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Screening for 3435CT and 2677GTA Polymorphisms of Multi- Drug.pdf


									Malaysian Journal of Biochemistry and Molecular Biology (2006) 14, 18-24                                                         18

    Screening for 3435C>T and 2677G>T/A Polymorphisms of Multi-
    Drug Resistance (MDR1) Gene in Malay Patients with Leukemia

Badrul Hisham Y1, Rosline H2, Mohd Ros S1, Norsa’adah B3,
Abdul Aziz B4 and Narazah MY5
 Human Genome Centre, 2Department of Haematology, 3Unit of Biostatistics & Research Methodology,
 Department of Medicine, School of Medical Sciences, Health Campus, Universiti Sains Malaysia,
16150 Kubang Kerian, Kelantan Malaysia. 6Advance Medical and Dental Institute (AMDI),
Universiti Sains Malaysia, Pulau Pinang Malaysia

The prevalence of 2677G>T and 3435C>T polymorphisms of the multi-drug resistance (MDR1) gene was found to be different in
many populations and it was significantly influenced by ethnicities. The mechanism on how these two single nucleotide
polymorphisms (SNPs) play a role in regulating the MDR1 expression especially in leukemia patients is still unclear and
controversial. The objective of this study was to evaluate the distribution of 3435C>T and 2677G>T/A polymorphisms among
Malay patients diagnosed to have leukemia (n=101) by using polymerase chain reaction-restriction fragment length polymorphism
(PCR-RFLP) based assays. The genotype frequencies for homozygous genotype GG, heterozygous genotype GT and genotype GA
and homozygous mutation genotype TT or AA in exon 21 were reported as 36.9%, 48.5%, 5.8% and 8.7%, respectively. In exon
26, the frequencies were 34.0%, 50.5% and 15.5% for the homozygous wild type CC, heterozygous mutation CT and homozygous
mutation TT, respectively. There were no association found between the distribution of SNPs in both exons with the types of
leukemia and sex of patients. Therefore, further clinical studies in a larger sample size should be carried out in order to find the
association between sex and types of leukemia in distribution of common SNPs in Malay patients with leukemia. The significant
implication of these common SNPs to the level of P-gp expression in Malay patients with leukemia that might contribute to the
chemotherapy resistance should be carried out as a future study.

Keywords: P-glycoprotein, Multi-drug resistance, SNPs, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML),
          chronic myeloid leukemia (CML)

Introduction                                                       the expression of MDR1 gene was increased in the stage
   Leukemia remains a difficult disease to treat despite           of relapsed or refractory disease compared to the patient
the improved clinical outcome by recent progress in                in diagnosis stage.
chemotherapy. One major problem was the emergence of
leukemic blast cells that were resistant to anticancer drugs,         Recently, several single nucleotide polymorphisms
which eventually will lead to treatment failure. A                 (SNPs) of MDR1 gene were identified with some
representative cause of multi-drug resistance (MDR) was            consequential protein amino acid changes [7]. As
the expression of the MDR1 gene product, P-glycoprotein            determined by Cascorbi et al., [8], the three most frequent
(P-gp) on the cell surface membrane[1]. The P-gp belongs           SNPs in the Caucasian population are located in exon 12,
to a family of ATP-binding cassette (ABC) transporters             exon 21 and exon 26 at position 1236, 2677 and 3435
that shared sequence and structure homology. The MDR1              respectively. Moreover, it was shown in healthy volunteers
gene mapped at position 7q21.1 of chromosome 7 and                 that these changes are in linkage disequilibrium and may
characterized as ATP-binding cassette (ABC) drug                   therefore be associated with transcriptional regulation of
pump[2].                                                           the MDR1 mRNA [9]. However, a mechanism on how
                                                                   these SNPs play a role in regulating the P-gp expression
   Over expression of the MDR1 gene had been reported              remains unclear.
in several human cancers [3] as well as acute leukaemia
[4]. The expression of P-gp had been reported in leukemia             To the best of knowledge, the 3435C>T and 2677G>T/
cells from about one-third of patients with acute myeloid          A polymorphisms of MDR1 gene has not been assessed
leukemia (AML) at the time of diagnosis, and more than             in Malay patients with leukemia. Therefore, the PCR-
50% of patients at relapsed; higher levels occurred in
certain subtypes including secondary leukemia. Expression          Author for correspondence: Badrul Hisham Yahaya,
of P-gp is correlated with reduction in complete remission         Human Genome Centre, School of Medical Sciences,
rates and a higher incidence of refractory disease [5]. In         Health Campus, 16150 Kubang Kerian, Kelantan, Malaysia.
acute lymphoblastic leukemia (ALL), expression of P-gp             Tel: 00 609 7664247/ 00 6012 5750013 Fax: 00 609 7658914
was observed in 38% of cases [6]. It was reported that             E-mail:
Screening for polymorphisms of multi-drug resistance gene                                                             19

RFLP based assays was developed to evaluate frequency           DNA Isolation:
of allelic variants of 3435C>T and 2677G>T/A                       Venous blood (200 µl) was obtained and genomic
polymorphisms in 101 Malay patients with leukemia               DNA was extracted using DNA Extraction Kit (QIAGEN,
admitted in Hospital Universiti Sains Malaysia (HUSM),          Germany). All procedures were followed as suggested by
Kota Bharu Kelantan, Malaysia. The results might provide        the manufacturer. The concentration and purity of DNA
basic information for treatment planning and valuable           samples were estimated spectrophotometrically.
tool to individualize pharmacotherapy especially in Malay
patients with leukemia.
                                                                PCR-RFLP assays:
                                                                   The reaction mixture for PCR amplification consisted
Materials and Methods:                                          of DNA template, 0.4 µM of each primers for both exons
Patients and samples collection:                                as listed in TABLE 1 (primers used in this study were
                                                                followed as suggested by Ilmer et al., [10] and Cascorbi
   A total of 101 Malay patients diagnosed to have              et al., [8], 3.0 mM MgCl2 (for exon 26) and 2.5 mM
leukaemia (ALL, AML and CML), who were admitted in              MgCl2 (for exon 21), 200 µM deoxynucleotide triphosphate
HUSM were enrolled in this study. Written consent was           (dNTPs), 10X AmpliTaq Gold PCR buffer and 1 unit
taken from all patients and for patients aged 16 and            AmpliTaq Gold (Applied Biosystems). PCR grade water
below, consent was taken from legal guardians. The study        was added to a final volume of 50 µl. PCR amplification
was approved by the local Ethics Committee of the School        conditions were as follows: initial denaturation at 95°C
of Medical Sciences (Reference No: USM/PPSP®/                   for 2 minutes, followed 32 cycles for denaturation at
EthicsCom./2004 (124.3[4]), Universiti Sains Malaysia,          95°C for 15 s, annealing at 60°C (exon 26) and 53°C
Kota Bharu Kelantan, Malaysia.                                  (exon 21) for 30 s and extension at 72°C for 30 s. The
                                                                terminal extension was performed at 72°C for 7 minutes.
   During the study period, out of 101 patients, 35 (34.7%)     The specific products were analyzed on standard 1.7%
were diagnosed as AML, 48 (47.5%) as ALL and 18                 agarose gels stained with SyBr Green (Amresco, Ohio).
(17.8%) as CML. Five out of 48 (10.4%) ALL patients             The type of enzymes used and DNA fragments generated
were diagnosed to have relapsed. There were 58 (57.4%)          after digestion are shown in TABLE 1; separated on 4%
males and 43 (42.6%) females. Age of the patients ranged        agarose gel, stained with SyBr Green and visualized under
from 1 to 75 years.                                             UV light.

Table 1: List of primers, type of enzymes used and fragment sizes for PCR-RFLP assays

Exon    Accession        Primer sequences         Restriction     Cutting     Recognition        SNPs          Fragment
          Code                                     enzyme          site        sequences                      length (bp)

                     F: 5' GTT TTG CAG
                     GCT ATA GGT TCC 3'
21                   R: 5' TTT AGT TTG
                     ACT CAC CTT CCC G 3'            BanI          2677     5' GG/PyPuCC 3'       G>T        228, 209, 19

                     F: 5' GTT TTG CAG
                     GCT ATA GGT TCC 3'
21                   R: 5' TTT AGT TTG
                     ACT CAC CTT 3'                  BsrI          2677     5' ACTGGN/ 3'        G>A         228, 210, 18

                     F: 5' GTT TTG CAG
                     GCT ATA GGT TCC 3'
21                   R: 5' TTT AGT TTG                                                         Wild type
                     ACT CAC CTT 3'                 BseYI          2677      5' CCCAGC 3'       (GG)            205, 23

                     F: 5' GAT CTG TGA
                     ACT CTT GTT TTC A 3'
26                   R: 5' GAA GAG AGA
                     CTT ACA TTA GGC 3'              MboI          3435       5' /GATC 3'         C>T        244, 172, 72
Screening for polymorphisms of multi-drug resistance gene                                                                 20

    In principle, there were three different enzymes used         Statistical analysis
in exon 21 in order to identify the type of allelic involved         Frequency and percentage were calculated for all the
for homozygous wild type, heterozygous for genotype               variables. The association between distribution of SNPs
GA and heterozygous for genotype GT. Two different                in both exons with type of leukaemia’s and sex of patients
reverse primers used resulting two different recognition          were analyzed using chi-square and Fisher Exact’s test
sites for BsrI and BanI enzymes. The additional base G            where p<0.05 considered as statistically significant.
was added at the 3' end of the reverse primer in order to
produce cutting site for BanI enzyme. In order to screen
for homozygous wild type for genotype GG, samples                 Results
were digested with BseY1 enzyme and the sizes of the                 As shown in TABLE 2, the genotype frequency for
fragments generated were 205 bp and 23 bp. Samples                homozygous mutation found lower (11.4%) in exon 26
showed persistent single band after digestion with BseY1          for AML patients while it was similar to ALL and CML
enzyme were classified having whether heteroduplexes              patients. The genotype frequency for homozygous wild
GT or GA or contains homozygous mutation for genotype             type found higher in exon 21 and exon 26 with 45.8%
TT or AA at position 2677. These samples were then                and 35.0%, respectively in ALL patients. Heterozygous
digested with BanI enzyme in order to screen for                  for genotype GA at position 2677 in exon 21 was only
heterozygous genotype GT. In this case, three fragments           found among Malay patients with AML and ALL with
generated 228 bp, 209 bp and 19 bp in sizes. Those                frequencies 5.7% and 8.3%, respectively while none of
samples did not digest with BanI enzyme was subjected             this genotype found in patients with CML. Overall, there
to BsrI enzyme in order to screen for heterozygous                was no association found between types of leukemia and
genotype GA. The fragments generated after 16 hours               sex of patients with the distribution of SNPs in both
incubation were 228 bp, 210 bp and 18 bp.                         exons where p>0.05.

    The PCR products for exon 26 contain the recognition          Discussion
site for MboI enzyme. In exon 26, all samples were
                                                                     Even though there is increased number of new
subjected to RFLP assays using MboI enzyme where 3
                                                                  technologies available for identification of specific genes
bands observed indicates the present of heterozygous
                                                                  contributing to diseases, it still lack of knowledge on
mutation at position 3435 with the sizes of 244 bp, 172
                                                                  how these genes function and play a role in developing a
bp and 72 bp. If the samples do not contain any mutation,
                                                                  disease. However, many of researchers are interested to
the sizes of fragments generated were 172 bp and
                                                                  study function of single base changes at the specific
72 bp while 1 band indicates the samples were
                                                                  location of whole coding region and their contribution to
homozygous mutation where the sizes of the fragments
                                                                  the disease development. Most human diseases influenced
were 244 bp.

Table 2: The distribution of common polymorphisms in exon 21 and exon 26 of MDR1 gene according to the types of
         leukemia and sex of the patients. These results were obtained based on the number of fragments generated after
         digestion with specific enzymes during PCR-RFLP based assays.

                                                                     Genotype frequency (%)

                                               Exon 21                                               Exon 26

Type of leukemia          GG           GT       GA       TT/AA         P-value       CC         CT         TT       P-value

      AML (n=35)           10           20        2        3                9        22         4
                         (28.6)       (57.1)    (5.7)    (8.6)                     (25.7)     (62.9)      (11.4)

      ALL (n=48)           22           19        4        3                         19         20          9
                         (45.8)       (39.6)    (8.3)    (6.3)          0.426*     (39.6)     (41.7)      (18.8)    0.455**

      CML (n=18)           6            9                  3                         6          9           3
                         (33.3)       (50.0)      0      (16.7)                    (33.3)     (50.0)      (16.7)

      Males (n=58)         24           26        4        4                         18         31          9
                         (41.4)       (44.8)    (6.9)    (6.9)          0.712*     (31.0)     (53.4)      (15.5)    0.769**

      Females (n=43)       14           22        2        5                         16         20          7
                         (32.6)       (51.2)    (4.7)    (11.6)                    (37.2)     (46.5)      (16.3)

*Statistical analysis was done using Fisher Exact’s test
**Statistical analysis was done using chi-square test
Screening for polymorphisms of multi-drug resistance gene                                                               21

by genes can be traced to SNPs, especially in important         lower compared to the Tang et al., [14] with the frequency
traits such as how diseases develop and how one responds        of 63.0%. On the other hand, the frequency for T-allele
to a pharmaceutical treatment. Variations in genome             was found to be higher (40.8%) compared to Tang et al
sequences underlie differences in one’s susceptibility or       [14] with frequency 37.0% and quite similar with Tang
protection from all kinds of diseases, age of onset and         et al., [13] with frequency 41.9%.
severity of illness [11].
                                                                   In terms of distribution of common alleles in exon 21,
   The 3435C>T polymorphism located in exon 26 is the           Tang et al., [14] reported the frequency for G-allele, T-
most popular SNP in the MDR1 gene that have thoroughly          allele and A-allele at position 2677 in percentage of
been studied by many researchers in various field of            57.5%, 36.0% and 6.5%, respectively though Tang et al.,
diseases. This polymorphism has been reported as a silent       [13] reported the frequency for G-allele, T-allele and A-
SNP which does not result in amino acid change and it           allele in percentage of 46.7%, 41.3% and 13.0%,
also been reported that the frequency of this SNP is            respectively. However, in this study, the genotype
significantly influenced by ethnicities [12]. In this type      frequencies were reported instead of reporting the allele’s
of polymorphism, T-allele has been found to be associated       frequencies where the frequency for homozygous
with reduced P-gp expression while the increased of C-          genotype GG, heterozygous genotype GT and genotype
allele frequency had been reported to give an impact on         GA and homozygous mutation genotype TT and AA were
high expression level of P-gp. There was also been              reported as 36.9%, 48.5%, 5.8% and 8.7%, respectively.
reported to play an important role in defence mechanism
against several toxins including bacteria and viral particles      The 3435C>T polymorphism is found to be common
[7]. Since this silent SNP is frequently correlated with        in all ethnicities; however the frequency is dependent on
the level of P-gp expression, many researchers are              racial background [9]. For example, 62% of European
interested to study on how this SNP can influence the           Americans and only 13% of African Americans carry at
regulation and expression of P-gp.                              least one such allele. Besides that, this polymorphism
                                                                also found to be linked to other non-synonymous
                                                                polymorphism in exon 21 (2677G>T) and synonymous
                                                                polymorphism in exon 12 (1236C>T). In the same study,
                                                                both polymorphisms in exon 26 and exon 21 were found
                                                                to be associated with altered fexofenadine disposition,
                                                                especially when both homozygous groups (CC and TT)
                                                                were compared. They found that subjects for homozygous
                                                                wild type in both exon 21 (homozygous GG) and exon
                                                                26 (homozygous CC) had significantly higher of
                                                                fexofenadine AUC (plasma concentration time) values
                                                                than homozygous TT subjects.

Figure 1: Electrophoresis patterns for 2677G>T/A
          polymorphism in exon 21 evaluated by PCR-RFLP
          based assay
          Lanes 1 and 2 : Shows digestion with BanI enzyme
          with the expected sizes 228, 209 bp
          Lanes 3 and 4 : Shows digestion with BsrI enzyme
          with expected sizes 228, 210 bp
          Lane 5 : Undigested PCR product (228 bp)
          Lane 6 : Shows sample uncut sample (228 bp)
          Lane 7 : Shows 100 bp DNA ladder
                                                                Figure 2: Electrophoresis patterns for 3435C>T
   In exon 26, the frequencies were 34.0%, 50.5% and                      polymorphism in exon 26 evaluated by PCR-RFLP
15.5% for the homozygous wild type CC, heterozygous                       based assay
mutation CT and homozygous mutation TT, respectively.                     Lane 1 : Shows undigested PCR product (244 bp)
                                                                          Lane 3 : Shows sample with homozygous wild
The frequency of polymorphisms at position 3435 in
                                                                          type CC (172 and 72 bp)
exon 26 was compared to the data published by Tang                        Lanes 2, 4, 6, 7 : Shows sample with heterozygous
et al., [13, 14] for Malay healthy volunteers in Asian                    mutation CT (244, 172 and 72 bp)
population. We found the frequency for C-allele in our                    Lane 8 : Shows sample with homozygous mutation
study quite similar to the frequency observed by Tang                     TT (244 bp)
et al., [13] which 59.2% and 58.1%, respectively while                    Lane 5 : Shows 100 bp DNA ladder
Screening for polymorphisms of multi-drug resistance gene                                                                 22

   Interestingly, several hypotheses have been made in         modalities and assessing patients’ response to therapy
order to a make an initial conclusion on how this 3435C>T      especially those receiving P-gp substrate.
polymorphism can influence the P-gp activity and
function. Firstly, the 3435C>T transition might have an           In other related study, Efferth et al., [17] showed no
impact on post-transcriptional modifications of the mRNA       significant correlation for drug resistance and prognosis
or secondly it might link to a sequence that is important      between homozygous genotype CC and heterozygous
for mRNA processing [15]. In this study, the correlation       genotype CT with MDR1 mRNA expression of cell lines
between 3435C>T and 2677G>T/A polymorphisms                    in ALL patients. They also found that there was no
(P<0.01) in terms of distribution of variations was thought    relationship between the response of the cell lines to
to be interesting as preliminary finding that these two        doxorubicin and the 3435C>T genotypes.
exons might play an important role in contributing to the
mRNA or P-gp regulation and expression in Malay                    Recent report indicates that 3435C>T polymorphism
leukaemia patients.                                            may be of clinical relevance where there was an association
                                                               between MDR1 3435C>T polymorphism and CNS (central
   As mentioned earlier, 3435C>T polymorphism might            nervous system) relapsed in childhood ALL patients. There
be linked with other polymorphisms in 1236C>T and              was 7-fold risk reduction of CNS relapsed observed in the
2677G>T/A. Ilmer et al., [10] had strongly agreed in           group of patients with intermediate or high risk of treatment
their study where in 405 patients with AML, three most         failure for patients with the TT and CT genotypes. This
frequent SNPs were found in exon 12, exon 21 and exon          data was consistent with study carried out by Jamroziak
26. They found that patients with the homozygous mutant        et al., [15] where increased frequency of C-allele carriers
genotype in exon 12 and exon 26 showed a lower median          among Polish children with ALL who relapsed.
age and was associated with poor risk cytogenetic
aberrations. On the other hand, there was a significant           However, a number of large-scale genotype-phenotype
association of the homozygous wild type genotype in            correlations are required in order to understand a
exon 21 and exon 26 with lower MDR1 expression,                correlation between SNPs and clinical outcome of Malay
whereas the heterozygous variants showed highest MDR1          leukaemia patients as future study. Laboratory testing of
values at all three investigated gene loci. The homozygous     common polymorphisms affecting activity of transporters
wild type in exon 26 was associated with lower overall         and enzymes involved in metabolism of a given drug
survival (OS) with P<0.05 and worse OS is likely linkage       might be useful to reduce toxicity and to increase efficacy
disequilibrium of the investigated SNPs. It was indicated      of therapy by individual dosage adjustment [18].
that combined polymorphisms could affect the regulation
of MDR1 and mRNA expression.                                      As mentioned earlier, the corresponding of C-allele was
                                                               associated with increased of P-gp levels [7]. In this study,
    However, the mechanism on how this linkage plays a         the genotype frequency for homozygous CC found to be
role in regulating the P-gp activity remains unclear. Unlike   higher in ALL and very low in AML patients. However,
what has been reported by Hoffmeyer et al., [7], the           when the distribution of genotype frequency is compared
linkage between polymorphisms in exon 21 and 26 is not         according to the type of leukemia, there was no significant
completely implying that these polymorphisms can act           difference. High frequency of homozygous CC in our ALL
independently in regulating the P-gp expression and            patients might have an association with chemotherapy
function. Lötsch et al., [16] discovered that                  response in these patients. This hypothesis however had
polymorphisms for 1236C>T, 3435C>T and 2677G>T                 been proved by Ilmer et al., [10] where they found that
occur together at a frequency of 62%, which was identical      higher frequency of homozygous CC in patients with ALL
to the value of 62% as previously reported by Kim et al.,      was found to be associated with worse prognosis in these
[9] in Caucasian population. Whereas, in Japanese              children and lower overall survival time in patients under
population, the 3435C>T and 2677G>T SNPs were                  age of 60 years old. However, larger sample size is required
reported to be linked in 94% of cases.                         to obtain statistically significant and further clinical study
                                                               should be carried out to come to this conclusion.
   In this study, combined polymorphisms was defined
when in these two exons, polymorphisms happened                    The TT genotype in exon 26 was found to be associated
concurrently whether homozygous wild type,                     with occurrence of ALL and the C-allele carriers were
heterozygous mutation or homozygous mutation.                  found to be reduced risk of developing ALL [15]. The
Therefore, when these polymorphisms were combined              homozygous genotype CC was associated with poor
and 39% link were found between 3435C>T and 2677G>T            outcome for the treatment. The T-allele carriers were
SNPs while 1% link were found between 3435C>T and              generally linked to weaker expression of P-gp in normal
2677G>A. On the other hand, there were 24% linked              tissues. Those homozygous for T-allele was found to
found between 3435C>C and 2677G>G among these                  have low intestinal expression of P-gp resulting in higher
studied samples. Thus, we would like to suggest that           intracellular concentrations of mutagens and eventually
study involves combination of these SNPs would be giving       leading to transformation of normal cell to cancer cells
a valuable results especially in initiating treatment          [19]. In contrast, homozygous for T-allele was found to
Screening for polymorphisms of multi-drug resistance gene                                                                             23

be associated with higher MDR1 mRNA levels in AML                     Conclusion
blasts cells [10]. The P-gp expression was found to be an                The clinical applications of genotyping studies demand
independent predictor of complete remission achievement               such methodology that would be rapid, simple and cost-
in adult with ALL. Furthermore, a hypothetic lower                    effective. Therefore, this study evaluated the PCR-RFLP
exposure of ALL cells to P-gp transported drugs in the                based assay as an effective method to detect known
CC homozygous individuals might be a consequence of                   polymorphisms at position 2677 and 3435 of MDR1
increased renal clearance, active secretion from                      gene. Even though this study only reported preliminary
enterocytes into the gut [20].                                        result, the results could serve as a basis knowledge for
                                                                      large scale correlation studies on relevance of 3435C>T
   Studies pertaining to linkage between 2677G>T and                  and 2677G>T/A genotypes in therapy of leukemia in
3435C>T polymorphisms are gaining increasing                          Malay population that was treated with drugs belongs to
significance especially as biomarkers to individualize                P-gp substrates as future studies. The clinical finding
pharmacotherapy in leukemia patients. This polymorphism               should be carried out to find a significant correlation
might play a role in patients who are not responsive to               between chemotherapy resistances with the increasing of
drug treatment. Furthermore, P-gp is essential in building            genotype CC in ALL subjects as well as patient with
blood-brain barrier (BBB) and as transporters among                   different types of leukemia in Malays.
other tissues as well as an important prognostic factor in
several tumour diseases [8]. On the contrary, there was
no significant difference when subjects were compared                 Acknowledgements
according to the gender and age of the patients where                    The study was supported by grant from Institute for
Jamroziak et al., [21] suggested that gender as well as               Graduate Studies (Tabung Siswazah 308/AIPS/415401),
other prognostic features might be treatment-dependent.               Universiti Sains Malaysia, 11800 Minden, Pulau Pinang
                                                                      Malaysia. The authors would like to express gratitude
   Even though the study on 2677G>T/A and 3435C>T                     to all postgraduate students in Human Genome Centre
polymorphisms and their effect on P-gp expression and                 and Haematology Lab, School of Medical Sciences,
regulation is still uncertain, further clinical correlation           Health Campus, Universiti Sains Malaysia, Kubang
should be studied since there was evidence that this                  Kerian Kelantan, Malaysia for their contribution to this
polymorphism may play a role in treatment and prognosis               research.
of patients with leukemia.

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