Department of Haematology, Institute of Pathology, University of Leeds, Leeds LS2 9JT and
1 Leukaemia Research Fund Centre for Clinical Epidemiology, 30 Hyde Terrace, Leeds LS9 2LN, UK
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Abstract |
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Abbreviations: ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; CI, confidence interval; FAB, French-American-British classification; GST, glutathione S-transferase; GST M1, glutathione S-transferase M1; GST P1, glutathione S-transferase P1; GST T1, glutathione S-transferase T1; OR, odds ratio.
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Introduction |
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The gene family of glutathione S-transferases (GSTs), including µ (GST M1), (GST T1) and
(GST P1), function in the detoxification of electrophilic intermediates and in the excretion of reactive species by the addition of glutathione. Both GST T1 and GST M1 have a null genotype due to an independent gene deletion (1), which results in a lack of active protein. The absence of these proteins has been shown to increase the susceptibility of cultured lymphocytes to exogenously added DNA damaging agents (2). Two alleles of GST P1 have been described (3), an A
G transition causes an Ile
Val substitution giving rise to the GST P1*B allele. This substitution occurs in close proximity to the substrate binding site of the molecule which results in the GST P1*B phenotype displaying an altered specific activity for substrates in comparison with the wild-type GST P1*A allele (4). Individuals with the GST P1*B allelic variant have also been shown to have a significantly higher level of hydrophobic DNA adducts (5). These observations provide a plausible biological role for polymorphisms within the GST enzyme family being potential candidate tumour susceptibility genes.
Polymorphisms within the GST T1, GST M1 and GST P1 families have been associated with several malignancies (3,513). However, few studies have investigated the aetiological role of GST polymorphisms in the development of haemopoietic neoplasms. It has been suggested, albeit inconclusively, that the null genotype of GST T1 is associated with myelodysplasia (14), although reports are conflicting (15,16). In the present study, PCR was used to examine the relationship between polymorphisms in the metabolizer enzymes GST T1, GST M1 and GST P1. The findings for these polymorphisms in 557 patients diagnosed with acute leukaemia and their corresponding controls are reported here.
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Materials and methods |
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Blood samples were taken from cases as soon as possible after diagnosis. If a pre-treatment sample could not be taken, samples were taken 10 days after transfusion with whole or packed cells, or 2 days after transfusion with platelets. For controls, blood was either taken at the time of interview or shortly after.
Overall, 1066 acute leukaemia patients were identified, of whom 838 (79%) were interviewed. Since the frequency of alleles has been shown to vary with race (18,19), the present analysis is restricted to Caucasian subjects. Of the 807 Caucasian cases interviewed, 592 (73%) had blood samples taken. Of these 592 cases, 557 had at least one matched Caucasian control who also gave blood: 395 cases had two matched Caucasian controls and 162 had only one.
Smoking history was collected at interview, with exposure to tobacco defined as the subject having smoked at least once a day for a minimum period of 6 months. Every change in habit, such as type of tobacco and the number of cigarettes per day, was recorded with corresponding start and stop dates. An area-based deprivation indicator was created by linking to the 1991 census and coding the Townsend score of the address at diagnosis (20).
Classification of acute myeloid leukaemia (AML) cases
Cases were grouped in three ways; the first based on the French-American-British classification (FAB). Cases were also grouped on case history: primary AMLs were defined as cases where AML was the first recorded malignancy (n = 430), while secondary AML were defined as cases having myelodysplasia (MDS) or a prior malignancy (n = 56). Cytogenetic classification was also carried out. Cases with reciprocal translocations clinically associated with good prognosis and a possible de novo origin including t(15;17), t(8;21) and inversion (16) (n = 96) were grouped together. Cases with a partial or complete deletion of chromosomes 5, 7, 12 and 17, cases with a translocation/inversion/deletion of chromosomes 3 and 11, trisomy at chromosomes 8, 11 and 21 were grouped together as cases possibly being associated with secondary AML (n = 75). If more than one abnormality was present, classification was by the primary abnormality or, if not known, by the following order, t(15;17) > Inv (16) > t(8;21) > 11q > 3q > 5q/7q > 12p > 17p > 20q. The remaining cases fell into three categories: 144 cases classified by cytogenetic analysis as normal, 120 cases where cytogenetics failed or were not carried out and 44 cases with other cytogenetic abnormalities which could not be readily classified in the grouping used.
Genotyping
Genomic DNA was extracted from whole frozen blood using a proteinase K treatment, followed by a series of phenol/chloroform extractions and ethanol precipitation (21).
GST T1 and GST M1
Primers specific for the 3' coding region of the GST T1 gene (22), which gave rise to a 480 bp product, were multiplexed with one primer specific for the GST M1 gene and a primer with homology to the GSTM gene family, which gave rise to a 230 bp product (9). The presence of amplification product corresponded to the homozygote/heterozygote form of the enzyme, the absence of the GST T1 or GST M1 amplification product corresponding to the null genotype. Differentiating between homozygotes and heterozygotes was not possible with this assay.
One additional primer specific for the GST M4 gene was included in the reaction. This primer, when paired with the GSTM gene-specific primer within the reaction, amplifies a 157 bp product specific for the GST M4 gene. GST M4 has no known polymorphisms and for all samples yielded an amplification product; as such this amplification product was used as an internal control to monitor each PCR reaction to test the integrity of the DNA. Reaction conditions were as described (9,22), however, this modified protocol used a `hot start' (23) and an annealing temperature of 56°C.
GST P1
The AG base pair polymorphism in exon 5 of the GST P1 gene was determined using the PCR with restriction fragment length polymorphism method of Harries et al. (3). Due to difficulty amplifying our DNA the primer sequences were redesigned as follows: sense, ACTGGTGTTGATCAGGCGCC; antisense, CCTTCTTGGGTCAGGGTGCAG. The 280 bp product amplified, which spans exon 5, was digested twice for 6 h with BsmA1 to ensure complete digestion and the products sized by electrophoresis on an 8% polyacrylamide gel. GST P1*A (wild-type) samples were undigested (280 bp), GST P1*B (homozygote mutation) samples gave two bands of 147 and 133 bp, while GST P1*AB (heterozygote) samples produced a digestion pattern of all three bands.
Statistical analysis
Odds ratios (OR) and 95% confidence intervals (CI) were calculated using conditional logistic regression, adjusting for deprivation (24). Analysis was conducted using Intercooled Stata 5.0 for Windows 95 (Stata Corp.). Subgroup analyses were performed by cell lineage, age (three groups, 1639, 4054 and 55+ years) and FAB type. For the purposes of the present analysis, a 2 year latent interval prior to diagnosis was assumed for tobacco exposure.
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Results |
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Distribution of GST genotypes
Table I shows the number of cases and controls by GST T1, GST M1 and GST P1 status. For acute leukaemias combined, the null genotype of GST T1 occurred in 104 (19%) cases and 135 (14%) of their corresponding controls, yielding a significantly elevated OR of 1.45 (95% CI 1.091.93). The more common GST M1 null genotype was found in 296 (53%) cases and 466 (49%) controls (OR 1.22, 95% CI 0.981.52). Compared with the GST P1*A wild-type, the heterozygote and homozygote loci show no increased risk of acute leukaemia. Both GST T1 null and GST M1 null were weakly associated with AML (GST T1 null, OR 1.32, 95% CI 0.971.79; GST M1 null, OR 1.24, 95% CI 0.981.56), whereas for ALL, being null at GST T1 conferred a 3-fold increase in risk (OR 3.28, 95% CI 1.318.26). The alleles of GST P1 were similar in cases and controls for AML and ALL.
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Cytogenetic classification
GST T1, GST M1 and GST P1 genotypes were compared between the two cytogenetic groups. A higher frequency of null genotypes was found in the balanced translocation group compared with the other major group (GST T1 null, 22.3 versus 17.56%; GST M1 null, 57.4 versus 51.3%). Due to the small numbers these values failed to reach significance (GST T1, OR 1.35, 95% CI 0.632.94; GST M1, OR 1.28, 95% CI 0.692.38). Comparing each group against the AML controls only GST T1 null in the balanced translocation group showed an increase in frequency compared with the AML study group as a whole (good prognosis 22.3%, controls 15.1%; OR 1.63, 95% CI 0.962.77). No differences were observed in GST P1 genotype distribution.
Age distribution
The age distribution of GST genotypes was investigated in the study group. Data were split into three age groups (1639, 4054 and 5569 years) and the effect of age compared for each polymorphic variant of GST T1, GST M1 and GST P1. No associations between age and genotype were found for AML or ALL (data not shown).
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Discussion |
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The GSTs may modulate the level of DNA damage in haemopoietic stem and progenitor cells resulting from reactive metabolites. In vitro GST T1 null status has been linked to an increased frequency of diepoxybutane-induced sister chromatid exchange in cultured lymphocytes, while GST M1 allele status has no effect on the DNA damage observed (25). GST T1 and GST M1 protein and mRNA have been detected within the bone marrow (26), as has mRNA for GST P1. There is therefore a plausible biological mechanism which would link an increased risk of leukaemia to a lack of activity of GST T1 and GST M1.
Our results suggest an association between lack of activity of the GST T1 enzyme and an increased risk of developing acute leukaemia (OR 1.45, 95% CI 1.091.93). We went on to examine the association with lymphoid and myeloid subtypes and found that AML was weakly associated with both the GST T1 and GST M1 null genotypes (GST T1, OR 1.32, 95% CI 0.971.79; GST M1, OR 1.24, 95% CI 0.981.56). Whilst in ALL the GST T1 null genotype conferred a 3-fold increased risk (OR 3.28, 95% CI 1.318.26), no effect was observed for GST M1. None of the polymorphic variants of GST P1 examined showed an association with acute leukaemia, either AML or ALL.
The acute leukaemias are associated with the transformation of haemopoietic precursor cells. AML and ALL are different diseases and they are thought to be the result of the transformation of an early myeloid or lymphoid progenitor cell. This may in fact not be true and it is equally possible that both adult AML and ALL are stem cell diseases but with different patterns of maturation. The GST T1 null genotype carries an OR of 3.28 for ALL and 1.32 for AML. While this may support different risks for these two entities the number of samples in the ALL group (n = 71) was too small to support the concept of different aetiologies for these diseases.
A number of environmental exposures, such as pesticides and tobacco smoke, have been suggested as risk factors for acute leukaemia (17). Although the risks associated with these exposures are small, it is possible that they could interact with the genotypes to influence susceptibility. When the GST T1 and GST M1 genotypes were analysed in combination, the risk of acute leukaemia was increased (GST T1 null + GST M1 homozygote/heterozygote, OR 1.81, 95% CI 1.152.85), suggesting that the additive effects of these polymorphisms are also important.
Although it is possible that the presented associations could have arisen from an unusual prevalence of null genotypes in our control population, this seems unlikely. Our population-based controls were individually matched to cases by age, sex, race and geographical area of residence. Further, within our control population, the genotype distributions of the polymorphisms examined did not vary with age or sex. More importantly, perhaps, is that no variations with the census-derived Townsend index (used here as an indicator of socio-economic status) were observed. To follow standard practice, the analyses presented here were adjusted to account for the potential confounding effects of socio-economic status, although, within our data, this had no effect on the results. However, it should be noted that among our controls, the proportion with GST T1 null (14%) is at the lower end of the published range (1324%) for Caucasian populations (11,12,14,18,27), while the percentage with the GST P1*B genotype (14%) is considerably greater than that reported for other studies (69%) (3,5,27,28). Only for GST M1 is the percentage of nulls (49%) within the published range of 4257% (9,11,18,27,29).
Elevated risk estimates were observed for GST T1 null among the M3 and M4 FAB types, while for GST M1 null M0 and M3 showed the strongest association: the same trend was observed in the cases with balanced translocations. Both classifications are associated with good prognosis, showing a good concordance between both classification systems. For de novo primary AMLs the GST M1 null genotype carried an increased risk of AML (OR 1.28, 95% CI 1.021.63), while including the secondary AMLs reduced this observed risk. It appears that de novo AML associated with balanced translocations is at increased risk of developing in individuals null for GST T1 and GST M1. These risks are less in secondary AML and other cytogenetic groups. The groups are, however, small, but nonetheless this study suggests that risks associated with specific genotypes should be analysed based on defined cytogenetic groups.
Tobacco smoking represents a defined exposure which can be quantified and we have examined the association of smoking with polymorphic status at the GST T1 and GST M1 loci. Although smoking, and in particular current smoking, has been positively associated with acute leukaemia amongst this study's interviewed subjects (17), no significant interaction was observed between smoking and the null genotype of either the GST T1 or GST M1 loci (data not shown). The GSTs, particularly GST P1, are important in the detoxification of compounds found in cigarette smoke (3). The GST P1*B allele has also been associated with an increased risk of smoking-related diseases such as lung cancer (5). Smoking is also a common source of exposure to benzene, which is also detoxified via GST T1. No increased risk was identified in this study and so it would seem that this mechanism is not significant in the generation of acute leukaemia in vivo.
A major evolutionary role for these enzyme systems is the prevention of oxidative DNA damage and the association described herein suggests that this mechanism should be examined further in haemopoietic stem cells, particularly in the context of environmentally encountered xenobiotics.
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Acknowledgments |
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Notes |
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References |
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