Ph.D. Theses

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Molecular analyses of germ line methylation patterns and of the common intron 22 inversion mutation in the factor VIII gene
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1998., 1998.) El-Maarri, Osman A.; Çağlayan, S. Hande.
The factor VIII gene includes two major mutation hotspots: the intron 22 inversion and transitions at CpG dinucleotides, both of which have been examined in the context of this thesis. A non-radioactive southern blot approach was implemented for the detection of the inversion, that allowed an accurate diagnosis for about 1/4 of the Turkish hemophilia A families. About 45 % of all point mutations in the factor VIII gene occur at CpG sites. Previous studies had pointed to a bias in paternal origin for transition mutation at CpG dinucleotides. This was believed to be a direct reflection of the hypomethylation in the female germ cells. However, to date there was no direct proof for a differential level of methylation in the germ cells, at human disease causing codons. In this study, an original investigation was undertaken to determine the methylation status of mature germ cells at selected sites in the human factor VIII and the FGFR3 genes. An equally high level of methylation, at non-cluster CpG sites, was observed in mature eggs, spermatocytes and polar bodies. This result shows that the higher mutation rate in the male germ line is apparently not a simple reflection of sex specific methylation differences.
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.
Molecular analysis of the factor IX gene in the Turkish population
(Thesis (Ph.D.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1999., 1999.) Onay, Ü. Venüs.; Çağlayan, S. Hande.
Hemophilia B, the third common hereditary coagulopathy, is very heterogeneous at both phenotypic and genotypic levels. This X-linked recessive disease is caused by the production of either reduced amounts or functionally defective forms of the coagulation factor IX. In order to undertake a comprehensive molecular analysis of the FIX gene in the Turkish population 41 hemophilia B patients were screened for mutations by ddF and direct DNA sequencing to contribute to the knowledge of genotype-phenotype correlations in hemophilia B and to construct a Turkish mutation profile. A hypervariable polymorphic site within the FIX gene have also been analyzed. Thirty-two mutations were identified in Turkish hemophilia B patients, including 4 novel single base changes and 2 novel gross rearrangements. The mutation profile of the Turkish population was found to be similar to the general profile observed worldwide. Haplotype analysis revealed the independent origin of 4 recurrent mutations. All patient data was compiled in a national database, which efficiently shows the mutation/phenotype/haplotype relations. The analysis of the hypervariable region in intron 1 in 85 Turkish individuals revealed the presence of a novel allele and showed that the Turkish population carries the Caucasian specific allele as the most frequent one.
SIK2 expression in retinal cells and its possible involvement along with PKA in FGF9 signal transduction
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Özmen, Yeşim.; Buğra, Kuyaş.
FGF signal transduction pathway activates a cascade of kinase and phosphatase dependent phosphorylation and dephosphorylation events. The pathway is subject to tight control mechanisms through negative feedback exerted by the the activated effector molecules, action of kinases and phosphatases. Besides the cellular context and interplay between the regulators, integration of heterologous signaling is also critical in shaping the cellular responses. In this study, we have shown FGF9 receptors FGFR2, FGFR3 and a putative modulator SIK2 is widely expressed in neuronal cells and Muller glia, FGF9 is detected in neuronal cell layers but not in Muller cells. Our data, when compared with the primary Muller cells, indicate that MIO-M1 cells are equipped with the components of signal transduction. Using these cells as model system we had shown the activation of FGF9 pathway and that the pathway is modulated by PKA activity. We had also shown that SIK2 is modulated both in response to PKA and FGF9 by phosphorylation and nuclear translocation. Regulation of SIK2 translocation correlates with levels of ERK phosphorylation. When both stimuli are present SIK2 tends to act in accordance with FGF9 but not PKA.
The effect of nNav1.5 gene expression in breast cancer metastasis
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Yamacı, Rezan Fahrioğlu.; Battaloğlu, Esra.
Breast cancer is the most common cancer among women. Its metastasis is lethal that can not be detected at microscopic levels using current techniques. Thus, there is need for reliable early metastasis markers. Neonatal Nav1.5 (nNav1.5) is a voltage-gated sodium channel (VGSC) and one of the potential early markers for breast cancer metastasis. In this study, we determined that nNav1.5 expression was in parallel with breast cancer metastasis and estrogen receptor (ER) expression in a group of patients. To provide data for future drug development, we analyzed the expression pattern of nNav1.5 protein in normal human tissues. The protein was not expressed in skeletal and heart muscle, brain, small intestine, colon, stomach, esophagus, urinary bladder and prostate but expressed in breast at basal level. We also investigated the distribution of VGSC in these non-excitable human tissues. Except urinary bladder, VGSC protein was determined mostly in secretory cells in all of the tissues above that may indicate a role in secretion. Upon identification of VGSC upregulation in tumor regions of different cancers including, colon, stomach, urinary bladder, kidney and lung it is possible that VGSC expression could be a widespread mechanism in cancer metastasis. Within the scope of this thesis, we also investigated the possible role of estrogen on nNav1.5 upregulation and activity in metastatic breast cancer. Estrogen had no effect on proliferation of cells but slightly increased motility through nNav1.5 in highly metastatic cells that express the protein. In weakly metastatic cells that do not posses nNav1.5, estrogen decreased motility slightly. The quantity of nNav1.5 protein was not affected by estrogen but functionally available form on the plasma membrane was increased only in the highly metastatic cells. These results may suggest that estrogen increases motility capacity of breast cancer cells by regulating nNav1.5 activity.
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.
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)
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.
Parkinson's disease in Turkish patients: molecular defects in familial and isolated cases
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2009., 2009.) Pirkevi, Caroline Selma.; Başak, A. Nazlı.
Parkinson's disease (PD) is worldwide the second most common neurodegenerative disorder after Alzheimer's disease. Recently, several genes have been shown to result in PD phenotype. Mutations in these genes may lead to autosomal dominant ( -synuclein, LRRK2), autosomal recessive (Parkin, PINK1, DJ1) or sporadic PD. In the framework of this study, the above five genes were investigated in Turkish patients. For the Parkin gene, we have performed dHPLC analysis prior to sequencing. The high percentage of abnormal dHPLC profiles observed in the patient population revealed only one mutation and 136 polymorphisms. Exon rearrangements were investigated by semi-quantitative multiplex PCR and by MLPA; 12 rearrangements were described in the Parkin gene. PINK1 and DJ1 exons were also sequenced; 75 variations were identified, two being heterozygous mutations. Point mutations in -synuclein are a rare cause of PD and were not found in our patient population. In contrast, triplications and duplications of the whole gene are found more frequently in autosomal dominant forms of the disease. A heterozygous duplication of exons 3 and 4 was described in several patients of a large kindred, and the whole family was subjected to haplotype analysis for chromosome 4q. LRRK2 consists of 51 exons and most mutations are seen in 15 of these. Sequencing of these exons revealed a novel S1508R and a G2019S mutation. G2019S frequency differs among populations and it is associated with three different haplotypes so far. The haplotype of our patient has been shown to have a great homology with the Japanese haplotype. Although an independent origin of the Turkish haplotype cannot be excluded at this point, the huge and centurieslong migration of the Turkic people in Central Asia rather supports a common origin.
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.
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.
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.
Mutation profile of hemophilia a patients with inhibitors and association of interleukin and cytokine gene polymorphisms with inhibitor development
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2010., 2010.) Fidancı, İnanç Değer.; Çağlayan, S. Hande.
Hemophilia A (HA) is an X linked recessive bleeding disorder characterized by qualitative and quantitative deficiency in the factor VIII (FVIII) protein, mainly caused by Factor 8 (F8) gene mutations. A severe complication in the replacement therapy of HA patients is the development of allo-antibodies (inhibitors) against FVIII which neutralize the substituted FVIII. Genetic risk factors along with F8 gene mutations influence the development of inhibitors. Interleukins and cytokines such as IL4, IL5, IL10, TGFB1 and IFNG that are involved in the regulation of B lymphocyte development are possible targets as other genetic risk factors. The aim of this dissertation was to reveal the F8 gene mutation profile of severe HA patients who developed inhibitors using various methods to assess the possible associations between 9 selected interleukin and cytokine gene polymorphisms with inhibitor development in HA patients with a null mutation in the F8 gene. The most prevalent mutation in inhibitor patients was intron 22 inversion followed by nonsense mutations and large deletions with major effects on FVIII function. Therefore, severe HA patients were screened for intron 22 inversion to constitute inhibitor (+) and inhibitor (–) patient subgroups to carry out a case-control association study. A significant association with the T-allele of rs2069812 located in IL5 gene promoter and patients with inhibitors was found with a p-value of 0.0251. The TT genotype was also significantly associated with the inhibitor (+) patient group with a p-value of 0.0082 and OR of about 7, suggesting that the T-allele as the recessive susceptibility allele and C-allele was the dominant protective allele. The present findings are highly informative about the role played by the polymorphisms in genes involved in B lymphocyte development as genetic risk factors in antibody development in severe HA patients with null mutations and paves the way for furthe studies in the field.
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.
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.
SIK2: a key player in FGF2-induced proliferation and insulin-induced survivale/hyperglycemia-dependent apoptosis in müller cells
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2011., 2011.) Küser, Gamze.; Bilge, Kuyaş Buğra.
Salt inducible kinase 2 (SIK2), a serine/threonine kinase, is primarily expressed in insulin-responsive tissues. It was suggested that SIK2 might be involved in type 2 diabetes by phosphorylating S789 residue of IRS1. In the initial experiments we observed rapid and transient increase of SIK2 activity upon FGF2 stimulation, confirming that the kinase is part of this pathway. Subsequently it was demonstrated that FGF2 dependent ERK and Akt activation was reduced significantly by SIK2 overexpression. SIK2 silencing, on the other hand, enhanced the intensity and the duration of active ERK and Akt levels, and led to a dramatic increase in FGF2-dependent Müller cell proliferation. Gab1 phosphorylation by SIK2 was verified in vitro, their interaction was revealed by coimmunoprecipitation. pSer of Gab1 was diminished significantly by SIK2 silencing. In the same time frame, pTyr levels and binding partner associations (p85/Gab1, Shp2/Gab1, Grb2/Gab1) were increased. Based on this data we propose that SIK2 involved in the negative feedback mechanisms acting on FGF/Ras/ERK and FGF/PI3K/Akt pathways and Müller cell proliferation at the level of Gab1 serine phosphorylation. In the context of insulin pathway we established enhanced tyrosine phosphorylation of IRS1, followed by Akt activation resulting in increased Müller cell survival. SIK2 knockdown leads to considerably earlier FGF2-dependent Akt activation. Its overexpression, hampers Akt activation and cell survival. Coimmunoprecipitation studies indicated IRS1 as an in vivo SIK2 substrate. We observed lower IRS1 expression and tyrosine phosphorylation, enhanced SIK2 activity/expression and downregulation of both basal and insulin-induced Akt activation in MIO-M1 cells under hyperglycemia. SIK2 gene silencing under hyperglycemia restored pAkt level, under normoglycemia in cells overexpressing SIK2, Akt phosphorylation was decreased and apoptosis increased. Based on these observations, we suggest that SIK2 is a negative modulator of insulin-induced IRS1/Akt mediated Müller glial survival pathway, which may contribute to the glial death observed in diabetes.
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.
Regulation of human BFK
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Uğurlu, Serkan.; Özören, Nesrin.
Bcl-2 protein family members are critical regulators for apoptosis. Reduced levels of apoptotic Bcl-2 family members are detected in gastrointestinal cancers which are responsible for a considerable part of the deaths from cancer. BFK (Bcl-2 Family Kin) is a novel pro-apoptotic Bcl-2 family member specifically expressed in the human gastrointestinal tract. BFK has the characteristic BH3 domain, which was shown to be essential for the apoptosis inducing activity of pro-apoptotic Bcl-2 family members. In the colon four alternatively spliced isoforms were identified. Human and mouse BFK genes share 70% homology at the DNA level and 68.7% homology at the aminoacid levels. Interestingly, human and mouse BFK genes show distinct expression patterns. To explain these differences, we performed gene evolution analyses on the promoter region as well as coding region of these genes. We found that the human BFK promoter experienced positive selective pressure and acquired a distinct set of repetetive elements and transcription factor binding sites. In this study, we identified several novel transcription factor candidates which may have roles in the transcriptional regulation of human BFK. As transcription factors, PARbZIP family members (especially TEF and NFIL3) regulate BFK upon binding to its promoter region. NFIL3 supresses TEF induced BFK transcription in HCT116 cells. We also studied hormonal regulation of human BFK. Tamoxifen, as a mixed agonist/antagonist of estrogen, upregulates human BFK levels in SW707 cells. We hope this study contributes to a better understanding of Bcl-2 family.
The myelination puzzle: do fibroblast growth factors and their receptors have regulatory roles in peripheral nerve myelination?
(Thesis (Ph.D.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2014., 2014.) Dağlıkoca, Emine Duygu.; Battaloğlu, Esra.
Axon-Schwann cell interaction is the key step in myelination and critical in the pathogenesis of peripheral neuropathies. Molecules mediating this interaction to promote the complex mechanism of myelination have not been elucidated properly yet. However, recent studies emphasized the importance of tyrosine kinase receptor signaling in the process. Although expression of FGFs and their tyrosine kinase receptors FGFRs have been shown in PNS, little information is available about their regulation during the myelination process. In this study, we aimed to investigate whether FGF1, FGF2, FGF9 that were previously implicated in CNS development, involved in peripheral nerve myelination and regulate axon-Schwann cell interactions, through their high affinity receptors FGFR1-3. For this purpose, we used dorsal root ganglia (DRG) and the sciatic nerve from mice as in vivo models and Schwann cell-neuron co-culture developed from fetal mouse DRG tissue as an in vitro model. The expression patterns and localization of the molecules were investigated both in vivo and in vitro. Among the three FGFs analysed, FGF1 was chosen as a candidate for further investigation because of its high level of expression in all tested tissue types with an axonal localization. In contrast, FGFR1-3 were found to be expressed by Schwann cells. Protein expression levels of FGF1 and FGFR1-3 were examined through the developmental stage to adulthood from sciatic nerves by Western blotting. Both FGF1 and its receptors were found to be modulated at key time points of the myelination route. Immunolabeling studies showed that FGF1 expression in neurons and FGFR1-3 expression in Schwann cells continued throughout the process. When FGF1 was blocked in DRG culture, a reduction in the levels of myelin proteins and in the number of myelinated axonal segments was observed. Our findings provide evidence for the first time for the involvement of FGF1 in peripheral nerve myelination and suggest that FGF1 signaling through FGFR1-3 have regulatory roles at the onset of myelination, in myelin compaction and protection of the stability of mature structure.

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