Novel RUNX1 variation in B-cell acute lymphoblastic leukemia

Egzona Qipa1, Muradiye Acar2, Sureyya Bozkurt3, Murat Buykdogan4, Hazal B. Sonmez1, Muge Sayitoglu5, Yucel Erbilgin5, Zeynep Karakaş6 and Veysel S. Hançer4.

1 Istinye University, Institute of Health Sciences, Department of Medical Biology and Genetics, Istanbul, Turkey.
2 Istinye University, Faculty of Medicine, Department of Medical Genetics, Istanbul, Turkey.
3 Istinye University, Faculty of Medicine, Department of Medical Biology, Istanbul, Turkey.
4 Donegen, Genetic Diseases Diagnosis Center, İstanbul. Turkey.
5 Aziz Sancar Institute of Experimental Medicine, Department of Genetics, Istanbul University, Istanbul, Turkey.
6 Istanbul University, Istanbul Faculty of Medicine Pediatric Hematology Oncology Department, Istan- bul, Turkey.

Correspondence to: Egzona Qipa. Istinye University, Institute of Health Sciences, Department of Medical Biology and Genetics, Istanbul, Turkey. E-mail: egzonna.q@gmail.com
Published: July 1, 2023
Received: December 22, 2022
Accepted: May 31, 2023 
Mediterr J Hematol Infect Dis 2023, 15(1): e2023036 DOI 10.4084/MJHID.2023.036

This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(https://creativecommons.org/licenses/by-nc/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Acute lymphoblastic leukemia (ALL) is a malignant disease of hematopoietic stem cells. B cell ALL (B-ALL) is characterized by highly proliferative and poorly differentiated progenitor B cells in the bone marrow. Chromosomal rearrangements, aberrant cell signaling, and mutations lead to dysregulated cell cycle and clonal proliferation of abnormal B cell progenitors. In this study, we aimed to examine hot spot genetic variations in the RUNX1, IDH2, and IL2RA genes in a group of (n=52) pediatric B-ALL. Sanger sequencing results revealed a rare RUNX1 variant p.Leu148Gln in one B-ALL patient with disease recurrence. Additionally, common intronic variations rs12358961 and rs11256369 of IL2RA were determined in two patients. None of the patients had the IDH2 variant.
RUNX1, IDH2, and IL2RA variations were rare events in ALL. This study detected a novel pathogenic RUNX1 variation in a patient with a poor prognosis. Examining prognostically important genetic anomalies of childhood lymphoblastic leukemia patients and the signaling pathway components will pilot more accurate prognosis estimations.



Introduction

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer caused by the malign transformation of hematopoietic progenitor B- or T-cells.[1] ALL is commonly associated with acquired chromosomal translocations and other genetic or epigenetic abnormalities, which lead to aberrant expression of transcription factors.[2] Eighty percent of ALL cases are B cell ALL, and five years survival rates exceed 90% in pediatrics in high-income countries.
One of the most frequent chromosome translocations is ETV6::RUNX1 in B-ALL patients.[3] RUNX1 is a transcription factor that regulates the maturation of blood cells.[4] The recurring genetic alteration of RUNX1 characterizes a rare poor prognostic subgroup of B-ALL. Additionally, germline RUNX1 mutations were associated with a predisposition to familial leukemia.[5]
IDH2 is an isocitrate dehydrogenase that catalyzes the oxidative decarboxylation of alpha-ketoglutarate. IDH2 gene variations are rare events in pediatric ALL (approx. 0.5%) and AML (3%) (acute myeloid IDH1/2 mutations in AML. and T-ALL were associated with a high cumulative incidence of relapse and poor response to therapy. Importantly targeted inhibitors of IDH1/2 are available, and preliminary clinical trials results are promising for refractory or relapsed AML patients.[6,7] IL2RA controls many different cellular functions, including proliferation, differentiation, and cell survival/apoptosis but are also involved in several pathological processes. IL2RA (CD25) expression has been associated with Ph-positive B-ALL in pediatrics and adults.[8]
The aim of this study was to determine the variants of RUNX1, IDH2, and IL2RA genes associated with leukemia development and to evaluate their association with the prognosis of B-ALL.

Materials and Methods

Patient Group. B-ALL cases (n=52) diagnosed in the pediatric hematology clinic of Istanbul University Medical Faculty were included in the study. Patients were treated with B.F.M. (Berlin-Frankfurt-Munich protocol for pediatric ALL) protocol. The median age of the patients was five years (min: 0,9 years and max: 15 years), and the gender distribution of male-female was 30:22 (1.3:1) (Table 1).
Table 1
Table 1. Clinical features of B-ALL patients.
Clinical characteristics such as Bone marrow (B.M.) blast percentage, white blood cell count (WBC) at diagnosis, hemoglobin levels (Hb), platelet count (Plt), translocation, and organ involvement of the cohort were given in Table 1. BCR::ABL1 (n=3), MLL::AF4B (n=1), and TEL::AML1 (n=3) fusions were detected in B-ALL patients. This study is approved by the Local Ethics Committee (Istinye University Clinical Research Ethics Committee (2017-KAEK-120)/2/2019.G-009).
D.N.A. isolation and Sanger Sequencing. ACCORDING TO THE MANUFACTURER'S INSTRUCTIONS, Genomic D.N.A. was isolated from diagnostic bone marrow and peripheral blood samples using a Gentra Puregene Blood Kit, Qiagen. Exxon's of the RUNX1, IDH2, and IL2RA genes were amplified with specific primers (Table 2). The PCR reactions comprised 50 ng of template D.N.A., 10 pmol of each primer, and 2×PCR master mix (Hibrigen, Turkey).
Table 2
Table 2. Primer sequences.
In a final volume of 50µl, PCR was performed using a T100 Thermal Cycler (Bio-Rad, U.S.A.) using the following conditions: 94°C denaturation for 5 minutes, 94°C denaturation for 30 seconds, 60°C of an- nealing for 30 seconds, 94°C of elongation for 30 seconds, 37 cycle in total and 5 minutes of 72°C for the last extension.
Sequential alterations were determined using bidirectional sequencing. PCR products have been treated with ExoSAP-IT (GML A.G., Wallerau, Switzerland) enzyme. Bigdye Terminator v3.1 cycle sequencing kit and A.B.I. 3500xL genetic analyzer device have been used for sequencing. Amplicon sequences were evaluated using a CLC workbench 3.6.1 (Denmark)(NM_001001890.3, NM_000417.3, NM_001289910).
Open source programs such as Sorting Tolerant From Intolerant release 63 (SIFT, http://sift.jcvi.org), Polyphen (http://genetics.bwh.harvard.edu/pph2), Combined Annotation Dependent Depletion (CADD, http://cadd.gs.washington.edu), and Mutation Taster (http://www.mutationtaster.org) were used to predict the functional impact of the gene variants. Also, the Database of Single Nucleotide Polymorphism (dbSNP, https://www.ncbi.nlm.nih.gov/SNP), 1000 Genomes Project samples (http://www.1000genomes.org), The Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/ac/ index.php), and the Exome Aggregation Consortium (ExAC, http://exac.broadinstitute.org) were used for frequency data.
 

Results

The hotspot regions of RUNX1, IDH2, and IL2RA genes were examined in 52 diagnostic samples of B-ALL cases. A pathogenic RUNX1 variation c.443T>A, p.Leu148Gln was detected in one B-ALL (1.9%) patient (Figure 1A). This novel variant, p.Leu148Gln was located in the Runt homology domain of the RUNX1 gene, and Figure 1B shows the RUNX1 gene variations distribution in the St. Jude‘s Children’s Research Hospital Pediatric Cancer Data Portal (PeCan). The Mutation Taster prediction was Disease Causing with a score of 1. Varsome (The Human Genomics Community) prediction was likely pathogenic; SIFT prediction was pathogenic with a score of 0, and the MutPred prediction was Pathogenic with a 0.87 score, PROEVAN (Protein Variation Effect Analyzer) prediction was Pathogenic with -5.31 score. Pathogenicity meta scores based on the combined evidence from multiple other in-silico predictors is 6 (BayesDel addAF, BayesDel noAF, MetaLR, MetaRNN, REVEL).
Figure 1
Figure 1.Sanger sequencing results for RUNX1 mutant patient. A) Electropherogram image of a patient with RUNX1 c.443T>A, p.Leu148Gln variant. B) Hotspot RUNX1 gene variations in PeCan (St.Judes Children’s Research Hospital Data Portal).
The patient with RUNX1 variation was 9 years old boy with high WBC counts (132400/mm3). He had lymphadenopathy at diagnosis. He had a 33-day treatment response to therapy without any BCR/ABL1, MLL-F4B, or TEL-AML1 translocations. He relapsed in 16 months and died due to the recurrence 30 months after diagnosis.
Two common intronic variants of IL2RA c.367+12A>T, rs12358961, and c.367+7G>C, rs11256369, were detected in two patients (Figure 2). According to the prediction tools, these variants were benign, and patients had a standard risk for B-ALL treatment response.
Additionally, we screened the hotspot region of IDH2, and none of the patients showed IDH2 variation.
Figure 2
Figure 2. Sanger sequencing results of patients with IL2RA variants. A) IL2RA c.367+12A>T, rs12358961, B) IL2RA c.367+7G>C, rs11256369 variants.

Discussion

In this study, we aimed to analyze RUNX1, IDH2, and IL2RA hotspot gene variations described as rare and poor prognostic events in ALL. Out of 52, one B-ALL patient was found to carry a pathogenic RUNX1 variation (1.9%). IL2RA and IDH2 genes did not found mutated in the pediatric B-ALL cohort. RUNX1 p.Leu148Gln variation is located on the Runt domain of the RUNX1 gene that mediates binding to the core-binding factor (CBFbeta). Heterodimerization of RUNX1 and CBFbeta increases the D.N.A. binding affinity. Previous studies showed that mutations on the Runt domain stabilize the D.N.A. binding ability of the RUNX1 gene. RUNX1 mutated B-ALL patient had a poor prognosis and dead after early relapse. It is known that RUNX1 mutations have been associated with poor prognosis in myeloid malignancies.[9] RUNX1 gene mutations were found in 18.3% of patients with T-ALL, 5-15% in cytogenetically normal acute myeloid leukemia (CN-AML), and 3.8% in patients with B-ALL.[10] Garza-Veloz et al. reported the association between the high IL2RA, SORT1, FLT3, and DEFA1 gene expression levels and relapse risk and poor survival rates in B-ALL patients.[11] We also found two common variations, rs12358961 and rs11256369 in the IL2RA gene in two B-ALL cases. Although both variations are classified as benign, both were placed in the splicing machinery sites, and the functional impacts are unknown. IDH2 mutation is rather more often seen in AML (8%) than in ALL patients (<1%).[12] None of our patients was found to carry pathogenic mutations in IL2RA and IDH2 genes, which is in concordance with the previous data.
Our study confirmed that the pathologic variants of RUNX1 act as a poor prognostic factor in pediatric B-ALL, which is uncommon. A limited number of B-ALL patients were reported for the hotspot regions of three genes (RUNX1, IL2RA, and IDH2), which took part in the early developmental stages of lymphocytes, particularly B-cells. According to the previously published manuscripts, mutation ratios are not significantly different compared to whole gene screening studies. Larger independent patient cohorts are required to confirm the findings of this study.
Gene expression profiling and genome-wide sequencing analyses have made great advancements in understanding B-ALL genetics over the past few years. High-throughput analysis of big ALL cohorts has been very helpful in subclassifying B-ALL patients with different risks, identifying novel therapeutic targets, and improving overall clinical outcomes. Biomarkers with prognostic and predictive value  and targeted therapeutic agents have emerged as promising approaches in the clinical care of B- in the era of personalized medicine.

References   

  1. Bellavia D, Palermo R, Pia Felli M, Screpanti I, Checquolo S. Notch signaling as a therapeutic target for Acute Lymphoblastic Leukemia. Expert Opin Ther Targets Sci 2018; 12 :331-342 11:32 https://doi.org/10.1080/14728222.2018.1451840 PMid:29527929
  2. Kruse A, Abdel-Azim N , Kim HN, Ruan Y, Phan V, Ogana H, Wang W, Lee R, Gang E, Khazal S, Kim YM. Minimal Residual Disease Detection in Acute Lymphoblastic Leukemia. Int Jou Mol Sci 2020; 6: 1054 https://doi.org/10.3390/ijms21031054 PMid:32033444 PMCid:PMC7037356  
  3. Cooper S L, Brown P A. Treatment of Pediatric Acute Lymphoblastic Leukemia. Pediatr Clin North Am 2015:19: 61-73. https://doi.org/10.1016/j.pcl.2014.09.006 PMid:25435112 PMCid:PMC4366417  
  4. Sun C, Chang L, Zhu X. Pathogenesis of ETV6/RUNX1-positive childhood acute lymphoblastic leukemia and mechanisms underlying its relapse. Oncotarget 2017:18: 35445-35459. https://doi.org/10.18632/oncotarget.16367 PMid:28418909 PMCid:PMC5471068  
  5. Lam K, Zhang D E. RUNX1 and RUNX1-ETO: roles in hematopoiesis and leukemogenesis.Front Biosci. 2012:1: 1120-1139. https://doi.org/10.2741/3977 PMid:22201794 PMCid:PMC3433167  
  6. Triplett T, Curti B D, Bonafede P R, Miller W L, Walker E B, Weinberg A D. Defining a functionally distinct subset of human memory CD4+ T cells that are CD25POS and FOXP3. Eur. J. Immunol 2012:5: 1893-1905. https://doi.org/10.1002/eji.201242444 PMid:22585674  
  7. Simonin M, Schmidt A, Bontoux C, Dourthe M E, Lengline E, Andrieu G P, Lhermitte L, Graux C, Grardel N, Cayuela J M, Huguet F, Arnoux I, Ducassou S, Macintyre E, Gandemer V, Dombret H, Petit A, Ifrah N, Baruchel A, Boissel N, Asnafi V. Oncogenetic landscape and clinical impact of IDH1 and IDH2 mutations in T-ALL. J Hematol Oncology 2021:5: 1-7. https://doi.org/10.1186/s13045-021-01068-4 PMid:33941203 PMCid:PMC8091755  
  8. Leonard W J, Donlon T A, Lebo R V, Greene W C. Localization of the Gene Encoding the Human interleukin-2 Receptor on Chromosome 10. Science.1985.28: 1547-1549. https://doi.org/10.1126/science.3925551 PMid:3925551  
  9. Smith BM, Arthur D , Camitta B, Carroll AJ, Crist W, Gaynon P, Gelber R, Heerema N, Korn EL, Link M, Murphy S, Pui CH, Pullen J, Reaman G, Sallan SE, Sather H, Shuster J, Simon R, Trigg M, Tubergen D, Uckun F, Ungerleider R. Uniform Approach to Risk Classification and Treatment Assignment for Children With Acute Lymphoblastic Leukemia. J Clin Oncol. 1996.14: 18-24 https://doi.org/10.1200/JCO.1996.14.1.18 PMid:8558195
  10. Grossmann V, Kern W, Harbich S, Alpermann T, Jeromin S, Schnittger S, Haferlach C, Haferlach T, Kohlmann A. Prognostic relevance of RUNX1 mutations in T-cell acute lymphoblastic leukemia. Haematologica. 2011:12:1874-1877. https://doi.org/10.3324/haematol.2011.043919 PMid:21828118 PMCid:PMC3232273
  11. Veloz IG, Martinez-Fierro ML, Jaime-Perez JC,MCarrillo-Sanchez K, Ramos-Del Hoyo MG, Lugo-Trampe A, Rojas-Martinez A, Gutierrez-Aguirre CH, Gonzalez-LlanoO, Salazar-Riojas R, Hidal- go-Miranda A, Gomez- Almaguer A, Ortiz-Lopez R. Identification of Differentially Expressed Genes Associated with Prognosis of B Acute Lymphoblastic Leukemia.Disease Markers.2015:24:1-11. https://doi.org/10.1155/2015/828145 PMid:25802479 PMCid:PMC4354728
  12. Andersson AK, Miller DW, Lynch JA, Lemoff AS, Cai Z, Pounds SB, Radtke I, Yan B, Schuetz JD, Rubnitz JE, Ribeiro RC, Raimondi SC, Zhang J, Mullighan CG, Shurtleff SA, Schulman BA, Downing JR. IDH1 and IDH2 mutations in pediatric acute leukemia. Leukemia.2011:7:1-15. https://doi.org/10.1038/leu.2011.133 PMid:21647154 PMCid:PMC3883450