Polymorphisms in transporter and phase II metabolism genes as potential modifiers of the predisposition to and treatment outcome of de novo acute myeloid leukemia in Israeli ethnic groups
Introduction
Despite aggressive treatment, adult acute myeloid leukemia (AML) has a poor prognosis, with most patients relapsing and succumbing to the disease [1]. The recent advent of genomic technologies has led to a large body of data regarding genes potentially involved in the prognosis of various malignancies, including AML. Among the most common genes which have been studied are drug metabolism and disposition genes [2]. The role of these genes in chemotherapy resistance in adult AML has not been fully elucidated.
While drug metabolism genes can affect prognosis by causing chemotherapy resistance, these types of genes may also influence the susceptibility to malignancies due to their role in processing carcinogens. Most procarcinogens are metabolically activated by phase I enzymes, such as those of the cytochrome P450 family, which modify functional groups on molecules. Then they are detoxified by phase II enzymes such as GST, NAT1/2, and others, which conjugate xenobiotics to endogenous substances to facilitate their excretion [2]. Alternatively, toxic exogenous substances may be removed from the cell by any of a number of transporters, such as those of the ATP-binding-cassette family (ABC family) of transporters [3]. Thus, these different metabolism and transporter genes may play a critical role not just in prognosis of but in predisposition to, various malignancies including AML.
The most common inherited sequence variations in the human genome are SNPs (single nucleotide polymorphisms), which have been found throughout the genome. The HapMap project has identified over 3.8 million SNPs in three major populations [4] some of which have been found to alter the activity of drug metabolism and disposition genes. Many studies have analyzed the relationship between various polymorphic markers and prognosis of AML (reviewed in Ref. [2]). Some of these studies have had conflicting results, and therefore, their confirmation is important in different populations.
Our previous study of the NQO1 polymorphism showed significant differences in genotypes between Arabs and Jewish individuals, however, this polymorphism did not predispose to AML in either of these ethnic groups [5]. The present study extends this work by examining additional SNPs and other genetic variations in drug metabolism and disposition genes. We again compared patients of the two major ethnic groups in Israel to each other and to normal controls. This would enable us to determine if drug metabolism and disposition SNPs or deletions influence either predisposition or prognosis of AML in these ethnic groups. We specifically wished to determine if there are genetic differences underlying the difference in prognosis which has been reported between different Israeli ethnic groups. Alcalai et al. [6] reported that Arab patients have a worse prognosis than Jewish patients of European origin (Ashkenazim) who received identical treatment, despite being younger in age than the Jewish patients [6].
In this study, we analyzed 7 different genes, 3 of whom are transporters and 4 of which are drug metabolism genes. Some of them have been previously studied in leukemia and others have not been previously studied in this disease. All of the polymorphisms investigated have either known or suspected phenotypic impact. The first gene we have studied is ABCC3, which is a member of the family of transporters originally known as multidrug resistance-associated protein (MRP). ABCC3 transports organic compounds conjugated to sulfate, glutathione or glucuronate [7], [8]. The expression of ABCC3 (MRP3) is associated with lower remission rate and decreased survival in adult AML [9] and poor prognosis in childhood [10] as well as adult ALL [11]. Lang et al. [12] described the C-211T polymorphism of the ABCC3 gene, located in the promoter region which correlates significantly with decreased mRNA expression in the human liver. Such a correlation of C-211T genotype with ABCC3 expression has not been found in a study of pediatric acute leukemia patients, which included a relatively small number (43) of AML patients [13]. Although no correlation was found between the C-211T genotype and treatment outcome in this group of childhood AML, this polymorphism has not been investigated in adult AML for possible predisposing or prognostic effects.
The second gene we have studied is ABCG2 (breast cancer resistant protein, BCRP), a transporter which has a wide range of substrates, including anticancer drugs and carcinogenic xenobiotics [14]. ABCG2 is known to actively transport carcinogenic heterocyclic amines, including the carcinogenic Aflatoxin B1 [15], and other food carcinogens [16] as well as metabolites of tobacco carcinogens [17]. Increased ABCG2 expression is associated with a poor prognosis in adult AML [9], [18], [19], [20], [21], with either lower rates of remission or shorter remission duration, and shorter survival. A polymorphism in this gene (ABCG2 C421A) was found to be common in Japanese normals [22]. This polymorphism was associated with low-level gene expression [22] and decreased transport activity [23]. Lockhart et al. demonstrated that transfected cells expressing this variant produced less ABCG2 protein [24] than those expressing the normal allele. This SNP has been studied in cell lines but not yet in clinical AML samples.
The third transporter we have studied is the concentrative nucleoside transporter, CNT1 (SLC28A1). This transporter mediates the uptake of pyrimidine nucleosides and analogs such as cytarabine or gemcitabine [25]. In gynecologic tumors, histological staining showed frequent protein reduction, with loss of CNT1 protein being associated with poor prognosis histological subtypes [26]. The polymorphism CNT1 G565A (Val189Ile) results in a reduced affinity for gemcitabine and possibly other substrates [27]. It has not been previously studied as a prognostic factor in AML, which could be significant since cytosine arabinoside is the major drug used for AML therapy.
The next two genes studied are two of the Glutathione S-transferases (GSTs) family of enzymes, which are known to be responsible for the metabolism of a broad range of xenobiotics and carcinogens. We studied two polymorphic genes, GSTT1 and GSTM1 which have null alleles due to gene deletion [28], such that 50% of Caucasians lack GSTM1, and about 20% lack GSTT1 due to homozygous deletion. GST null alleles have been found to be associated with poor survival in adult AML [29], and may confer increased risk of adult AML [30], [31], [32], though a meta analysis found that null alleles more frequently predispose to ALL [33].
The last two genes studied here are two additional drug metabolism genes, N-acetyltransferases 1 and 2 (NAT1/2). NAT1 and NAT2 are two isoenzymes with genetically determined activity [34]. Both enzymes are involved in either activation or inactivation of numerous chemicals and carcinogens. Enzyme activity (“fast” or “slow”) was found to correlate with several polymorphisms (haplotypes), and therefore, determination of gene activity by analysis of these SNPs is technically complex. Acetylation status of both NAT1 and NAT2 (“slow” or “fast”) was previously suggested to alter the risk of different malignancies [34]. Furthermore, NAT1 and NAT2 may have opposing effects, and therefore, it is important to study both simultaneously. Slow NAT2 acetylators were found to have an increased risk of ALL [35].
We conducted this pilot study to investigate the allelic frequencies of these various genetic variants in these 7 transporters or drug metabolism genes in Israeli Jewish and Arab populations and their impact on both the risk of developing AML and the outcome of AML therapy.
Section snippets
Patients and therapy
DNA samples were prepared from blood or bone marrow of de novo AML patients who were treated at Hadassah University Hospital during the period between 1996 and 2005. We analyzed all samples with sufficient DNA for genetic studies, without selection. Mononuclear blood cells were isolated from patient samples and from control peripheral blood, using standard Ficoll separation. Some of the control DNA samples were anonymized samples obtained from healthy Jewish and Arab hospital controls, referred
Results
Patient data is presented in Table 1 and frequencies and genotyping data are presented in Table 3, Table 4, Table 5, Table 6.
All SNP-genotypes were found to be in accordance with the Hardy–Weinberg equilibrium (within cases and controls for both ethnic groups all p > 0.05). Hardy–Weinberg equilibrium for GST genotypes cannot be calculated from our results according to the Hardy–Weinberg rule, since the assay detects only homozygotes for the null alleles and heterozygotes cannot be identified.
Discussion
This study presents data on genotypes of AML patients and controls of both Jewish and Arab ethnicity. The individual SNPs investigated are ABCC3 C-211T, ABCG2 C421A, CNT1 G565A. NAT1 and NAT2 haplotypes and the GSTT1 and GSTM1 null alleles were also analyzed. These genetic markers were primarily selected because of the evidence that either the expression of these genes themselves (ABCC3 or ABCG2) or the variant alleles have been implicated in the etiology and/or prognosis of AML. New
Acknowledgments
Supported by grant number 590/03 from the Israel Science Foundation (to DR and SBC), grant number CA 113/1851 from the Cooperative Program in Cancer Research of the Ministry of Science and Technology, Israel and the Deutsches Krebsforschungszentrum (DKFZ) (to PM, AR, DR and SBC), the Israel Ministry of Health (DR) and the Caesaria Edmond Benjamin de Rothchild Foundation (DR and SBC). We also thank Olfert Landt (TIB MOLBIOL, Berlin, Germany) for helping with the LightCycler probe design as well
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These authors contributed equally to this work.