MTHFR and TYMS genotypes influence TPMT activity and its differential modulation in males and females
Introduction
Thiopurine S-methyltransferase (TPMT) (EC 2.1.1.67) catalyzes the S-methylation of the thiopurine antimetabolic drug, 6-mercaptopurine (6-MP), used in treatment of cancer and autoimmune diseases [1]. The methyl moiety donor in this reaction, S-adenosyl methionine (SAM), is synthesized from methionine, whose production is tightly connected to folate metabolism and homocysteine (Fig. 1). Patients with low levels of TPMT more often experience severe toxic effects when treated with standard doses of 6-MP than patients with high levels [1]. TPMT activity is controlled by common genetic polymorphisms, 460 G > A and 719 A > C, that are responsible for inter-individual differences in thiopurine toxicity and efficacy. The two polymorphisms correspond to alleles ⁎3B (460 G > A), ⁎3C (719 A > C) and, most common, ⁎3A (460 G > A and 719 A > C in cis), which all result in decreased TPMT activity [2]. The TPMT genotype to phenotype correlation is quite high, but not 100% [3], [4], [5], [6]. This may be the consequence of the presence of rarer mutations in the TPMT gene, as well as of other TPMT activity-regulating factors. It is therefore of great importance to identify factors, other than TPMT genotype, that may influence TPMT activity.
One such promising novel factor is SAM and its analogue from Pseudomonas syringae, sinefungin, which have been shown to prevent degradation of the variant TPMT enzyme in vitro, by stabilizing its native structure [7], [8]. Thus, TPMT activity could be affected by endogenous SAM levels and factors influencing SAM synthesis, including 5,10-methylenetetrahydrofolate reductase (MTHFR) (EC 1.5.1.20) and thymidylate synthase (TYMS) (EC 2.1.1.45) (Fig. 1).
MTHFR catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyl-tetrahydrofolate, the methyl donor in the biosynthesis of SAM precursor methionine from homocysteine (Fig. 1). Consequently, SAM concentration in vivo might be affected by MTHFR activity. The MTHFR gene is polymorphic, with two most common variants being 677 C > T and 1298 A > C, which both result in lower enzyme activity. The two polymorphisms are rarely present at the same allele (in cis) [9]. Recently, it has been shown, that the 677 TT genotype results in lower SAM concentrations than the CC genotype and that this difference is more pronounced under conditions of folate restriction [10].
TYMS is one of key enzymes controlling DNA replication. It catalyzes the transformation of dUMP to dTMP, which is coupled with the conversion of one of the SAM precursors, 5,10-methylenetetrahydrofolate, back to dihydrofolate (Fig. 1). The TYMS promoter region is polymorphic. It contains double or triple tandem repeats of a 28 bp sequence. The double repeat results in lower gene expression than the triple repeat [11]. The homozygous triple repeat genotype (TR 3/3) is associated with reduced levels of SAM precursor 5-methyl-tetrahydrofolate [12], [13].
To investigate the effect of TPMT, MTHFR and TYMS genotypes on TPMT activity, we measured RBC TPMT activity and genotyped 86 healthy individuals. We hypothesized that a low activity MTHFR genotype and/or high activity TYMS genotype would result in decreased synthesis of 5-methyl-tetrahydrofolate, decreased production of methionine and consequently SAM, thus leading to decreased TPMT activity.
Section snippets
Study subjects
Venous blood was obtained from 95 healthy Slovenian university students, who were at the time undergoing an annual physical examination. Nine subjects were later excluded from the study due to unsuccessful genotype analysis. The final study group consisted of 86 healthy Caucasians, 55% male and 45% female, aged from 18 to 27 years (mean age 19.7 ± 1.4). In one subject gender and in four subjects age was unknown. The study was approved by ethical committee of Slovenia and written informed consent
Results
TPMT enzyme activity was measured in 86 healthy individuals. It ranged from 7.3 to 28.5 (mean 15.5 ± 4.6) pmol/107 RBC/h.
Genotype analysis of TPMT, MTHFR and TYMS genes was performed in all study subjects. Nine percent of participants were heterozygous for one of the three most common TPMT mutated alleles (⁎3A, ⁎3C or ⁎3B), while 91% were wild-type. Genotype frequencies and linkage data are presented in Table 1. High values of D′ and r2 indicate that mutations ⁎3B and ⁎3C are present together in
Discussion
The distribution of TPMT, MTHFR and TYMS genotypes reported in our study was in agreement with published results on Caucasian populations [9], [14], [15].
The TPMT genotype to phenotype correlation is strong, but not complete [3], [4], [5], [6], due mostly to the lack of concordance between genotype and phenotype in the heterozygous range. We propose that genetic variation in folate metabolism genes, such as MTHFR and TYMS, also influences TPMT activity and contributes to the overlap of
Acknowledgments
We thank Ursa Zager and Ana Marta Pacheco de Sousa for technical assistance and Professor Roger Pain for advice on the English language.
References (28)
- et al.
The relationship between thiopurine methyltransferase activity and genotype in blasts from patients with acute leukemia
Blood
(1998) - et al.
Ethnic variation in the thymidylate synthase enhancer region polymorphism among Caucasian and Asian populations
Genomics
(1999) - et al.
Determination of thiopurine S-methyltransferase (TPMT) activity by comparing various normalization factors: reference values for Estonian population using HPLC-UV assay
J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci.
(2006) - et al.
Determinants of plasma total homocysteine concentration in the Framingham Offspring cohort
Am. J. Clin. Nutr.
(2001) - et al.
Major lifestyle determinants of plasma total homocysteine distribution: the Hordaland Homocysteine Study
Am. J. Clin. Nutr.
(1998) - et al.
Interaction between dietary methionine and methyl donor intake on rat liver betaine-homocysteine methyltransferase gene expression and organization of the human gene
J. Biol. Chem.
(1999) - et al.
Betaine-homocysteine methyltransferase-2: cDNA cloning, gene sequence, physical mapping, and expression of the human and mouse genes
Genomics
(2000) - et al.
Human thiopurine methyltransferase pharmacogenetics: gene sequence polymorphisms
Clin. Pharmacol. Ther.
(1997) - et al.
Human thiopurine S-methyltransferase pharmacogenetics: variant allozyme misfolding and aggresome formation
Proc. Natl. Acad. Sci. U. S. A.
(2005) - et al.
Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine
J. Clin. Oncol.
(2001)
Analysis of thiopurine methyltransferase variant alleles in childhood acute lymphoblastic leukaemia
Br. J. Haematol.
Thiopurine S-methyltransferase pharmacogenetics: genotype to phenotype correlation in the Slovenian population
Pharmacology
Consequences of binding an S-adenosylmethionine analogue on the structure and dynamics of the thiopurine methyltransferase protein backbone
Biochemistry
Enhanced proteolysis of thiopurine S-methyltransferase (TPMT) encoded by mutant alleles in humans (TPMT⁎3A, TPMT⁎2): mechanisms for the genetic polymorphism of TPMT activity
Proc. Natl. Acad. Sci. U. S. A.
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