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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 43  |  Issue : 2  |  Page : 45-48

Screening of single nucleotide polymorphism in CD95 (APO-1/FAS) promoter region (G-1377A) in children with acute leukemia


1 Department of Medical Biology, Faculty of Medicine, Niğde Ömer Halisdemir University, NNiğdee, Turkey
2 Vocational School of Health Service, Okan University, İstanbul, Turkey
3 LÖSANTE Children’s and Adult Hospital, Ankara, Turkey
4 TOBB-ETU Hospital, Ankara, Turkey

Date of Submission12-Dec-2017
Date of Acceptance25-Mar-2018
Date of Web Publication7-Aug-2018

Correspondence Address:
Dilara F Akin
Department of Medical Biology, Faculty of Medicine, Niğde Ömer Halisdemir University, Niğde
Turkey
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_55_17

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  Abstract 


Background CD95 is a cell surface receptor involved in apoptotic signal transmission. Deregulation of this pathway results in downregulation of apoptosis and subsequent persistence of a malignant clone. A single nucleotide polymorphism resulting in guanine-to-adenine (G>A) transition in the CD95 promoter region (position −1377) is thought to reduce stimulatory protein 1 transcription factor binding and decrease CD95 expression. The purpose of this study is to examine a genetic polymorphism in the core promoter region of CD95 and to evaluate association between its frequency and clinical findings.
Patients and methods G-1377A in the CD95 promoter region was genotyped by polymerase chain reaction and restriction endonuclease analysis finally were sequenced by Sanger Sequecing.
Results Among 146 patients, CD95 G-1377A (rs2234767) single nucleotide polymorphism carriers frequencies have been identified as 25% (n=37) GA and AA 4% (n=6). This polymorphism of the distribution of the CD95 gene in children with acute leukemia will be a guide for future studies.
Conclusion This polymorphism of the distribution of the CD95 gene in children with acute leukemia will be a guide for future studies.

Keywords: acute lymphoblastic leukemia, acute myeloblastic leukemia, CD95 polymorphism


How to cite this article:
Akin DF, Özkan DT, Kürekçi E, Akar N. Screening of single nucleotide polymorphism in CD95 (APO-1/FAS) promoter region (G-1377A) in children with acute leukemia. Egypt J Haematol 2018;43:45-8

How to cite this URL:
Akin DF, Özkan DT, Kürekçi E, Akar N. Screening of single nucleotide polymorphism in CD95 (APO-1/FAS) promoter region (G-1377A) in children with acute leukemia. Egypt J Haematol [serial online] 2018 [cited 2018 Aug 18];43:45-8. Available from: http://www.ehj.eg.net/text.asp?2018/43/2/45/238770




  Introduction Top


The CD95 (Apo-1/Fas) receptor-ligand system is one of the key regulators of apoptosis and is particularly important for the maintenance of lymphocyte homeostasis. The CD95 receptor (also termed Apo-1 or Fas) is a type I transmembrane protein that belongs to the tumor necrosis factor receptor superfamily [1]. In humans, germline and somatic mutations of the CD95 gene are associated with a high risk of both lymphoid and solid tumors. The CD95 receptor is expressed in most human cells and signals apoptosis upon interaction with the CD95 ligand [2]. Binding to the receptor by the CD95 ligand triggers receptor trimerization and subsequent assembly of the death-inducing signaling complex [3]. This mechanism of CD95-mediated apoptosis is believed to be involved in the removal of autoreactive lymphocytes during normal development of the immune system [4]. Dysregulation of this pathway is believed to result in downregulation of apoptosis, allowing subsequent persistence of a malignant clone [5]. Germline mutations or deletions within CD95, resulting in a loss or a reduction in receptor function, have been shown to cause autoimmune lymphoproliferative syndrome as well as an overall increased risk of hematological malignancies [6].

CD95 expression levels may also be affected by mutations or polymorphisms in the promoter region of CD95, when they affect the transcription binding sites. A single nucleotide polymorphism (SNP) resulting in guanine-to-adenine (G>A) transition in the CD95 promoter region occurs at position-1377, affecting a stimulatory protein 1 (SP1) transcription factor binding site [5]. An adenine residue at this position significantly reduces SP1 binding compared with guanine residue, causing a decrease in CD95 expression [5]. Many cell types express CD95 including cells of B-lineage [7], T-lineage, [8] and acute leukemias. Myeloblasts are known to express high levels of CD95, and functional deficiencies of CD95 signaling have been shown to be important in several subtypes of acute myeloid leukemia (AML), providing further evidence for CD95-mediated apoptosis in the etiology of AML [9]. CD95 expression has been associated with a better prognosis in B-cell lymphomas and with responsiveness to chemotherapy in AML, whereas most primary T-cell leukemias are constitutively resistant against CD95-induced apoptosis [10].

The purpose of the study was to assess patients with acute leukemia in childhood regarding G-1377A SNP and to investigate the relationship between its frequency and clinical findings.


  Patients and methods Top


A written informed consent for genetic analysis was obtained from the patients. The patient blood was collected at Losante Children Hospital, Turkey. A total of 146 children with leukemia were included in the study. We evaluated 146 patients with leukemia and control sample using PCR analysis and DNA sequencing study (Beckman Coulter, California, USA). When selecting control samples, the ones with leukemia were omitted. The healthy group were comprised healthy unrelated individuals from Turkey (n=102). An informed written consent was obtained from all the patients’ parents. The study is carried out in accordance with the code of ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans.

In the patient group, 123 (84%) children were diagnosed as having acute lymphoblastic leukemia (ALL), 13 (9%) as AML, seven (5%) as biphenotypic leukemia, and three (2%) acute promyelocytic leukemia. DNA was extracted from blood samples using conventional methods. The following primers were employed according to the previously described method—forward: 5′-TGTGTGCACAAGGCTGGCGC-3′ and reverse: 5′-TGCATCTGTCACTGCACTTACCACCA-3′ [11]. The restriction enzyme BstUI (Fermantas, Lithuania) was used to detect the CD95, G-1377A SNP, in which the loss of a BstUI restriction site occurs in the polymorphic G allele. The homozygous polymorphic GG genotype produces a single band representing 118-bp fragment. The homozygous AA wild-type genotype produces two bands of 98 and 20-bp fragments, and the heterozygous AG genotype produces three bands (118, 98, and 20 bp). BstUI-digested PCR products were separated on 4% agarose gels, stained with ethidium bromide, and visualized under ultraviolet light. Furthermore, final confirmation of G-1377A genotyping assay was obtained in 40 samples using DNA sequencing (CEQ 8000; Beckman Coulter, California, USA) and analyzed using the sequence analysis program.


  Results and discussion Top


The analysis of the promotor region of CD95 gene in 146 Turkish children with acute leukemia revealed the prevalence of CD95 G-1377A (rs2234767) polymorphisms. Of the 146 childhood leukemia patients, we identified that 37 (25%) were heterozygous and six (4%) were homozygotes. Five patients were diagnosed as having ALL and one patient was diagnosed as having biphenotypical acute leukemia among CD95 G-1377A homozygous patients. All previous investigations have shown that the mutation caused CD95 downregulation. A revision has been made on the participant in the conclusion section of the article. In the literature, there is no study that investigates CD95 polymorphism in children with acute leukemia.

Heterozygous patients were diagnosed as follows: 29 with ALL, four AML, three biphenotypic acute leukemia, and one patient diagnosed with acute promyelocytic leukemia. Overall, 31% (n=4) of the 13 patients with AML were found to be heterozygous, whereas the heterozygous percentage of patients with ALL was 24% (n=29). The prevalence of CD95 G-1377A polymorphisms was determined in 102 healthy individuals. Among 102 healthy individuals, CD95 G-1377A polymorphisms were found in 12 cases (11.7%; [Table 1] and [Table 2]). There were significant differences in the genotype frequency for CD95 G-1377A polymorphism between the groups ([Table 2], [Figure 1]).
Table 1 Genotype distributions in childhood leukemia patients and healthy groups

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Table 2 Comparison of CD95 G-1377A in between childhood leukemia and healthy groups

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Figure 1 (a) Sequencing analysis of CD95 gene in healthy individual; (b) Sequencing analysis of CD95 gene in childhood leukemia patients.

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Functional G-1377A SNP in CD95 gene, which plays an important role in the development of apoptosis and cancer, have been reported in many cancer studies. The G-1377A polymorphism in the promoter region of the CD95 gene which SNP affects a SP1 transcription factor binding site provides evidence the risk of developing AML.

Individuals homozygous for adenine at G-1377A are almost twice as likely to develop AML, with heterozygotes having an intermediate risk compared with individuals homozygous for guanine [12]. In this study, we expected to find a value in a statistical significant difference between CD95 SNP and clinical data in patients with acute leukemia. However, there was no evidence of an increased risk of AML associated with the G-1377A polymorphism. Mata et al reported that lower gene expression levels of the pro-apoptotic genes might affect poor response and prognosis in children with ALL studied in univariable and multivariable analyses and with a lower chance of an unfavorable event [13]. In childhood pre-B ALL, higher CD95 antigen expression was associated with longer survival in complete remission [13],[14]. Other studies, however, did not find any association between constitutive expression or CD95 function and prognosis in ALL [14],[15],[16],[17].

When SNPs detected in the promoter region of the gene probe are assessed in databases and supported by literature surveys, it is very clear that this is the guideline for disease progression and further studies.

Our work carries scientific importance in three ways. Firstly, the screened polymorphism in this present article seems to be the first result of CD95 promotor region in children with leukemia patients. Second, we found that G-1377A polymorphism may be associated with development of leukemia as genetic risk factor. Three, the present study is helpful to provide knowledge to other researchers about the included gene variations analyzed in this pathway in children with leukemia.

In conclusion, the results of the current study showed that CD95 gene polymorphisms might have an implication in pediatric leukemias; however, large-scale studies are needed. In addition, studies aiming to define the functionality of CD95 gene polymorphism will help clarify the importance of variants at this site.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Oehm A, Behrmann I, Falk W, Pawlita M, Maier G, Klas C et al. Purification and molecular cloning of the APO-l cell surface antigen, a member of the tumor necrosis factor nerve growth factor receptor family. J Biol Chem 1992; 267:10709.  Back to cited text no. 1
    
2.
Leithauser F, Dhein J, Mechtersheimer G, Koretz K, Bruderlein S, Henne C et al. Constitutive and induced expression of APO-1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab Invest 1993; 69:415–429.  Back to cited text no. 2
    
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Muschen M, Warskulat U, Beckmann MW. Defining CD95 as a tumor suppressor gene. J Mol Med 2000; 78:312–325.  Back to cited text no. 3
    
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Watanabe-Fukunaga R, Brannan CI, Copeland NG, Jenkins NA, Nagata S. Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis. Nature (Lond) 1992; 356:314–317.  Back to cited text no. 4
    
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Mehta PA, Gerbing RB, Alonzo TA, Elliott JS, Zamzow TA, Combs M et al. FAS promoter polymorphism: outcome of childhood acute myeloid leukemia. A children’s oncology group report. Clin Cancer Res 2008; 14:7896–7899.  Back to cited text no. 5
    
6.
Rieux-Laucat F, Le Deist F, Hivroz C, Roberts IA, Debatin KM, Fischer A, de Villartay JP. Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity. Science 1995; 268:1347–1349.  Back to cited text no. 6
    
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Owen-Schaub LB, Meterission S, Ford RJ. Fas/APO-1 expression and function on malignant cells of hematologic and non-hematologic origin. J Immunother Emphasis Tumor Immunol 1993; 14:234–241.  Back to cited text no. 7
    
8.
Debatin KM, Krammer PH. Resistance to APO-1 (CD95) induced apoptosis in T-ALL is determined by a BCL-2 independent antiapoptotic program. Leukemia 1995; 9:815.  Back to cited text no. 8
    
9.
Komada Y, Zhou YW, Zhang XL, Xue HL, Sakai H, Tanaka S et al. Fas receptor (CD95)-mediated apoptosis is induced in leukemic cells entering G1B compartment of the cell cycle. Blood 1995; 86:3848–3860.  Back to cited text no. 9
    
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Beltinger C, Kurz E, Böhler T, Schrappe M, Ludwig WD, Debatin KM. CD95 (APO-1/Fas) mutations in childhood T-lineage acute lymphoblastic leukemia. Blood 1998; 91:3943–3951.  Back to cited text no. 10
    
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Hu Z, Li C, Chen K, Wang LE, Sturgis EM, Spitz MR, Wei Q. Single nucleotide polymorphisms in selected apoptotic genes and BPDE-induced apoptotic capacity in apparently normal primary lymphocytes: a genotype-phenotype correlation analysis. J Cancer Epidemiol 2008; 2008:147905  Back to cited text no. 11
    
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Sibley K, Rollinson S, Allan JM, Smith AG, Law GR, Roddam PL et al. Functional FAS promoter polymorphisms are associated with increased risk of acute myeloid leukemia. Cancer Res 2003; 63:4327–4330.  Back to cited text no. 12
    
13.
Mata JF, Silveira VS, Mateo EC, Cortez MA, Queiroz RG, Yunes JA, Lee ML. Low mRNA expression of the apoptosis-related genes CASP3, CASP8, and FAS is associated with low induction treatment response in childhood acute lymphoblastic leukemia (ALL). Pediatr Blood Cancer 2010; 55:100–107.  Back to cited text no. 13
    
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Baryshnikov A, Polosukhina ER, Tupitsin NN, Gavrikova NV, Andreeva LYu, Zabotina TN et al. CD95 (FAS/APO-1) antigen is a new prognostic marker of blast cells of acute lymphoblastic leukaemia patients. Adv Exp Med Biol 1999; 457:251–258.  Back to cited text no. 14
    
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Aref S, Salama O, Al-Tonbary Y, Mansour A. Assessment of bcl-2 expression as modulator of fas mediated apoptosis in acute leukemia. Hematology 2004; 9:113–121.  Back to cited text no. 15
    
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Wuchter C, Karawajew L, Ruppert V, Buchner T, Schoch C, Haferlach T et al. Clinical significance of CD95, Bcl-2 and Bax expression and CD95 function in adult de novo acute myeloid leukemia in context of P-glycoprotein function, maturation stage, and cytogenetics. Leukemia 1999; 13:1943–1953.  Back to cited text no. 16
    
17.
Wuchter C, Karawajew L, Ruppert V, Schrappe M, Harbott J, Ratei R et al. Constitutive expression levels of CD95 and Bcl-2 as well as CD95 function and spontaneous apoptosis in vitro do not predict the response to induction chemotherapy and relapse rate in childhood acute lymphoblastic leukaemia. Br J Haematol 2000; 110:154–160.  Back to cited text no. 17
    


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