Kronik Miyeloid Lösemi (KML) hastalarında DNA Metil Transferaz 3A (DNMT3A) kodlayan gende R882H mutasyon varlığının araştırılması

Yıl 2020, Cilt: 34 Sayı: 3, 271 - 278, 19.01.2021

Nazlı Şirin Bengüsu Aydın Melek Pehlivan Hakkı Sercan

https://doi.org/10.5505/deutfd.2020.16362

Öz

Amaç: Kronik Miyeloid Lösemi (KML), hematopoetik kök hücre (HKH) orijinli klonal miyeloproliferatif bir hastalıktır. KML’deki Bcr-Abl kimerik geni ile birlikte diğer ek mutasyonlar ve epigenetik modifikasyonlar hastalığın blastik faza doğru ilerleyişi için gereklidir. DNA metiltransferazlar (DNMTler), epigenetik olarak düzenlenmiş genlerin ifade edilmesinde ve baskılanmasında önemli rol oynayan genom metilasyonu için anahtar proteinlerdir. Hematolojik malignitelerde DNMT3A ve diğer DNA metilasyon düzenleyicilerinde mutasyonlar tanımlanmıştır. R882H mutasyonu DNMT3a’da en sık gözlenen mutasyondur. Bu çalışmanın amacı DNMT3A R882H mutasyonunun KML hastalarındaki görülme oranını araştırmaktır.
Gereç ve Yöntem: Bcr/Abl kimerik gen analizi amacıyla gönderilmiş KML hastalarının kemik iliklerinden elde edilmiş olan cDNA örneklerinde, DNMT3A R882H mutasyonunu taramak için, AciI restriksiyon enzim kesimi kullanıldı. Kesim sonrası Bcr/Abl+ ve Bcr/Abl- örneklerden random seçilerek DNMT3A için DNA dizi analizi yapıldı. Elde edilen verilerin analizleri istatistiksel olarak değerlendirildi.
Bulgular: 35 Bcr/Abl+ ve 60 Bcr/Abl- örneğin AciI restriksiyon enzim kesimi sonrası bu mutasyonu taşımadıkları gözlendi. Bu örneklerden random seçilerek DNA dizi analizi ile değerlendirilen örneklerde de R882H mutasyonunun olmadığı doğrulandı.
Sonuç: Elde edilen sonuçlarımıza göre DNMT3A-R882H mutasyonunun KML hastalarında gözlenmediği, Bcr/Abl+ ve Bcr/Abl- bireyler arasında mutasyon görülme oranı açısından herhangi bir farklılık olmadığı gözlenmiştir. Hematopoetik hücrelerin proliferasyon potansiyelini artırmakta rol oynadığı bilinen DNMT3A R882H mutasyonunun KML progresyonu açısından etkili olmadığı ortaya konmuştur.

Anahtar Kelimeler

Kronik Myeloid Lösemi, Bcr/Abl, DNMT3A, R882H mutasyonu

Destekleyen Kurum

Dokuz Eylül Üniversitesi Bilimsel Araştırmalar Projeleri Koordinatörlüğü

Proje Numarası

2019.KB.SAG.045

Teşekkür

Bu çalışma Dokuz Eylül Üniversitesi Bilimsel Araştırmalar Projeleri Koordinatörlüğü tarafından (2019.KB.SAG.045 ) desteklenmiştir.

INVESTIGATION OF R882H MUTATION IN THE GENE CODING DNA METHYL TRANSFERASE 3A (DNMT3A) IN CHRONIC MYELOID LEUKEMIA (CML) PATIENTS

Öz

Objective: Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disease of hematopoietic stem cell (HKH) origin. Along with the Bcr-Abl chimeric gene in KML, other additional mutations and epigenetic modifications are necessary for the disease to progress to the blastic phase. DNA methyltransferases (DNMTs) are key proteins for genome methylation, which play an important role in the expression and suppression of epigenetically regulated genes. Mutations in DNMT3A and other DNA methylation regulators have been identified in hematological malignancies. R882H mutation is the most common mutation in DNMT3a. The aim of this study is to investigate the incidence of DNMT3A R882H mutation in CML patients.
Materials and Methods: AciI restriction enzyme cutting was used to screen the DNMT3A R882H mutation in cDNA samples obtained from bone marrow samples of CML patients sent for Bcr/Abl chimeric gene analysis. DNA sequence analysis was performed for DNMT3A after random selection from Bcr/Abl + and Bcr/Abl- samples after cutting. Analyzes of the data obtained were evaluated statistically.
Results: It was observed that 35 Bcr/Abl + and 60 Bcr/Abl- samples did not carry this mutation after AciI restriction enzyme cutting. It was confirmed that there was no R882H mutation in the samples evaluated randomly by DNA sequence analysis from these samples.
Conclusion: According to our results, it was founded that DNMT3A-R882H mutation was not observed in CML patients and there was no difference in the rate of mutation among Bcr/Abl + and Bcr/Abl- individuals. It has been demonstrated that the DNMT3A R882H mutation, which is known to play a role in increasing the proliferation potential of hematopoietic cells, is not effective for CML progression.

Anahtar Kelimeler

Chronic Myeloid Leukemia, Bcr/Abl, DNMT3A, R882H mutation

Proje Numarası

2019.KB.SAG.045

Kaynakça

• Di Bacco A, Keeshan K, McKenna SL, Cotter TG. Molecular Abnormalities in Chronic Myeloid Leukemia: Deregulation of Cell Growth and Apoptosis. Oncologist. 2000;5(5):405–15.
• Quintás-Cardama A, Kantarjian HM, Cortes JE. Mechanisms of primary and secondary resistance to imatinib in chronic myeloid leukemia. Cancer Control. H. Lee Moffitt Cancer Center and Research Institute. 2009;16: 122–31.
• Von Bubnoff N, Duyster J. Chronische Myeloische Leukämie - Therapie und Monitoring. Dtsch Arztebl Int. 2010; 107: 114–21.
• Feinberg AP, Vogelstein B. Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature. 1983; 301(5895):89–92.
• Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet. 1999;21:163–7.
• Okano M, Xie S, Li E. Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases. Nat Genet. 1998;19:219–20.
• Chen T, Ueda Y, Xie S, Li E. A novel Dnmt3a isoform produced from an alternative promoter localizes to euchromatin and its expression correlates with active de novo methylation. J Biol Chem. 2002;277(41):38746–54.
• Li KK, Luo L-F, Shen Y, Xu J, Chen Z, Chen S-J. DNA Methyltransferases in Hematologic Malignancies. Semin Hematol. 2013;50(1):48–60.
• Ayyanathan K, Lechner MS, Bell P, Maul GG, Schultz DC, Yamada Y, et al. Regulated recruitment of HP1 to a euchromatic gene induces mitotically heritable, epigenetic gene silencing: A mammalian cell culture model of gene variegation. Genes Dev. 2003;17(15):1855–69.
• Weinstein JN, Collisson EA, Mills GB, Shaw KRM, Ozenberger BA, Ellrott K, et al. The cancer genome atlas pan-cancer analysis project Nat Genet. 2013; 45: 1113–20.
• Yokochi T, Robertson KD. Preferential methylation of unmethylated DNA by mammalian de novo DNA methyltransferase Dnmt3a. J Biol Chem 2002;277(14):11735–45.
• Drabik A, Bodzoń-Kułakowska A, Silberring J. Gel Electrophoresis. In: Proteomic Profiling and Analytical Chemistry: The Crossroads: Second Edition. Elsevier Inc.; 2016. p. 115–43.
• Optimizing Restriction Endonuclease Reactions | NEB [Internet]. New England BioLabs. 2018. Erişim tarihi: 24.11.2020. Erişim adresi: https://international.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions
• Pehlivan M, Caliskan C, Yuce Z, Sercan HO. Forced expression of Wnt antagonists sFRP1 and WIF1 sensitizes chronic myeloid leukemia cells to tyrosine kinase inhibitors. Tumour Biol. 2017;39(5):1010428317701654.
• Emperle M, Dukatz M, Kunert S, Holzer K, Rajavelu A, Jurkowska RZ, et al. The DNMT3A R882H mutation does not cause dominant negative effects in purified mixed DNMT3A/R882H complexes. Sci Rep. 2018;8(1).
• Shlush LI, Zandi S, Mitchell A, Chen WC, Brandwein JM, Gupta V, et al. Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia. Nature. 2014;506(7488):328–33.
• Yang L, Rau R, Goodell MA. DNMT3A in haematological malignancies. Nat Rev Cancer [Internet]. 2015 Feb 24 [cited 2020 Nov 17];15(3):152–65.
• Mayle A, Yang L, Rodriguez B, Zhou T, Chang E, Curry C V, et al. Hematopoiesis and stem cells: Dnmt3a loss predisposes murine hematopoietic stem cells to malignant transformation. Blood. 2015;125(4):629–38.
• Celik H, Mallaney C, Kothari A, Ostrander EL, Eultgen E, Martens A, et al. Hematopoiesis and stem cells: Enforced differentiation of Dnmt3a-null bone marrow leads to failure with c-Kit mutations driving leukemic transformation. Blood. 2015;125(4):619–28.
• Anteneh H, Fang J, Song J. Structural basis for impairment of DNA methylation by the DNMT3A R882H mutation. Nat Commun. 2020;11:2294.
• Koya J, Kataoka K, Sato T, Bando M, Kato Y, Tsuruta-Kishino T, et al. DNMT3A R882 mutants interact with polycomb proteins to block haematopoietic stem and leukaemic cell differentiation. Nat Commun. 2016;7:10924.
• Holz-Schietinger C, Matje DM, Reich NO. Mutations in DNA methyltransferase (DNMT3A) observed in acute myeloid leukemia patients disrupt processive methylation. J Biol Chem. 2012;287(37):30941–51.
• Russler-Germain DA, Spencer DH, Young MA, Lamprecht TL, Miller CA, Fulton R, et al. The R882H DNMT3A Mutation Associated with AML Dominantly Inhibits Wild-Type DNMT3A by Blocking Its Ability to Form Active Tetramers. Cancer Cell. 2014;25(4):442–54.
• Meyer SE, Qin T, Muench DE, Masuda K, Venkatasubramanian M, Orr E, et al. DNMT3A haploinsufficiency transforms FLT3ITD myeloproliferative disease into a rapid, spontaneous, and fully penetrant acute myeloid leukemia. Cancer Discov. 2016;6(5):501–15.
• Yang L, Rodriguez B, Mayle A, Park HJ, Lin X, Luo M, et al. DNMT3A Loss Drives Enhancer Hypomethylation in FLT3-ITD-Associated Leukemias. Cancer Cell. 2016;29(6):922–34.
• Xu J, Wang YY, Dai YJ, Zhang W, Zhang WN, Xiong SM, et al. DNMT3A Arg882 mutation drives chronic myelomonocytic leukemia through disturbing gene expression/DNA methylation in hematopoietic cells. Proc Natl Acad Sci U S A. 2014;111(7):2620–5.
• Elsayed GM, Fahmi AEA, Shafik NF, Elshimy RAA, Abd Elhakeem HK, Attea SA. Study of DNA methyl transferase 3A mutation in acute myeloid leukemic patients. Egypt J Med Hum Genet. 2018;19(4):315–9.
• Kim TH, Tyndel MS, Zhang Z, Ahn J, Choi S, Szardenings M, et al. Exome sequencing reveals DNMT3A and ASXL1 variants associate with progression of chronic myeloid leukemia after tyrosine kinase inhibitor therapy. Leuk Res. 2017;59:142–8.
• Schmidt M, Rinke J, Schäfer V, Schnittger S, Kohlmann A, Obstfelder E, et al. Molecular-defined clonal evolution in patients with chronic myeloid leukemia independent of the BCR-ABL status. Leukemia. 2014;28(12):2292–9.
• Ciftciler R, Saglam EA, Inanc A, Ozcebe O, Haznedaroglu IC. A unique case of complex variant translocation of t(6;9;22)(p22;q34;q11.2), der(19) in a newly diagnosed patient with chronic myeloid leukemia. Cancer Genet. 2019;237:78–81.