Moleküler Biyoloji ve Genetik

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Escherichia coli r-plazmitlerinin direnç genlerinin genetik analizi
(Thesis (Assoc. Prof.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1982., 1982.) Kadıkıran, Ahmet.
A molecular investigation of β-thalassemia in the Aegean and Mediterranean coasts of Turkey :|is there a region-dependent specificity?
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1994., 1994.) Senga, Edward Bisweck.; Başak, A. Nazlı.
As in many other Mediterranean countries, β-Thalassemia is also a major public health concern in Turkey. The average gene frequency.is estimated to be two per cent, and regions with higher figures are known to exist. Country scale frequencies of β-thalassemia mutations have been established in Turkey by several investigators in the past, but mutational data maps at regional scale are not available yet. The present study was designed to investigate the presence of a possible locus-specific heterogeneity of β-thalassemia mutations at the Aegean and the Mediterranean coasts of Turkey. Blood samples of patients sent to our laboratory from the Medical Schools in Izrnir, Antalya and Adana were chosen as being representative for the Western and Southern parts of the country. The method of choice for screening a large number of chromosomes for point mutations, involves the PCR amplification of the gene under investigation, followed by hybridization of the amplified DNA to Allele Specific Oligonucleotide (ASO) probes. A total of 191 chromosomes were analyzed in the framework of this thesis using 19 oligonucleotide probes specific for the Mediterranean countries. The results obtained do confirm the remarkable molecular heterogeneity of β-thalassemia in all three districts investigated. Although a marked locus-specific heterogeneity of mutations was not observed, different patterns of mutational distribution were obvious, which may help in the elucidation of the molecular heterogeneity of Turkey in certain cases.
Screening for point mutations in the factor VIII gene by denaturing gradient gel electrophoresis
(Thesis (M.S.)- Bogazici University. Institute for Graduate Studies in Science and Engineering, 1994., 1994.) Aksu, A. Anıl.; Çağlayan, S. Hande.
In genetic disorders such as hemophilia A, accurate carrier identification and prenatal diagnosis is achieved by molecular approaches namely linkage analysis and direct identification of mutations: DNA linkage analysis is the major diagnostic approach because of the large size of the FVlll gene causing hemophilia A. However, linkage analysis has a number of limitations such as the requirement of the participation of many family members and the need of an informative and preferably intragenic marker for the family. Family members of five hemophilia A afflicted families that requested carrier identification andlor prenatal diagnosis have been analyzed with three markers that had previously been used in linkage analysis of hemophilia A. In three families expecting mothers were diagnosed as noncarriers by exclusion analysis. The two prenatal diagnosis revealed that one fetus was a normal female and the other one was an affected male. In order to improve the effectiveness of linkage analysis in families afflicted with hemophilia A, the preliminary analysis of a hypervariable (CA), repeat polymorphism located at lntron 13 of the Factor Vlll gene was carried out. Seven families were analyzed using (CA), repeat polymorphisms. In two families carrier identification and prenatal diagnosis carried out with previous markers were confirmed with (CA), repeat analysis. Denaturing ~radienGt el Electrophoresis (DGGE) is one of the screening methods used to identify point mutations rapidly, in large genes. It involves the separation of DNA fragments according to their melting properties in a gel system that contains a linear gradient of DNA denaturants. Partially melted fragments are required for separation of normal and mutant DNA, where mutations fall in the melted region of the fragments. Ten per cent of the FVlll gene was screened by analyzing exons 11, 23, and 24 of 78 Turkish patients. One putative mutation in Exon 11 and two putative mutations in Exon 23 were detected. Partial sequencing of Exon 11 from the patient presumed to have a base change did not reveal any sequence alteration when compared with the sequence of Exon 11 in a normal individual. This work has initiated and established the use of DGGE analysis in screening for mutations in the factor Vlll gene of Turkish patients with the aim of providing accurate prenatal diagnosis and studying the molecular biology of the gene.
Analysis of Turkish cystic fibrosis chromosomes
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1995., 1995.) Akarsubaşı, Alper Tunga.; Tolun, Aslı.
Cystic fibrosis (CF results from mutations in the gene encoding the cystic fibrosis transmembrane regulator (CFTR), a protein that regulates chloride ion transport in exocrine gladns. Since the cloning of the gene, more than 375 disease-causing mutations have been identified. The major mutation in the gene is DF508, a deletion of three bp in exon 10 that removes a phenylalanine residue at position 508 and is found in 68 per cent of CF chromosomes worldwide. In this study, heteroduplex analysis was performed for detection of the mutations DF508 and 1677 delTA. The former was found in 15.5 per cent of CF chromosomes in Turkey. The latter was found in relatively high frequency (4.5 per cent) with respect to other mutations in our population. Further, DGGE analysis was used to detect mutations in exon 10, a mutation hot spot. A rare mutation, S466X, found in one patient, was the first case reported in the Turkish population. The DGGE migration pattern for the polymorphism 1540A/G was identified by DNA sequencing. It was confirmed with Hph I enzyme digestion and found with high frequency (37 per cent). Also, again by using Hph I digestion, the CF chromosomes were screened for an interesting mutation, 3849+10 kb C->T, which is associated with a mild type of disease. None of the patients were found to carry this mutation.
The molecular basis of alpha-thalassemia in Turkey: |establishment and application of PCR-based methods
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1996., 1996.) Taştan, A. Özlem.; Başak, A. Nazlı.
As in many other Mediterranean countries, hemoglobinopathies, especially thalassemias pose a major health concern in the Turkish population. Alpha-thalassemias, which may be the most common genetic disorders of human being, result from underproduction of the alpha-globin chains. Reviewing the thalassemia studies at molecular level, it is observed that beta-thalassemia has attracted much more attention than alpha-thalassemia in Turkey. The lack of essential molecular studies in alpha-thalassemia may likely be due to diagnosis difficulty of the alpha-thalassemia symptoms; furthermore, a variety of technical and economical reasons may have prevented the establishment of large-scale population screening programs to detect the alpha-thalassemia mutations in Turkey thus far. A breakthrough in the molecular diagnosis of alpha-thalassemia mutations occured with the advent of PCR technology. The present thesis is focused on the establishment of PCRbased methods and their applications to the detection of the four most common alpha-thalassemia determinants in the Turkish population. The introduction of PCR-based methods for direct and specific detection of the most frequently encountered alpha-thalassemia determinants are very promising in screening programs; because PCR-based methods are faster, more specific, cheaper and simpler than other methods that are used to detect the athalassemia deletions. Making use of two different PCR strategies, 32 Turkish Hb H patients and a-thalassemia carriers were studied in the framework of this thesis. It was observed that the most common genotype in Hb H disease was associated with a combination of 20.5 kb and 3.7 kb deletions. Moreover, the systematic investigation of cord-blood samples from newborn babies revealed the presence of a potential alpha-thal-2 carrier population in Antalya. Due to their relatively common presence in the world, both alpha and beta-thalassemia mutations can be coinherited in the same individual. In this thesis, such a case was observed in a family.
A study for the detection of known and novel mutations in the cystic fibrosis transmembrane conductance regulator gene
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1996., 1996.) Kılınç, Okyay.; Tolun, Aslı.
Cystic fibrosis (CF) is one of the most common and severe autosomal recessive genetic disorders worldwide. It results from mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The gene has 27 exons which encode a 1480 amino acid transmembrane ion channel protein. The major CF mutation F508 accounts for about 67% of the 30000 CF Caucasian chromosomes screened worldwide. Moreover, more than 600 other mutations have been identified, each with a frequency of at the most a few percent. The mutations responsible for CF in the Turkish population are not yet known. In the Turkish patients, F508 is found at a significantly lower frequency (13%), which indicates that CF is caused predominantly by other mutations. A systematic study was initiated to characterize the CF mutations. First, we screened for four mutations which are frequent in neighboring geographical areas, and found them to be infrequent. It became obvious that, an efficient, quick and reliable mutation screening method which would cover all of the coding region was needed. We applied to 124 CF chromosomes the DGGE technique to analyze all of the gene except for the first and last exons with the purpose of detecting any variants in the gene. These variants would later be subjected to DNA sequence analysis to determine mutations or polymorphisms which they represent. Our previous hypothesis that the profile of CF mutations in the Turkish population shows great heterogeneity is consistent with the data obtained. As expected, several mutations and variants were identified which were distributed randomly throughout 14 exons in the coding regions of the CFTR gene, each of which would account for a relatively small fraction of the CF mutations.
Detection of DNA duplications in charcot-marie-tooth type 1 (CMT1) patients by DNA analysis
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Science and Engineering, 1997., 1997.) Bissar-Tadmouri, Nisrine.; Battaloğlu, Esra.
Charcot-Marie-Tooth neuropathy (CMT), also known as Hereditary Motor and Sensory Neuropathy (HMSN), is a heterogeneous group of inherited diseases of peripheral nerves. It is estimated that 112500 persons have a form of CMT. CMT1, which typically shows dominant inheritance, has been associated with at least 4 distinct loci: The CMTlA locus on chromosome 17, the CMTlB locus on chromosome 1, the CMTlC locus, yet unmapped, and the CMTX locus on the X chromosome. CMTlA appears to be the most prevalent form of CMTl and is associated with a 1.5 Mb tandem DNA duplication of p 11.2-p12 locus on chromosome 17. The duplications in various ethnic groups have similar frequencies suggesting that it is the most prevalent cause of CMTlA. Unequal crossing over during male gametogenesis is thought to be the mechanism leading to the CMTlA duplication. Evidence showed the responsibility of dosage effect of the PMP22 gene, which maps within the CMTlA duplication interval, for the disease phenotype. The aim of this study was to accomplish molecular analysis of CMT in Turkey. Since CMTlA is the most common type of CMT for which the genetic defect has already been identified, the analysis was started by detection of duplication in CMTl patients. Detection of the duplication is an important requirement for the differential diagnosis of CMTlA, especially for patients with clinical symptoms similar to those typical for other peripheral neuropathies. In this study, sixty nine per cent of CMTl patients were found to carry the duplication by using a CA repeat polymorphism. Presence of the duplications in these patients was confirmed by Southern analysis using two different markers. These markers were shown to be highly informative for the detection of duplications, correlating with the previously published data for other populations.
Expression of aFGF and FGF9 in the developing rat retina
(Thesis (M.S.) - Bogazici University. Institute for Graduate Studies in Sciences and Engineering, 1995., 1997.) Çayırlıoğlu, Pelin.; Buğra, Kuyaş.
aFGF, an FGF gene family member, has been postulated to be important in the physiology and development of vertebrate retina. The distribution and relative levels of aFGF mRNA in the external photoreceptors layer, and in the internal layer, containing the interneurons, Muller and the ganglion cells, were analyzed by RT-PCR and Southern hybridization techniques during development. This approach provides high specificity defined by specific primers, characterization of the PCR product by restriction digestion and/or hybridization with nested oligonucleotide probes. To compare the transcript levels, amplifications were performed at exponential phases. aFGF was detected in both layers, which unequivocally places aFGF expression to the inner layer cells. Its expression pattern suggests that aFGF may be involved in developmental events subsequent to the photoreceptor differentiation and in the further maturation of the retina. The difference in the aFGF expression patterns in two layers suggest aFGF may play different roles in different layers. The widespread expression of FGF9 in the central nervous system raises the possibility of its presence in the retina. Using RT-PCR we show that FGF9 is present in all postnatal days and embryonic day 17. Thus, it might be mitogenic, and survival factor for the retinal cells.
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.
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.
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.
Genetic analyses in Seckel syndrome and azoospermia
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2004., 2004.) Korur, Serdar.; Tolun, Aslı.
The autosomal recessive genetic disorders Seckel syndrome and azoospermia were studied. In Seckel syndrome, the two known loci were tested for linkage in 15 Seckel families. In addition, four ATR interacting and fifteen DNA repair gene loci were tested for linkage. In azoospermia, the locus which was previously identified in our laboratory was refined with additional markers, and computer based parametric tests were applied to evaluate the linkage information obtained. The strongest candidate genes were selected using databases. MNAT1 and RAD51L1 were assessed as the strongest candidates for Seckel syndrome and P2RX1 and NYD-SP20 for azoospermia. Those genes were analyzed for mutations in relevant families by direct sequencing and/or SSCP in order to identify any disease causing defects. No mutations were found in any of the genes, but a novel polymorphism was identified in MNAT1.
Cloning, expression and purfication of the recombinant DNA polymerase I from the hyperthermophilic bacteria geobacillus anatolicus
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2005., 2005.) Çağlayan, Melike.; Bilgin, Neşe.
The DNA polymerase I gene of a recently described Geobacillus species, Geobacillus anatolicus from a terrestrial hydrothermal vent has been cloned and expressed in Escherichia coli. Evolutionarily conserved regions of DNA polymerase I genes from related organisms were used for designing oligonucleotide primers for the amplification of the unknown DNA polymerase I gene from Geobacillus anatolicus by polymerase chain reaction (PCR) and for its DNA sequencing. Geobacillus anatolicus DNA polymerase I gene contains a long open reading frame of 2637 bases that encodes 878 amino acid residues. Similarity analyses suggested that Geobacillus anatolicus DNA polymerase I may not contain a putative 3'-5' exonuclease activity. However, the conserved regions related to 5'-3' exonuclease activity were observed in the amino acid sequence of Geobacillus anatolicus DNA polymerase I. The entire DNA polymerase I gene excluding the start codon was cloned into pCR-T7/NT-TOPO expression vector and was expressed in Eschericha coli JM109(DE3) strain. The recombinant Geobacillus anatolicus DNA polymerase I fusion protein including an His6-tag at its N terminal part was obtained. The recombinant protein was purified using Ni-affinity and gel filtration chromatography.
Cardiovascular disease in the Turkish population: molecular analysis of the renin-angiotensin system and its associated genes with premature myocardial infarction
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Gencer, Pınar.; Başak, A. Nazlı.
Cardiovascular disease (CVD), which is a complex multifactorial disease with many environmental and genetic factors, is the leading cause of death worldwide. Extensive research is ongoing for the identification of the genetic risk factors with association studies between patients and controls. As a result, several polymorphisms in several genes were identified as risk factors for CVD, but the results are mostly not reproducible within different study groups. In the framework of this thesis, selected polymorphisms in angiotensin 1 receptor (AT1R), angiotensin converting enzyme (ACE), angiotensinogen (AGT), bradykinin B2 receptor (BKB2), endothelial nitric oxide synthase (eNOS) and G-protein beta-3 subunit (GNB3) genes were investigated to study the significance of the polymorphisms between myocardial infarction (MI) patients and healthy controls. Moreover, the LightCycler system was optimized for genotyping of the ACE and eNOS genes and the genotyping results wer compared with the conventional PCR and restriction enzyme (RE) analysis. Screening of 84 premature MI patients and 60 age-matched healthy controls with RE analysis, DNA sequencing and LightCycler did not demonstrate any significant association between the studied polymorphims in the six genes mentioned above and premature MI. However, this is the first study of its kind in Turkey, and we hope that it paves the way for more extensive studies with larger patient cohorts and healthy controls to unravel the complex interaction between genes and myocardial infarction.
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.
Gene localization studies in two neurological disorders
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Duru, Nadire.; Tolun, Aslı.
Autozygosity mapping is a frequently used powerful method for discovering loci for autosomal recessive diseases resulting from consanguineous marriages. Gene localization is an initial step for the identification of genes responsible for such disorders. Identification of a disease gene would enable us to better understand its functions, investigate the underlying cellular mechanisms, and provide critical insights into the pathogenesis of the disease. It can also improve the drug development and enable diagnostic and prenatal genetic testing. Computer programs developed to analyze data obtained by such studies are very helpful in finding the most likely gene locus, supplying ease and speed to studies. In the framework of this study, autozygosity mapping was performed to identify the gene loci for two rare autosomal recessive disorders: early onset progressive encephalopathy with myoclonus and dystonia (PEMD) and infantile neuroaxonal dystrophy (INAD). Computer based parametric tests including two and multi-point lod score analyses were also performed to assess the significance of the linkage data generated by autozygosity mapping and further candidate locus evaluation. The gene responsible for PEMD was localized to a maximum 3.26 Mb-interval on telomere of chromosome 16p. Among the 173 genes residing in the identified locus, 25 could be assigned as candidate genes. For INAD, on the other hand, no significant genetic linkage to any locus could be detected.
Characterization of yellow rust (Puccinia Striiformis) resistance in A F6 durum wheat population
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Tufan, Hale Ann.; Sayar, Müge.
Nearly half of the agricultural land in Turkey is devoted to wheat. Turkey is one of the leading durum wheat producers in the Middle East and one of the most important biotic stress factors restricting the production is yellow rust. An F6 recombinant inbred line durum wheat population from a Kunduru-1149 X Cham-1 cross was previously characterized for field resistance by Göçmen et al. (2003). The 150 lines of the population showed differing reactions to yellow rust in the field. The work done in this M.S. thesis was aimed at characterizing the yellow rust seedling resistance of this population and developing markers linked to this resistance. The 150 lines were tested for seedling resistance using a yellow rust isolate virulent on yellow rust resistance genes Yr2, Yr6, Yr7 and Yr9 representing the virulence profile of the yellow rust isolate used by Göçmen et al. (2003). The 150 lines were categorized based on their seedling resistances, 4 categories were formed, the majority of the lines being resistant. Based on the ratios observed in the seedling test, 53 of the 150 lines were chosen to constitute a sub-population. This subpopulation was used to screen for SSR and NBS-profiling markers. Three linkage groups were formed and alleles of the SSR and NBS-profiling markers were compared to seedling and adult phenotypes (Göçmen et al., 2003) using the Kruskal-Wallis single marker regression analysis program in MapQTL version 5 for Windows. Two QTLs were found on chromosome 1BL where the NBS-profiling band NBS3 290 was linked to three SSR markers. Three of the NBS-profiling bands were excised and cloned into a pGEM-T Easy vector and sequenced. The obtained sequences were analyzed using the BLASTX program. These sequences show homology to known NBS-LRR resistance genes in plants. Due to this sequence homology and results of the linkage analysis, we predict the presence if two QTLs on chromosome arm 1BL.
Identification of the gene responsible for pulmonary alveolar microlithiasis and possibly associated with testicular microlithiasis
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Corut, Ayşe.; Tolun, Aslı.
In this study two inherited disorders, namely pulmonary alveolar microlithiasis (PAM) and testicular microlithiasis (TM), were studied. PAM is a rare autosomal disease characterized by the deposition of calcium phosphate microliths throughout the lungs. TM is a more common disorder that is thought to follow a complex pattern of inheritance often associated with infertility and cancer. Autozygosity (homozygosity) mapping was used to fine-map the disease gene responsible for PAM resulting from parental consanguinity in a large Anatolian family. Computer based parametric tests were applied to evaluate the linkage information obtained by autozygosity mapping and confirmed gene localization. Later, positional candidate gene approach assessed SLC34A2 or type IIb sodium-phosphate cotransporter as the gene responsible for PAM. In this study, SSCP assay was employed to screen SLC34A2 for disease causing mutations in PAM patients. A total of five homozygous exonic mutations were identified in six unrelated PAM patients showing that impaired activity of the phosphate transporter SLC34A2 is presumably responsible for the alveolar microliths. Since the gene is also expressed in testis, using SSCP and subsequent DNA sequencing analysis, mutations were searched in 15 TM subjects and two rare variants identified.
Analysis of the Cx32, MPZ and PMP22 mutations in the Turkish Charcot-Marie-Tooth patients
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Maracı, Nazmiye Öncü.; Battaloğlu, Esra.
Charcot-Marie-Tooth disease is the most common hereditary peripheral neuropathy with a prevalence rate of one in 2500. Although the disease is classified as demyelinating and axonal CMT based on clinical investigations, genetic studies revealed more than 28 genes and subtypes for both axonal and demyelinating forms. In a total of 161 unrelated CMT patients were analyzed in this study. The incidence of CMT1A duplication in the Turkish population was found to be significantly lower (41.6 per cent) compared to that of other populations (70 per cent). The frequency of the HNPP deletion was found to be 66.6 per cent. Although this rate is lower than that of other populations (86 per cent), the difference was not statistically significant. In this study, five nucleotide variations were identified; a novel and a previously reported mutation in Cx32, a previously reported polymorphism in MPZ and two novel mutations in PMP22. Phenotype/genotype correlations were performed for the identified mutations and the observed phenotype of the patients was found to be compatible with the mutations they carry. Identification of causative mutations in the patients verifies the clinical diagnosis and leads the clinicians for choosing the therapeutic approach. However, for the patients that could not be genetically diagnosed further research is required to identify the pathogenic mutations in other CMT genes. On the other hand, identification of further mutations and genotype/phenotype correlations are of critical importance and will help elucidation of the pathogenic mechanisms leading to the CMT disease.
Screening of autosomal recessive Charcot-Marie-Tooth (ARCMT2), distal hereditary motor neuropathy (dHMN) and hereditary spastic paraplegia (HSP) patients for mutations in axonal neuropathy genes
(Thesis (M.S.)-Bogazici University. Institute for Graduate Studies in Science and Engineering, 2006., 2006.) Şenergin, H. Başak.; Battaloğlu, Esra.
Charcot-Marie-Tooth Disease (CMT), distal Hereditary Motor Neuropathy (HMN) and Hereditary Spastic Paraplegia (HSP) constitute the largest group of inherited diseases affecting the peripheral nervous system. In the scope of this thesis, the genetic background of autosomal recessive CMT2 disease was investigated in a cohort of Turkish families and isolated cases. For this purpose, GDAP1 and LMNA genes were investigated by SSCP analyses, and linkage to 19q13.1 locus was tested by homozygosity mapping. The study was extended to screening of the small heat shock protein genes, HSPB1, and HSPB8, that have been recently shown to be responsible for autosomal dominant CMT2 and distal HMN. The involvement of another heat shock protein gene, HSP60, was screened for mutations in a small group of Hereditary Spastic Paraplegia patients. Exclusion of all known loci responsible for ARCMT2 in a family demonstrated that there is at least one more locus responsible for the disease phenotype. Only one of the CMT4 patients was found to be mutated in GDAP1. Another causative mutation was identified in HSPB1 in a CMT2 patient. Clinical diagnoses of the patients were found to be compatible with the genetical diagnoses. All other patients tested negative for mutations in the GDAP1, LMNA, HSPB1, HSPB8, and HSP60 genes although several novel or previously repoted polymorphisms were detected. Absence of mutations in these patients indicated that the contribution of these genes to the appropriate disease phenotypes is low in the Turkish population. v

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