A relationship between methylenetetrahydrofolate reductase variants and the development of invasive cervical cancer

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Gynecologic Oncology 90 (2003) 560 –565

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A relationship between methylenetetrahydrofolate reductase variants and the development of invasive cervical cancer D.S. Gerhard,a,b,* L.T. Nguyen,a Z.Y. Zhang,c I.B. Borecki,a,d B.I. Coleman,a and J.S. Radera,c a

Department of Genetics, Washington University School of Medicine, St. Louis, MO 63110, USA Department of Psychiatry, Washington University School of Medicine, St. Louis, MO 63110, USA c Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO 63110, USA d Department of Biostatistics, Washington University School of Medicine, St. Louis, MO 63110, USA b

Received 21 August 2002

Abstract Objective. Low red blood cell folate levels have been associated with hypomethylation of DNA in dysplastic tissue and an increased risk for cervical intraepithelial neoplasia in human papillomavirus (HPV)-infected women. Methylenetetrahydrofolate reductase (MTHFR) is a critical enzyme regulating the metabolism of folate and methionine, the important components of DNA synthesis and methylation. Two common genetic polymorphisms, causing reduced MTHFR activity, have been identified. Therefore, the goal of this study was to evaluate these MTHFR variations as risk factors for invasive cervical cancer. Methods. To overcome the failure to properly match cases and controls that can cause false-positive inferences due to population stratification and unrecognized variables in a traditional case– control study, a family-based transmission/disequilibrium test (TDT) was used. We obtained samples from nuclear families of 102 women with invasive cervical cancer (ICC). One polymorphism was typed by a PCR-RFLP method, while a template-directed dye-terminator assay was developed for the other. Results and conclusions. We were unable to confirm a strong association of MTHFR polymorphisms and ICC using family-based controls and a transmission/disequilibrium test. The overall results of the TDT showed ␹2 (1 df) of 0.28 (P ⫽ 0.60) for exon 4, ␹2 (1 df) of 0.81(P ⫽ 0.37) for exon 7, and ␹2 (3 df) of 2.56 (P ⫽ 0.46) for the haplotype, meaning that there was no transmission of those alleles significantly in excess of Mendelian expectations to affected women. In addition, there was no effect of these variants with increased parity or infection with high-risk-type human papillomavirus. © 2003 Elsevier Inc. All rights reserved.

Introduction Invasive cervical cancer (ICC) is one of the most common malignancies of women worldwide. It is second only to breast cancer as the most common malignancy in both incidence and mortality [1]. Infection by human papillomavirus (HPV) increases a woman’s risk for the development of dysplasia and cancer [2,3]; however, it is not sufficient for the development of ICC [4]. Other risk factors have been

* Corresponding author. Office of Cancer Genomics; NCI; Building 31, Room 10A07; Bethesda, MD 20892-2582, USA. Fax: ⫹1-301-480-4368. E-mail address: [email protected] (D.S. Gerhard). 0090-8258/03/$ – see front matter © 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0090-8258(03)00368-8

identified, including smoking, contraceptive use, low socioeconomic status, and HLA class II subtypes, [1,5–7]. In the 1970s, Whitehead and colleagues noted that megaloblastic changes (nuclear enlargement) in cervical cytological specimens, characteristic of folate deficiency, could be reversed by administration of folic acid [8]. However, two epidemiological intervention trials evaluating folic acid supplementation failed to show regression of cervical intraepithelial neoplasia (CIN) or inhibit progression (reviewed in [9]). An effect of folate in the later stages of tumorigenesis was not supported by the few case– control studies of ICC [9]. A study of colposcopy-directed biopsies from women with abnormal Pap smears showed increased hypomethylation of DNA associated with the grade of cervical

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neoplasia from low-grade to invasive cancer [10]. Fowler and colleagues demonstrated that dysplastic tissue is hypomethylated when compared with normal tissue, although they did not find an increase in hypomethylation with increased grades of CIN [11]. They also found a correlation between cervical tissue and serum folate levels together with hypomethylation, although neither hypomethylation nor folate status was associated with smoking, parity, and HPV infection. Rosl et al. demonstrated that integration of HPV DNA into host cells is facilitated by hypomethylation at specific promoter sites and that DNA methylation is an important regulatory pathway in the modulation of HPV expression [12]. Low tissue folate levels increase the frequency of fragile sites on DNA [13]. Popescu et al. showed that HPV-18 integrates preferentially near known folate sensitive fragile sites, thereby implicating suboptimal cellular folate levels and the methylenetetrahydrofolate reductase (MTHFR) polymorphisms as risk factors for ICC [14]. The interest in methylation as an important mechanism in tumor progression has led to the evaluation of enzymes in the folate metabolic pathways. MTHFR is a critical enzyme in both DNA synthesis and methylation, and thereby it affects DNA stability and gene expression. MTHFR is a flavoprotein dimer responsible for generating circulating form of folate, 5-methyltetrahydrofolate (5-methylTHF), by reduction from 5,10-methylenetetrahydrofolate (5,10-methyleneTHF). The 5,10-methyleneTHF is a substrate for purine and thymidine synthesis. 5-MethylTHF is necessary for methionine synthesis, which in turn is a substrate for Sadenosyl methionine (SAM). SAM is the universal donor in methyl transfer reactions including those of DNA. High MTHFR function leads to low levels of 5,10-methyleneTHF, thereby causing dUTP incorporation into DNA, which in turn results in double strand breaks [15]. The gene for MTHFR is on chromosome 1p36, has 11 exons [16] and a number of single nucleotide polymorphisms (SNPs) within the coding region. Four SNPs change the protein’s amino acids, while another four are synonymous substitutions (dbSNP, http://www.ncbi.nlm.nih.gov/ SNP/snp_ref.cgi?locusId ⫽ 4524). Two of the SNPs are variants that influence MTHFR’s function. One, a C to T transition at nucleotide 677 (C677T), changes an alanine at position 222 to valine and affects the catalytic domain of the enzyme [17]. This form of the protein is thermolabile and has a reduced activity [18]. The enzyme levels are 70% lower than the common form and individuals homozygous and heterozygous for this mutation have an increased amount of homocysteine [19]. Folate acts to stabilize the protein and neutralize the effect of the variant. The frequency of the variant is heterogeneous in the different regions of the world; the lowest frequency is found in sub-Saharan Africa and northern Canada, while the highest is in peoples of Southern Europe and South America [5,20 – 22]. The second variant is an A to C transversion at position 1298 (A1298C) which changes a glutamine to alanine at position 429 [23]. This amino acid is within the regulatory

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domain of the protein [24]. By itself it does not seem to affect the enzyme’s function, but when it is heterozygous in trans with the 677T allele, the enzymatic levels of MTHFR are reduced [23,25]. A positive, monotonic trend in the odds ratio for cervical squamous intraepithelial lesions associated with the number of variant MTHFR T alleles, after multivariate adjustment, was reported [26]. A study by Giuliano and co-workers also found an increased risk in women with at least 1 MTHFR T allele resulting in an odds ratio (OR) of 2.9 (95% confidence interval 1.2–7.9) [27]. The risk was even higher in women who have had children (OR ⫽ 23, 95% confidence interval 2.3–225, P ⫽ 0.02). Pregnancy stresses folate status and could therefore increase the risk of DNA damage and cause cancer. HPV infection stayed a strong risk factor for cervical dysplasia, particularly among women with the variant T allele. Here we report the examination of the two MTHFR variants affecting its function in a set of 102 parent–ICC patient trios by the transmission/disequilibrium test (TDT). We did not find evidence of excess transmission to the affected women of a particular allele at either of the two polymorphic sites we examined. These results were not altered when we restricted our attention to families whose probands had high-risk HPV infections or who were parous. Therefore, our data do not provide supporting evidence that either of the MTHFR variants significantly effects the susceptibility alleles for cervical cancer.

Material and methods Subjects Women undergoing treatment for ICC in the Division of Gynecologic Oncology at Washington University in St. Louis were recruited to the study. Eligibility for study enrollment included living parents who were willing to participate. The majority of the individuals were described in a study assessing HLA class II involvement of ICC risk [6]. Blood was obtained from the proband and her parents whenever possible. If participants were unable or unwilling to give blood, a buccal cell sample was obtained. These samples were acquired by either of two ways: (1) scraping the inner cheek for 5 s with a cytology brush and then washing off the cells in a tube with 10 ml 10 mM Tris–Cl, pH 8.0, or (2) washing the inner checks with ⬃40 ml of Scope mouthwash (Proctor and Gamble) and collecting the solution in a 50-ml conical tube. The Human Studies Committee at Washington University approved this study. HPV typing Tissue was obtained from formalin-fixed, paraffin-embedded or frozen OCT-embedded (a mixture of polyvinyl alcohol and polyethylene glycol) cervical specimens from

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the patients. HPV typing was done by PCR amplification of the E6-E7 gene using the degenerate consensus primers pU-1M/pU-2R and restriction fragment length polymorphism analysis (RFLP) as previously described [6]. MTHFR genotyping Blood or buccal cells were obtained from 102 patients and both biological parents whenever possible or one parent and any sisters. The DNA was extracted by a proteinase K digestion, phenol/chloroform extraction, and ethanol precipitation [28]. The DNA of the patients and their parents was set up in a 96-well tray format and the PCR, restriction digests, and gel electropheresis were performed with multichannel pipets. The C677T genotyping (dbSNP rs1801133, http://www.ncbi.nlm.nih.gov/SNP/) was done by a PCR-RFLP protocol essentially as reported [19], except that we designed new primers by Oligo 3.0 (http:// www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). The PCR was performed with 1.25 ␮M of MTHFRe45 5⬘tccctgtggtctcttcatcc-3⬘ and MTHFRe43 5⬘-caaagcggaagaatgtgtca-3⬘ primers, 1.5 mM MgCl2 standard buffer, and 0.7 U of Taq polymerase in 10 ␮l vol for 35 cycles. The DNA was first denatured at 94°C for 2 min and then amplified at 92°C for 30 s, 55°C for 1 min, and 72°C for 1 min, repeated 35 times. A total of 2 ␮l of each reaction was electrophoresed in 2% 3:1 NuSieve agarose to determine the presence of a product. When a product was seen, the rest of the reaction was digested overnight at 37°C with 2 U of HinfI (New England Biolabs). The 677T HinfI digested fragments were 150 and 56 bp, while the 677C was 206 bp. In each tray 2 of 96 DNAs were controls (either CEPH parents or parents from large multigenerational families) and 34 patient or parent samples were genotyped twice. The repeated genotypes were identical. The A1298C SNP (dbSNP rs1801131, http://www. ncbi.nlm.nih.gov/SNP/) had a confusing pattern when digested with MboII [29]; therefore, we developed a templatedirected dye incorporation assay for this locus [30]. Briefly, 5 ng of genomic DNA was amplified with 0.125 ␮M of MTHFRe75 5⬘-gagtgggacgagttccctaa-3⬘ and MTHFRe73 5⬘-tttggttctcccgagaggta-3⬘ in 1.5 mM MgCl2 standard PCR buffer. The cycling conditions were the same as for exon 4, except that the reaction was done for 40 cycles in a final volume of 7 ␮l using 50 ␮M of nucleotides and 0.25– 0.5 U of Platinum Taq polymerase (Invitrogen, Inc.). Two microliters of each reaction was electrophoresed in 2% 3:1 NuSieve agarose to determine the presence of a 302-bp product. When a product was seen, unincorporated nucleotides and primers were digested with 0.1 U of exonuclease I (USB) and 0.1 U of shrimp alkaline phosphatase (USB) at 37°C for 30 min and then these enzymes were heat inactivated at 80°C for 15 min. To each product was added primer that abuts the polymorphic site, MTHFRe7R 5⬘GAACRAAGACTTCAAAGACACTT-3⬘, to a final concentration of 0.25 ␮M. The primer is a mixed population; at the fifth position 50% of the molecules have an A and the

other 50% have a G. This is a silent polymorphism [29]. We used the AcycloPrime-FP SNP Detection kit (PerkinElmer Life Sciences, Boston, MA) with R110 –acyG and TamraacyT labeled derivatized Acyclo terminators. The template was first denatured at 95°C for 2 min and then amplified at 95°C for 15 s and 55°C for 30 s. The reaction proceeded for 45– 60 cycles depending on the efficiency of dye incorporation. The dye incorporation was determined on an LJL Analyst fluorescence reader (Molecular Devices, Sunnyvale, CA). The nucleotide calls were determined using a macro written by B. Coleman for this purpose. In each tray were 2 control DNAs and 87 patient or parent samples were genotyped twice. The repeated genotypes were identical. Analysis One hundred two families, each ascertained through a woman with ICC, were genotyped. In 30 families, the father was either unavailable or biological paternity could be excluded on the basis of mostly the HLA genotypes [6]; the mothers were unavailable in 6 families. Siblings were routinely typed to provide additional information for the reconstruction of parental genotypes and haplotypes, maximizing the segregation information in the trios. The genotypes of trios (an affected daughter with both parents) were analyzed using a TDT, as described in [31]. The TDT enables an evaluation of the association between specific alleles at a candidate locus and ICC. Since the family trios are ascertained through an ICC patient, a risk allele would be expected to segregate to these affected offspring more often than the alternative allele from heterozygous parents. Under the null hypothesis, if the allele has no impact on disease risk, the allele would simply segregate to affected offspring 50% of the time, consistent with Mendelian expectations. An association between a putative risk allele and the disease is supported when the allele appears significantly more than 50% of the time in these subjects. A ␹2 [32] test is used that is equivalent to McNemar’s ␹2 [32] for diallelic loci. The test is slightly different in the case of haplotype analysis: the global null hypothesis that none of the haplotypes is associated with ICC is tested, which conserves power because multiple testing of each allele in turn is avoided. Thus, the test of the diallelic SNPs is 1 degree of freedom ␹2, and the exons 4/7 haplotype tests have 3 degrees of freedom, since there are four possible haplotypes. Calculations were carried out by the computer program TRANSMIT [31]; significance levels were checked by bootstrap simulations when the expected values for certain haplotypes were small. The disequilibrium between the exon 4 and 7 polymorphisms was assessed as the correlation among alleles at the adjacent sites using the formula: r2 ⫽ (␲AB ⫺ ␲A␲B)2/ (␲A␲a␲B␲b), where ␲A and ␲a are the frequencies of the two alleles at the first locus, ␲B and ␲b are the frequencies of the two alleles at the other locus, and ␲AB is the estimated haplotype frequency [33].

D.S. Gerhard et al. / Gynecologic Oncology 90 (2003) 560 –565 Table 1 Clinical characteristics of patients Mean age of probands Caucasian, non-hispanic Other Tumor histology Squamous Adenocarcinoma Adenosquamous Neuroendocrine FIGO stage I II III IV Parity 0 ⬍2 ⬎2 HPV subtype 16 HPV subtype 18 HPV subtype 31 HPV subtype 33 HPV subtype other HPV multiple subtypes HPV negative Unknown virus status

35 (21–55) years 92% 8% 77% 13% 5% 5% 84% 11% 4% 1% 23.4% 53.2% 23.4% 53 (66.3%) 8 (10.0%) 7 (8.8%) 3 (3.8%) 1 (1.2%) 7 (8.8%) 15 (18.8%) 22

Note. Only 80 patients had HPV typing performed; the rest were either missing the tissue or the lab procedure failed. Some tumors have multiple HPV types and therefore the numbers add up to over 80.

Results The clinical characteristics of the subjects are given in Table 1. The allele frequencies are as follows: 677C 70.4% and 677T 29.6%; 1298A 66.7% and 1298C 33.3%. The frequency of the haplotypes is given in Table 2. The double variant haplotypes are much rarer than would be expected given their respective allele frequency, r2 ⫽ 0.183. The double variant haplotype is 16.5 times less frequent that would be expected if the two alleles were in linkage equilibrium (see Discussion). The overall TDT results were ␹2 (1 df) ⫽ 0.28 (P ⫽ 0.60) for exon 4, ␹2 (1 df) ⫽ 0.81 (P ⫽ 0.37) for exon 7, and ␹2 (3 df) ⫽ 2.56 (P ⫽ 0.46) for the haplotype. When the patient sample was divided by the parity of the proband analyzing only those families in which the affected woman had at least one pregnancy yielded a ␹2 (3 df) of 2.65 (P ⫽ 0.45). Similarly, considering only families in which the proband had a high-risk HPV infection yielded a ␹2 (3 df) of 2.30 (P ⫽ 0.51) analyzing the haplotypes for the two polymorphisms.

red blood cell folate levels have been associated with an increased risk for development of ICC in HPV-infected women. In addition, two reports [27,28] found an association between CIN and the T allele of MTHFR’s exon 4 in women from Hawaii and Arizona. Therefore, we tested the hypothesis that the MTHFR variants are associated with ICC. For the analysis, the family-based transmission/disequilibrium test was used on genotypes of both MTHFR polymorphisms, either of which can lead to decreased activity of the enzyme. Our results do not support a strong association for either, or both, of the MTHFR polymorphisms and ICC. However, we cannot exclude the possibility of a weak effect given our sample size. A number of studies have reported an association of the MTHFR variants and the modification of risk of tumor development. Skibola et al. reported a decreased frequency of either of the two homozygous variant alleles (i.e. 677TT or 1298CC) in patients with acute lymphocytic leukemia, although not in acute myeloid leukemia [34]. A similar decrease of the T allele was reported by a number of investigators for colorectal cancer (reviewed in [35]). Most of the studies, though, were not large enough to detect effects twofold in size, while one report [36] may have found an effect of the MTHFR mutations that was the result of population stratification. However, the meta analysis of the combined colorectal data could not reject the trend of decreased 677TT frequency in the patients with cancer [35]. In contrast, Song et al. reported a sixfold increase in risk of developing esophageal squamous cell carcinoma in patients that were homozygous for the 677T allele compared to those homozygous for the 677C allele in a large Chinese case– control study [37]. Furthermore, they reported an elevated risk for cancer development associated with the 677 variant in an allele– dose relationship. Finally, they found an indication of increased risk with the 1298C mutation, although this effect was smaller (the authors suggest that it was due to the lower allele frequency of 1298C in the Chinese population). In a case– control study of Chinese gastric cancers, Shen et al. found a slightly increased risk of tumor development when the patients were 677TT, but not when the patients were 1298CC [38]. Our study was done using family-based controls, thereby greatly reducing the risk of false-positive results attributable to population or ethnic stratification. However, a limitation of the present study is that it is adequately powered to detect Table 2 Expected and observed haplotype frequencies of two variants in MTHFR Position 677

Discussion The goal of this study was to evaluate two MTHFR polymorphisms as risk factors for cervical cancer since low

563

C C T T

Expected frequency

Observed frequency

0.243 0.470 0.099 0.197

0.330 0.372 0.006 0.290

1298 C A C A

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associations of moderate or large effect; modest effects would be detected at a low rate in studies of this size. The exact power of the TDT is a function of many parameters including the frequency of the susceptibility allele, its mode of inheritance, the size of its effect on disease susceptibility, and its disequilibrium relationship with the true susceptibility locus if the polymorphism itself is not a functional variant. Assuming the observed frequency of approximately 30% for the minor allele, and that this is the functional susceptibility variant, we would be able to detect an association at a 5% significance level having a minimum genotype relative risk of ⬃2.5–3 with this sample, depending on the assumed genetic model. Thus, this study is adequately powered for our objective of finding risk alleles with effects that are potentially clinically important, although we may fail to detect the effect of alleles with modest effect on disease risk. Previously, we have detected and reported and association with HLA in a smaller sample size [6] using the TDT approach, demonstrating the feasibility of achieving our objective. The question remains of how to explain the previous positive association results with CIN. The experiments of Piyathilake and colleagues [27] suffer from a lack of appropriate controls. First, they analyzed only 31 controls as opposed to 64 cases and, second, their controls are made up of 83.9% “non-Hispanics” while in the cases the “nonHispanics” constitute only 62.5%. There is a significant difference in allele frequencies between the exon 4 polymorphism in the world populations with allele T being higher in Hispanics than in the EuroAmerican population [20 –22]. Piyathilake et al. [27] considered this bias, but they argued that the increased risk found in women with one or more child should be real. The study by Goodman et al. [26] does not suffer from this stratification problem. Therefore, a possible explanation is that just like HPV infection, the lower levels of 5-methylTHF increase the risk for CIN, but not ICC. A second possible explanation is that the effect of the MTHFR variants is lower than previously reported and that additional studies may be warranted in the future. Isotalo and colleagues, in a study of MTHFR of 119 neonates and 161 fetal samples, did not find triple (677TT/ 1298C or 677T/1298CC) or quadruple (677TT/1298CC) variant combinations in the neonates, although they were present in the fetal samples [39]. The authors suggest that the triple and quadruple variant combinations may decrease the viability of fetuses and have a possible selection disadvantage with respect to fetuses with increased number of mutant MTHFR alleles. Rosenberg et al. reported that the 677T allele arose on a chromosome wild type for the 1298 mutation (1298A); thereby, one can infer that the 1298 mutation in MTHFR must have occurred independently of the 677 mutation and it arose on the 677C background [40]. Therefore, the chromosomes that have the 677T/1298C genotype probably arose by recombination within the 2.1-kb stretch that separates these two polymorphic sites. Two other studies also did not observe doubly homozygous in-

dividuals in their studies of 525 and 353 individuals, respectively [41,42]; they suggest that there is a selection against these individuals. We have one patient who has three of the lower frequency alleles that affect the protein’s function. Therefore, we can confirm that recombination occurs in the 2.1 kb of DNA that separates these two polymorphic sites, although we cannot provide evidence that cis combination of mutations completely compromises fetal viability. In summary, we examined the hypothesis that enzyme variants that influence the levels of 5-methylTHF (MTHFR), and thereby DNA methylation, could affect the risk of invasive cervical cancer. We genotyped the DNA from 102 trios for two variants affecting the enzyme’s function and performed the analysis with a state-of-the-art methodology of transmission/disequilibrium test in our study, using the nontransmitted parental alleles from the parents as the controls to avoid the problems of population stratification. We could not confirm a strong association of MTHFR polymorphisms and ICC. In addition, there was no disease association of these variants with increased parity or infection with high-risk-type human papillomavirus.

Acknowledgments We thank members of the Kwok laboratory for letting us use their equipment and providing us with advice when needed and Erin Watson for DNA extraction. This work was supported by grants from ACS (RSG-96-088-08-CCE) to JSR and DSG, NIH Grant CA094141 to DSG, and NIH Grant GM28719 supporting IBB.

References [1] Petro R. Introduction: geographic patterns and trends. In: Petro R, zur Hausen H, editors. Viral etiology of cervical cancer. New York: Cold Spring Harbor Laboratory press, 1986. pp. 3–15. [2] Bosch FX, Munoz N, Shah KV, Meheus A. Second international workshop on te epidemiology of cervical cancer and human papillomavirus. Int J Cancer 1992;52:171–3. [3] Schiffman MH, Bauer HM, Hoover RN, Glass AG, Cadell DM, Rush BB, Scott DR, Sherman ME, Kurman RJ, Wacholder S. Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 1993;85: 958 – 64. [4] Cain JM, Howett MK. Preventing cervical cancer. Science 2000;288: 1753–5. [5] Koutsky LA, Galloway DA, Holmes KK. Epidemiology of genital human papillomavirus. Epidemiol Rev 1988;10:122– 63. [6] Neuman RJ, Huettner PC, Li L, Mardis ER, Duffy BF, Wilson RK, Rader JS. Association between DQB1 and cervical cancer in patients with human papillomavirus and family controls. Obstet Gynecol 2000;95:134 – 40. [7] Syrja¨ nen S. Epidemiology of human papillomavirus infections and genital neoplasia. J Infect Dis Suppl 1990;69:7–17. [8] Whitehead N, Reyner F, Lindenbaum J. Megaloblastic changes in the cervical epithelium: association with oral contraceptive therapy and reversal with folic acid. J. Am. Med. Assoc. 1973;226:1421– 4.

D.S. Gerhard et al. / Gynecologic Oncology 90 (2003) 560 –565 [9] Eichholzer M, Luthy J, Moser U, Fowler B. Folate and the risk of colorectal, breast and cervix cancer: the epidemiological evidence. Swiss Med. Weekly 2001;131:539 – 49. [10] Kim YI, Giuliano A, Hatch KD, Schneider A, Nour MA, Dallal GE, Selhub J, Mason JB. Global DNA hypomethylation increases progressively in cervical dysplasia and carcinoma. Cancer 1994;74:893–9. [11] Fowler BM, Giuliano AR, Piyathilake C, Nour M, Hatch K. Hypomethylation in cervical tissue: is there a correlation with folate status. Cancer Epidemiol Biomarkers Prev 1998;7:901– 6. [12] Rosl F, Arab A, Klevenz B, zur Hausen H. The effect of DNA methylation on gene regulation of human papillomaviruses. J. Gen. Virol. 1993;74:791– 801. [13] Giuliano AR, Gapstur S. Can cervical dysplasia and cancer be prevented with nutrients. Nutr Rev 1998;56:9 –16. [14] Popescu NC, DiPaulo JA, Amsbaugh SC. Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes. Cytogenet Cell Genet 1987;44:58 – 62. [15] Blount BC, Mack MM, Wehr CM, MacGregor JT, Hiatt RA, Wang G, Wickramasinghe SN, Everson RB, Ames BN. Folate deficiency causes uracil misincorporation into human DNA and chromosome breakage: implications for cancer and neuronal damage. Proc Natl Acad Sci USA 1997;94:3290 –5. [16] Goyette P, Pai A, Milos R, Frosst P, Tran P, Chen Z, Chan M, Rozen R. Gene structure of human and mouse methylenetetrahydrofolate reductase (MTHFR). Mamm. Genome 1998;9:652– 6. [17] Guenther BD, Sheppard CA, Tran P, Rozen R, Matthews RG, Ludwig ML. The structure and properties of methylenetetrahydrofolate reductase from Escherichia coli suggest how folate ameliorates human hyperhomocysteinemia. Nat. Struct. Biol. 1999;6:359 – 65. [18] Yamada K, Chen Z, Rozen R, Matthews RG. Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase. Proc Natl Acad Sci USA 2001;98: 14853– 8. [19] Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3. [20] Botto LD, Yang Q. 5,10-Methylenetetrahydrofolate reductase gene variants and congenital anomalies: a HuGE review. Am. J. Epidemiol 2000;151:862–77. [21] Pepe G, Vanegas OC, Giusti B, Brunell T, Marcucci R, Attanasio M, Rickards O, DeStefano GF, Prisco D, Gensini GF, Abbate R. Heterogeneity in world distribution of the thermolabile C677T mutation in 5,10-methylenetetrahydrofolate reductase. Am. J. Hum. Genet. 1998; 63:917–20. [22] Stevenson RE, Schwartz CE, Du YZ, Adams MJ. Differences in methylenetetrahydrofolate reductase genotype frequencies between Whites and Blacks. Am. J. Hum. Genet. 1997;60:229 –30. [23] van der Put NMJ, Gabreels F, Stevens EMB, Smeitink JAM, Trijbels FJM, Eskes TKAB, van den Heuvel LP, Blom HJ. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects. Am J Hum Genet 1998;62: 1044 –51. [24] Summer J, Jencks DA, Khani S, Matthews RG. Photoaffinity labeling of methylenetetrahydrofolate reductase with 8-azido-S-adenosylmethionine. J. Biol. Chem. 1986;261:7697–700. [25] Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity. Mol Genet Metab 1998;64:169 –72.

565

[26] Goodman MT, McDuffie K, Hernandez B, Wilkens LR, Bertram CC, Killeen J, Le Marchand L, Selhub J, Murphy S, Donlon TA. Association of methylenetetrahydrofolate reductase polymorphism C677T and dietary folate with the risk of cervical dysplasia. Cancer Epidemiol Biomarkers Prev 2001;10:1275– 80. [27] Piyathilake CJ, Macaluso M, Johanning GL, Whiteside M, Heimburger DC, Giuliano A. Methylenetetrahydrofolate reductase (MTHFR) polymorphism increases the risk of cervical intraepithelial neoplasia. Anticancer Res 2000;20:1751– 8. [28] Radford D, Ashley SW, Wells SA, Gerhard DS. Loss of heterozygosity of markers on chromosome 11 in tumors from patients with multiple endocrine neoplasia syndrome type 1. Cancer Res. 1990;50: 6529 –33. [29] Sibani S, Leclerc D, Weisberg II, Rozen R. The silent T1317C mutation of methylenetetrahydrofolate reductase should not interfere with MboII restriction isotyping of the reported A1298C mutation. Mol Genet Metab. 1999;68:512. [30] Chen Z, Levine L, Kwok P-Y. Fluorescence polarization in homogeneous nucleic acid analysis. Genome Res 1999;9:492– 8. [31] Clayton D, Jones H. Transmission/disequilibrium tests for extended marker haplotypes 22. Am. J. Hum. Genet. 1999;65:1161–9. [32] Spielman RS, McGinnis RE, Ewens WJ. Transmission test for linkage disequilibrium: the insulin gene region and insulin-dependent diabetes mellitus (IDDM) 8. Am. J. Hum. Genet. 1993;52:506 –16. [33] Pritchard JK, Przeworski M. Linkage disequilibrium in humans: models and data. Am J Hum Genet 2001;69:1–14. [34] Skibola CF, Smith MT, Kane E, Roman E, Rollinson S, Cartwright RA, Morgan G. Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with susceptibility to acute leukemia in adults. Proc Natl Acad Sci USA 1999;96:12810 –5. [35] Houlston RS, Tomlinson IPM. Polymorphisms and colorectal tumor risk. Gastroenterology 2001;121:282–301. [36] Ma J, Stampfer MJ, Giovannucci E, Artigas C, Hunter DJ, Fuchs C, Willett WC, Selhub J, Hennekens CH, Rozen R. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer. Cancer Res 1997;57:1098 –102. [37] Song C, Xing D, Tan W, Wei Q, Lin D. Methylenetetrahydrofolate reductase polymorphisms increase risk of esophageal squamous cell carcinoma in a Chinese population. Cancer Res 2001;61:3272–5. [38] Shen H, Xu Y, Zheng Y, Qian Y, Yu R, Qin Y, Wang X, Spitz MR, Wei Q. Polymorphisms of 5,10-methylenetetrahydrofolate reductase and risk of gastric cancer in a Chinese population: a case– control study. Int. J. Cancer (Pred. Oncol.) 2001;95:332– 6. [39] Isotalo P, Wells G, Donnelly J. Neonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations. Am. J. Hum. Genet 2000;67:986 –90. [40] Rosenberg N, Murata M, Ikeda Y, Opare-Sem O, Zivelin A, Geffen E, Seligsohn U. The frequent 5,10-methylenetetrahydrofolate reductase C677T polymorphism is associated with a common haplotype in whites, Japanese, and Africans. Am. J. Hum. Genet 2002;70:758 – 62. [41] Chen J, Giovannucci E, Kelsey K, Rimm EB, Stampfer MJ, Colditz GA, Spiegelman D, Willett WC, Hunter DJ. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer. Cancer Res 1996;56:4862– 4. [42] Stegmann K, Ziegler A, Ngo ET, Kohlschmidt N, Schroter B, Ermert A, Koch MC. Linkage disequilibrium of MTHFR genotypes 677C/ T-1298A/C in the German population and association studies in probands with neural tube defects (NTD). Am J Med Genet 1999;87: 23–9.