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Original article
NT5C3 polymorphisms and outcome of first induction chemotherapy in acute myeloid leukemia Hyun Sub Cheonga,d, Youngil Kohb, Kwang-Sung Ahne, Chansu Leea, Hyoung Doo Shind,f and Sung-Soo Yoona,b,c Aims The cytosolic 5′-nucleotidase-III (NT5C3) is involved in the metabolism of the nucleoside analog, cytosine arabinose (AraC), and the expression level of NT5C3 is correlated with sensitivity to AraC in acute myeloid leukemia (AML) patients. The current study examined whether the NT5C3 polymorphisms could affect chemotherapy outcomes in 103 Korean AML patients. Methods Forty-seven single nucleotide polymorphisms in NT5C3 were genotyped using the Illumina GoldenGate genotyping assay. The genetic effects of the polymorphisms on the outcome of chemotherapy were analyzed using χ2 and logistic regression models. Results Although none of the NT5C3 polymorphisms was associated with a complete remission rate, a common single nucleotide polymorphism, rs3750117, showed a significant association with induction rate after the first course of chemotherapy (Pcorr = 0.004 and odds ratio = 11.28) in AML patients. In addition, NT5C3 expression levels were significantly increased in patients with risk allele homozygote.
Introduction Acute myeloid leukemia (AML) is a rare cancer in Korea and worldwide. Among adults with AML, less than 20% survive 5 years after the initial diagnosis. Despite recent scientific advances in understanding the molecular biology of AML and mechanisms of drug metabolism, new successful therapeutic methods still need to be developed. The identification of patients who would not benefit from aggressive chemotherapy regimens requires more knowledge from pharmacogenetic studies. This strategy is especially important for elderly patients, who comprise the major age category for AML and whose capability to withstand intensive treatment is often limited. The 5′-nucleotidases are a family of enzymes that catalyze the dephosphorylation of various nucleoside 5′monophosphates [1]. One family member, the cytoplasmic 5′-nucleotidase-III (NT5C3), mainly catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates, including nucleoside analog, cytosine arabinose (AraC), that are used to treat AML. This reaction results in the deactivation of active phosphorylated Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.pharmacogeneticsandgenomics.com). 1744-6872 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Conclusions The data suggest that genotyping the NT5C3 polymorphism may have the potential to identify patients more likely to respond to AraC-based chemotherapy. Pharmacogenetics and Genomics 24:436–441 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Pharmacogenetics and Genomics 2014, 24:436–441 Keywords: acute myeloid leukemia, chemotherapy, cytosine arabinose, NT5C3, single nucleotide polymorphism a
Cancer Research Institute, bDepartment of Internal Medicine, Seoul National University College of Medicine, cClinical Research Institute, Seoul National University Hospital, dDepartment of Genetic Epidemiology, SNP Genetics Inc., e Functional Genome Institute, PDXen Biosystem Inc. and fDepartment of Life Science, Sogang University, Seoul, Korea Correspondence to Sung-Soo Yoon, MD, Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehang-ro, Jongno-gu, Seoul 110-744, Korea Tel: + 82 2 2072 3079; fax: + 82 2 762 9662; e-mail: [email protected] Received 20 February 2014 Accepted 26 May 2014
metabolites [2,3]. Large variations have been observed in response to AraC, and one possible explanation is that genetic variation in genes involved in the activation and inactivation of these drugs might contribute toward differences in drug response. A series of functional analyses were carried out for NT5C3. Gene expression level of NT5C3 was significantly associated with cytotoxicity for AraC in human lymphoblastoid cell lines and knockdown of NT5C3 altered cancer cell sensitivity to this drug. Furthermore, higher NT5C3 expression correlated with lower intracellular AraC phosphorylated metabolite concentration [4]. Genetic variations in NT5C3 have shown to be associated with protein function and a potential effect on response to AraC. In a previous study, among 61 NT5C3 polymorphisms, Asp283His was correlated with decreased level of enzyme activity and I4(-114) and I6(9) showed altered DNA–protein-binding patterns [5]. An intronic single nucleotide polymorphism (SNP), rs4316067, was also associated with the mRNA expression level of NT5C3 in leukoblast from AML patients [6]. However, to our best knowledge, the genetic effect of NT5C3 polymorphisms on the outcome of chemotherapy has never been investigated in AML patients. To define the nature and extent of common genetic polymorphisms in NT5C3 and the possible functional DOI: 10.1097/FPC.0000000000000072
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NT5C3 polymorphisms and AML Cheong et al. 437
effect on the gene, we carried out a genetic association study using 103 Korean AML patients. The results from this study provide information on common genetic polymorphisms in NT5C3 as well as the functional consequences of the sequence variations.
Methods Patient characteristics and treatment protocol
From those with marrow samples available for DNA isolation, a total of 103 newly diagnosed AML patients (60 men and 43 women) from Seoul National University Hospital, Seoul, Korea, between December 2001 and September 2009 were enrolled in the study. The patient characteristics were as follows: the median age was 50.4 years (range 16–76 years) and the male to female ratio was 58 : 42 (n = 60 : 43). The FAB classification showed the following distribution of subtypes: M0, two patients (2%); M1, 14 (14%); M2, 47 (46%); M4, 33 (32%); M5, five (5%); and M7, two (2%). Patients diagnosed with acute promyelocytic leukemia (M3 FAB subtype) were excluded from the study. The criteria used to describe a cytogenetic clone and karyotype followed the recommendations of the International System for Human Cytogenetic Nomenclature [7]. Cytogenetic data were available for 95 patients. The cytogenetic risk groups were classified according to the MRC10 criteria, as described previously [8] [favorable risk: AML associated with t(8;21), t(15;17), or inv(16); unfavorable risk: the presence of a complex karyotype, − 5, del(5q), − 7, or abnormalities of 3q; intermediate risk: the remaining group of patients, including those with 11q23 abnormalities, + 8, + 21, + 22, del(9q), del(7q), or other miscellaneous structural or numerical defects not included in the favorable or unfavorable risk groups]. The favorable, intermediate, and unfavorable cytogenetic groups were 23 (22%), 69 (67%), and three (3%), respectively. The frequencies of FLT3-ITD and NPM1 mutations were 19.7 and 19.0%, respectively. Information on first induction chemotherapy outcome was available in 90 patients. All patients were treated with standard induction chemotherapy (idarubicin 12 mg/m2/day intravenously for 3 consecutive days and AraC 200 mg/m2/day intravenously for 7 consecutive days). In the case of patients older than 55 years of age, the dose of idarubicin was modified to two-thirds of the total dose. Consolidation therapies were performed on the basis of two more cycles of high-dose AraC (3 g/m2/day intravenously twice a day on D1, 3, 5). The institutional review board of Seoul National University Hospital approved the study protocol, and all patients provided informed consent. Single nucleotide polymorphism genotyping
Among 487 dbSNPs detected in NT5C3, 92 were reported in two Asian groups (Han Chinese and Japanese) in the HapMap database (release #27). Only 52 SNPs were selected on the basis of their minor allele frequencies (>5%) in the HapMap database. When we selected
SNPs in the HapMap database, linkage disequilibriums (LDs) among Asian groups were not considered because, although there is a high concordance between Korean and the two Asian samples, the tagging SNP selection strategy could miss an untagged SNP that may be potentially important in Koreans. In addition, we also genotyped one nonsynonymous SNP (Asp283His) and two intronic SNPs [I4(-144) and I6(9)], that were previously known to affect protein function and potentially influence drug response [5]. Genotyping was performed at a multiplex level using the Illumina GoldenGate genotyping system (Illumina, San Diego, California, USA) [9]. The genotype quality score for retaining data was set to 0.25. A total of 47 SNPs were successfully genotyped. Real-time quantitative PCR
Total RNA was extracted from bone marrow plasma of AML patients (n = 31) using the Qiagen RNeasy kit (Qiagen, Hilden, Germany) and measured its concentration using an ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, USA). RNA (1 μg) per sample was reverse transcribed with SuperScript reverse transcriptase (Invitrogen, Carlsbad, California, USA) using random hexamers. The cDNA were synthesized by incubation at 45°C for 1 h. Then, the reaction was inactivated by heating at 95°C for 5 min. The PCR process was performed in 20 μl volume for 95° C for 5 min, 30 cycles at 95°C for 1 min, 48°C for 30 s, and 72°C for 1 min, and finally 10 min at 72°C. To perform real-time qPCR utilizing an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, California, USA), TaqMan Gene Expression Assays (Applied Biosystems) were used as a probe/primer set specified for NT5C3 (Assay ID: Hs00826433_m1) and a probe/primer set for the ACTB gene, a housekeeping gene, encoding β-actin (Assay ID: 4326315E). PCR was performed in a final volume of 20 μl containing TaqMan Gene Expression Master Mix (Applied Biosystems), 1 μl TaqMan Gene Expression Assay, 1 μl cDNA as the template, and up to 20 μl distilled H2O. The PCR program was as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. The expression level of NT5C3 was normalized to that of the ACTB gene for each sample. Experiments were conducted in duplicate (separate experiments) for each sample, and averaged values were calculated for normalized expression levels [10]. Statistics
To determine whether the individual variant was in equilibrium at each locus in the population (Hardy–Weinberg equilibrium), χ2-tests were performed. We examined a widely used measure of LD between all pairs of biallelic loci, D' [the correlation coefficient (Delta | D'|)], logarithm of odds, and r2 using Haploview (Broad Institute of Harvard and MIT Cambridge, MA, USA) [11].
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438 Pharmacogenetics and Genomics 2014, Vol 24 No 9
To determine the genetic association of NT5C3 polymorphisms, Fisher’s exact test and logistic regression analysis were carried out to calculate odds ratio, 95% confidence intervals, and corresponding P-values. Student’s t-test was used to compare NT5C3 expression levels between the rs3750117 genotype subgroups. Pairwise comparisons of the nine SNPs [only one SNP if there were absolute LDs (r2 = 1)], haplotypes were inferred using PHASE algorithm ver. 2.0 (Matthew Stephens Lab, Chicago, Illinois, USA) [12], and subsequent statistical analysis was carried out using SAS version 9.1 (SAS Inc., Cary, North Carolina, USA). P-values were corrected by Bonferroni correction for multiple testing [the threshold of raw P-value significance was 0.001 (47 polymorphisms analyzed)]. A type I error of 5% was used to obtain the Bonferroni-corrected P-value.
Results The current study examined the association of NT5C3 polymorphisms with chemotherapy outcomes in 103 Korean AML patients. Forty-seven SNPs in NT5C3 were genotyped using the Illumina GoldenGate genotyping assay. Genotype distributions were in Hardy–Weinberg equilibrium (P > 0.05) (Supplementary Table 1, Supplemental digital content 1, http://links.lww.com/FPC/ A741). Pairwise comparisons among SNPs showed five sets of absolute LDs (|D'| = 1 and r2 = 1), and SNPs in NT5C3 were in one LD block in Koreans (Fig. 1a and c). Nine SNPs (rs10266244, rs6976843, rs7800610, rs4720100, rs6946062, rs4316067, rs17170218, rs17170180, and rs3750117) were selected [only one SNP if there were absolute LDs (r2 = 1)] for haplotype construction and statistical analysis. Among eight haplotypes constructed, five common haplotypes (frequency > 10%) were used for further analysis (Fig. 1b). Allele frequencies of each of nine SNPs and five haplotypes were compared between (a) remission failure and complete remission groups and (b) induction failure and induction success groups after the first course of chemotherapy. None of the SNPs and haplotypes showed a significant association with complete remission rate by intensive chemotherapy (Supplementary Table 2, Supplemental digital content 2, http://links.lww.com/FPC/ A742). SNPs were also not associated with the risk of relapse, relapse-free survival, and overall survival (Supplementary Table 3, Supplemental digital content 3, http://links.lww.com/FPC/A743 and Supplementary Fig. 1, Supplemental digital content 4, http://links.lww.com/FPC/ A744). However, a common synonymous SNP, rs3750117 (Y131Y), showed a significant association with induction rate after the first course of chemotherapy (Pcorr = 0.02 and odds ratio = 4.10). The frequency of the G allele of rs3750117 was higher in induction failure group than in the induction success group (0.735 vs. 0.404) (Table 1). The susceptible effect of the rs3750117 G allele was clearer in the subsequent genotype test. The P-values of
rs3750117 retained significance after multiple comparisons in codominant and recessive models (Pcorr = 0.03 and 0.004, respectively). The frequency of the G/G genotype was higher in the induction failure group than in the induction success group (0.647 vs. 0.123). In contrast, the frequency of A allele-bearing genotypes, A/A + A/G, was high in the induction success group (0.353 vs. 0.877). Odds ratios were 4.56 and 11.28 in codominant and recessive models, respectively (Table 2). To estimate the impact of the rs3750117 polymorphism on gene expression level, the NT5C3 mRNA expression level was compared between the genotype subgroups of the AML samples in the real-time qPCR. The expression levels (mean ± SEM) were 0.024 ± 0.013 and 0.039 ± 0.024 for the A/A + A/G and G/G genotypes, respectively (Fig. 2). The gene expression level was 1.61-fold higher in the risk allele homozygote, G/G, than in the combined A/G and A/A genotypes. The difference was significant between the G/G genotype and the combined A/A and A/G genotypes (P = 0.019).
Discussion The 5′-nucleotidases are a family of enzymes that catalyzes the dephosphorylation of nucleoside monophosphates, including nucleoside analogs that are used to treat a variety of diseases [3,13]. NT5C3 is a cytosolic enzyme that catalyzes the dephosphorylation of pyrimidine analogs [13], thus playing an important role in both endogenous nucleoside and nucleotide pool balance as well as response to pyrimidine analogs such as gemcitabine and AraC. In previous studies, genetic variations in NT5C3 were determined and its effects on NT5C3 function were reported [5,6]. However, the genetic effect of NT5C3 polymorphisms on the outcome of chemotherapy has never been investigated in AML patients. This study is the first to report genotypes of NT5C3 and outcome of chemotherapy for AML. An exonic SNP, rs3750117, was associated with short-term treatment outcome, but was not associated with long-term outcomes such as complete remission, risk of relapse, relapse-free survival, and overall survival. Findings from the current study describe inferior outcome for patients with the minor allele homozygote, G/G of rs3750117, caused largely by an increase in the failure of the first course of chemotherapy. Treatment of AML requires intensive myelosuppressive induction chemotherapy to achieve remission and continuing postremission treatment to maintain a durable long-term remission. The G allele of rs3750117 may reduce or delay metabolism of the chemotherapy drug, AraC, used for AML and may lead to a reduced induction rate. It is possible that the NT5C3 genotype influences excretion of drug metabolites that contribute toward inactivation to a greater degree than it influences metabolites that have an antileukemic effect.
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NT5C3 polymorphisms and AML Cheong et al. 439
Fig. 1
(a) r2=1
r2=1
E1
rs12155477 T>C (0.382)
rs6942974 C>G (0.208) rs2893457 T>C (0.383)
rs2392209 G>A (0.387)
rs11532669 C>T (0.150)
rs3750118 T>C (0.466) rs3750117 A>G (0.466)∗
rs3750119 T>C (0.471)
rs17170180 T>C (0.191)∗
rs6955792 G>A (0.383)
rs4236335 A>C (0.466)
E2
E3 E4 E5 E6 E7 E8 E9
Hap.
rs6976843
rs7800610
rs4720100
rs6946062
rs4316067
rs17170218
rs17170180
rs3750117
(c)
rs10266244
(b)
rs7793793 T>C (0.383) rs2392210 T>C (0.218)
rs4562213 C>T (0.379)
rs6462450 T>C (0.218)
rs6462449 C>A (0.379)
rs2049758 T>G (0.383)
rs12668520 A>G (0.277) rs4720097 T>C (0.377) rs17170218 A>T (0.184)∗
rs6956397 C>T (0.379)
rs7806813 T>C (0.376)
rs6462453 G>T (0.379)
rs4720098 C>G (0.373)
rs10085768 A>G (0.377)
rs6948212 T>G (0.379)
rs10230500 C>T (0.379)
rs4316067 A>G (0.039)∗
rs7778958 C>T (0.379)
rs6946062 T>C (0.432)∗
rs7801986 T>G (0.379)
rs10486512 T>C (0.377)
rs4723240 A>G (0.383)
rs10231011 A>G (0.383)
rs4723241 T>A (0.466)
rs10951370 T>C (0.379) rs4720100 A>G (0.150)∗
rs4445128 C>G (0.373)
rs4338000 C>T (0.379)
rs4723242 G>A (0.377)
rs10251079 C>T (0.471)
rs10281012 G>A (0.371)
rs10256717 C>T (0.379) rs7800610 A>G (0.218)∗
rs6954923 T>G (0.379)
rs10266244 T>C (0.379)∗ rs6976843 C>T (0.277)∗
r2=1 2 r =1 r2=1
Freq.
ht1 ht2 ht3 ht4 ht5
T C C T T
T C C C C
A G A A A
A A A G A
C T T T C
A A A A A
A A A A T
T T T T C
G A A A G
0.277 0.218 0.160 0.150 0.150
ht6 ht7 ht8
T T T
C C C
A A A
A A A
T C T
G A G
T A A
C T C
G A G
0.034 0.005 0.005
Gene map of NT5C3. (a) Map of NT5C3 (5′-nucleotidase, cytosolic III) on chromosome 7p14.3. Coding exons are marked by black blocks, and 5′ and 3′ UTRs by white blocks (Ref. NM#. NM_001002010). The frequencies in parentheses were based on the Korean acute myeloid leukemia patients (n = 103). Asterisks indicate single nucleotide polymorphisms (SNPs) that were used in statistical analysis. (b) Haplotypes in NT5C3. Nine NT5C3 polymorphisms were used for haplotype construction. Five haplotypes with over 10% of frequency were used in statistical analyses. (c) Linkage disequilibriums (LDs) among NT5C3 polymorphisms. All 47 SNPs were in one LD block.
Allele tests of nine NT5C3 single-nucleotide polymorphisms and haplotypes with the outcome of first induction chemotherapy in Korean acute myeloid leukemia patients
Table 1
MAF rs# rs10266244 rs6976843 rs7800610 rs4720100 rs6946062 rs4316067 rs17170218 rs17170180 rs3750117 ht1 ht2 ht3 ht4 ht5
Allele test
Position (AA change)
Allele
Induction failure (n = 17)
Induction success (n = 73)
Promoter Promoter Intron 1 Intron 1 Intron 1 Intron 1 Intron 1 Intron 2 Exon 5 (Y131Y)
T>C C>T A>G A>G T>C A>G A>T T>C A>G
0.206 0.441 0.206 0.059 0.618 0.118 0.294 0.294 0.735 0.441 0.206 0 0.059 0.177
0.425 0.233 0.226 0.164 0.390 0.021 0.164 0.174 0.404 0.233 0.226 0.199 0.164 0.151
OR (95% CI) 0.35 2.60 0.89 0.32 2.52 6.36 2.12 1.98 4.10 2.60 0.89
(0.14–0.86) (1.19–5.66) (0.35–2.22) (0.07–1.42) (1.17–5.43) (1.35–29.88) (0.90–4.99) (0.84–4.66) (1.79–9.40) (1.19–5.66) (0.35–2.22) NA 0.32 (0.07–1.42) 1.21 (0.45–3.26)
P
Pcorr
0.02 0.01 0.80 0.17 0.02 0.02 0.08 0.11 0.0005 0.01 0.80 0.002 0.17 0.71
NS NS NS NS NS NS NS NS 0.02 NS NS NS NS NS
The values highlighted in bold are statistically significant (P< 0.05). CI, confidence interval; MAF, minor allele frequency; NA, not available; NS, not significant; OR, odds ratio.
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Pharmacogenetics and Genomics 2014, Vol 24 No 9
Table 2
Association analysis of rs3750117 with the outcome of first induction chemotherapy in Korean acute myeloid leukemia patients
Analyzing model
Genotype
Codominant
A/A A/G G/G A/A A/G + G/G A/A + A/G G/G
Dominant Recessive
Induction failure (n = 17) [n (%)] 3 3 11 3 14 6 11
Induction success (n = 73) [n (%)]
(17.6) (17.6) (64.7) (17.6) (82.4) (35.3) (64.7)
23 41 9 23 50 64 9
(31.5) (56.2) (12.3) (31.5) (68.5) (87.7) (12.3)
OR (95% CI)
P
Pcorr
4.56 (1.81–11.24)
0.0009
0.03
2.02 (0.68–7.31)
0.65
11.28 (3.69–35.27)
0.0001
NS 0.004
Genotype distributions and P-values of three alternative models (codominant, dominant, and recessive models) are shown. Logistic regression analysis was used to calculate the odds ratio (95% confidence intervals) and corresponding P-values controlling for cytogenetics risk groups and mutation status of FLT3-ITD and NPM1 as covariates. The values highlighted in bold are statistically significant (P< 0.05). CI, confidence interval; OR, odds ratio.
SNP, rs3750117 (Y131Y), was in absolute LD (|D'| = 1 and r2 = 1) with five intronic SNPs (rs10251079, rs4723241, rs4236335, rs3750119, and rs3750118). The mechanisms involved in the association of these silent SNPs with the outcome of chemotherapy are not currently understood. We can hypothesize that synonymous/intronic SNPs might impact gene function by affecting the mRNA stability as well as regulatory region. In addition, although it is true that the allele itself may be functional and directly affect the expression of the phenotype, a more likely event is that the allele is in LD with another allele at a nearby locus that is the true causal allele.
Fig. 2
P = 0.019
Normalized gene expression level
0.10
0.08
0.06
0.04
0.02
0.00 A/A (n=10)
A/G (n=13)
G/G (n=8)
NT5C3 rs3750117 genotype NT5C3 mRNA expression level between each genotype subgroup of the acute myeloid leukemia samples.
To further infer the precise mechanism underlying the antileukemic effect in the G/G patients in rs3750117, we compared the NT5C3 mRNA expression level between the genotype subgroups of the AML samples in the real-time qPCR. A significant difference in expression level was observed between the G/G genotype and the combined A/A and A/G genotypes, consistent with the results of the association study, in which the patients with the G/G genotype had a significantly higher risk of reduced induction rate than the other genotypes. The rs3750117 G/G patients failed first induction chemotherapy, probably because of higher NT5C3 gene expression levels and consequently lower drug effects. However, the data in this study do not indicate any reduced frequency of relapse to counterbalance the increase in death in remission. We do not have any evidence to explain how rs3750117 contributes toward increased expression level and increased risk of chemotherapy failure. The synonymous
In previous studies, one nonsynonymous SNP (Asp283His) and three intronic SNPs [I4(-144), I6(9) and rs4316067], were known to affect protein function and potentially influence drug response [5,6]. However, in our study, Asp283His, I4(-144) and I6(9) were monomorphic and rs4316067 showed low frequency (0.039) in Korean AML patients. In addition, in our statistical analysis, rs4316067 showed a marginal association with the induction success rate (P = 0.02), but this association could not retain significance after multiple corrections. As the purpose of the first course of chemotherapy is to potentially achieve a remission of AML and remission is necessary before other treatments, such as bone marrow transplants, can be performed, the NT5C3 genotype might be useful in selecting appropriate chemotherapy regimens for AML patients. Further research should be carried out to overcome the limitation of the relatively small number of patients. Also, validation of the current results will be needed in patients of different ethnicities.
Acknowledgements H.S.C. designed and performed research, analyzed data, and wrote the paper; Y.K., K.-S.A. and H.D.S. designed research and interpreted data; C.L. collected data; and S.-S.Y. designed research, interpreted data, and wrote the paper. This work was supported by the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (PGM21-A111218); the Basic Science
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NT5C3 polymorphisms and AML Cheong et al. 441
Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0008846).
7
Conflicts of interest
There are no conflicts of interest.
References 1 Chiarelli LR, Fermo E, Abrusci P, Bianchi P, Dellacasa CM, Galizzi A, et al. Two new mutations of the P5'N-1 gene found in Italian patients with hereditary hemolytic anemia: the molecular basis of the red cell enzyme disorder. Haematologica 2006; 91:1244–1247. 2 Donadelli M, Costanzo C, Beghelli S, Scupoli MT, Dandrea M, Bonora A, et al. Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine. Biochim Biophys Acta 2007; 1773:1095–1106. 3 Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 2002; 3:415–424. 4 Li L, Fridley B, Kalari K, Jenkins G, Batzler A, Safgren S, et al. Gemcitabine and cytosine arabinoside cytotoxicity: association with lymphoblastoid cell expression. Cancer Res 2008; 68:7050–7058. 5 Aksoy P, Zhu MJ, Kalari KR, Moon I, Pelleymounter LL, Eckloff BW, et al. Cytosolic 5'-nucleotidase III (NT5C3): gene sequence variation and functional genomics. Pharmacogenet Genomics 2009; 19:567–576. 6 Jordheim LP, Nguyen-Dumont T, Thomas X, Dumontet C, Tavtigian SV. Differential allelic expression in leukoblast from patients with acute myeloid
8
9
10
11 12
13
leukemia suggests genetic regulation of CDA, DCK, NT5C2, NT5C3, and TP53. Drug Metab Dispos 2008; 36:2419–2423. Schoch C, Schnittger S, Kern W, Dugas M, Hiddemann W, Haferlach T. Acute myeloid leukemia with recurring chromosome abnormalities as defined by the WHO-classification: incidence of subgroups, additional genetic abnormalities, FAB subtypes and age distribution in an unselected series of 1897 patients with acute myeloid leukemia. Haematologica 2003; 88:351–352. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998; 92:2322–2333. Oliphant A, Barker DL, Stuelpnagel JR, Chee MS. BeadArray technology: enabling an accurate, cost-effective approach to high-throughput genotyping. Biotechniques 2002; Suppl:56–58, 60–61. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25:402–408. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005; 21:263–265. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001; 68: 978–989. Hunsucker SA, Mitchell BS, Spychala J. The 5'-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacol Ther 2005; 107:1–30.
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Original article
NT5C3 polymorphisms and outcome of first induction chemotherapy in acute myeloid leukemia Hyun Sub Cheonga,d, Youngil Kohb, Kwang-Sung Ahne, Chansu Leea, Hyoung Doo Shind,f and Sung-Soo Yoona,b,c Aims The cytosolic 5′-nucleotidase-III (NT5C3) is involved in the metabolism of the nucleoside analog, cytosine arabinose (AraC), and the expression level of NT5C3 is correlated with sensitivity to AraC in acute myeloid leukemia (AML) patients. The current study examined whether the NT5C3 polymorphisms could affect chemotherapy outcomes in 103 Korean AML patients. Methods Forty-seven single nucleotide polymorphisms in NT5C3 were genotyped using the Illumina GoldenGate genotyping assay. The genetic effects of the polymorphisms on the outcome of chemotherapy were analyzed using χ2 and logistic regression models. Results Although none of the NT5C3 polymorphisms was associated with a complete remission rate, a common single nucleotide polymorphism, rs3750117, showed a significant association with induction rate after the first course of chemotherapy (Pcorr = 0.004 and odds ratio = 11.28) in AML patients. In addition, NT5C3 expression levels were significantly increased in patients with risk allele homozygote.
Introduction Acute myeloid leukemia (AML) is a rare cancer in Korea and worldwide. Among adults with AML, less than 20% survive 5 years after the initial diagnosis. Despite recent scientific advances in understanding the molecular biology of AML and mechanisms of drug metabolism, new successful therapeutic methods still need to be developed. The identification of patients who would not benefit from aggressive chemotherapy regimens requires more knowledge from pharmacogenetic studies. This strategy is especially important for elderly patients, who comprise the major age category for AML and whose capability to withstand intensive treatment is often limited. The 5′-nucleotidases are a family of enzymes that catalyze the dephosphorylation of various nucleoside 5′monophosphates [1]. One family member, the cytoplasmic 5′-nucleotidase-III (NT5C3), mainly catalyzes the dephosphorylation of pyrimidine nucleoside monophosphates, including nucleoside analog, cytosine arabinose (AraC), that are used to treat AML. This reaction results in the deactivation of active phosphorylated Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.pharmacogeneticsandgenomics.com). 1744-6872 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins
Conclusions The data suggest that genotyping the NT5C3 polymorphism may have the potential to identify patients more likely to respond to AraC-based chemotherapy. Pharmacogenetics and Genomics 24:436–441 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Pharmacogenetics and Genomics 2014, 24:436–441 Keywords: acute myeloid leukemia, chemotherapy, cytosine arabinose, NT5C3, single nucleotide polymorphism a
Cancer Research Institute, bDepartment of Internal Medicine, Seoul National University College of Medicine, cClinical Research Institute, Seoul National University Hospital, dDepartment of Genetic Epidemiology, SNP Genetics Inc., e Functional Genome Institute, PDXen Biosystem Inc. and fDepartment of Life Science, Sogang University, Seoul, Korea Correspondence to Sung-Soo Yoon, MD, Department of Internal Medicine, Seoul National University Hospital, Seoul National University College of Medicine, 101 Daehang-ro, Jongno-gu, Seoul 110-744, Korea Tel: + 82 2 2072 3079; fax: + 82 2 762 9662; e-mail: [email protected] Received 20 February 2014 Accepted 26 May 2014
metabolites [2,3]. Large variations have been observed in response to AraC, and one possible explanation is that genetic variation in genes involved in the activation and inactivation of these drugs might contribute toward differences in drug response. A series of functional analyses were carried out for NT5C3. Gene expression level of NT5C3 was significantly associated with cytotoxicity for AraC in human lymphoblastoid cell lines and knockdown of NT5C3 altered cancer cell sensitivity to this drug. Furthermore, higher NT5C3 expression correlated with lower intracellular AraC phosphorylated metabolite concentration [4]. Genetic variations in NT5C3 have shown to be associated with protein function and a potential effect on response to AraC. In a previous study, among 61 NT5C3 polymorphisms, Asp283His was correlated with decreased level of enzyme activity and I4(-114) and I6(9) showed altered DNA–protein-binding patterns [5]. An intronic single nucleotide polymorphism (SNP), rs4316067, was also associated with the mRNA expression level of NT5C3 in leukoblast from AML patients [6]. However, to our best knowledge, the genetic effect of NT5C3 polymorphisms on the outcome of chemotherapy has never been investigated in AML patients. To define the nature and extent of common genetic polymorphisms in NT5C3 and the possible functional DOI: 10.1097/FPC.0000000000000072
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NT5C3 polymorphisms and AML Cheong et al. 437
effect on the gene, we carried out a genetic association study using 103 Korean AML patients. The results from this study provide information on common genetic polymorphisms in NT5C3 as well as the functional consequences of the sequence variations.
Methods Patient characteristics and treatment protocol
From those with marrow samples available for DNA isolation, a total of 103 newly diagnosed AML patients (60 men and 43 women) from Seoul National University Hospital, Seoul, Korea, between December 2001 and September 2009 were enrolled in the study. The patient characteristics were as follows: the median age was 50.4 years (range 16–76 years) and the male to female ratio was 58 : 42 (n = 60 : 43). The FAB classification showed the following distribution of subtypes: M0, two patients (2%); M1, 14 (14%); M2, 47 (46%); M4, 33 (32%); M5, five (5%); and M7, two (2%). Patients diagnosed with acute promyelocytic leukemia (M3 FAB subtype) were excluded from the study. The criteria used to describe a cytogenetic clone and karyotype followed the recommendations of the International System for Human Cytogenetic Nomenclature [7]. Cytogenetic data were available for 95 patients. The cytogenetic risk groups were classified according to the MRC10 criteria, as described previously [8] [favorable risk: AML associated with t(8;21), t(15;17), or inv(16); unfavorable risk: the presence of a complex karyotype, − 5, del(5q), − 7, or abnormalities of 3q; intermediate risk: the remaining group of patients, including those with 11q23 abnormalities, + 8, + 21, + 22, del(9q), del(7q), or other miscellaneous structural or numerical defects not included in the favorable or unfavorable risk groups]. The favorable, intermediate, and unfavorable cytogenetic groups were 23 (22%), 69 (67%), and three (3%), respectively. The frequencies of FLT3-ITD and NPM1 mutations were 19.7 and 19.0%, respectively. Information on first induction chemotherapy outcome was available in 90 patients. All patients were treated with standard induction chemotherapy (idarubicin 12 mg/m2/day intravenously for 3 consecutive days and AraC 200 mg/m2/day intravenously for 7 consecutive days). In the case of patients older than 55 years of age, the dose of idarubicin was modified to two-thirds of the total dose. Consolidation therapies were performed on the basis of two more cycles of high-dose AraC (3 g/m2/day intravenously twice a day on D1, 3, 5). The institutional review board of Seoul National University Hospital approved the study protocol, and all patients provided informed consent. Single nucleotide polymorphism genotyping
Among 487 dbSNPs detected in NT5C3, 92 were reported in two Asian groups (Han Chinese and Japanese) in the HapMap database (release #27). Only 52 SNPs were selected on the basis of their minor allele frequencies (>5%) in the HapMap database. When we selected
SNPs in the HapMap database, linkage disequilibriums (LDs) among Asian groups were not considered because, although there is a high concordance between Korean and the two Asian samples, the tagging SNP selection strategy could miss an untagged SNP that may be potentially important in Koreans. In addition, we also genotyped one nonsynonymous SNP (Asp283His) and two intronic SNPs [I4(-144) and I6(9)], that were previously known to affect protein function and potentially influence drug response [5]. Genotyping was performed at a multiplex level using the Illumina GoldenGate genotyping system (Illumina, San Diego, California, USA) [9]. The genotype quality score for retaining data was set to 0.25. A total of 47 SNPs were successfully genotyped. Real-time quantitative PCR
Total RNA was extracted from bone marrow plasma of AML patients (n = 31) using the Qiagen RNeasy kit (Qiagen, Hilden, Germany) and measured its concentration using an ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, Delaware, USA). RNA (1 μg) per sample was reverse transcribed with SuperScript reverse transcriptase (Invitrogen, Carlsbad, California, USA) using random hexamers. The cDNA were synthesized by incubation at 45°C for 1 h. Then, the reaction was inactivated by heating at 95°C for 5 min. The PCR process was performed in 20 μl volume for 95° C for 5 min, 30 cycles at 95°C for 1 min, 48°C for 30 s, and 72°C for 1 min, and finally 10 min at 72°C. To perform real-time qPCR utilizing an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems, Foster City, California, USA), TaqMan Gene Expression Assays (Applied Biosystems) were used as a probe/primer set specified for NT5C3 (Assay ID: Hs00826433_m1) and a probe/primer set for the ACTB gene, a housekeeping gene, encoding β-actin (Assay ID: 4326315E). PCR was performed in a final volume of 20 μl containing TaqMan Gene Expression Master Mix (Applied Biosystems), 1 μl TaqMan Gene Expression Assay, 1 μl cDNA as the template, and up to 20 μl distilled H2O. The PCR program was as follows: 50°C for 2 min, 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. The expression level of NT5C3 was normalized to that of the ACTB gene for each sample. Experiments were conducted in duplicate (separate experiments) for each sample, and averaged values were calculated for normalized expression levels [10]. Statistics
To determine whether the individual variant was in equilibrium at each locus in the population (Hardy–Weinberg equilibrium), χ2-tests were performed. We examined a widely used measure of LD between all pairs of biallelic loci, D' [the correlation coefficient (Delta | D'|)], logarithm of odds, and r2 using Haploview (Broad Institute of Harvard and MIT Cambridge, MA, USA) [11].
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438 Pharmacogenetics and Genomics 2014, Vol 24 No 9
To determine the genetic association of NT5C3 polymorphisms, Fisher’s exact test and logistic regression analysis were carried out to calculate odds ratio, 95% confidence intervals, and corresponding P-values. Student’s t-test was used to compare NT5C3 expression levels between the rs3750117 genotype subgroups. Pairwise comparisons of the nine SNPs [only one SNP if there were absolute LDs (r2 = 1)], haplotypes were inferred using PHASE algorithm ver. 2.0 (Matthew Stephens Lab, Chicago, Illinois, USA) [12], and subsequent statistical analysis was carried out using SAS version 9.1 (SAS Inc., Cary, North Carolina, USA). P-values were corrected by Bonferroni correction for multiple testing [the threshold of raw P-value significance was 0.001 (47 polymorphisms analyzed)]. A type I error of 5% was used to obtain the Bonferroni-corrected P-value.
Results The current study examined the association of NT5C3 polymorphisms with chemotherapy outcomes in 103 Korean AML patients. Forty-seven SNPs in NT5C3 were genotyped using the Illumina GoldenGate genotyping assay. Genotype distributions were in Hardy–Weinberg equilibrium (P > 0.05) (Supplementary Table 1, Supplemental digital content 1, http://links.lww.com/FPC/ A741). Pairwise comparisons among SNPs showed five sets of absolute LDs (|D'| = 1 and r2 = 1), and SNPs in NT5C3 were in one LD block in Koreans (Fig. 1a and c). Nine SNPs (rs10266244, rs6976843, rs7800610, rs4720100, rs6946062, rs4316067, rs17170218, rs17170180, and rs3750117) were selected [only one SNP if there were absolute LDs (r2 = 1)] for haplotype construction and statistical analysis. Among eight haplotypes constructed, five common haplotypes (frequency > 10%) were used for further analysis (Fig. 1b). Allele frequencies of each of nine SNPs and five haplotypes were compared between (a) remission failure and complete remission groups and (b) induction failure and induction success groups after the first course of chemotherapy. None of the SNPs and haplotypes showed a significant association with complete remission rate by intensive chemotherapy (Supplementary Table 2, Supplemental digital content 2, http://links.lww.com/FPC/ A742). SNPs were also not associated with the risk of relapse, relapse-free survival, and overall survival (Supplementary Table 3, Supplemental digital content 3, http://links.lww.com/FPC/A743 and Supplementary Fig. 1, Supplemental digital content 4, http://links.lww.com/FPC/ A744). However, a common synonymous SNP, rs3750117 (Y131Y), showed a significant association with induction rate after the first course of chemotherapy (Pcorr = 0.02 and odds ratio = 4.10). The frequency of the G allele of rs3750117 was higher in induction failure group than in the induction success group (0.735 vs. 0.404) (Table 1). The susceptible effect of the rs3750117 G allele was clearer in the subsequent genotype test. The P-values of
rs3750117 retained significance after multiple comparisons in codominant and recessive models (Pcorr = 0.03 and 0.004, respectively). The frequency of the G/G genotype was higher in the induction failure group than in the induction success group (0.647 vs. 0.123). In contrast, the frequency of A allele-bearing genotypes, A/A + A/G, was high in the induction success group (0.353 vs. 0.877). Odds ratios were 4.56 and 11.28 in codominant and recessive models, respectively (Table 2). To estimate the impact of the rs3750117 polymorphism on gene expression level, the NT5C3 mRNA expression level was compared between the genotype subgroups of the AML samples in the real-time qPCR. The expression levels (mean ± SEM) were 0.024 ± 0.013 and 0.039 ± 0.024 for the A/A + A/G and G/G genotypes, respectively (Fig. 2). The gene expression level was 1.61-fold higher in the risk allele homozygote, G/G, than in the combined A/G and A/A genotypes. The difference was significant between the G/G genotype and the combined A/A and A/G genotypes (P = 0.019).
Discussion The 5′-nucleotidases are a family of enzymes that catalyzes the dephosphorylation of nucleoside monophosphates, including nucleoside analogs that are used to treat a variety of diseases [3,13]. NT5C3 is a cytosolic enzyme that catalyzes the dephosphorylation of pyrimidine analogs [13], thus playing an important role in both endogenous nucleoside and nucleotide pool balance as well as response to pyrimidine analogs such as gemcitabine and AraC. In previous studies, genetic variations in NT5C3 were determined and its effects on NT5C3 function were reported [5,6]. However, the genetic effect of NT5C3 polymorphisms on the outcome of chemotherapy has never been investigated in AML patients. This study is the first to report genotypes of NT5C3 and outcome of chemotherapy for AML. An exonic SNP, rs3750117, was associated with short-term treatment outcome, but was not associated with long-term outcomes such as complete remission, risk of relapse, relapse-free survival, and overall survival. Findings from the current study describe inferior outcome for patients with the minor allele homozygote, G/G of rs3750117, caused largely by an increase in the failure of the first course of chemotherapy. Treatment of AML requires intensive myelosuppressive induction chemotherapy to achieve remission and continuing postremission treatment to maintain a durable long-term remission. The G allele of rs3750117 may reduce or delay metabolism of the chemotherapy drug, AraC, used for AML and may lead to a reduced induction rate. It is possible that the NT5C3 genotype influences excretion of drug metabolites that contribute toward inactivation to a greater degree than it influences metabolites that have an antileukemic effect.
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NT5C3 polymorphisms and AML Cheong et al. 439
Fig. 1
(a) r2=1
r2=1
E1
rs12155477 T>C (0.382)
rs6942974 C>G (0.208) rs2893457 T>C (0.383)
rs2392209 G>A (0.387)
rs11532669 C>T (0.150)
rs3750118 T>C (0.466) rs3750117 A>G (0.466)∗
rs3750119 T>C (0.471)
rs17170180 T>C (0.191)∗
rs6955792 G>A (0.383)
rs4236335 A>C (0.466)
E2
E3 E4 E5 E6 E7 E8 E9
Hap.
rs6976843
rs7800610
rs4720100
rs6946062
rs4316067
rs17170218
rs17170180
rs3750117
(c)
rs10266244
(b)
rs7793793 T>C (0.383) rs2392210 T>C (0.218)
rs4562213 C>T (0.379)
rs6462450 T>C (0.218)
rs6462449 C>A (0.379)
rs2049758 T>G (0.383)
rs12668520 A>G (0.277) rs4720097 T>C (0.377) rs17170218 A>T (0.184)∗
rs6956397 C>T (0.379)
rs7806813 T>C (0.376)
rs6462453 G>T (0.379)
rs4720098 C>G (0.373)
rs10085768 A>G (0.377)
rs6948212 T>G (0.379)
rs10230500 C>T (0.379)
rs4316067 A>G (0.039)∗
rs7778958 C>T (0.379)
rs6946062 T>C (0.432)∗
rs7801986 T>G (0.379)
rs10486512 T>C (0.377)
rs4723240 A>G (0.383)
rs10231011 A>G (0.383)
rs4723241 T>A (0.466)
rs10951370 T>C (0.379) rs4720100 A>G (0.150)∗
rs4445128 C>G (0.373)
rs4338000 C>T (0.379)
rs4723242 G>A (0.377)
rs10251079 C>T (0.471)
rs10281012 G>A (0.371)
rs10256717 C>T (0.379) rs7800610 A>G (0.218)∗
rs6954923 T>G (0.379)
rs10266244 T>C (0.379)∗ rs6976843 C>T (0.277)∗
r2=1 2 r =1 r2=1
Freq.
ht1 ht2 ht3 ht4 ht5
T C C T T
T C C C C
A G A A A
A A A G A
C T T T C
A A A A A
A A A A T
T T T T C
G A A A G
0.277 0.218 0.160 0.150 0.150
ht6 ht7 ht8
T T T
C C C
A A A
A A A
T C T
G A G
T A A
C T C
G A G
0.034 0.005 0.005
Gene map of NT5C3. (a) Map of NT5C3 (5′-nucleotidase, cytosolic III) on chromosome 7p14.3. Coding exons are marked by black blocks, and 5′ and 3′ UTRs by white blocks (Ref. NM#. NM_001002010). The frequencies in parentheses were based on the Korean acute myeloid leukemia patients (n = 103). Asterisks indicate single nucleotide polymorphisms (SNPs) that were used in statistical analysis. (b) Haplotypes in NT5C3. Nine NT5C3 polymorphisms were used for haplotype construction. Five haplotypes with over 10% of frequency were used in statistical analyses. (c) Linkage disequilibriums (LDs) among NT5C3 polymorphisms. All 47 SNPs were in one LD block.
Allele tests of nine NT5C3 single-nucleotide polymorphisms and haplotypes with the outcome of first induction chemotherapy in Korean acute myeloid leukemia patients
Table 1
MAF rs# rs10266244 rs6976843 rs7800610 rs4720100 rs6946062 rs4316067 rs17170218 rs17170180 rs3750117 ht1 ht2 ht3 ht4 ht5
Allele test
Position (AA change)
Allele
Induction failure (n = 17)
Induction success (n = 73)
Promoter Promoter Intron 1 Intron 1 Intron 1 Intron 1 Intron 1 Intron 2 Exon 5 (Y131Y)
T>C C>T A>G A>G T>C A>G A>T T>C A>G
0.206 0.441 0.206 0.059 0.618 0.118 0.294 0.294 0.735 0.441 0.206 0 0.059 0.177
0.425 0.233 0.226 0.164 0.390 0.021 0.164 0.174 0.404 0.233 0.226 0.199 0.164 0.151
OR (95% CI) 0.35 2.60 0.89 0.32 2.52 6.36 2.12 1.98 4.10 2.60 0.89
(0.14–0.86) (1.19–5.66) (0.35–2.22) (0.07–1.42) (1.17–5.43) (1.35–29.88) (0.90–4.99) (0.84–4.66) (1.79–9.40) (1.19–5.66) (0.35–2.22) NA 0.32 (0.07–1.42) 1.21 (0.45–3.26)
P
Pcorr
0.02 0.01 0.80 0.17 0.02 0.02 0.08 0.11 0.0005 0.01 0.80 0.002 0.17 0.71
NS NS NS NS NS NS NS NS 0.02 NS NS NS NS NS
The values highlighted in bold are statistically significant (P< 0.05). CI, confidence interval; MAF, minor allele frequency; NA, not available; NS, not significant; OR, odds ratio.
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Pharmacogenetics and Genomics 2014, Vol 24 No 9
Table 2
Association analysis of rs3750117 with the outcome of first induction chemotherapy in Korean acute myeloid leukemia patients
Analyzing model
Genotype
Codominant
A/A A/G G/G A/A A/G + G/G A/A + A/G G/G
Dominant Recessive
Induction failure (n = 17) [n (%)] 3 3 11 3 14 6 11
Induction success (n = 73) [n (%)]
(17.6) (17.6) (64.7) (17.6) (82.4) (35.3) (64.7)
23 41 9 23 50 64 9
(31.5) (56.2) (12.3) (31.5) (68.5) (87.7) (12.3)
OR (95% CI)
P
Pcorr
4.56 (1.81–11.24)
0.0009
0.03
2.02 (0.68–7.31)
0.65
11.28 (3.69–35.27)
0.0001
NS 0.004
Genotype distributions and P-values of three alternative models (codominant, dominant, and recessive models) are shown. Logistic regression analysis was used to calculate the odds ratio (95% confidence intervals) and corresponding P-values controlling for cytogenetics risk groups and mutation status of FLT3-ITD and NPM1 as covariates. The values highlighted in bold are statistically significant (P< 0.05). CI, confidence interval; OR, odds ratio.
SNP, rs3750117 (Y131Y), was in absolute LD (|D'| = 1 and r2 = 1) with five intronic SNPs (rs10251079, rs4723241, rs4236335, rs3750119, and rs3750118). The mechanisms involved in the association of these silent SNPs with the outcome of chemotherapy are not currently understood. We can hypothesize that synonymous/intronic SNPs might impact gene function by affecting the mRNA stability as well as regulatory region. In addition, although it is true that the allele itself may be functional and directly affect the expression of the phenotype, a more likely event is that the allele is in LD with another allele at a nearby locus that is the true causal allele.
Fig. 2
P = 0.019
Normalized gene expression level
0.10
0.08
0.06
0.04
0.02
0.00 A/A (n=10)
A/G (n=13)
G/G (n=8)
NT5C3 rs3750117 genotype NT5C3 mRNA expression level between each genotype subgroup of the acute myeloid leukemia samples.
To further infer the precise mechanism underlying the antileukemic effect in the G/G patients in rs3750117, we compared the NT5C3 mRNA expression level between the genotype subgroups of the AML samples in the real-time qPCR. A significant difference in expression level was observed between the G/G genotype and the combined A/A and A/G genotypes, consistent with the results of the association study, in which the patients with the G/G genotype had a significantly higher risk of reduced induction rate than the other genotypes. The rs3750117 G/G patients failed first induction chemotherapy, probably because of higher NT5C3 gene expression levels and consequently lower drug effects. However, the data in this study do not indicate any reduced frequency of relapse to counterbalance the increase in death in remission. We do not have any evidence to explain how rs3750117 contributes toward increased expression level and increased risk of chemotherapy failure. The synonymous
In previous studies, one nonsynonymous SNP (Asp283His) and three intronic SNPs [I4(-144), I6(9) and rs4316067], were known to affect protein function and potentially influence drug response [5,6]. However, in our study, Asp283His, I4(-144) and I6(9) were monomorphic and rs4316067 showed low frequency (0.039) in Korean AML patients. In addition, in our statistical analysis, rs4316067 showed a marginal association with the induction success rate (P = 0.02), but this association could not retain significance after multiple corrections. As the purpose of the first course of chemotherapy is to potentially achieve a remission of AML and remission is necessary before other treatments, such as bone marrow transplants, can be performed, the NT5C3 genotype might be useful in selecting appropriate chemotherapy regimens for AML patients. Further research should be carried out to overcome the limitation of the relatively small number of patients. Also, validation of the current results will be needed in patients of different ethnicities.
Acknowledgements H.S.C. designed and performed research, analyzed data, and wrote the paper; Y.K., K.-S.A. and H.D.S. designed research and interpreted data; C.L. collected data; and S.-S.Y. designed research, interpreted data, and wrote the paper. This work was supported by the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (PGM21-A111218); the Basic Science
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NT5C3 polymorphisms and AML Cheong et al. 441
Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2011-0008846).
7
Conflicts of interest
There are no conflicts of interest.
References 1 Chiarelli LR, Fermo E, Abrusci P, Bianchi P, Dellacasa CM, Galizzi A, et al. Two new mutations of the P5'N-1 gene found in Italian patients with hereditary hemolytic anemia: the molecular basis of the red cell enzyme disorder. Haematologica 2006; 91:1244–1247. 2 Donadelli M, Costanzo C, Beghelli S, Scupoli MT, Dandrea M, Bonora A, et al. Synergistic inhibition of pancreatic adenocarcinoma cell growth by trichostatin A and gemcitabine. Biochim Biophys Acta 2007; 1773:1095–1106. 3 Galmarini CM, Mackey JR, Dumontet C. Nucleoside analogues and nucleobases in cancer treatment. Lancet Oncol 2002; 3:415–424. 4 Li L, Fridley B, Kalari K, Jenkins G, Batzler A, Safgren S, et al. Gemcitabine and cytosine arabinoside cytotoxicity: association with lymphoblastoid cell expression. Cancer Res 2008; 68:7050–7058. 5 Aksoy P, Zhu MJ, Kalari KR, Moon I, Pelleymounter LL, Eckloff BW, et al. Cytosolic 5'-nucleotidase III (NT5C3): gene sequence variation and functional genomics. Pharmacogenet Genomics 2009; 19:567–576. 6 Jordheim LP, Nguyen-Dumont T, Thomas X, Dumontet C, Tavtigian SV. Differential allelic expression in leukoblast from patients with acute myeloid
8
9
10
11 12
13
leukemia suggests genetic regulation of CDA, DCK, NT5C2, NT5C3, and TP53. Drug Metab Dispos 2008; 36:2419–2423. Schoch C, Schnittger S, Kern W, Dugas M, Hiddemann W, Haferlach T. Acute myeloid leukemia with recurring chromosome abnormalities as defined by the WHO-classification: incidence of subgroups, additional genetic abnormalities, FAB subtypes and age distribution in an unselected series of 1897 patients with acute myeloid leukemia. Haematologica 2003; 88:351–352. Grimwade D, Walker H, Oliver F, Wheatley K, Harrison C, Harrison G, et al. The importance of diagnostic cytogenetics on outcome in AML: analysis of 1612 patients entered into the MRC AML 10 trial. The Medical Research Council Adult and Children’s Leukaemia Working Parties. Blood 1998; 92:2322–2333. Oliphant A, Barker DL, Stuelpnagel JR, Chee MS. BeadArray technology: enabling an accurate, cost-effective approach to high-throughput genotyping. Biotechniques 2002; Suppl:56–58, 60–61. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001; 25:402–408. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005; 21:263–265. Stephens M, Smith NJ, Donnelly P. A new statistical method for haplotype reconstruction from population data. Am J Hum Genet 2001; 68: 978–989. Hunsucker SA, Mitchell BS, Spychala J. The 5'-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacol Ther 2005; 107:1–30.
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