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

Altıntaş Nuray; Aşçı Mustafa; Sarıca Yılmaz Özge; Miskioğlu Mine; Kızıldağ Sefa

doi:https://dx.doi.org/10.26650/JARHS2021-869345

Research Article

DOI :10.26650/JARHS2021-869345 IUP :10.26650/JARHS2021-869345 Full Text (PDF)

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

Nuray Altıntaş, Mustafa Aşçı, Özge Sarıca Yılmaz, Mine Miskioğlu, Sefa Kızıldağ

Objective: Objective: In our study, we aimed to detect Ph (Philadelphia) chromosome and BCRABL 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

DOI :10.26650/JARHS2021-869345 IUP :10.26650/JARHS2021-869345 Full Text (PDF)

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

Nuray Altıntaş, Mustafa Aşçı, Özge Sarıca Yılmaz, Mine Miskioğlu, Sefa Kızıldağ

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

References

1. Rowley JD. The Philadelphia chromosome translocation. A paradigm for understanding leukemia. Cancer 1990;15;65(10):2178-84. google scholar
2. Rowley JD. Chromosome abnormalities in leukemia and lymphoma. Ann Clin Lab Sci 1983;13(2):87-94. google scholar
3. Drexler HG, MacLeod RA, Uphoff CC. Leukemia cell lines: in vitro models for the study of Philadelphia chromosome-positive leukemia. Leuk Res 1999;23(3):207-15. google scholar
4. Abdullah MA, Amer A, Nawaz Z, Abdullah AS, Al-Sabbagh A, Kohla S, Nashwan AJ, Yassin MA. An uncommon case of chronic myeloid leukemia with variant cytogenetic. Acta Biomed 2018:3;89(3-S):28-32. google scholar
5. Kang ZJ, Liu YF, Xu LZ, Long ZJ, Huang D, Yang Y, Liu B, Feng JX , Pan YJ, Yan JS. Liu Q. The Philadelphia chromosome in leukemogenesis. Chin J Cancer 2016;27;35-48. google scholar
6. Hagemeijer A. Chromosome abnormalities in CML. Baillieres Clin Haematol 1987;1(4):963-81. google scholar
7. Barnes DJ, Melo JV. Cytogenetic and Molecular Genetic Aspects of Chronic Myeloid Leukaemia. Acta Haematol 2002;108:180-202. google scholar
8. Costello R, Bouabdallah R, Sainty D, Gastaut JA, Gabert J. Chronic myeloid leukemia, biological aspects. Rev Med Interne 1996;17(3):213-23. google scholar
9. Sercan HO, Eresen Ç, Yüksel E, Sercan Z, Paralı F, Altungöz O, Sakızlı M. Molecular detection of bcr/abl m-RNA and cytogenetic analysis in CML patients: Comparison of results and factors affecting the methodologies used. Turk J Cancer 1998;28(4):166-73. google scholar
10. Larson RA, Golomb HM, Rowley JD. Chromosome changes in hematologic malignancies. CA Cancer J Clin 1981;31(4):222-38. google scholar
11. Szanto A, Pap Z, Denes L, Lazar EB, Horvath A, Tunyogi AB, Baroti BA, Pavai Z. Real-time quantitative PCR detection of WT1 and M-BCR-ABL expressions in chronic myeloid leukemia. Rom J Morphol Embryol 2015;56(2):703-7. google scholar
12. Arslan S. KML ve ALL tanılı hastalarda bcr/abl füzyon geni mutasyonlarının taranması.T.C.Eskişehir Osmangazi Üniversitesi Sağlık Bilimleri Enstitüsü Tıbbi Genetik Anabilim Dalı Yüksek Lisans Tezi 2014. google scholar
13. Özbey Ü, Yüce H, Elyas H. Kronik Myeloid Lösemili Hastalarda Kromozomal Anomalilerin Belirlenmesi için Sitogenetik ve FISH Tekniği Uygulamaları. FÜ Sağ Bil Derg 2006;20(6):383-9. google scholar
14. Morgan GJ, Wiedemann LM. Molecular biology of the Philadelphia positive leukaemias. Recenti Prog Med 1989:80(10):508-19. PMID: 2690217. google scholar
15. Laurent E, Talpaz M, Kantarjian H, Kurzrock R. The BCR Gene and Philadelphia Chromosome-positive Leukemogenesis. Cancer Research 2001;61(6):2343-55. google scholar
16. Bilen Y. Philadelphia Pozitif Kronik Myelositer Lösemi Hastalarında İmatinib Mesilata Alınan Klinik, Hematolojik, Sitogenetik Ve Moleküler Yanıtın Değerlendirilmesi. T.C. Atatürk Üniversitesi Tıp Fakültesi İç Hastalıkları Anabilim Dalı Uzmanlık Tezi 2009. google scholar
17. Varmius H, Weinberg RA. Genes and Biology of Cancer, New York; Scientific American Library Series, No.42; 1993. p.147-65. google scholar

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APA

Altıntaş, N., Aşçı, M., Sarıca Yılmaz, Ö., Miskioğlu, M., & Kızıldağ, S. (2021). Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique. Journal of Advanced Research in Health Sciences, 4(2), 1-6. https://doi.org/10.26650/JARHS2021-869345

AMA

Altıntaş N, Aşçı M, Sarıca Yılmaz Ö, Miskioğlu M, Kızıldağ S. Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique. Journal of Advanced Research in Health Sciences. 2021;4(2):1-6. https://doi.org/10.26650/JARHS2021-869345

ABNT

Altıntaş, N.; Aşçı, M.; Sarıca Yılmaz, Ö.; Miskioğlu, M.; Kızıldağ, S. Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique. Journal of Advanced Research in Health Sciences, [Publisher Location], v. 4, n. 2, p. 1-6, 2021.

Chicago: Author-Date Style

Altıntaş, Nuray, and Mustafa Aşçı and Özge Sarıca Yılmaz and Mine Miskioğlu and Sefa Kızıldağ. 2021. “Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique.” Journal of Advanced Research in Health Sciences 4, no. 2: 1-6. https://doi.org/10.26650/JARHS2021-869345

Chicago: Humanities Style

Altıntaş, Nuray, and Mustafa Aşçı and Özge Sarıca Yılmaz and Mine Miskioğlu and Sefa Kızıldağ. “Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique.” Journal of Advanced Research in Health Sciences 4, no. 2 (Sep. 2024): 1-6. https://doi.org/10.26650/JARHS2021-869345

Harvard: Australian Style

Altıntaş, N & Aşçı, M & Sarıca Yılmaz, Ö & Miskioğlu, M & Kızıldağ, S 2021, 'Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique', Journal of Advanced Research in Health Sciences, vol. 4, no. 2, pp. 1-6, viewed 8 Sep. 2024, https://doi.org/10.26650/JARHS2021-869345

Harvard: Author-Date Style

Altıntaş, N. and Aşçı, M. and Sarıca Yılmaz, Ö. and Miskioğlu, M. and Kızıldağ, S. (2021) ‘Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique’, Journal of Advanced Research in Health Sciences, 4(2), pp. 1-6. https://doi.org/10.26650/JARHS2021-869345 (8 Sep. 2024).

MLA

Altıntaş, Nuray, and Mustafa Aşçı and Özge Sarıca Yılmaz and Mine Miskioğlu and Sefa Kızıldağ. “Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique.” Journal of Advanced Research in Health Sciences, vol. 4, no. 2, 2021, pp. 1-6. [Database Container], https://doi.org/10.26650/JARHS2021-869345

Vancouver

Altıntaş N, Aşçı M, Sarıca Yılmaz Ö, Miskioğlu M, Kızıldağ S. Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique. Journal of Advanced Research in Health Sciences [Internet]. 8 Sep. 2024 [cited 8 Sep. 2024];4(2):1-6. Available from: https://doi.org/10.26650/JARHS2021-869345 doi: 10.26650/JARHS2021-869345

ISNAD

Altıntaş, Nuray - Aşçı, Mustafa - Sarıca Yılmaz, Özge - Miskioğlu, Mine - Kızıldağ, Sefa. “Detection of Philadelphia Chromosome and BCR-ABL Fusion Gene in Patients with Chronic Myeloid Leukemia by Cytogenetic Analysis and RT-PCR Technique”. Journal of Advanced Research in Health Sciences 4/2 (Sep. 2024): 1-6. https://doi.org/10.26650/JARHS2021-869345

Volume 4, Issue 22021, P. 1-6

TIMELINE

Submitted	27.01.2021
Accepted	17.04.2021
Published Online	19.06.2021

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