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Cell Biology International (2012) 36, 79–86 (Printed in Great Britain)
ABCC1 polymorphisms in anthracycline-induced cardiotoxicity in childhood acute lymphoblastic leukaemia
Agnes F. Semsei*, Daniel J. Erdelyi*†, Ildiko Ungvari*, Edit Csagoly*, Marta Z. Hegyi†, Petra S. Kiszel*, Orsolya Lautner‑Csorba*, Judit Szabolcs†, Peter Masat‡, Gyorgy Fekete†, Andras Falus*, Csaba Szalai1§¶‖ and Gabor T. Kovacs†
*Department of Genetics, Cell and Immunobiology, Semmelweis University, Budapest, Hungary, †Second Department of Pediatrics, Semmelweis University, Budapest, Hungary, ‡Markusovszky Hospital of Vas County Council, Szombathely, Hungary, §Heim Pal Children Hospital, Budapest, Hungary, ¶Inflammation Biology and Immunogenomics Research Group, Hungarian Academy of Sciences, Semmelweis University, Budapest, Hungary, and ‖Csertex Research Laboratory, Budapest, Hungary


Anthracyclines are potent cytostatic drugs, the correct dosage being critical to avoid possible cardiac side effects. ABCC1 [ATP-binding cassette, sub-family C, member 1; also denoted as MRP1 (multidrug resistance-associated protein 1)] is expressed in the heart and takes part in the detoxification and protection of cells from the toxic effects of xenobiotics, including anthracyclines. Our objective was to search for associations between LV (left ventricular) function and single-nucleotide polymorphisms of the ABCC1 gene in children receiving anthracycline chemotherapy. Data of 235 paediatric patients with acute lymphoblastic leukaemia was analysed. Patients were followed-up by echocardiography (median follow-up 6.3 years). Nine polymorphisms in the ABCC1 gene were genotyped. The ABCC1 rs3743527TT genotype and rs3743527TT–rs246221TC/TT genotype combination were associated with lower LVFS (left ventricular fractional shortening) after chemotherapy. The results suggest that genetic variants in the ABCC1 gene influence anthracycline-induced LV dysfunction.


Key words: ABCC1, cardiotoxicity, fractional shortening, leukaemia, pharmacogenetics, side effects

Abbreviations: ABC, ATP-binding cassette, ABCC1, ABC, sub-family C, member 1, ALL, acute lymphoblastic leukaemia, BFM, Berlin–Frankfurt–Münster, ECHO, echocardiography, HWE, Hardy–Weinberg equilibrium, LV, left ventricular, LVEDD, left ventricular end-diastolic-dimension, LVESD, left ventricular end-systolic dimension, LVFS, left ventricular fractional shortening, MRP1, multidrug resistance-associated protein 1, NHL, non-Hodgkin lymphoma, SNP, single-nucleotide polymorphism, 3′-UTR, 3′-untranslated region

1To whom correspondence should be addressed (email genomika.cs@gmail.com).


1. Introduction

Childhood ALL (acute lymphoblastic leukaemia) is cured in ∼80–90% of the patients, but a significant number of survivors suffer from chemotherapy-induced side effects (Mody et al., 2008). These can appear during treatment or become clinically evident years after therapy. Follow-up studies indicate that survivors of childhood leukaemia often have cardiac problems as late side effects of chemotherapy (Shankar et al., 2008; Mulrooney et al., 2009). These can be due to the most cardiotoxic anthracyclines, but other drugs used in chemotherapy regimens can also damage the heart (Viale and Yamamoto, 2008).

Anthracyclines, such as doxorubicin and daunorubicin, are highly effective anti-neoplastic drugs used in a wide range of cancers (Johnson and Richardson, 1998). Anthracycline-induced cardiotoxicity can be divided into 3 types. Acute anthracycline-induced cardiotoxicity occurs during treatment, often immediately after the administration of the first dose. In contrast, early onset chronic progressive anthracycline-induced cardiotoxicity presents within 1 year after anthracycline administration, and late-onset chronic progressive cardiotoxicity develops years or even decades later (Lipshultz et al., 2008). Anthracycline-induced cardiotoxicity can be a mild, transient condition, characterized by asymptomatic electrocardiographic changes, e.g., arrhythmias. More severe toxicities include LV (left ventricular) dysfunction, decreased exercise capacity, decreased LVFS (left ventricular fractional shortening), late onset cardiomyopathy and congestive heart failure. Heart dysfunction is associated with cardiomyocyte loss, LV wall thinning and LV dilation (Wojtacki et al., 2000; Adams and Lipshultz, 2005; Iarussi et al., 2005).

Late-onset anthracycline-induced cardiotoxicity is by far the most frequent cardiac consequence of the therapy. In a study of 115 survivors of childhood ALL, several patients showed abnormal LV structure or function years after anthracycline treatment, while early-onset anthracycline-induced cardiotoxicity was usually experienced only in a few patients (Lipshultz et al., 2005). Subclinical reduction of heart function during or right after therapy might indicate increased susceptibility to late-onset cardiomyopathy (Kremer et al., 2002; Scully and Lipshultz, 2007).

Several risk factors for cardiotoxicity have been identified, including age at treatment (<4 years), concomitant therapy (irradiation and other anti-neoplastic drugs), gender (female) and cumulative dose of drug. Nevertheless, the fact that there is no safe dose of anthracyclines and the wide inter-patient variability in the time and seriousness of this adverse effect suggest that genetic background is a major determinant of drug response and toxicity (Lipshultz et al., 2008).

ABC (ATP-binding cassette) transporters have an important role in the protection of the body against xenobiotics (Borst et al., 2000; Sparreboom et al., 2003). These membrane-localized efflux pumps export a wide range of chemotherapeutic agents using the energy of ATP hydrolysis. ABC transporters are expressed in the heart, some of which have anthracyclines among their substrates (Couture et al., 2007). The ABCC1 [ATP-binding cassette, sub-family C, member 1; also denoted as MRP1 (multidrug resistance-associated protein 1)] transporter was first described in a doxorubicin-resistant cell line and is expressed ubiquitously in the body. Tissues showing high levels of ABCC1 expression include the heart, lung, testis, kidney and placenta. ABCC1 participates in detoxification, protects the cells from toxic effects of xenobiotics and is also involved in the defence mechanisms against oxidative stress (Bakos and Homolya, 2007). ABCC1 expression increases after doxorubicin treatment in cardiac tissue of mice (Jungsuwadee et al., 2009), and experiments with Abcc1-knockout mice have shown an important role of this protein in the efflux of drugs from the heart. Finally, several studies indicated that ABCC1 gene polymorphisms can influence the function of the transporter (Kerb et al., 2001).

Our objective has been to examine potential associations between subclinical reduction of cardiac function and the genetic background of childhood ALL patients, focusing the investigation of the polymorphisms of ABCC1, covering all haplotype blocks in this gene in order to gain improve our understanding on the genetic background of the anthracycline cardiotoxicity.

2. Patients and methods

2.1. Study population and definitions

DNA were collected retrospectively from children with ALL who had undergone chemotherapy between 1990 and 2002, aged 1–18 years at diagnosis, treated according to the ALL BFM (Berlin–Frankfurt–Münster) 90 or 95 study protocols in 6 Hungarian paediatric oncology centres. A total of 235 children were included in the analysis, with their characteristics being shown in Table 1. According to the data of the Hungarian Paediatric Cancer Registry, 337 cases were diagnosed with ALL in these hospitals during the same period who had survived at least until the end of chemotherapy (Table 1). The selected cohort represents 70% of all the cases. There was no difference in the distribution of the characteristics of the patients between the 2 groups. Informed consent was requested from the parents. The study was conducted according to the principles expressed in the Declaration of Helsinki (2000) and approved by the Ethics Committee of the Hungarian Medical Research Council.


Table 1 Characteristics of the study population and all cases diagnosed with ALL between 1990 and 2002 in Hungary in the hospitals included in the study from the Hungarian Paediatric Cancer Registry

Characteristics of patients Study population Whole population P-value
Number of patients 235 337
Gender n (%)
    Male 126 (54) 190 (56)
    Female 109 (46) 147 (44) 0.6
Age at diagnosis [means±S.D., years (range)] 5.7±3.8 (1.2–18.0) 6.2±4.1 (1.0–18.0) 0.1
Risk group n (%)
    LR 61 (26) 69 (20)
    MR 155 (66) 231 (69)
    HR 19 (8) 37 (11) 0.2
Chemotherapy protocol n (%)
    ALL BFM 90 (before 1995) 83 (35) 146 (44)
    ALL BFM 95 (after 1995) 152 (65) 189 (56) 0.06
Dexrazoxane usage n (%)
    No 164 (70) 179 (70)
    Yes 69 (30) 76 (30) 0.9
Anthracycline dose (range, mg/m2)
    During ALL BFM treatment 120–360 120–360
    Including cumulative doses of those treated for relapse 120–840 120–840
LVFS (means±S.D., %)
    At the diagnosis 39.8±6.0 (n = 133) n.d.a.
    End of the treatment 39.0±6.0 (n = 173) n.d.a.
    Latest ECHO 38.6±5.2 (n = 168) n.d.a.
Time of the ECHO from the diagnosis (median, years)
    End of the treatment (range) 2.0 (0.7–3.4) n.d.a.
    Latest ECHO (range) 6.3 (2.4–13.7) n.d.a.


Abbreviations: HR, high risk; LR, low risk; MR, medium risk; n.d.a., no data available.



The patients were followed-up by ECHO (echocardiography) to assess LV function by measuring LVEDD (left ventricular end-diastolic-dimension) and LVESD (left ventricular end-systolic dimension). LVFS was calculated from these 2 data: LVFS % = (LVEDD−LVESD)/LVEDD×100%. We analysed the LVFS at 3 time-points, first, at the time of diagnosis, secondly at the end of the treatment, that is, at a median of 2 years after diagnosis, while the third data-point was determined at the time of the latest follow-up. ECHO was performed with varying frequency at the different centres, so LVFS data were not present at all time points for all the children. In the case of 7 patients with relapsed leukaemia, we included 2 separate ‘latest’ datasets. These were the last ECHO results before starting relapse chemotherapy and the latest after relapse, featured by different cumulative anthracycline doses respectively.

The chemotherapy regimen included repeated doses of intravenous vincristine, l-asparaginase, daunorubicin, doxorubicin, methotrexate, cyclophosphamide, cytosine arabinoside, oral prednisone, dexamethasone, mercaptopurine, thioguanine, intrathecal methotrexate, intrathecal prednisone and ifosfamide (the latter only in patients with high-risk leukaemia). The protocol in the low-risk and medium-risk arms differed only in the number of anthracycline doses, the cumulative anthracycline doses being between 180 and 300 mg/m2. The high-risk arm differed considerably from the low-risk and medium-risk arms in terms of the applied therapy dosage.

2.2. Laboratory methods

DNA was extracted from peripheral blood taken in remission (n = 206) from the survivors. DNA was obtained from bone marrow smears (n = 19), from neonatal Guthrie spots (n = 4), or from stored buffy coats (n = 6) from patients who were deceased before sample collection was carried out. We extracted genomic DNA from blood using the QIAmpBlood DNA Maxi Kit (Qiagen) according to the manufacturers' instructions; from smears and from lymphocytes with HighPure PCR Template Preparation Kit (Roche Diagnostics) with minor alterations (see the Supplementary online data available at http://www.cellbiolint.org/cbi/036/cbi0360079add.htm): and from Guthrie spots using Chelex 100 reagent (Bio-Rad Laboratories).

SNPs (single nucleotide polymorphisms) were selected prioritized on the basis of their estimated functionality in this order: non-synonymous SNPs, SNPs in promoter and 3′-UTR (3′-untranslated region) region, synonymous SNPs and intronic SNPs. Our goal was to cover every haplotype block in the gene defined by the Haploview 4.1 software (http://www.broad.mit.edu/mpg/haploview/) (Barrett et al., 2005) with 1 or 2 SNPs. Detailed information on the selected SNPs is shown in Table 2.


Table 2 Genotyped SNPs and LVFS in the three time points in different genotype groups

SNP Alleles on the forward strand Position according to NCBI Genome Build 36.3 B* Function MAF Deviation from the HWE (χ2 test) LVFS time N all LVFS 11 S.D. N 11 LVFS 12 S.D. N 12 LVFS 22 S.D. N 22 P-value
rs215060 A/G 15984788 4 Intron 0.14 0.51 LVFS dg. 130 39.1 5.7 93 41.5 6.7 37 0.1
LVFS e.t. 168 38.8 6.0 118 39.6 5.9 50 0.8
LVFS l. 163 38.4 4.8 126 39.7 5.4 37 0.2
rs246219 C/T 15993136 5 Intron 0.14 0.02 LVFS dg. 128 39.2 6.0 91 40.9 6.2 37 0.1
LVFS e.t. 166 38.7 6.0 119 39.8 6.3 47 1
LVFS l. 161 38.3 5.1 123 39.1 5.1 38 0.6
rs246221 T/C 16045823 10 V275V 0.35 0.08 LVFS dg. 132 40.0 6.0 57 39.3 6.1 65 42.5 5.0 10 0.1
LVFS e.t. 171 39.4 6.5 69 38.4 5.6 84 40.0 6.4 18 0.2
LVFS l. 164 38.4 4.8 62 38.5 5.2 86 40.7 5.6 16 0.027
rs45511401 G/T 16080733 12 G671V 0.05 0.41 LVFS dg. 129 39.7 6.2 114 41.3 5.2 15 0.3
LVFS e.t. 164 39.0 6.1 147 39.2 5.3 17 1
LVFS l. 155 38.7 5.0 140 38.1 5.8 15 0.3
rs4148358 C/T 16094676 13 Intron 0.2 0.76 LVFS dg. 130 39.6 6.1 80 40.2 6.0 50 0.8
LVFS e.t. 168 39.3 6.4 103 38.7 5.5 65 0.3
LVFS l. 162 38.5 5.3 112 38.5 4.5 50 0.9
rs11864374 A/G 16109386 14 Intron 0.24 0.56 LVFS dg. 129 39.0 6.1 72 41.1 5.7 49 39.3 6.8 8 0.1
LVFS e.t. 165 39.0 5.7 95 39.0 6.5 57 39.4 6.6 13 0.5
LVFS l. 162 38.5 4.6 97 38.3 5.2 53 41.5 6.1 12 0.2
rs6416666 A/G 16121463 15 Intron 0.07 0.28 LVFS dg. 131 39.7 6.2 112 39.9 5.3 19 0.7
LVFS e.t. 170 39.1 6.0 144 38.5 6.4 26 0.9
LVFS l. 166 38.7 5.1 148 37.4 5.7 18 0.3
rs3743527 C/T 16143182 16 3′-UTR 0.22 0.49 LVFS dg. 131 39.7 5.6 79 40.1 7.1 43 38.5 4.5 9 0.5
LVFS e.t. 168 39.5 5.8 103 39.3 6.4 54 34.0 4.4 11 0.001
LVFS l. 161 38.7 4.9 108 38.9 5.1 47 35.3 3.6 6 0.2
rs212097 A/G 16151630 16 3′-UTR 0.47 0.43 LVFS dg. 131 39.9 6.0 38 39.6 6.2 69 40.0 5.9 24 0.9
LVFS e.t. 170 38.0 6.3 45 39.4 6.0 87 39.5 5.7 38 0.2
LVFS l. 166 37.8 5.8 40 38.8 4.9 88 38.8 5.2 38 0.8


B: haplotype blocks determined by Haploview 4.1 using the HapMap data.


Mean LVFS of the patients in the different genotype groups: 11, homozygote for the frequent allele; 12, heterozygote; 22, homozygote for the rare allele. If there were not enough data for the rare 22 genotype groups, the 12 and 22 data were merged for the analysis. This is indicated by a dash in column LVFS 22.; LVFS is indicated in mean %; LVFS dg., LVFS at the time of diagnosis; LVFS e.t., LVFS at the end of the treatment; LVFS l., LVFS at the time of the latest echocardiography; N, number of patients in the genotype group.



The ABCC1 rs45511401 genotypes were determined by multiplex single base extension using a SNaPshot Multiplex Kit (Applied Biosystems) followed by mini-sequencing on an ABI 310 genetic analyser (Applied Biosystems). All other ABCC1 SNPs were genotyped using the GenomeLab SNPstream genotyping platform (Beckman Coulter) according to the manufacturer's instructions. Detailed description of these procedures can be found in the Supplementary online data.

2.3. Statistical analysis

Allele frequencies were calculated by allele counting. The HWE (Hardy–Weinberg equilibrium) was tested by using on-line software (http://ihg2.helmholtz-muenchen.de/cgi-bin/hw/hwa1.pl, and significant violation of HWE was considered where P<0.01). We performed the statistical analysis to assess the effect of the genetic background on cardiac parameters with multi-adjusted general linear model procedures. The multi-adjusted models included the following potential confounders: gender (male–female), age at the time of diagnosis (years), clinical centre (6 centres), total anthracycline dose (mg/m2), dexrazoxane administration (yes–no), and chemotherapy protocol (ALL BFM 90–ALL BFM 95). Regarding the data at the time of diagnosis, the multi-adjusted models included only gender, age at the time of diagnosis, clinical centre and chemotherapy protocol. Three genotype groups were analysed separately when the number of patients was sufficient in each group.

Alpha levels of P<0.005, that is, 0.05 after Bonferroni correction considering multiple testing for 9 SNPs, were considered significant. The analyses were performed using the SPSS 15.1 (SPSS Inc.) and MedCalc 10.0.2 (MedCalc Software) software.

3. Results

3.1. Study population and allele frequencies

As mentioned above, the characteristics of the study population are shown in Table 1. Genotyping for 9 SNPs in ABCC1 gene was performed. The positions of the investigated SNPs in the gene can be seen in Figure 1, and minor allelic frequencies in Table 2. Genotype distributions were in HWE (Table 2).

3.2. The effect of genotypes on LVFS

With the help of the 9 SNPs in the ABCC1 gene, we investigated whether these polymorphisms influence cardiac functioning in children with ALL after anthracycline therapy, LVFS being used to measure cardiac function.

There were no significant differences in the LVFS in the genotype groups at the time of diagnosis (Table 2; LVFS dg.). At later time points, however, LVFS was influenced by ABCC1 rs246221 and rs3743527 polymorphisms. The ABCC1 rs3743527TT genotype group had decreased LVFS at the end of treatment ECHO (34.0%) compared with patients with CC (39.5%) or CT (39.3%) genotype (P = 0.001; Table 2 and Figure 2). LVFS was 35.3, 38.9 and 38.7% at the time of the latest ECHO of the patients with TT, CT and CC genotypes respectively, although due to the low number of patients being homozygote for the rare allele, this difference was not statistically significant.

Furthermore, we found an association between harbouring the rs246221 T allele and LVFS at the time of the latest ECHO (6.6±2.7 years after the diagnosis). Patients with TC and TT genotype displayed reduced LVFS (38.4 and 38.5% respectively) compared with patients with CC 40.7%, which was only nominally significant (P = 0.027; Table 2 and Figure 2).

Among the clinical factors included in the analysis of LVFS, the age at the time of diagnosis, clinical centre, chemotherapy protocol were significant cofactors (data not shown). In the case of the 2 significant SNPs, neither of these parameters influenced the effect of the genotypes.

3.3. The effect of genotype combinations on LVFS

Because rs3743527TT, rs246221TT and TC genotypes were associated with lower mean LVFS, we were interested in the LVFS data of patients who had both the rs3743527TT and rs246221TC or rs246221TT genotypes, and therefore analysed the effect of these genotype combinations on the LVFS. We compared the LVFS data of patients with both the rs3743527TT and rs246221TT or rs246221TC genotypes to the LVFS data of patients with any other genotype for these 2 SNPs. Patients harbouring the rs3743527TT and rs246221TC or TT genotypes (group 2) did not differ in their mean LVFSs before chemotherapy from the patients with other genotypes (group 1). However, after the chemotherapy the mean LVFS was significantly lower in patients with the TT-TC/TT genotype combination (34.0%) compared with other patients (39.4%) (P = 0.001; Table 3 and Figure 3).


Table 3 LVFS in patients with rs3743527TT and rs246221TC or rs246221TT genotypes

LVFS time Patients involved LVFS group 1* S.D. Number of patients in group 1 LVFS group 2 S.D. Number of patients in group 2 P-value
LVFS dg. 131 39.8 6.2 122 38.5 4.5 9 0.4
LVFS e.t. 169 39.4 6 158 34.0 4.4 11 0.001
LVFS 1. 163 38.7 4.9 158 35.6 4 5 0.1


Group 1: patients with genotype other than group 2 patients.


Group 2: patients with rs3743527TT and rs246221TC or TT genotypes; LVFS is indicated in mean %; LVFS dg., LVFS at the time of diagnosis; LVFS e.t., LVFS at the end of the treatment; LVFS 1., LVFS at the time of the latest echocardiography.



4. Discussion

Childhood ALL is highly curable today, but the survivors may suffer from severe late side effects of the chemotherapy. One of the most important late adverse effects is anthracycline cardiotoxicity. The development and progression of this toxicity varies between patients, which suggests that drug toxicity might be influenced by genetic background. Patients can survive for decades, so it is particularly important to prevent these late side effects of therapy, identify early markers of these late problems, and identify patients with elevated susceptibility to development of late cardiac problems. We accordingly examined the association of genetic polymorphisms in the ABCC1 gene with LV function after chemotherapy.

Patients with the ABCC1 genotype rs3743527TT had reduced LVFS at the end of the treatment. Moreover, the genotype combination TT-TC/TT (rs3743527–rs246221) was associated with decreased LVFS.

A possible limitation of these findings is that patients who died before the period of sample collection were under-represented in our cohort. However, in our opinion, this is not a relevant bias, as late effects are only manifest and have relevance in survivors. Furthermore, according to the data stored at the Hungarian Paediatric Cancer Registry, these patients did not die due to cardiac-related events, and thus these limitations are insufficient to question the results of our study.

It has also to be mentioned that patients with rs3743527TT genotype have rs246221TT or rs246221TC genotypes, except for one patient with rs246221CC genotype. The reason for this is the low number of patients (n = 11) with rs3743527TT genotype, but this may also cause some bias.

So far, there has been only one study by Wojnowski et al. (2005) examining the role of ABC transporters in anthracycline-induced cardiotoxicity, in which 206 SNPs were examined in 82 genes comparing 87 Caucasian patients with NHL (non-Hodgkin lymphoma) experiencing cardiac problems with 363 NHL patients without any cardiac malfunction. This study found an association between chronic anthracycline-induced cardiotoxicity and a polymorphism in the NAD(P)H oxidase subunit NFC4 (rs1883112), and between: acute anthracycline-induced cardiotoxicity and the NAD(P)H oxidase subunits CYBA (rs4673) and RAC2 (rs13058338); and the ABC transporter ABCC1 Gly671Val variant (which is rs45511401) and the Val1188Glu–Cys1515Tyr (rs8187694–rs8187710) haplotype of the ABCC2 gene.

Interestingly, we did not find an association between ABCC1 rs45511401 and reduced LVFS, a fact that must be interpreted carefully, as there were differences in the chemotherapy protocols and the age of the study populations; and also because this SNP has a low minor allele frequency, which, together with the investigated number of patients, makes this comparison under-powered. In addition, Wojnowski et al. (2005) defined the acute anthracycline-induced cardiotoxicity with parameters we did not analyse. They defined cardiotoxicity on the basis of the following criteria: cases of arrhythmia, myocarditis, pericarditis, and acute heart failure as acute anthracycline-induced cardiotoxicity. Reduction of the ejection fraction to <50%, or of fractional shortening to <25%, was classified as chronic anthracycline-induced cardiotoxicity. We did not analyse their first 3 parameters regarding acute cardiotoxicity and we had only one patient with fractional shortening of <25%. They found no association with ABCC1 rs45511401 and chronic anthracycline-induced cardiotoxicity which is similar to our study as we examined the reduction in LVFS. In summary, the important similarity between the 2 results is that they found an association between cardiac problems and ABCC1 gene variations. Naturally for a more decisive statement, both studies ought to be repeated on independent and larger populations with more SNPs in the ABCC1 gene involved in the analysis.

The ABCC1 transporter is important in the protection of the cell from distinct types of chemical stress. In most polarized cells, it is localized in the basolateral membrane, contralateral to the other ABC transporters involved in the detoxification. This suggests that the main role of ABCC1 is the protection of the cells against xenobiotics (Bakos and Homolya, 2007). The anthracycline doxorubicin does not require a transporter to enter the cell; thus a sufficient efflux of this drug is very important to protect the cell (Borst et al., 2000). This fact is in agreement with our observation regarding the role of ABCC1 in the protection of cardiomyocytes.

Also the ABCC1 transporter plays an important role in oxidative stress. It is involved in the maintenance of sufficient levels of glutathione, which is necessary for the defence against reactive oxygen species. Moreover, ABCC1 also requires glutathione for transport of anthracyclines (Borst et al., 2000; Kruh and Belinsky, 2003; Bakos and Homolya, 2007), which can also influence the response in oxidative stress induced by anthracyclines (Wojtacki et al., 2000).

In our study, 2 SNPs, rs3743527 and rs246221, influenced cardiac function after anthracycline treatment. Unfortunately, there is no data on the potential function of these SNPs. There are studies investigating the role of other known ABCC1 SNPs (Wang et al., 2006; Huang, 2007), but not of these ones. Rs246221 is an exonic synonymous polymorphism, which does not influence the amino acid sequence. It is located in the linker region of the ABCC1. The rs3743527 SNP is located in the 3′-UTR of the ABCC1 gene. In the literature, there is only one study involving this SNP. Studying the susceptibility to lung cancer in Chinese patients, Wang et al. (2009) investigated SNPs located in the 3′-UTR of ABCB1 and ABCC1, including rs3743527. They found no association with ABCC1 rs3743527, but with ABCC1 rs212090 and ABCB1 rs3842. The ABCC1 rs212090 is only 300 bp distance away from rs3743527.

Further studies are important to reveal the exact role of these polymorphisms. Haplotype analysis may be useful to discover possible linked functional SNPs, because not these but others in the same haplotype may block or influence the function of ABCC1. Also it is possible that there are some as yet unknown regulatory elements in this region that could be affected by this SNP. According to the miRDB microRNA database (http://mirdb.org/miRDB/), there are predicted hsa-miR-185, hsa-miR-548o and hsa-miR-1254 binding sites in a 500 bp region around rs3743527 that might have regulatory roles. Other polymorphisms in the same haplotype block with rs3743527 can influence the binding sequence of the regulatory elements. The ENCODE project (Birney et al., 2007) identified regulatory sites in the genome also in the proximity of SNPs in the ABCC1 gene. There are predicted transcription factor binding sites near rs3743527 and rs246221. The CTCF transcription factor binds 1235 bp downstream from rs3743527 and BAF155 binds 893 bp downstream from rs246221. DNase hypersensitivity assays also show regulatory regions within 3000 bp to these SNPs.

In our study, the anthracycline dose did not influence LVFS. This is probably because the dose range was relatively narrow (180–240 mg/m2) in ∼80% of the samples of the cohort. The threshold for LVFS associated with LV systolic dysfunction is LVFS <30% according to the National Cancer Institute Common Terminology Criteria for Adverse Events [v.3.0] (http://ctep.info.nih.gov/protocolDevelopment/electronic_applications/ctc.htm). It must be noted that the reduced LVFS values in this study associated with certain genetic background were in the ‘normal’ range. According to several studies, normal range or subclinical reduction of LVFS in patients after anthracycline therapy might have prognostic value for late onset more serious cardiotoxicity (Kremer et al., 2002; Lipshultz et al., 2008).

It is also important to note that some drugs used in the chemotherapy protocols, like Vinca alkaloids and methotrexate, may be cardiotoxic (Floyd et al., 2005; Simbre et al., 2005). They are also substrates of ABCC1 and might also have contributed to the associations identified in our study. However, it is generally accepted that the cardiotoxic effect of anthracyclines considerably exceeds that of these other drugs.

5. Conclusions

Our results indicate that certain genotypes of the ABCC1 rs3743527 or rs246221 SNPs might influence the development of cardiotoxicity after anthracycline treatment in the patient population studied. Common genetic polymorphisms have weak effects; each one contributes only slightly to the susceptibility to a disease. The effect might be stronger if genotype associations or haplotypes are investigated. According to these and earlier findings the ABCC1 transporter is important in limiting the anthracycline exposure of cardiomyocytes, and this protective mechanism may be influenced by genetic polymorphisms. Further studies with longer follow-up involving other genes and more patients are necessary to examine the exact genetic background of anthracycline-induced late onset cardiomyopathy. Such studies are needed for establishing possible individualized chemotherapies.

Author contribution

Agnes Semsei performed the genotyping, evaluated the results and wrote the paper. Daniel Erdelyi organized the whole project, and organized and collected patients. Ildiko Ungvari performed statistical evaluation. Edit Csagoly and Marta Hegyi collected the clinical data. Petra Kiszel evaluated the results. Orsolya Lautner-Csorba performed the genotyping. Judit Szabolcs and Peter Masat performed the clinical measurements. Gyorgy Fekete evaluated the clinical data. Andras Falus organized the project. Csaba Szalai organized the laboratory work and wrote the paper. Gabor Kovacs collected and evaluated the clinical data. All of the authors read the paper, and contributed to the evaluation of the results.

Acknowledgements

We are grateful to all the nurses, physicians and patients who took part in the study. We thank Anna Zalka, Eniko Kamory, Bela Csokay, Andras Kiss, Dora Lippai, Xenia Majorosi, Melinda Racz, Veronika Galasz, Erika Toth, Gabor Varadi, Krisztina Toth and Judit Bali for technical assistance and Zoltan Pos for help with writing the article prior to submission.

Funding

The majority of the SNP genotyping was carried out in the Semmelweis University-SNP Core Facility (http://www.dgci.sote.hu/en/snpcorefacilityen) which is supported by the Richter Gedeon PLC Company. This study was funded by the Hungarian Scientific Research Fund [grant number T042500], the Economic Competitiveness Operational Programme, Hungary [grant number GVOP 3.1.1-2004-05-0022/3.0] and NKTH (National Research and Technology) [grant number TECH_08-A1/2-2008-0120].

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Received 2 May 2011/7 July 2011; accepted 20 September 2011

Published as Cell Biology International Immediate Publication 20 September 2011, doi:10.1042/CBI20110264


© The Author(s) Journal compilation © 2012 Portland Press Limited


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)