Ege Bölgesindeki beta talasemi vakalarında ASO prob hibridizasyon tekniği ile 8 mutasyonun analizi

Abstract

A. ÖZET EGE BÖLGESİNDEKİ BETA TALASEMİ VAKALARINDA ASO PROB HİBRİDİZASYON TEKNİĞİ İLE 8 MUTASYONUN ANALİZİ SUNAY GÜLEŞKEN Anahtar Kelimeler ?-Talasemi, (3-Globiıı Gen Mutasyonlan, ASO Prob Hibridizasyoıra Bu çalışmada, Ege Bölgesindeki beta talasemi hastalarında yaygın görülen 8 mutasyonun gözlenme sıklıklarının saptanması amaçlandı. Beta globin gen bölgelerindeki mutasyonlan saptamada Multipleks-PCR ve ASO(Ailel-Specifıc-01igonücieotide)Prob Hibridizasyon teknikleri kullanıldı. Hibridizasyon sonuçlan ELISA(Enzyme-Linked-İmmunosorbent Assay) metodu kullanılarak, 450-655nm. çift dalga boylarındaki absorbans ölçümleri ile kantitatif olarak değerlendirildi. Normal ve Mutant ASO problan için okunan değerler oranlanarak mutasyon tipleri belirlendi. Toplam 68 beta talasemi vakasının 49'unda(%72) mutasyon tipi saptandı. Mutasyon tipleri saptanan hastaların 47'si klinik olarak talasemi major, geri kalan 2`si ise talasemi minördür.Mutasyon tipi saptanan 47 talasemi major hastasının 19'u (%40.5) genotip olarak tek bir mutasyon için homozigot, 16' sı (%34) iki farklı mutasyon için bileşik heterozigot bulundu. Hastaların 12'sinde(%25.5) ise tek bir gendeki mutasyon saptanırken, diğer gendeki mutasyon çalıştığımız yöntemle gösterilemedi. Talasemi major'lu 19 hastada (%28) bu yöntemle mutasyon tipi saptanamadı. Testin duyarlılığım ve hassasiyetini değerlendirmek amacıyla, Hacettepe Üniversitesinde ASO-Dot-Blot ve DNA dizi analizi gibi metodlarla mutasyon tipleri belirlenmiş olan. ancak çalışma sırasında sonuçlarını bilmediğimiz 4 hasta çalışıldı. Bu hastalardan elde ettiğimiz sonuçların Hacettepe verileri ile uyumlu olduğu görüldü. Çalışılan 134 kromozomda, 8 lokusda bulunan mutasyonların frekansları IVS1-110(G->A)(%30), rVSl-l(G->A)(%16.4), ıVSİ-6(T->C){%8.2), IVS2-745(C-»G)(%5.2), IVS2-1(G-»A)(%1.5), CD-39(C->T)(%1.5), -87(C-»G)(%0), CD6-A(%0) olarak bulundu.B. ABSTRACT MUTATIONAL ANALYSES AT EIGHT LOCI OF BETA THALASSEMIA CASES FROM AGEAN REGION BY USING ASO PROB HYBRIDIZATION TECHNIQUE SUNAY GÜLEŞKEN Key Words ?-Thalassemia, P-GIobin Gene Mutations, ASO Prob Hybridization The main perspective of this study is to determine point mutations frequently observed at eight hot spot regions of p-globin gene in (3-thalassemia patients from Agean Region of Turkey. A combination of multiplex-PCR and ASO(Aileie Specific Oiigonucieotide) prob hybridization approach has been utilized to determine putative mutations in P-globin gene. Hybridization results have been evaluated by using ELISA (Enzyme-Linked- Immunosorbent Assay) method quantitatively, measuring absorbance at 450 and 655nm. Mutation types were determined based on the ratio O.D. readings for normal and mutant ASO probes. We found mutations in total of 49 (%72)patients out of 68 P-thalassemia cases. 47 of these found to be mutants clinically were thalassemia major from which 19(%40.5) patients were homozygotes for one mutation and from which 16 (%34) patients were compound heterozygotes for two mutations. Out of 47 beta thalassemia major patients, 12(%25. 5) patients were found to be mutant for one gene in spite of being compound heterozygotes. No mutation has been found in 19(%28) P-thalasscmia major patients with the probes used in this study. To evaluate the sensitivity and the specificity of this methodology, we compared four cases that had been previously studied in Hacettepe University with ASO-DOT BLOT and DNA SEQUENCING anaiyses.Our results were consistent with that of obtained at Hacettepe University.The frequency distribution of mutations for eight loci was IVSi-110(G->A)(%30), IVS1-1(G->A)(%16.4). IVSi- 6(T->C)(%8.2), IVS2-745(C->-G)(%5.2), iVS2-l(G-»A)(%L5), CD-39(C-»T)(%1.5), -87(C->G)(%0), CD6-A(%0).

B. ABSTRACT MUTATIONAL ANALYSES AT EIGHT LOCI OF BETA THALASSEMIA CASES FROM AGEAN REGION BY USING ASO PROB HYBRIDIZATION TECHNIQUE SUNAY GÜLEŞKEN Key Words ?-Thalassemia, P-GIobin Gene Mutations, ASO Prob Hybridization The main perspective of this study is to determine point mutations frequently observed at eight hot spot regions of p-globin gene in (3-thalassemia patients from Agean Region of Turkey. A combination of multiplex-PCR and ASO(Aileie Specific Oiigonucieotide) prob hybridization approach has been utilized to determine putative mutations in P-globin gene. Hybridization results have been evaluated by using ELISA (Enzyme-Linked- Immunosorbent Assay) method quantitatively, measuring absorbance at 450 and 655nm. Mutation types were determined based on the ratio O.D. readings for normal and mutant ASO probes. We found mutations in total of 49 (%72)patients out of 68 P-thalassemia cases. 47 of these found to be mutants clinically were thalassemia major from which 19(%40.5) patients were homozygotes for one mutation and from which 16 (%34) patients were compound heterozygotes for two mutations. Out of 47 beta thalassemia major patients, 12(%25. 5) patients were found to be mutant for one gene in spite of being compound heterozygotes. No mutation has been found in 19(%28) P-thalasscmia major patients with the probes used in this study. To evaluate the sensitivity and the specificity of this methodology, we compared four cases that had been previously studied in Hacettepe University with ASO-DOT BLOT and DNA SEQUENCING anaiyses.Our results were consistent with that of obtained at Hacettepe University.The frequency distribution of mutations for eight loci was IVSi-110(G->A)(%30), IVS1-1(G->A)(%16.4). IVSi- 6(T->C)(%8.2), IVS2-745(C->-G)(%5.2), iVS2-l(G-»A)(%L5), CD-39(C-»T)(%1.5), -87(C->G)(%0), CD6-A(%0).