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Technical Briefs |
1 Purine Research Laboratory, Department of Chemical Pathology, and3 Department of Gastroenterology, Guy’s and St. Thomas’ Hospital NHS Trust, London, United Kingdom;2 Department of Medical and Molecular Genetics, GKT School of Medicine, King’s College, London, United Kingdom
aaddress correspondence to this author at: Purine Research Laboratory, Department of Chemical Pathology, 5th Floor Thomas Guy House, Guy’s Hospital, London SE1 9RT, United Kingdom; fax 44-207-188-1280, e-mail tony.marinaki{at}kcl.ac.uk
The immunosuppressive drug 6-mercaptopurine (6-MP) and its prodrug azathioprine are used in the treatment of inflammatory bowel disease and other disorders of immune regulation (1). Thiopurine methyltransferase (TPMT) inactivates 6-MP by methylation. The genetic variants TPMT*2 to *19 are associated with decreased TPMT activity (2), and TPMT*3A, *3C, and *2 are the most common deficiency-associated variants (1). A heterozygous TPMT genotype (1 in 10 individuals from the general population) is associated with an increased risk of myelosuppression with standard-dose azathioprine therapy (3) and a favorable response to reduced-dose thiopurine therapy (1). Patients with complete TPMT deficiency (1 in 300 individuals from the general population) are at high risk for myelosuppression (4).
The erythrocyte TPMT activity distribution is continuous, and concordance between genotype and phenotype in the carrier range varies, depending on where the cutoff is established between the ranges for carriers and noncarriers. We propose that genetic variation in folate metabolism influences TPMT activity and contributes to the lack of concordance between genotype and phenotype in the carrier range.
TPMT irreversibly transfers a methyl group from S-adenosylmethionine (SAM) to 6-MP, forming 6-methylmercaptopurine (6-MeMP) and S-adenosylhomocysteine (SAH). The adenosyl moiety of SAH is subsequently cleaved, and homocysteine is remethylated to methionine. The methyl donor for this folate-dependent remethylation cycle is 5-methyltetrahydrofolate, which is formed from 5,10-methylenetetrahydrofolate (MTHF) in a reaction catalyzed by 5,10-MTHF reductase (MTHFR). The MTHFR 677C>T (A222V) thermolabile variant (5) and the 1298A>C (E429A) variant (6) are associated with decreased MTHFR activity. The homozygous MTHFR 677TT genotype occurs in 8%–10% of the population (7), shows 30%–50% of wild-type activity in lymphocytes (8)(9), and is associated with hyperhomocysteinemia (10), DNA hypomethylation (11), increased risk of neural tube defects(12), and decreased risk of some cancers (13)(14). The homozygous 1298CC genotype (frequency, 10%) is associated with 60% of wild-type activity in lymphocytes (6)(7).
MTHF dehydrogenase/methenyltetrahydrofolate cyclohydrolase/formyltetrahydrofolate synthetase (MTHFD1) catalyzes the conversion of tetrahydrofolate to N10-formyltetrahydrofolate, N5,N10-methenyltetrahydrofolate, and 5,10-MTHF. The MTHFD1 1958G>A (R653Q) allele is associated with neural tube defects (15).
The Ethics Committee of Guy’s and St. Thomas’ Hospitals NHS Trust approved this study, which used leftover blood samples. Erythrocyte TPMT activity was measured in EDTA blood as the conversion of 6-MP to 6-MeMP (16) and expressed as picomoles of 6-MeMP formed per hour per milligram of hemoglobin [pmol 6-MeMP · h–1 · (mg Hb)–1]. Completely deficient TPMT activity was defined as <2.5 pmol 6-MeMP · h–1 · (mg Hb)–1, and the carrier and noncarrier ranges were 2.5–7.5 and >7.5–14.5 pmol 6-MeMP · h–1 · (mg Hb)–1, respectively.
Patients were genotyped for the MTHFR 677C>T and 1298A>C variants and the TPMT*3A, *3C, and *2 variants (16). The MTHFD1 1958G>A mutation (R653Q) destroys an HpaII site and was amplified with the primers 1958for (5'-TTCTTCTCATTCTTCCTCACACCTG-3') and 1958rev (5'-CAATGTCTGCTCCAAATCCTGC-3'). The thermocycler profile was 35 cycles of 94 °C for 30 s, 52 °C for 30 s, and 72 °C for 30 s. The 421-bp fragment digested by HpaII yielded fragments of 253 and 168 bp for the wild-type allele.
All variants were tested for Hardy–Weinberg equilibrium. Genotype frequencies were compared by use of 2 x 2 contingency tables, with recessive and dominant models applied. Significance was determined by 2-tailed Fisher exact tests. Because the analyses were not independent, no correction was applied for multiple testing of variants or models. The linkage disequilibrium coefficient between the MTHFR 677T>C and 1298A>C variants was calculated, and haplotype models were fitted by use of a log-linear model in COCAPHASE (17). Models were also fitted with MTHFD1 included to determine any multilocus effects across MTHFR and MTHFD1.
Concordance between TPMT genotype and phenotype in the laboratory carrier range [2.5–7.5 pmol 6-MeMP · h–1 · (mg Hb)–1] was determined from a series of 1575 predominantly Caucasian patients. Of the 185 (11.7%) patients with a carrier phenotype, 158 were heterozygous for a common TPMT mutation, a concordance between genotype and phenotype of 85.4%, in good agreement with a previous study reporting 86% concordance in the carrier range (18).
To test the hypothesis that a polymorphism in folate metabolism influences TPMT activity, we defined 2 groups in the TPMT carrier range: patients with a carrier TPMT phenotype and a heterozygous genotype (66 patients), and individuals with carrier activity but a wild-type TPMT genotype (n = 70). These groups were compared with a well-separated group of patients (n = 83) with TPMT activity in the narrow interval of 12.0–14.5 pmol 6-MeMP · h–1 · (mg Hb)–1 at the upper limit of the laboratory noncarrier range of >7.5–14.5 pmol 6-MeMP · h–1 · (mg Hb)–1. The study populations were enriched, and although the groups were recruited sequentially on the basis of TPMT genotype and phenotype, the sizes are not proportional to those found in the general population.
All 83 patients with TPMT activity in the noncarrier range [12–14.5 pmol 6-MeMP · h–1 · (mg Hb)–1] were wild type for TPMT*3A, *3C, and *2 mutations. The median TPMT activity of patients with a heterozygous genotype was 6.3 (range, 5.0–7.5) pmol 6-MeMP · h–1 · (mg Hb)–1, which was significantly different from the value of 7.1 (range, 4.0–7.5) pmol 6-MeMP · h–1 · (mg Hb)–1 obtained for wild-type patients with TPMT activity in the carrier range (P <0.0001, Mann–Whitney rank-sum test).
The genotype frequencies for MTHFR and MTHFD1 in the 3 groups are shown in Table 1
. All variants were in Hardy–Weinberg equilibrium. The MTHFR 677TT homozygous genotype frequency of 23% in the carrier range/wild-type TPMT genotype group was significantly greater than the frequency of 8% found in the noncarrier range group [odds ratio (OR) = 3.2; 95% confidence interval (CI), 1.2–8.3; P = 0.0216]. We found no significant differences for the MTHFR 677T/1298C compound heterozygous genotype (OR = 1.2; 95% CI, 0.5–2.5; P = 0.7047), the MTHFR 1298CC variant genotype (OR = 0.2; 95% CI, 0.1–1.3; P = 0.1106), or the MTHFD1 1958AA variant genotype (OR = 1.0; 95% CI, 0.5–2.1; P = 1.0000).
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The MTHFR 677C>T and 1298A>C variants were in strong linkage disequilibrium (D' = 1), and only 3 haplotypes were observed. The MTHFR haplotype frequencies in the group with TPMT activity in the noncarrier range were as follows: 677C-1298A, 37%; 677C-1298C, 30%; and 677T-1298A, 33%. There was no difference in haplotype frequencies in the wild-type patient groups with high or low TPMT activity (P = 0.32), and the fit of the model was not improved by including MTHFD1 1958G>A.
The finding that the MTHFR 677TT homozygous genotype is significantly associated with erythrocyte TPMT activity in the carrier range suggests that polymorphisms in folate metabolism play a role in modulating TPMT enzyme activity. We postulated that mutations occurring simultaneously in both the MTHFR and MTHFD1 genes could impair 5,10-MTHF and 5-methyltetrahydrofolate formation and hence lead to reduced SAM concentrations and decreased TPMT stability. SAM is known to stabilize variant TPMT enzymes from degradation in vitro (19). Moreover, a recent study of the binding of the SAM analog sinefungin to TPMT from Pseudomonas syringae suggested that substrate binding leads to conformational stability of peripheral structural elements of the enzyme as well as an increase in backbone mobility that may protect variant and, by implication, wild-type TPMT enzymes from ubiquitylation and degradation (20). Studies of the MTHFR-deficient mouse model have predicted decreased SAM concentrations and an altered SAM/SAH ratio (21)(22), although recently a slight but significant increase in erythrocyte SAM concentrations in individuals with an MTHFR 677CT heterozygous genotype has been reported (23). Little is known about the interplay between plasma and intracellular concentrations of SAM and SAH and how these are affected by polymorphisms in folate-metabolizing enzymes.
The coinheritance of variant MTHFR and MTHFD1 alleles did not have an additive effect on TPMT activity, consistent with reports concluding that MTHFD1 does not directly influence reactions for which a methyl group is required and does not affect SAM concentrations through 5,10-MTHF pools (15). In a pharmacogenetic context, inheritance of an MTHFR 677T-1298A haplotype and an MTHFD1 1958A allele was associated with a lower probability of event-free survival in children treated with the antifolate drug methotrexate for acute lymphoblastic leukemia, particularly when in combination with the thymidylate synthase triple repeat associated with increased thymidylate synthase concentrations (24). Polymorphisms in both genes thus have a demonstrable effect on folate metabolism within the cell, with the potential to affect intracellular SAM concentrations and hence, indirectly, TPMT activity.
TPMT activity in individuals heterozygous for a variant TPMT allele was significantly lower than in those with a wild-type TPMT genotype and activity in the carrier range. Concordance between TPMT genotype and phenotype in the carrier range will thus be determined essentially by the limits of the carrier range. Rare polymorphisms in the TPMT gene may also contribute to the lack of concordance.
The TPMT phenotype is determined in erythrocytes, and it is not known how a MTHFR 677TT genotype will affect TPMT activity or thiopurine metabolism in nucleated cells, which unlike erythrocytes are able to continuously synthesize protein. Methylated thiopurine metabolites have been reported to be immunosuppressive (25), and high concentrations of 6-MeMP have been implicated in hepatoxicity (26). It is thus possible that patients with an MTHFR 677TT genotype may be poor responders to therapy or, conversely, may be protected against methylated metabolite–mediated toxicity.
In conclusion, polymorphisms in the MTHFR gene may play an important role in determining the erythrocyte TPMT phenotype, but this effect needs to be replicated in further genetic association studies. We speculate that decreased intracellular SAM pools lead to enhanced TPMT enzyme degradation. Further studies should investigate the effect of an MTHFR 677TT genotype on clinical responses to thiopurine drug therapy.
Acknowledgments
This study was funded by the National Association for Colitis and Crohn’s Disease, UK.
Footnotes
1 current address: School of Microbial and Molecular Sciences, University of Queensland, Australia; 
References
T and 1298AC mutations on plasma homocysteine and 5,10-methylenetetrahydrofolate reductase activity in healthy subjects. Br J Nutr 2000;83:593-596.[ISI][Medline]
[Order article via Infotrieve]
T and 1298AC mutations are associated with DNA hypomethylation. J Med Genet 2004;41:454-458.T mutation in the methylenetetrahydrofolate reductase gene a risk factor for neural tube defects? A meta-analysis. QJM 1997;90:111-115.The following articles in journals at HighWire Press have cited this article:
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  N. B. Y. Tsui Multiple Thiopurine S-Methyltransferase Variation Detection: A Step toward Personalized Medicine Clin. Chem., October 1, 2008; 54(10): 1598 - 1599. [Full Text] [PDF]
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