Denizli yöresinde glukoz-6-fosfat dehidrogenaz (G6PD) enzim yetmezlikli bireylerde Akdeniz mutasyonu sıklığının ve eritrositlerdeki ince yapı değişikliklerinin araştırılması
Küçük Resim Yok
Tarih
2001
Yazarlar
Dergi Başlığı
Dergi ISSN
Cilt Başlığı
Yayıncı
Ege Üniversitesi
Erişim Hakkı
info:eu-repo/semantics/closedAccess
Özet
IV ÖZET DENİZLİ YÖRESİNDE GLUKOZ- 6- FOSFAT DEHİDROGENAZ ENZİM YETMEZLİKLİKLİ BİREYLERDE AKDENİZ MUTASYONU SIKLIĞININ VE ERİTROSİTLERDEKİ İNCE YAPI DEĞİŞİKLİKLERİNİN ARAŞTIRILMASI KESKİN, Nazan Doktora Tezi, Biyoloji Bolümü Tez Yöneticileri: Prof. Dr. Sabire KARAÇALI Prof. Dr. Hüseyin BAĞCI Mart 2001, 77 Sayfa Bu çalışmada, Denizli il merkezi ve ilçelerindeki G6PDakdeniz 563T mutasyonu sıklığı ile, mutant eritrositlerde meydana gelen ince yapı değişiklikleri araştırılmıştır. Bu amaçla, orta dereceli okullarda yaşlan 14-17 arasında değişen 918'i kız, 1032 si erkek olmak üzere toplam 1950 kişi taranmıştır. Kan örneklerinin kalitatif ve kantitatif ölçüm sonuçlarına göre 24 birey G6PD yetmezlikli bulunmuştur. PCR-RFLP çalışmasına, G6PD yetmezlikli olarak bulunan 24 birey ile, akrabalarından (anne-baba-kardeş) oluşan 12 birey olmak üzere, toplam 36 birey alınmıştır. Ekson VI ve VII'ye özgül primer çifti, sırasıyla AKA ve AKB kullanılarak 563T'nin amplifikasyon ve Mbo II restriksiyon endonükleaz enzimi ile kesim sonuçlarına göre, 24 bireyde G6PD)Akdeniz 563T mutasyonu saptanmıştır. Çalışma grubundaki kalitatif ve kantitatif ölçüm sonuçlarına göre belirlenen 24 öğrencinin 19'unda (%79) G6PD Akdeniz563T mutasyonusaptanmıştır. 19 bireyin 13'ü hemizigot, 2'si homozigot, 4'ü ise heterozigot olarak belirlenmiştir. Normal enzim aktivitesine sahip ancak heterozigot olan bireylerin saptanması kalitatif ve kantitatif yöntemlerin heterozigotlar için güvenilir olmadığını ortaya koymuştur. Kalitatif ve kantitatif ölçüm değerleri hemizigot bireylerinkine benzeyen ancak mutant bulunmayan bir erkek birey, bölgede başka mutasyonların varlığına işaret etmektedir. Normal ve mutant bireylerin eritrosit morfolojileri ışık mikroskobu (İM), taramalı (scanning) elektron mikroskobu (SEM), geçirimli (transmission) elektron mikroskobu (TEM) ile, yüzey anyonik yerlerin dağılışı da katyonik bir boya olan rutenyum kırmızısı (RR) kullanarak TEM mikroskobu ile karşılaştırılmıştır. İM ile toluidin mavisi ile boyanan mutant bireylere ait kesitlerde hipokromik ve hedef hücreler ayırt edilmiştir. Ayrıca hücrelerin içinde mavi mor renkte granüller şeklinde Heinz cisimcikleri görülmektedir. Mutant eritrositler SEM ile normal bikonkav disk şekillerinden oldukça farklı olarak, düzensiz şekillerde görülmektedir. Eritrositlerin çentikler şeklinde görülen kenarları TEM mikrograflarında hücre zarının sitoplazmaya doğru yaptığı geniş çöküntüler şeklinde ayırt edilmektedir. TEM mikrograflarında mutant eritrosit zarında erime ve zardan ayrılan parçaların görülmesi, hücrelerdeki yıpranmayı göstermesi bakımından anlamlıdır. Ayrıca, hücrelerin sitoplazmasında dejeneratif halkasal yapılar, elektronca yoğun kitleler ve vakuoller görülmektedir. Hemi/homozigot ve heterozigot bireylerin eritrositlerinde meydana gelen morfolojik değişiklikler bakımından fark bulunmamıştır. Tüm bireylere ait eritrositlerin yüzey anyonik yerleri RR ile pozitif reaksiyon vermiştir. Normal eritrosit yüzeyinde RR pozitif reaksiyon homojen olarak dağılım göstermiştir. Bununla birlikte reaksiyon mutant eritrosit yüzeyinde bölgesel olarak farklı yoğunluklarda ve heterojendir.VI PCR-RFLP ve mikroskobik sonuçlar; Denizli yöresinde G6PD yetmezliğinin ileride de önemli bir sağlık sorunu olacağını, eritrosit genotipinde meydana gelen değişikliklerle eritrosit morfolojisi ve yüzey değişikliklerinin ilişkili olduğunu göstermektedir. Eritrositlerin makrofajlarca tanınarak zamanından önce dolaşımdan alınmasına neden olan glukokonjugatlardaki değişikliklerin araştırılması ve yörede başka mutasyonların saptanmasıyla ilgili olarak, yeni. çalışmaların yapılması gerektiği ortaya çıkmıştır. Anahtar kelimeler G6PDAkdfiniz563T mutasyonu, PCR-RFLP, Eritrosit, Işık mikroskobu, Taramalı (Scanning) Elektron Mikroskobu, Geçirimli (Transmission) Elektron Mikroskobu, Rutenyum kırmızısı
vn ABSTRACT THE INVESTIGATION OF GLUCOSE- 6-PHOSPHATE DEHYDROGENASE (G6PD)-MEDITERRANEAN MUTATION FREQUENCY AND ULTRASTRUCTURAL CHANGES ON ERYTHROCYTES IN G6PD DEFICIENT SUBJECTS IN DENİZLİ REGION KESKİN, Nazan Ph.D. in Biology Supervisors: Prof. Dr. Sabire KARAÇALI Prof. Dr. Hüseyin BA?CI March 2001, 77 pages In this study, the frequency of G6PD Mediterranean 563T mutation along with ultrastructural changes in mutant erythrocytes was investigated. In regard of this purpose, a survey has been carried out among the secondary schools' students in Denizli city and its municipial area (total of 1950: 918 female, 1032 male; age varies in between 14-17). The total of 24 G6PD deficient subjects were spotted and identified based upon qualitative and quantitative tests of the blood samples. The total of 36 subjects (24 from the deficient subjects and the remaining 12 from their relatives-mother, father, brother/sister) were investigated for the PCR-RFLP study. 24 out of the 36 subjects were identified to have G6PD Mediterranean 563T mutation based on amplification of 563 T and Mbo II restriction endonuclease digestion, using consecutively AKA and AKB, which are spesific primers on exon VI, VII.vm G6PD Mediterranean 563T mutation was identified on the total 19 out of 24 (79 %) subjects. Among those 19 subjects, it was found that 13 of them were hemizygotes, 2 of them were homozygotes and 4 of them were heterozygotes. It was found that qualitative and quantitative methods have limitations for the identification of the heterozygote subjects who have normal enzyme activity. It should also be noted that there exists other mutations in the region due to the qualitative and quantitative tests results identical to hemizygote subjects on a non-mutant male subject. The erythrocyte morphologies of normal and mutant subjects were compared and evaluated using Light Microscopy (LM), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). Distribution of surface anionic locations was also evaluated and compared using TEM technique on cationic ruthenium red (RR) stained samples. Hypochromic and target cells were distinguished by light microscope on the toluidine blue stained sections of mutant subjects. Bluish-purple colored granular Heinz bodies are also observed in the cells. In SEM analysis, mutant erythrocytes were observed as irregular shaped forms differ from biconcave disk-shaped normals. Notched sides of erytrocytes are distinguieshed by TEM micrographs as wide holes in cell membranes towards cytoplasm. TEM micrographs showing dissolved cell membrane and particles separated from the membrane clearly indicate distinct degradation within the cells. In addition, there are electron-dense masses and degenerative circular forms and vacuoles within the mutant erythrocytes. Findings indicates that morphological changes in the erythrocytes of hemi/homozygote and heterozygote subjects are identical and do not differ.IX Surface anionic locations of erythrocytes for all the subjects show positive reaction with RR. RR-positive reaction has homogeneous distribution on normal erythrocyte surface. However, the reaction was locally different in densities and heterogeneous in mutant erythrocyte surfaces. The results of PCR-RFLP and microscopic evaluations show that G6PD deficiency will be a health problem in future within the Denizli region. It is also shown that change in erythrocyte genotypes is correlated with erythrocyte morphology and change in surface characteristics. Further studies are also needed to conduct on changes of glycoconjugates having an active role early taking the erythrocytes out from circulation by recognition of macrophages and determination of other mutations in the region. Key Words: G6PDMedilaiaQean563T mutation, PCR-RFLP, Erythrocyte, Light microscope, Scanning Electron Microscope, Transmission Electron Microscope
vn ABSTRACT THE INVESTIGATION OF GLUCOSE- 6-PHOSPHATE DEHYDROGENASE (G6PD)-MEDITERRANEAN MUTATION FREQUENCY AND ULTRASTRUCTURAL CHANGES ON ERYTHROCYTES IN G6PD DEFICIENT SUBJECTS IN DENİZLİ REGION KESKİN, Nazan Ph.D. in Biology Supervisors: Prof. Dr. Sabire KARAÇALI Prof. Dr. Hüseyin BA?CI March 2001, 77 pages In this study, the frequency of G6PD Mediterranean 563T mutation along with ultrastructural changes in mutant erythrocytes was investigated. In regard of this purpose, a survey has been carried out among the secondary schools' students in Denizli city and its municipial area (total of 1950: 918 female, 1032 male; age varies in between 14-17). The total of 24 G6PD deficient subjects were spotted and identified based upon qualitative and quantitative tests of the blood samples. The total of 36 subjects (24 from the deficient subjects and the remaining 12 from their relatives-mother, father, brother/sister) were investigated for the PCR-RFLP study. 24 out of the 36 subjects were identified to have G6PD Mediterranean 563T mutation based on amplification of 563 T and Mbo II restriction endonuclease digestion, using consecutively AKA and AKB, which are spesific primers on exon VI, VII.vm G6PD Mediterranean 563T mutation was identified on the total 19 out of 24 (79 %) subjects. Among those 19 subjects, it was found that 13 of them were hemizygotes, 2 of them were homozygotes and 4 of them were heterozygotes. It was found that qualitative and quantitative methods have limitations for the identification of the heterozygote subjects who have normal enzyme activity. It should also be noted that there exists other mutations in the region due to the qualitative and quantitative tests results identical to hemizygote subjects on a non-mutant male subject. The erythrocyte morphologies of normal and mutant subjects were compared and evaluated using Light Microscopy (LM), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). Distribution of surface anionic locations was also evaluated and compared using TEM technique on cationic ruthenium red (RR) stained samples. Hypochromic and target cells were distinguished by light microscope on the toluidine blue stained sections of mutant subjects. Bluish-purple colored granular Heinz bodies are also observed in the cells. In SEM analysis, mutant erythrocytes were observed as irregular shaped forms differ from biconcave disk-shaped normals. Notched sides of erytrocytes are distinguieshed by TEM micrographs as wide holes in cell membranes towards cytoplasm. TEM micrographs showing dissolved cell membrane and particles separated from the membrane clearly indicate distinct degradation within the cells. In addition, there are electron-dense masses and degenerative circular forms and vacuoles within the mutant erythrocytes. Findings indicates that morphological changes in the erythrocytes of hemi/homozygote and heterozygote subjects are identical and do not differ.IX Surface anionic locations of erythrocytes for all the subjects show positive reaction with RR. RR-positive reaction has homogeneous distribution on normal erythrocyte surface. However, the reaction was locally different in densities and heterogeneous in mutant erythrocyte surfaces. The results of PCR-RFLP and microscopic evaluations show that G6PD deficiency will be a health problem in future within the Denizli region. It is also shown that change in erythrocyte genotypes is correlated with erythrocyte morphology and change in surface characteristics. Further studies are also needed to conduct on changes of glycoconjugates having an active role early taking the erythrocytes out from circulation by recognition of macrophages and determination of other mutations in the region. Key Words: G6PDMedilaiaQean563T mutation, PCR-RFLP, Erythrocyte, Light microscope, Scanning Electron Microscope, Transmission Electron Microscope
Açıklama
Bu tezin, veri tabanı üzerinden yayınlanma izni bulunmamaktadır. Yayınlanma izni olmayan tezlerin basılı kopyalarına Üniversite kütüphaneniz aracılığıyla (TÜBESS üzerinden) erişebilirsiniz.
Anahtar Kelimeler
Biyoloji, Biology, Denizli, Denizli, Enzimler, Enzymes, Eritrositler, Erythrocytes, Glükozfosfat dehidrogenaz, Glucosephosphate dehydrogenases, Mutasyon, Mutation
