[CANCER RESEARCH 62, 4519 – 4524, August 1, 2002] Germ-Line Variants in Methyl-Group Metabolism Genes and Susceptibility to DNA Methylation in Normal Tissues and Human Primary Tumors 1 Maria F. Paz, 2 Sonia Avila, 2 Mario F. Fraga, Marina Pollan, Gabriel Capella, Miquel Angel Peinado, Montserrat Sanchez-Cespedes, James G. Herman, and Manel Esteller 3 Cancer Epigenetics Laboratory, Molecular Pathology Program, Spanish National Cancer Research Center (CNIO), 28029 Madrid, Spain [M. F. P., S. A., M. F. F., M. S-C., M. E.]; Cancer Epidemiology Unit, National Center for Epidemiology, Carlos III Institute of Health, 28029 Madrid, Spain [M. P.]; Institut de Recerca Oncologica-Institut Catala d’Oncologia, 08907 Barcelona, Catalonia, Spain [G. C., M. A. P.]; and The Johns Hopkins Oncology Center, Baltimore, Maryland 21231 [J. G. H.] ABSTRACT Aberrant DNA methylation is recognized as being a common feature of human neoplasia. CpG island hypermethylation and global genomic hy- pomethylation occur simultaneously in the cancer cell. However, very little is known about the interindividual inherited susceptibility to these epigenetic processes. To address this matter, we have genotyped in 233 cancer patients (with colorectal, breast, or lung tumors), four germ-line variants in three key genes involved in the metabolism of the methyl group, methylene-tetrahydrofolate reductase, methionine synthase, and cystathionine -synthase, and analyzed their association with DNA meth- ylation parameters. The epigenetic features analyzed were the 5-methyl- cytosine content in the genome of the tumors and their normal counter- parts, and the presence of CpG island hypermethylation of tumor suppressor genes (p16 INK4a , p14 ARF , hMLH1, MGMT, APC, LKB1, DAPK, GSTP1, BRCA1, RAR2, CDH1, and RASSF1). Two positive associations were found. First, carriers of genotypes containing the methylene-tetrahy- drofolate reductase 677T allele show constitutive low levels of 5-methylcy- tosine in their genomes (P  0.002), and tumors in these patients do not achieve severe degrees of global hypomethylation (P  0.047). Second, tumors occurring in homozygous carriers of the methionine synthase 2756G allele show a lower number of hypermethylated CpG islands of tumor suppressor genes (P  0.029). The existence of these associations may provide another example of the interplay between genetic and epi- genetic factors in the cancer cell. INTRODUCTION Disruption of the normal DNA methylation patterns is an estab- lished common hallmark of human cancer cells. In a healthy cell, the DNA methylation patterns are conserved through cell divisions, al- lowing the expression of the particular set of cellular genes necessary for that cell type and blocking the expression of exogenous-inserted sequences (1–3). Cancer cells often exhibit the dual phenomenon of global hypomethylation accompanied by hypermethylation of several small regions rich in CpGs called CpG islands (1–3). The generalized loss of 5-methylcytosine in malignant cells occurs mainly in the CpGs scattered in the bodies of the genes and also in repetitive sequences (4). The aberrant methylation of the CpG island located in the 5'- promoter region of several tumor suppressor genes such as hMLH1, BRCA1, VHL, CDH1, p16 INK4a , and APC shuts down the expression of these contiguous genes (1–3). Although many tumors share this change for a given gene, unique profiles of promoter hypermethyla- tion do exist for each tumor type with important biological and clinical consequences (5, 6). However, one of several questions remain unanswered: is there a susceptibility factor that predisposes certain genes and/or particular tumors to possessing different degrees of global hypomethylation or local hypermethylation? This question has been approached from different experimental angles in the past. From the study of the detailed structure of the CpG island, it has been proposed that Sp1 binding sites may serve as protective factors against methylation (7, 8); however, Sp1 knockout mice show no evident alteration in the CpG island methylation patterns (9). On the other hand, certain CpG islands may be more prone to being methylated because they are located near or between regions that are normally methylated, such as Alu sequences and other repetitive elements, from where the methy- lation may be propagated (10). A similar propagation hypothesis has also recently been postulated as affecting methylation of the borders of CpG islands in an age-dependent manner (11). However, to date, the factors that have gained the widest acceptance as affecting DNA methylation are genetically based. First, germ-line mutations in DNMT3b 4 , which occurs in the Immunodeficiency-Centromeric Instability-Facial anomalies syndrome, cause hypomethylation of pericentromeric satellites of chromosomes 1, 9, and 16 (reviewed in Refs. 12, 13). Second, germ-line mutations in the chromatin-remod- eling factor ATRX, which occurs in the ATRX syndrome (X-linked -thalassemia/mental retardation), cause methylation changes in ribo- somal DNA arrays, a Y-specific satellite and subtelomeric repeats (14). Third, the generation of a somatic knockout of the DNMT1 in a cancer cell line causes demethylation of juxtacentromeric satellites (15). Finally, knockout mice of the three most recognized DNMTs, DNMT1, DNMT3a, and DNMT3b, suffer several degrees of hypo- methylation (reviewed in Refs. 12, 13). Nevertheless, are there any other more common and naturally occurring genetic factors that may affect the degree of methylation of normal and cancer cells? The genes involved in the metabolism of the methyl group may represent good candidates and allow the interaction of environmental factors in the process. For example, it has long been known that diets deficient in methyl-group donors such as choline and methionine or in coenzymes of methyl-group metabolism such as folate and vitamin B12 disrupt the levels of intracellular SAM causing DNA hypomethylation (16 –19). Fig. 1 illustrates the enzymatic com- ponents and metabolic pathways of the methyl-group network. DNA methylation patterns depend on a sufficient cellular supply of the methyl-group donor SAM, which is synthesized using dietary methi- onine, but also using methionine recycled from the methylation reac- tion product S-adenosylhomocysteine. Three candidate genes emerge from this picture: MTHFR that supplies methyltetrahydrofolate as a methyl-group donor; MS that remethylates homocysteine to generate methionine; and CBS that conjugates homocysteine to serine. We have chosen these three enzymes because they have been found to be relatively commonly polymorphic in the general population because the germ-line variants generate less active alleles that lead to higher levels of homocysteine and a deficit in methyl-group donors, and Received 4/4/02; accepted 6/3/02. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by Investigacion + Desarrollo Grant SAF2001-0059 and the International Rett Syndrome Association. 2 These two authors contributed equally to this work. 3 To whom all correspondence and requests for reprints should be addressed, at Cancer Epigenetics Laboratory, 3 rd Floor, Molecular Pathology Program, Spanish National Can- cer Center (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spain. Phone: 34-91- 2246940; Fax: 34-91-2246923; E-mail: mesteller@cnio.es. 4 The abbreviations used are: DNMT, DNA methyltransferase; SAM, S-adenosylme- thionine; MTHFR, methylene-tetrahydrofolate reductase; MS, methionine synthase; CBS, cystathionine -synthase. 4519 Research. on January 9, 2015. © 2002 American Association for Cancer cancerres.aacrjournals.org Downloaded from