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Keywords:

  • methionine synthase;
  • MTHFR;
  • polymorphisms;
  • non-Hodgkin's lymphoma;
  • multiple myeloma

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgments
  6. References

Summary. Lymphoproliferative diseases are characterized by chromosomal aberrations, and susceptibility may depend on inherited activity of enzymes required for DNA synthesis and methylation. We analysed genetic polymorphisms for methionine synthase (MS) A2756G, methylenetetrahydrofolate reductase (MTHFR) C677T and MTHFR A1298C in Caucasians with non-Hodgkin's lymphoma (NHL; n = 151), multiple myeloma (MM; n = 90) and 299 control subjects. The MS 2756 AG/GG genotypes were significantly under-represented in NHL (26·2%) vs control subjects (37·2%; P = 0·02), and conferred a 2·4-fold lower risk of follicular (odds ratio = 0·41, 95% confidence interval: 0·19–0·88, p = 0·02) but not diffuse large B-cell lymphoma. MM patients showed no significant difference in the polymorphisms compared with control subjects.

Susceptibility to genomic damage has been linked to folate deficiency as this metabolic pathway is important for both nucleotide synthesis and DNA methylation. The enzymes methylenetetrahydrofolate reductase (MTHFR) and methionine synthase (MS) play key roles in these processes and both genes display single nucleotide polymorphisms (MTHFR C677T, MTHFR A1298C and MS A2756G) that decrease the enzymatic activity of their respective encoded proteins (Frosst et al, 1995; Leclerc et al, 1996; van der Put et al, 1998). Reduction in MTHFR activity facilitates thymidylate synthesis, lowering the incidence of uracil misincorporation and effectively decreasing the risk of chromosomal damage caused during subsequent excision and repair processes (Blount et al, 1997). In support of this theory, MTHFR polymorphic variants have been associated with a decreased risk of colon cancer (Ma et al, 1997), NHL (Matsuo et al, 2001) and acute leukaemia (Skibola et al, 1999). In addition, MTHFR and MS activity can affect gene expression by controlling the amount of methionine available for DNA methylation. Thus, we hypothesized that polymorphisms which decrease MTHFR and/or MS enzyme activity would also lessen the risk of chromosomal translocations and DNA hypermethylation that are common features of lymphoproliferative diseases. We, therefore, conducted a retrospective study of these polymorphisms using DNA from stored tissue samples from patients with confirmed diagnoses of multiple myeloma (MM) or non-Hodgkin's lymphoma (NHL), and compared the results with a group of normal healthy control subjects.

Subjects.  The study population consisted of 241 Caucasian individuals diagnosed with either MM (n = 90, 62 men and 28 women, median age = 47 years, range 41–95 years), according to the United Kingdom Medical Research Council (MRC UK) definition, or non-Hodgkin's lymphoma (n = 151, 82 men and 69 women, median age = 62 years, range 16–89 years), grouped according to the revised European–American classification of lymphoid neoplasms, as diffuse large B-cell (DLBC; n = 74), follicular (FL; n = 48), small lymphocytic (n = 16), T-cell (n = 10) or mantle cell (n = 3) lymphoma. The control group consisted of 299 healthy Caucasian volunteer bone marrow donors aged 18–65 years. The research protocol was approved by the Hunter Area Health Service Research Ethics Committee and informed consent was received from all living participants.

Genotyping.  DNA was extracted from frozen bone marrow mononuclear cell pellets (MM, n = 90; NHL, n = 61) or lymph node tissue biopsy samples (NHL, n = 90), and genotyped for MTHFR C677T, MTHFR A1298C and MS A2756G by polymerase chain reaction (PCR)-restriction fragment length polymorphism using previously published methods (Frosst et al, 1995; Skibola et al, 1999; Matsuo et al, 2001). All PCRs were performed in a microtube thermocycler (Corbett Research, Sydney, Australia), all oligonucleotide primers were synthesized by Geneworks (Adelaide, Australia), and all buffers and enzymes were purchased from Promega (Madison, WI, USA).

Statistical analysis.  Odds ratios (OR) and 95% confidence intervals (CI) were calculated to express the relative risk of disease associated with a specific genotype. Genotype frequencies were compared using the X2 test, and differences were considered statistically significant when P < 0·05. All statistical analyses were performed using statistica v.4·1 software (StatSoft, Tulsa, OK, USA).

Results and discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgments
  6. References

The variant allele frequencies for MTHFR 677T, MTHFR 1298C and MS 2756G in our control group were calculated to be 0·292, 0·341 and 0·206, respectively, and all genotypes were in accordance with the Hardy–Weinberg equilibrium. The NHL population showed a significantly reduced incidence of MS 2756 AG/GG genotypes (26·2% for NHL vs 37·2% for control subjects, P = 0·02), but no difference from control frequencies for MTHFR C677T and MTHFR A1298C polymorphisms (Table I). In contrast, the MM group showed no significant difference from the control subjects for any of the three polymorphisms. When the NHL group was analysed by histological subtype, the MS 2756 AG/GG genotypes were found to confer a 2·4-fold lower risk of FL (OR = 0·41, 95% CI 0·19–0·88, P = 0·02) but no protective effect for DLBC (Table II). For the latter NHL subtype, the frequencies of all three polymorphisms were similar to those of control subjects. Thus we found the MS 2756 AG/GG low-activity genotypes to be protective against FL, but not DLBC or MM.

Table I.  Comparison of MTHFR C677T, MTHFR A1298C and MS A2756G polymorphism frequencies in control subjects and patients with NHL and MM.
Polymorphism/genotypeControl subjects (n = 299) All NHL patients (n = 151) vs control subjects MM patients (n = 90) vs control subjects
n*(%)n*(%)OR(95% CI)Pn*(%)OR(95% CI)P
  • *

    Totals vary as a result of incomplete genotyping data for some individuals.

MTHFR 677 CC145(48·5)73(49·3)1·00reference38(42·2)1·00reference
CT133(44·5)58(39·2)0·87(0·57–1·32)0·5044(48·9)1·26(0·77–2·07)0·35
TT21(7·0)17(11·5)1·61(0·80–3·23)0·188(8·9)1·45(0·60–3·57)0·41
CT/TT154(51·5)75(50·7)0·97(0·65–1·43)0·8752(57·8)1·29(0·80–2·07)0·29
MTHFR 1298AA124(42·2)64(44·1)1·00reference29(35·8)1·00reference
AC139(47·3)68(46·9)0·95(0·62–1·44)0·8043(53·0)1·32(0·78–2·25)0·30
CC31(10·5)13(9·0)0·81(0·40–1·66)0·579(11·2)1·24(0·53–2·89)0·62
AC/CC170(57·8)81(55·9)0·92(0·62–1·38)0·6952(64·2)1·31(0·79–2·18)0·30
MS 2756AA187(62·8)110(73·8)1·00reference51(63·8)1·00reference
AG99(33·2)34(22·8)0·58(0·37–0·92)0·0225(31·3)0·93(0·54–1·58)0·78
GG12(4·0)5(3·4)0·71(0·24–2·06)0·524(5·0)1·22(0·38–3·95)0·73
AG/GG111(37·2)39(26·2)0·60(0·39–1·08)0·0229(36·3)0·96(0·57–1·60)0·87
Table II.  Comparison of MTHFR C677T, MTHFR A1298C and MS A2756G polymorphism frequencies in control subjects and patients with NHL FL and NHL DLBC.
Polymorphism/genotypeControl subjects (n = 299) FL patients (n = 48) vs control subjects DLBC patients (n = 74) vs control subjects
n*(%)n*(%)OR(95% CI)Pn*(%)OR(95% CI)P
  • *

    Totals vary as a result of incomplete genotyping data for some individuals.

MTHFR 677 CC145(48·5)21(44·7)1·00reference37(51·4)1·00reference
CT133(44·5)20(42·5)1·04(0·53–2·00)0·9125(34·7)0·74(0·42–1·29)0·28
TT21(7·0)6(12·8)1·97(0·71–5·45)0·1810(13·9)1·87(0·81–4·30)0·10
CT/TT154(51·5)26(55·3)1·17(0·63–2·16)0·6235(48·6)0·89(0·53–1·49)0·66
MTHFR 1298AA124(42·2)22(50·0)1·00reference31(44·3)1·00reference
AC139(47·3)19(43·2)0·77(0·40–1·49)0·4431(44·3)0·89(0·51–1·55)0·68
CC31(10·5)3(6·8)0·55(0·15–1·94)0·348(11·4)1·03(0·43–2·47)0·95
AC/CC170(57·8)22(50·0)0·73(0·39–1·38)0·3339(55·7)0·92(0·54–1·55)0·75
MS 2756AA187(62·8)37(80·4)1·00reference51(68·9)1·00reference
AG99(33·2)9 (19·6)0·46(0·21–0·99)0·0419(25·7)0·70(0·39–1·26)0·23
GG12(4·0)0(0·0)0·000·000·124(5·4)1·22(0·38–3·95)0·73
AG/GG111(37·2)9 (19·6)0·41(0·19–0·88)0·0223(31·1)0·76(0·44–1·31)0·32

Methylation of the CgP islands in the 5′ promotor region of genes involved in tumour suppression, cell cycle regulation, DNA repair and apoptosis is a common method of gene silencing that is essential for neoplastic cell proliferation (Rhee et al, 2002). It has recently been shown that both the overall number and particular profile of methylation sites are correlated with cancer cell type, such that haematological malignancies have a high frequency of hypermethylation of the tumour suppressor genes p73 and p15INK4b that is rarely observed in solid tumours (Esteller et al, 2001). Differential DNA methylation status between NHL subtypes has not been thoroughly investigated, but is likely to reveal particular variances between classifications. An independent study has shown that the number of hypermethylated CpG islands in tumour tissue from cancer patients correlates with inheritance of the MS 2756A allele, suggesting that germline variation in this gene may predispose individuals to cancers that are characterized by high numbers of DNA methylation sites (Paz et al, 2002). Studies in colorectal cancer support this theory (Ma et al, 1999; Esteller et al, 2001) and our results suggest that a similar mechanism of carcinogenesis may apply to FL.

Our study showed no correlation between MM and any of the polymorphisms studied. The possible association with the MS A2756G variant has not been previously investigated, but our results for the MTHFR C677T and MTHFR A1298C polymorphisms are in accordance with a recently published Spanish study (Gonzalez-Fraile et al, 2002). Together, these results argue against a role for folate metabolism in the pathogenesis of MM in Caucasians. However, investigations into the risk of colon cancer have shown that the protective effect of the MTHFR C677T polymorphism can be negated by a low dietary intake of folate (Ma et al, 1997). It is, therefore, possible that the addition of folate to many foods in Westernised countries may mask the impact of differential enzyme activities along this pathway. Thus a role for folate metabolism in the pathogenesis of MM cannot be excluded until further studies are done which incorporate measures of dietary intake of folate, family history and environmental exposures.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgments
  6. References

This study was funded in part by a research grant from the Newcastle Mater Misericordiae Hospital Margaret Mitchell Grant Scheme. A.S. is supported by the Anderson Trust and R.P.′s participation in the project was made possible through the Bannerman Summer Scholarship. The authors would like to thank Rhonda Holdsworth for assistance in providing control samples and Dr Philip Rowlings for help with statistical analysis.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results and discussion
  5. Acknowledgments
  6. References
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