Browsing by Author "Akar, Nejat"
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Item Ailevi Akdeniz ateşi hastalığı taşıyan bireylerde MEFV geninde R202Q gen değişiminin taranması(Biyoteknoloji Enstitüsü, 2009) Yıldız, Cennet; Akar, Nejat; BiyoteknolojiFamilial Mediterranean Fever (FMF) is an autosomal recessive genetic disordercharacterised by recurrent acute self-limited episodes of fever and serosal inflammationmanifested by sterile peritonitis, pleuritis and synovitis. FMF mainly affects populationsaround the eastern Mediterranean origin, especially Sephardic Jews, Armenians, Turks andArabs. The most serious complication of FMF is the development of amyloidosis.The gene causing FMF, MEFV, is located on the short arm of chromosome 16. MEFVgene encodes a protein named Pyrin/Marenostrin. It has been thought that pyrin acts as anegative regulator of the inflammatory response. Mutations in the MEFV gene have beenidentified in the majority of FMF patients. The M694V mutation in exon 10 is the mostcommon among Turkish FMF patients.After amplification of exon 2 region with polimerase chain reaction (PCR), we analyseR202Q at exon 2 with PvuII restriction endonuclease enzyme digestion method.R202Q polymorphism is located in cis position with M694V mutation. In case of R202Qexistinced at exon 2, It was observed that there is one more haplotype which is not inlinkage with M694V mutation. Our study revealed that there is another haplotype ofcarrying R202Q in trans with M694V mutation. This indicates that R202Q alteration mightbe a disease-causing mutation. When combined with other disease-causing mutation, theclinical spectrum appears. This emphasizes that R202Q alteration is important fordiagnosis.Our results show that R202Q gene alteration should be included in routine FMF geneanalysis.Item Blood group genotyping in multi-transfused patients(2013-02-25) İnce, Elif; Dil ve Tarih-Coğrafya Fakültesi; Bakanay, Şule; Arslan, Önder; Öztürk, Ayşenur; İleri, Talia; Yavaşoğlu, Suzan; Akar, Nejat; Uysal, ZümrütBackground: In chronically transfused patients, the classical hemagglutinati on assays may be inaccurate in defining the RBC phenotypes of the patients due to previous transfusions. Design: DNA samples from 39 multi-transfused patients including thalassemia and sickle cell disease were used for red blood cell genotyping. The Rh-Type and KKD-Type (BAGene, BAG Healthcare) were used to determine the polymorphisms associated with antigen expression for RHD, RHCE and Kell, Kidd, Duffy blood group systems, respectively. Results were compared with previously determined phenotyping results for RhD, RhCcEe and Kell by hemagglutinati on method.v Results: Nineteen out of the 37(51%) patients had discrepancies between genotyping and phenotyping results in a total of 25 alleles. In 12 patients, the discrepanc ies had the potential of alloimmunization . Conclusion: Blood group genotyping has vital importance in trans fusion management of chronically transfused patients especially if the patients were not phenotyped before starting the initial transfusions .Item Çeşitli klinik örneklerden izole edilen candida albicans suşlarının genotipik dağılımı(Sosyal Bilimler Enstitüsü, 2004) Karahan, Zeynep Ceyhan; Akar, Nejat; Çocuk Sağlığı ve HastalıklarıGenotypic Distribution of Clinical Isolates of Candida albicans With the emergence of more aggresive chemotherapy protocols to cancer patients and transplant recipients, and the increase in the number of AIDS patients and long term survival of these immunsuppressed patients, an increase in the number of fungal infections was observed, with Candida albicans as the leading etiological agent. Molecular typing of the infectious agent is especially important for epidemiological studies and for the development of appropriate infection control strategies. In this study, a total of 401 C. albicans isolates, 81 invasive (blood culture isolates) and 320 non-invasive (sputum, wound, urine, vagen, feaces and throat culture isolates), were genotyped according to the presence of the transposable group 1 mtron in the 25S rDNA gene according to the method developed by McCullough et al. (1999). The frequencies of genotypes A, B and C were found to be 51.9%, 65% and 31.9% respectively. Genotypes D and E which belong to C dubliniensis were not found. When the isolates were evaluated as invasive and non-invasive, the genotype frequencies were found as follows: Genotype: 65.4%-48.4%; Genotype B: 8.6%- 18.1%; Genotype C: 26%-33.4%. Statistically significant differences were observed between the isolation sites (p=0.07) and between invasive and non-invasive isolates (p=0.015), genotype A being more prevalent among invasive isolates and genotypes B and C being more prevalent among non-invasive isolates. When the groups were compared according to the presence of the group 1 intron, the differences became even more significant (p=0.001 and p=0.006 respectively). These results show that, this region can be important in determining invasiveness and the presence of group 1 intron may have an importance in determining invasiveness besides resistance to flucytosine. In this study, different subgroups with slightly different molecular wights were observed among genotype A isolates. These groups were identified by restriction endonuclease and sequence analayses and an easy differentiation scheme using HaeE and Mspl restriction andonucleases was made. As a result, 8 different subtypes (subtype a-»h) were identified among genotype A isolates and the sequences of these subtypes were submitted to the Genbank. Subtype a, which gives a 460bp PCR product, was found to carry the same sequence with the previously submitted sequence. The 46()bp subtype c and 459bp subtype e (which carries a lbp deletion), differed from subtype a with single base substitutions. The 478bp subtype b, h and h, and 468bp subtype d were found to carry some insertions among their sequences. Subtype f, which gives a PCR product of 447bp, was found to carry a 13bp deletion among with many single base substitutions. When the subtypes were evaluated according to their isolation sites and the hospitals which they were recovered; subtypes a, b, and c were found to account for 86.1% of the isolates, and recovered from various infection sites of the patients hospitalized in different hospitals. Subtype d was recovered only from blood cultures and this subtype accounted for 6.2% of all isolates, and 25% of blood culture isolates. This subtype may be important for its invasive nature Subtypes e and g were obtained from patients hospitalized in the same department and subtype h was isolated from only one patient. These three subtypes were thought to be strains causing infection in the same hospital department with microevalutionary changes As a result, the genotypic distribution of C. albicans isolates has a unique pattern in Turkey, the main difference being the high frequency of genotype C isolates. Among invasive isolates, genotype A was found to be significantly more prevalent; for this reason in determining the therapy protocols of patients with genotype A C. albicans as the ethnological agent, resistance to flucytocine and risk for invasiveness must be taken into consideration. In order to understand the role of different genotype A subtypes in different infection types and their importance in epidemiological studies, more sophisticated and large scale studies must be performed. Key Words : Candida albicans, DNA sequence analysis, Genotyping, PCR, Restriction endonuclease analysis.Item Dejenerasyon sürecindeki kas dokusunda housekeeping genlerin ekspresyon düzeyinin incelenmesi(Biyoteknoloji Enstitüsü, 2008) Yüzbaşıoğlu, Ayşe; Akar, Nejat; BiyoteknolojiRT-PCR and quantitative PCR applications are widely used for the assessment of transcriptional response that cells and tissues develop for external stimuli. For samples that have been collected over a time period or under various physiological conditions, RT-PCR technique is employed to observe any gene of interest. A housekeeping gene whose expression is believed to be stable is used to normalize the expression of the gene of interest. A large battery of genes are being used for the normalization of gene expression in short-term studies where tissue architectural changes are not observed. But under extreme conditions such as the chronic muscle degeneration, the tissue architecture is deteriorated and the cell population exhibit important variations. Thus the gene of choice for normalization has to be carefully assessed under such conditions. The aim of this study is to assess and compare the expression variations of various housekeeping genes on the course of two diverse models of muscle degeneration where large tissue architectural changes are observed. Denervation and tenotomy are the models that are chosen for the study where experimental interventions have been made on the right extremity of rats and the contralateral-side muscles have been utilized as control. The expression of B-actin, TBP, GAPDH and HPRT have been investigated. In order to assess the amount of tissue deterioration, a structural gene of muscle contractile apparatus, the myosin heavy chain is also analyzed.Item Dev trombositli hastalarda MYH9 geninde mutasyon taraması(Biyoteknoloji Enstitüsü, 2009) Erik, Zafer; Akar, Nejat; Biyoteknolojilatelets are small, disk-shaped cells with an average diameter of 2 to 3µm. They are derived from cytoplasmic fragmentation of megakaryocytes. Their mean life-span is 7?10 days. Lots of cytokines (thrombopoietin and others) are responsible for megakaryocyte development and platelet formation. Platelets have two forms; one is resting platelet which is circulating during normal circulation and the other is active platelet. When the vascular injury occurs, platelets are rapidly activated and aggregate with each other to form a plug on the vessel wall that prevents vascular leakage.An abnormality or disease of the platelets is called a thrombocytopathy which could be either a decrease (thrombocytopenia) or an increase in the number of (thrombocytosis). In this study patients with giant platelet syndrome which is usually accompanied by thrombocytopenia were analyzed. Hence, it is also called macrotrombocytopenia. Macrotrombocytopenia is characterized by the presence of abnormally large platelets this dissertation is focused on the analysis of Myosin Heavy Chain 9, Nonmuscle (MYH9)-related mactrombositopenia. MYH9 gene, located on chromosome 22q12?13, encodes for a non muscle myosin heavy chain type II-A (NMMHC II-A). Mutations in this gene are responsible for May-Hegglin anomaly, Sebastian, Fechtner and Epstein syndromes. These four diseases are autosomal dominant macrotrombocytopenias distinguished by different combinations of clinical and laboratory signs (such as sensorineural hearing loss, cataract, nephritis, and Döhle-like bodies).Consequently, we aimed to investigate the relationship between a possible mutation in MYH9 gene and the diseases via mutation scanning of patients with macrotrombositopenia. 122 patients with macrotrombositopenia were examined by SSCP and DNA sequencing of the MYH9 gene. After mutation scanning of all patients, a single base alteration (c.366C>T, p.45Ser>Ser) which causing a silent mutation in exon 1 in an affected individualItem EPCR genindeki olası değişimlerin trombozlu olgularda sEPCR seviyeleri üzerindeki etkisinin araştırılması(Biyoteknoloji Enstitüsü, 2009) Karabıyık, Afife; Akar, Nejat; BiyoteknolojiThe protein C pathway is a physiologically important mechanism for the regulation of the coagulation process. The pathway is triggered when thrombin binds to the endothelial cell surface receptor thrombomodulin. The thrombin-thrombomodulin complex activates protein C. Activated protein C (APC), together with its cofactor protein S, limits the amplification and progression of the coagulation cascade by proteolytic inactivation of factors (F)Va and FVIIIa. This process occurs on the negatively charged surface of activated platelets.Endothelial cell protein C receptor (EPCR) is a type I transmembrane protein that is almost exclusively expressed on the endothelium of large vessels. Binding of protein C (PC) to EPCR stimulates PC activation by increasing the affinity of PC for the thrombin-thrombomodulin complex. EPCR is similar to molecules of the class I major histocompatibility complex, in particular the CD1-subfamily. In addition to the extracellular domains, EPCR has a transmembrane domain and a very short cytoplasmic tail. EPCR gene is located on chromosome 20q11.2. It consists of four exons. A soluble form of this receptor (sEPCR) circulates in plasma and inhibits both PC activation and APC anticoagulant activity.The aim of this study was to investigate the detection of the variations in the EPCR gene which has a role for thrombosis and the effects of this variations on the plasma sEPCR levels. We have 42 thrombotic patients and 29 healthy subject samples that have lower sEPCR levels (sEPCR< 50 ng / ?l) and 74 thrombotic patients and 24 healthy subject samples that have higher sEPCR levels (sEPCR>130 ng / ?l). Following DNA extraction, PCR, SSCP and DNA sequencing analysis of EPCR gene were performed. We detected 3998C>T and 4678 C>G polymorphisms in patients that have known sEPCR levels. The role of 3?UTR at RNA stability is known. Thus, EPCR 3?UTR 4678G>C polymorphism might have important effect on the thrombotic risk.Key WordsItem Faktör V 4070 A-G değişiminin trombozlu hastalarda incelenmesi(Biyoteknoloji Enstitüsü, 2010) Ballı, Sezen; Akar, Nejat; BiyoteknolojiTrombosis is a multigenic disease and APC resistance is major risk factor for causing thrombosis. A common polymorphism (FVL) in the FV gene that causes a missense mutation, FV Arg506Gln, results in the loss of one activated protein cleavage site of FV and APC resistance. Recently, a complex haplotype of FV (HR2), which includes 13 different polymorphisms throughout the gene, has been reported. One of these polymorphisms is factor V 4070 A(R1) ?G(R2) substitution which also gives its name to the haplotype. Factor V with HR2 possesses decreased co-factor activity to APC in the degradation of FVIIIa and an increased ratio of the more procoagulant isoform FV1 compared to FV2. Contrasting results on whether the haplotype induces a significant risk of venous thromboembolism have been reported. There are several individuals who have FVL, a common risk factor related to thrombosis, didn? t experienced thrombosis. Contrast to R2 allele which contributed to thrombosis risk, R1 allele is thought to be protective associated with the disease and the goal of the study is to determine effects of FV 4070 A (R1) ?G(R2) substitution in patients with thrombosis.In the study, in the range of 0-18 years and 70 and over 70 years patients diagnosed with thrombosis and the same two age groups healthy controls were screened. DNA isolation was carried out with phenol-chloroform method from blood samples taken from individuals. Amplification of exon 13 of the factor V gene was performed by polymerase chain reaction (PCR) with appropriate primers. PCR products was digested with RsaI restriction endonuclease enzyme for detecting gene variation with agarose gel electrophoresis.Effects of factor V 4070 A-G substitution were determined in 0-18 and 70 and older age groups included patient and healthy individuals. Carrying R1 allele in heterozygous or homozygous state was not associated with any protective effect in thrombosis. It was found that R2 allele was not an independent risk factor for thrombosis in the same age groups and genotype states.Item FVII promotor bölge gen değişimlerinin Behçet hastalarında tromboz oluşumundaki rolünün araştırılma(Biyoteknoloji Enstitüsü, 2009) Veli, Zehra; Akar, Nejat; BiyoteknolojiBehçet disease is a multisystem disorder which causes damage at different organs. Vascular involvement is the major complication of Behçet disease. Venous and arterial thrombosis is the most common type of the vascular involvement.Coagulation system defects are the major reasons of thrombosis. There are two common pathyways which play a role in this system (intrinsic and extrinsic). When the vassel wall is damaged, Tissue Factor synthesized by the endothel and then activated FVII. Thus coagulation system is started with extrinsic pathway activation. As a result protrombin turns into trombin and cause that all other coagulation stage improvement.Coagulation factor VII plays a role in the coagulation cascade which is synthesized at the liver and secreted into blood as an inactive single chain glycoprotein. FVII gene is located on chromose 13q.34. FVII gene polymorphisms at promotor region influence thrombosis involvement. The ?401G/T and -323ins10bp polymorphisms are associated with a reduced basal rate of FVII transcription; the ?402G/A polymorphism confers increased transcriptional activity of FVII.In this study we investigated -323ins10bp, -401G/T, -402G/A polymorphisms on FVII promotor region which may effect thrombosis risk in Behçet?s disease, 77 Behçet disease patients and 101 control healthy individuals were included in this study. The Fenole-chloroform method was used for DNA isolation, suitable primers were used to amplificate the region, then a different band profile samples with the single strand conformation polymorhisms technique were created. We performed a DNA sequencing for each band profile.We found six different band profiles in our research. One band profile which is heterozygote for three polymorphisms was shown in this study firstly. Also, it was detected with statistical analysis that this profile has a 11.5 fold risk for development of thrombosis in BehcetItem FVII promotor bölge polimorfizmlerinin aterosklerozlu hastalarda haplotip gruplandırılmasıyla tromboz oluşumunda rolünün araştırılması(Biyoteknoloji Enstitüsü, 2009) Cumaoğulları, Özge; Akar, Nejat; BiyoteknolojiCoagulation factor VII (FVII), a vitamin K-dependent zymogen of a serin protease, plays an important role in initiating thrombosis through the interaction with tissue factor at a site of blood vessel injury. Recent studies have provided evidence for association between common polymorphic markers in the FVII gene and plasma FVII levels. FVII plasma levels are influenced by enviromental and genetic factors. Several polymorphisms in the promoter associated with decrease or increase of FVII plasma levels have been previously identified. Some studies have reported altered plasma FVII levels in groups with manifest or risk of coronary arterial disease (CAD), whereas other did not. One of the genetic factor is the -323ins10bp polymorphism, known to be associated with low FVII levels and has suggest to protect agains CAD. This polymorphism is in complete allelic association with -401G/T polymorphism in Caucasians. The -402 G/A promoter polymorphism, that has been associated opposite effect, is clinically less well studied. Also the effect of the three FVII polymorphisms are ethnicity-dependent.In this study we aimed to find the effect of three FVII gene polymorphisms on the risk of CAD in Turkish population and investigate haplotypes.Three polymorphisms of the FVII gene were studied in 101 healthy control and 224 patient with coronary arterial disease. These polymorphisms of the FVII gene were studied using Polymerase Chain Reaction(PCR), SSCP and DNA sequencing technigues were used for the analysis.We detected a new Haplotype in a Turkish patient. The promoter polymorhisms, -323ins10bp, -401G/T, -402G/A, were not associated with a risk of an initial coronary event. -323ins10bp allel frequency was similar to North European, Italians, Etiopians and Indians. -401G/T allel frequency similar to Italians and Etiopians. -402G/A allel frequency was find similar to Italians and Etiopians.Item Genç ve yaşlı trombozlu hasta gruplarında olası genetik risk etmenlerinin değerlendirilmesi(Biyoteknoloji Enstitüsü, 2010) Akın, Dilara Fatma; Akar, Nejat; BiyoteknolojiAccording to The World Health Organization (WHO)?s data, cardiovascular diseases the leading cause of death With respect to WHO?s estimate, worldwide 17,5 million people has lost their lifes because of cardiovascular diseases in 2005. In Turkey cardiovascular diseases is one of the deadly diseases as all over the world. Thrombosis is a disease with many factors where inherited and acquired factors is acting together or alone. The aim of this study was to evaluate the effect of some cardiovascular, thrombotic risk factors on longevity such as FV 1691 G-A, PT 20210 G-A, MTHFR 677 C-T and ACE I/D and to determine the role of combination of mutations in young and old groups.In our study as a clinical thrombosis of age between 0?18, 362 individual, 70 years and over 209 individual, with 332 individual in the 0?18 age range do not have thrombosis 70 years and over 266 individuals were included. DNA was extracted by classical phenol-clorofrom method and genotyping of FV 1691 G-A, PT 20210 G-A, MTHFR 677 C-T polymorphisms were performed by Real-Time PCR technique. Genotyping of ACE I/D polymorphism, perfect- matched primers used to amplify the target region and agarose gel electrophoresis was performed for the identification of this polymorphism.In the two age groups between healty 8 patient groups FV 1691 G-A, PT 20210 G-A, MTHFR 677 C-T and ACE I/D polymorphisms were not found statistically significant when analyzed one by one. But our study showed that when analyzed together these polymorphisms are thrombotic risk factor in the studied groups.Item Hemoglobin alpha 2 gene +861 G>A polymorphism in Turkish population(2011) Özdag, Hilal; Akar, Nejat; http://orcid.org/0000-0001-7940-2499; Biyoteknoloji Enstitüsü; Dungul, Dilay CiglidagThalassemia is an inherited blood disorder which is divided into two groups: alpha and beta. HBA1 and HBA2 are the two genes associated with alpha thalassemia. The aim of this study is to investigate abnormal hemoglobin variants of alpha globin gene in healthy abnormal hemoglobin carrying individuals with intact beta globin gene. DNA was extracted from peripheral blood samples of seven healthy carrier individuals who have abnormal hemoglobin variants and 16 control individuals from Turkey. Complete coding and intronic sequences of HBA1 and HBA2 genes were amplified by polymerase chain reaction (PCR) and PCR products of HBA1 and HBA2 were sequenced. We were unable to find any base change in our carrier group in the HBA1 gene. We have observed an A/G polymorphism in the downstream untranslated region (+861 G>A) of the HBA2 gene. Our study showed that 14.29% (1/7 carriers) of the carrier group and 37.50% (6/16 controls) of the control group were heterozygous for the +861 G>A polymorphism. The distribution of allele frequencies and genotypes of HBA2 between carrier and control samples were analyzed and it is seen that the distribution of allele frequencies and that of genotypes were not statistically significant between carrier and control samples (P-value = 0.4131, P-value = 0.366, respectively). HBA2 +861 G>A nucleotide substitution is a neutral polymorphism previously reported in other populations. This is the first report in Turkish population.Item Homosistein metabolizmasında rol oynayabilecek gen değişimlerinin incelenmesi(Biyoteknoloji Enstitüsü, 2007) Koç, Lütfiye Yasemin; Akar, Nejat; BiyoteknolojiIn this study, CT replacement in the 677th nucleotide in MTHFR enzyme involved in homocysteine metabolism, 68 bp insertion in the 844th nucleotide in CBS enzyme, 6bp insertion/deletion in the region of 3?UTR in TYMS enzyme and 19 bp deletion in DHFR In this study, CT replacement in the 677th nucleotide in MTHFR enzyme involved in homocysteine metabolism, 68 bp insertion in the 844th nucleotide in CBS enzyme, 6bp insertion/deletion in the region of 3?UTR in TYMS enzyme and 19 bp deletion in DHFR enzyme were investigated. The effects of these mutations over homocysteine levels were also studied. In previous studies the main subjects were mutations in MTHF, CBS and TYMS enzymes. However, DHFR enzyme of Turkish society was studied first time in this work. Although the mutations have been related with illnesses in the previous works, 296 individuals with known homocysteine levels and individuals who?s any mutations not related with any illnesses were used in this study. Real-Time PCR method was used for a MTHFR 677 CT replacement. The CBS844ins68 and DHFR 19 bp deletion were studied using Polymerase Chain Reactions. TYMS 3?UTR 6bp insertion/deletion mutations were studied using Polymerase Chain Reaction based Restriction Fragment Polymorphism (PCR-RFLP). Obtained data was evaluated with non-parametric analyze methods. All enzymes were studied and correlation analyzes were tested between enzymes. As a result; it is discovered that TT genotype could be a risk factor for mutant genotype MTHFR 677 CT over homocysteine levels. There was no statistical meaning in other analyzes.Item Homozigot FVL taşıyıcılarında F5 geni promotor bölgesinde yer alan -426G/A gen değişiminin etkisi(Biyoteknoloji Enstitüsü, 2009) Esmael, Arjan Jalil; Akar, Nejat; BiyoteknolojiBlood clotting factor V is a protein that plays an important role in both procoagulant and anticoagulant pathways of hemostasis. Genetic and acquired disorders can lead to thrombotic and hemorrhagic events by affecting the activity or expression of FV molecule. FVL mutation is seen in 20% of patients with venous thrombosis and this proportion rises to 50% in selected thrombophilia patients. FVL mutation is a risk factor with the same effects as coagulation inhibitor deficiencies. The risk of thrombosis increases 5-fold with heterozygote mutation while a 50-100 fold increase in thrombosis is seen with homozyogote mutation.In this study, real time PCR technique is used to detect FVL mutation. F5 gene is amplified by polimerase chain reaction (PCR) using appropriate primers for promotor region. -426 G/A change is detected by imaging band profiles obtained by RFLP (Restriction Fragment Length Polimorfizm) in 3% agaroz gel.The objective of this study is to find the effect of -426 G/A gene change present in the promotor region of F5 gene on thrombosis. The effect of -426 G/A gene change on thrombosis in individuals who are homozygote for FVL mutation and have or have not had a thrombotic episode is investigated. No significant difference with respect to genotype and allele distribution of -426 G/A polimorfizm was detected between homozygote carriers of the FVL mutation who did or did not suffer a thrombotic episodeItem İnme geçirmiş olan çocuklarda, trombomodulin geninin taranması(Biyoteknoloji Enstitüsü, 2009) Kızıl, Hamit Emre; Akar, Nejat; BiyoteknolojiStroke is the third most common cause of mortality and of morbidity in the world. In the past decade, the mortality rate from stroke has declined, but the risk factors which may cause morbidity have increased.Thrombomodulin (TM) is a 557 amino acid a glycosylated transmembrane protein and is a thrombin receptor located on endothelial cell surface. It was named as thrombomodulin due to its modulator role on the functions of thrombin. While a part of TM?s physiological functions are through the activity of the TM-trombin complex on protein C and on thrombin activated fibrinolysis inhibitor (TAFI), a substantial number TM functions are independent from thrombin.The pediatric stroke is the most observed type of thrombosis in the pediatric age we aimed to study the thrombomodulin gene which play a balancing role of coagulation and fibrinolysis. We screened 190 samples of pediatric stroke patients. Following DNA extraction, PCR, SSCP and DNA sequencing analysis of Thrombomodulin gene was performed. We have identified a patient with c.519 C>G polymorphism in the thrombomodulin gene.Item Light cycler real time PCR teknolojisi ile faktör V geninde yeni mutasyon taraması(Biyoteknoloji Enstitüsü, 2009) Sanlıdilek, S. Duygu; Akar, Nejat; BiyoteknolojiThe risk of venous thrombosis is increased when the hemostatic balance between pro- and anticoagulant forces is shifted in favor of coagulation. If this is caused by an inherited defect, the resulting hypercoagulablestate conveys a lifelong increased risk of thrombosis. Mutations in the genes coding for proteins in the coagulation cascade were found to be associated with increased risk for venous thrombosis. Dahlbäck et al. described a predisposition to thrombosis caused by a poor anticoagulant response to activated protein C (APC). A year later Bertina et al., reported that a point mutation in exon 10 of the gene encoding blood coagulation factor V.Real Time PCR is a system, which analyses a mutation Factor V Leiden. In this technique hybridization probe can be used to detect and monitor single nucleotide mutation. The probe has 2 part and its specific to mutation. With the melting curve analysis the hybridization probes melt off, two fluorescent dyes are no longer in close proximity and the fluorescence will degrees. The resulting melting peaks allow discrimination between the homozygous as well as the heterozygous genotype. The anchor probe has a higher Tm than the mutation probe. During the experiment tm degree of the unknown samples may vary ±2ºC from the tm degree of positive control. Differences in base composition, nucleotide position, and nearest-neighbor environments all affect Tm.There were 4100 diagnostic samples for thrombophilia screening starting 2002 in our department. We found 13 patients with a variation of Tm degree more than ±2ºC. Results of DNA sequencing showed only FVL.In order to delineate the reason for the artifact of the Tm degree, DNA of the patients were isolated again and the study was repeated by LC and sequencing. The shift of the LC experiment was within normal range indicating that they are FVL.Our study showed that DNA quality and quantity is important and needs to be analyzed prior to laboratory procedures and archival and not well-kept DNA samples may cause the same variation, which causes an artifactItem Nöral tüp defektli çocuklar ve annelerinde ZIP14 (SLC39A14) geninin taranması(Biyoteknoloji Enstitüsü, 2008) Torun, Didem; Akar, Nejat; BiyoteknolojiThe neural tube defects are congenital malformations caused by missclosure of the neural tubethat result in serious complications. Various studies have shown the high incidence of neuraltube defects (NTD) in Turkey. The estimated incidence is around 3 per 1000 live births.Several factors influence the development of neural tube defects. Zinc deficiency is one of theflawed factor for the pathogenesis of NTD?s. Eithernutritional factors and /or genetic defectsrelated zinc may cause zinc deficiency among women which may predispose to NTD?s. SLC(Solute carrier family) gene family in which Zip14 provides uptake of zinc into the cell.So, we investigated a zinc related gene, i.e. ZIP14. 70 NTD mothers, 70 NTD babies and 35Algerian samples were included. Following DNA extraction, PCR, SSCP and DNA sequencingof the promotor and HHH motife were analyzed. However, no relation of neural tube defects andZIP14 was detected in Turkish and Algerian NTD patientsItem Otozomal resesif işitme kayıplı ailelerde otozigozite taraması ile MYO7A mutasyonlarının gösterilmesi(Biyoteknoloji Enstitüsü, 2011) Duman, Duygu; Akar, Nejat; BiyoteknolojiHearing loss is the most common sensorial disorder. Congenital or prelingual hearing loss occurs approximately in one case per 1000 live births. Genetic causes account for 50% of cases. Additional findings are present in 30% of cases leading to the diagnosis of a syndrome . Autosomal recessive transmission occurs in 80% of hereditary deafness. To date, 38 genes in which mutations are responsible for autosomal recessive deafness have been identified. Mutations in GJB2, encoding connexin 26, are the most commonly identified cause of sensorineural hearing loss in Caucasians. Our data and the results of other studies show that the autosomal recessive inheritance accounts for more than 90% of genetic cases in Turkey. The GJB2 gene is the most common cause accounting for approximately 20% of cases.Due to the high rate of consanguineous marriages in Turkey as well as the traditional settlements in isolated small villages, rare autosomal recessive deafness alleles are frequently present in affected individuals . One of the previously identified deafness genes, MYO7A , is on the q13.5 region of chromosome 11. This gene consists of 48 coding exon, and codes a protein containing 2215 amino acids. Mutations in this gene cause Usher syndrome type 1B, atypical Usher syndrome, as well as non-syndromic recessive (DFNB2) and dominant (DFNA11) sensorineural hearing loss.This study included 55 unrelated multiplex families with sensorineural hearing loss. Autosomal recessive inheritance was evident with multiple siblings being affected as well as with the presence of parental consanguinity. The phenotypical and audiological characteristics of these families were evaluated and and no syndromic findings or environmental cause for hearing loss were detected. Mutations in GJB2 were negative in all families. Whenever genomewide SNP-based autozygozity mapping showed an autozygous segment flanking MYO7A in a family, mutation analysis was performed. The aim of this study was to reveal the spectrum of MYO7A mutations in Turkey to facilitate molecular diagnosis in clinical setting.MYO7A mutations were detected in 5 out of 7 families, where an autozygotic region demonstrated by microarrays was found to flank the gene. In the remaining two families no change has been identified in this gene.The following homozygous mutations were found in affected individuals : c.5824 G>A (p.G1924R) (one family), in c.5838delT (p.F1946LfsX24) (one family), c.6487 G>A (p.G2163S) (one family), c.5581 C>T (p.R1861X) (one family), c.5660 C>T (p.P1887L) (one family).This study shows that MYO7A mutations have a considerably high frequency in families with hearing loss in Turkey. Identifiying genes that cause hearing loss in Turkey will accelerate the molecular diagnosis and would improve accurate genetic counseling .Item Pediatrik obezitede melanokortin 4 reseptör geni,plazminojen aktivatör inhibitör 1 geni (-675 4g/5g), tümör nekrozis faktör alpha (-308 g/a), yağ asiti bağlama proteini -87 T/C ve interlökin -6 (-174 G/C) gen değişimlerinin önemi ve siRNA yaklaşım ile fonksiyon tayini(Biyoteknoloji Enstitüsü, 2006) Demiralp, Fatma Duygu Özel; Akar, Nejat; BiyoteknolojiObesity is a complex, multi-factorial chronic disease, frequently associated with cardiovascular risks, hypertriglyceridaemia, low HDL-cholesterol, high blood pressure and the insulin resistance that appears to be central to the pathogenesis of type II diabetes. Obesity occures when the energy in take from foods is over than energy used and balance is break-down (WHO 2000). Obesity Over the last several decades, cases of obesity and Type II diabetes have reached epidemic proportions and threaten to over burden the world?s health care systems. The incidance of obesity seems over 315 million in the world. This effective increase of obesity is also affect Turkey. In our country; 1/3 of women and 1/5 of men are obese (satman i 2001). There is considerable evidence to suggest that, like height, weight is a heritable trait. Traditionally the most favoured model for seperation of the genetic component of variance is based on studies of twins , as monozygotic co- twins share 100% of their genes and dizygotes 50% on average. Overall, data from twin and adoption studies are consistent with a genetic contribution for body mass index of between 60-84%. It is clear that different individuals have a certain genetic propensity to store excessive caloric intake as fat(Maes, Neale et al. 1997). However the role of PAI-1 in adipogenesis is still unknown, circulating PAI-1 levels are elevated at an early stage of impaired glucose tolerance and continue to be elevated as diabetes and metabolic syndrome develop humans. In our study we investigated PAI-1 promoter -675 4G/5G polymorphism in pediatric obese group as a risk factor with 2.8 fold (Berberoglu, Evliyaoglu et al. 2006). And we demonstrated that the 4G/4G genotype increase the PAI-1 promoter activity, PAI 1 increases the differentiation of preadipocytes to adipocytes. In this present studie, IL-6 -174 G/C and FABP4 T/C polymorphisms were investigated as protector gene alternations and TNF-a -308 G/A polymorphism was not significant in our obese group. Tha last gene we focus on was melanocortin 4 receptor gene that in 18 obese patients, 2 different mutations were found. One of these mutations was a new mutation. So that we conclude, Melanocortin 4 receptor gene and plasminogen activator inhibitor-1 genes modulate the obesity and thought to continue focus on these genes with additional studies. Key Words: Plasminogen activator inhibitor-1 (PAI-1), Melanocortin 4 Reseptor gene (MC4R), Tumor Necrosis faktor ?alpha (TNF-a), Interleukin-6 (IL-6), Obesity, Diabetes mellitusItem Plazminojenaktivatör inhibitor-1(PAI-1) 4g/5g gen değişiminin uzun yaşama etkisi(Biyoteknoloji Enstitüsü, 2010) Gülbahar, Z. Gülin; Akar, Nejat; BiyoteknolojiStudies related to longevity genes gained a new perspective with the ?damage due to response? theory in recent years. The most important of these in the theory formation are metabolic events. According to the theory, accumulation of damage process formed response. Genes that ensure genetic control in these mechanisms are called ?longevity genes?. Thrombosis is a disease that has a multifactorial nature. Although it is thought that Factor V 1691 mutation has the leading effect for thrombosis, there are many other genetic effects play role together. Previous studies indicate that the increased plasminogen activator inhibitor-1 levels in thrombosis patients, fibrinolytic activity is decreased. The increased PAI-1 plasma level is related with the 4G/5G insertion/deletion polymorphism that found 675 upstream of the transkription start sequence in the promoter region. In this content, the aim of this study is to search the effect of PAI-1 675 4G/5G insertion/deletion alone and in combination with FVL on longevity.The healthy and patient subjects were divided into mainly two groups, i.e. 1 to 18 years and 70 and older. Each group was also divided into two as thrombotic and non-thrombotic. Peripheral venous blood samples were collected and phenol- chloroform method was used to extract DNA. Analysis of PAI?1 4G/5G polymorphism was carried out by PCR with the forward 5?CACAGAGAGAGTCTGGCCACGT3?and reverse 5?CCAACAGAGGACTCTTGGTCT3? primers. The amplified 98/99 bp PCR products were controlled by running them on 3% agarose gel. BseLI restriction endonuclease enzyme used to detect 4G/5G polymorphism. band profiles were seen in the 7% polyacrylamide gel electrophoresis to detect gene variations.For the PAI-1 4G/5G gene variation, when 70 and older patients compared to healthy group 4G allele heterozoygosity is found to have has a protective role(OR:0.45, p:0.003). Homozygosity for 4G allele seen as also a protective factor for 70 and older patients groups(OR:0.50, p:0.03). Moreover 5G allele homozygosity is a risk factor for the disease with 1.98 odds ratio and 0.03 p-value. PAI-1 4G/5G gene variation in combination with FVL mutation also have protective role for 70 and older in case of 4G heterozygosity and homozygosity(p:0.004 OR:0.44, p:0.03 OR:0.48).It is observed that increased plasma PAI-1 activity responsible for decreased fibrinolytic activity for the individuals that escape from angina pectoris, diabetes, myocardial infarction. 4G/5G polymorphism is related to transcriptional activation changes in the PAI-1 gene. PAI-1 gene 4G motif is determined to has more transcriptional activity than 5G motif and in correspondence with the experimental results 4G allele homozygosity cause increased PAI-1 protein levels and activity than 5G allele. Some studies suggested that 4G allele that relates with increased PAI-1 levels is a sign of increasing thrombotic risks. As PAI-1 4G/5G poymorphism is thought to play a role in pathogenesis of the mentioned fatal diseases, it is considered that the survival of individuals can be affected by this gene variation. In the previous thesis study was done in our department, FVL mutation shown to be a risk factor for thrombosis after birth and have effects on morbidity. Therefore, FVL mutation are expected to shorten the life span. However, examined our DNA samples showed although some individuals carry FVL mutation, they were identified as long-lived. The contribution of the study hypothesis that FVL and PAI-1 4G/5G has a relation and by this relation 0-18 and 70 and over individuals been included in the study, as a result it is determined that, 4G allele found to have a protective role for the eldery group whereas 5G allele is a risk factor for the disease.Key words: Thrombosis, PAI-1 4G/5G ins/del, FV1691 G-A, Longevity GenesItem Relation of Soluble Endothelial Protein C Receptor and Cytokines After Allogeneic Hematopoietic Stem Cell Transplantation(2009-08-18) İnce, Elif; Tıp Fakültesi; Azık, Fatih; Ertem, Mehmet; İleri, Talia; Uysal, Zümrüt; Eğin, Yonca; Akar, NejatAim: The objective of this study was to elucidate the effects of tumor necrosis factor a (TNF-a), interleukin 1b (IL-1b), interleukin 2 (IL-2), interleukin 6 (IL-6), and interleukin 8 (IL-8) on the expression of soluble endothelial protein C receptor (sEPCR) in the pathogenesis of thrombotic complications after hematopoietic stem cell transplantation (HSCT). Methods: The relationship between plasma concentrations of proinflammatory cytokines (TNF-a, IL-1b, IL-2, IL-6, and IL-8) and sEPCR was evaluated in 32 consecutive allogeneic hematopoietic stem cell–transplanted patients prior to conditioning regimen and randomly once between þ5 and þ30 days after transplantation and compared these results with 20 healthy controls. Results: Soluble endothelial protein C receptor levels did not indicate any significant difference between pre- and posttransplantation period, and sEPCR levels showed a significantly negative correlation between IL-6 and IL-8 (sEPCR and IL-6, r ¼ .43, P < .01; sEPCR and IL-8, r ¼ .57, P < .01). There was no correlation between sEPCR levels and TNF-a, IL-1b, or IL-2 (sEPCR and TNF-a, r ¼ .13, P > .05; sEPCR and IL-1b, r ¼ .1, P .05; sEPCR and IL-2, r ¼ .07, P > .05). Conclusions: Our results suggest that the production of sEPCR was not affected by allogeneic HSCT. Soluble endothelial protein C receptor did not show any positive correlation between these proinflammatory cytokines (TNF-a, IL-1b, IL-2, IL-6, and IL-8), on the contrary a significantly negative correlation was determined between sEPCR and either IL-6 or IL-8. This negative correlation may be a protective mechanism in the pathway of protein C activation.