. Furthermore, candidate loci identified were further investigated by fine-mapping and candidate gene approach. Autosomal Recessive Ataxia (ARA) is a subgroup of hereditary ataxias, and characterized by slowly progressive impaired coordination of gait, hands, eye movements and speech. Nine patients from two consanguineous families were included in the study. We found that Aprataxin gene was mutated in six patients. Three patients were homozygous for a known nonsense mutation, and three patients for a novel missense mutation. For the three remaining patients clinical reevaluation revealed that they were afflicted with another disease, but no responsible locus could be identified. Autosomal recessive Larsen Syndrome (LRS) is characterized by congenital largejoint dislocations and craniofacial abnormalities. Seven consanguineous families were analyzed. In four families disease was mapped to 17q25.3 and homozygous mutations were found in Calcium Activated Nucleotidase 1. Three patients had the same known missense mutation. In one family, a novel missense mutation was identified. No common candidate region could be defined for the remaining three families. Juvenile Parkinsonism (JP) is characterized by classical triad of parkinsonism signs, bradykinesia, rigidity and resting tremor and disease onset before the age of 40 years. A missense variant in AK3L1 was identified in homozygous state in all patients but also in a healthy individual in the family. This variant was not detected in any of the 130 control individuals. We propose that an undetermined second locus either contributes to the disease manifestation in patients or protects the homozygous healthy individual. Lastly, autosomal recessive Congenital Cerebellar Hypoplasia (CCLH), characterized by nonprogressive congenital cerebellar ataxia with mental retardation, delayed motor development, disturbed coordination, hypotonia and cerebellar Hypoplasia was studied in two families. In one of the families we mapped the disease to 6q16.1-22.1. No candidate gene could be defined in the region. In the other family, several candidate loci at other chromosomal regions were found.
Genetic and molecular analyses of Turkish patients with pelizaeus - merzbacher disease
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2007., 2007.) Bilir, Birdal.; Battaloğlu, Esra.
Pelizaeus-Merzbacher disease (PMD) is a rare inherited leukodystrophy with Xlinked recessive segregation. About 80 per cent of patients with PMD have been associated with mutations of the PLP1 gene on Xq21.3-Xq22 which encodes two proteins, PLP1 and DM20, expressed abundantly in oligodendrocytes. Mutations of GJA12 gene on 1q41-42 are responsible for some of the PMD cases with autosomal recessive inheritance. In the framework of this study, the molecular basis of PMD was investigated in a cohort of 21 Turkish families with PMD. In total, pathogenic mutations were identified in 57 per cent of the families, 19 per cent of which were due to PLP1 duplications, and nine and 29 per cent were due to mutations in the PLP1 and GJA12 genes, respectively. The distribution of the mutations identified in our cohort of patients was different from those reported in the literature, which may result due to the high frequency of consanguinity and autosomal recessive cases in our population. Absence of mutations in PLP1 or GJA12 genes in 43 per cent of the cases analyzed suggests presence of further genetic heterogeneity in PMD. In vitro immunocytochemical analyses of two PLP1 mutations identified in our cohort revealed that accumulation of mutant proteins in the endoplasmic reticulum, leading to UPR activation and subsequent apoptosis were observed for the mutant proteins. However, one of the mutations showed a different pattern of localization for DM20 isoform. Since patients present similar clinical features, the results implicate that PLP1 and DM20 may have different roles in myelin.|Keywords: Pelizaeus-Merzbacher Disease, Dysmyelination, Proteolipid Protein 1 (PLP1) Gene, Gap Junction Protein Alpha12 (GJA12) Gene, Unfolded Protein Response (UPR)
Identification of novel genes involved in the etiology of benign neonatal / infantile epilepsy syndromes and genetic epilepsy with febrile seizures plus (GEFS+)
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2015., 2015.) Usluer, Sunay.; Çağlayan, S. Hande.
Epilepsy is among the most prevalent episodic neurological disorders. Genetic factors play a major role in the etiology of epilepsy. This thesis included analysis of families with distinct epilepsy phenotypes in order to delineate their complex genetic background using advanced and highthroughput current technologies. The first part of the thesis comprised a large family with BFIS phenotype which was analyzed and found to have a synonymous change in the SCN1B gene affecting splicing efficiency as shown in neuronal cell culture and by in silico tools. It was the first time SCN1B gene was shown to be associated with the BFIS phenotype. Several patients in the family also had KCNQ2 gene copy number gain and a frameshift mutation in the PRRT2 gene. The lower penetrance of these two BFIS associated gene mutations indicated the oligogenic nature of the disease. Additional families with BFIS phenotype were also analyzed for point mutations in the SCN1B and PRRT2 genes. A frameshifting 2 bp deletion in the PRRT2 gene was found in one family and rare SNPs in SCN1B genes were identified in other families. Five BFNS patients with neonatal disease onset, on the other hand, had inherited KCNQ2 gene copy number gain mutations suggesting that KCNQ2 duplications mutations may also be implicated in the etiology of BFIS/BFNS phenotypes. In the second part of the study a large multiplex, multigenerational kindred with epilepsy similar to GEFS+ phenotype and with patients having idiopathic generalized or partial epilepsy subtypes was analyzed by current genomic technologies and found to have a VNTR expansion on the mir137 gene in significantly higher numbers in individuals with epilepsy phenotypes. The VNTR expansion disrupts the expression of mir137 that targets all the genes involved in schizophrenia, synapses formation and important ion channel genes involved in epilepsy.

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