Kronik Miyeloid Lösemili Hastalarda Sitogenetik Analiz ve RT-PCR Tekniği ile Philadelphia Kromozomu ve BCR-ABL Füzyon Geninin Saptanması

Abstract

Amaç: Çalışmamızda 25 KML (Kronik Miyeloid Lösemi) hastasında Sitogenetik Analiz ve RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) Tekniği ile Ph (Philadelphia) kromozomu ve BCR-ABL füzyon geninin saptanması amaçlanmıştır.

Gereç ve Yöntemler: Çalışmamıza dahil edilen hastalardan kemik iliği veya kan örnekleri alındı. RNA izolasyon kiti kullanılarak periferik kan, kemik iliği ve hücre hattından total RNA izolasyonu yapıldı. RNA saflığı spektrofotometre ile ölçülerek, çalışma için uygun saflıkta RNA’ lar seçildi. Random hekzamer primerleri ve reverse transkriptaz enzimi kullanılarak mRNA’ dan cDNA sentezi gerçekleştirildi. Hedef DNA oligonukleotid primerler kullanılarak iki basamaklı Nested PCR yöntemi ile çoğaltıldı. K562 hücre hattı pozitif kontrol olarak, distile su negatif kontrol olarak kullanıldı. PCR ürünleri ethidium bromide ile boyanmış agaroz jelde yürütülerek, jel görüntüleme sisteminde görüntülendi. Sitogenetik analiz için kemik iliği materyalinden 24 saatlik hücre kültürü yapıldı. Preparatlara G-Bantlama yapılarak mikroskobik incelemeleri gerçekleştirildi.

Bulgular: Çalışmamıza 10 kadın, 15 erkek hasta dahil edildi. Hasta grubunun yaş ortalaması 53,24 olarak hesaplandı. PCR sonuçlarına göre; M-BCR/ABL 5 hastada negatif, 20 hastada pozitifti. Sitogenetik analizde her hasta preparatında 20 metafaz alanı incelendi. 5 hastada üreme gerçekleşmedi. 5 hastada 46,XY karyotip, 1 hastada 46,XX karyotip, 9 hastada 46,XY t (9;22) (q34, q11) karyotip, 5 hastada 46,XX (9;22) (q34;q11) karyotipi saptandı. Sitogenetik analizde üreme olmayan ve Ph kromozomu negatif olan hastalar moleküler analiz ile incelendi. 6 hastada M-BCR/ABL pozitif tespit edildi. Sonuçlarımıza göre M-BCR/ABL oranı 20/25 (% 80), Ph kromozom pozitifliği oranı % 80 olarak bulundu.

Sonuç: KML hastalığında teşhis ve tedavide yanıtın izlenmesinde sitogenetik analiz ile daha hızlı ve daha duyarlı RT-PCR tekniği birlikte uygulanarak sonuçlar irdelenmiştir.

Keywords

• BCR-ABL geni
• RT-PCR
• KML
• Ph kromozomu

Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique

Abstract

Objective: Objective: In our study, we aimed to detect Ph (Philadelphia) chromosome and BCR-ABL fusion gene in 25 patients with CML (Chronic Myeloid Leukemia) by Cytogenetic Analysis and RT-PCR (Reverse Transcriptase Polymerase Chain Reaction).

Materials and Methods: Bone marrow or blood samples were obtained from the patients included in our study. Total RNA was isolated from peripheral blood, bone marrow and cell lines using an RNA isolation kit. RNA purity was measured by spectrophotometer and RNAs of appropriate purity were selected for the study. cDNA synthesis was performed from mRNA using random hexamer primers and reverse transcriptase enzyme. Target DNA was amplified by the two-step Nested PCR method using oligonucleotide primers. The K562 cell line was used as a positive control and distilled water was used as a negative control. PCR products were run on agarose gel stained with ethidium bromide and displayed on the gel imaging system. Cell culture was made from bone marrow material over 24 hours for cytogenetic analysis. Microscopic examinations were performed by applying G-banding s to the preparations.

Results: Ten female and 15 male patients were included in our study. The average age of the patient group was calculated as 53,24. According to the PCR results; M-BCR/ABL was negative in 5 patients and positive in 20 patients. In cytogenetic analysis, 20 metaphase areas were examined in each patient preparation. No growth occurred in 5 patients. 46,XY karyotype in 5 patients, 46,XX karyotype in one patient, 46,XY t (9; 22) (q34, q11) karyotype in 9 patients, and 46.XX (9; 22) (q34; q11) karyotype in 5 patients was found. The patients with no reproduction in cytogenetic analysis and negative for the Ph chromosome were examined by molecular analysis. M-BCR / ABL was positive in 6 patients. According to our results, it was found the M-BCR / ABL ratio was 20/25 (80%), and the Ph chromosome positivity rate was 80%.

Conclusion: In CML disease, for monitoring diagnosis and treatment response, cytogenetic analysis and the faster and more sensitive RT-PCR technique were applied together and the results were examined.

Keywords

• Physician
• BCR-ABL gene
• RT-PCR
• CML
• Ph chromosome

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