1.1. INTRODUCTION Although assessing DNA methy-lation has not yet become commonplace in the diagnostic molecular pathology laboratory, many tests involving methyla-tion analysis will become part of routine practice. In this chapter, a practical approach will be taken toward evaluating DNA methylation. The principal question likely to be asked in the pathology laboratory is whether a specific gene or region is methylated in a specific pathological situation; for example, is the MLH1 gene promoter methylated in a colorectal cancer specimen showing microsatellite instability? This chapter will deal with methods that examine specific sequences for methy-lation rather than those examining total genomic methylation or those screening for new sites of recurrent methylation.
The various methods used to study methylation each have their particular advantages and disadvantages. The methods chosen should be dictated by the nature of the sample that needs to be analyzed and the information required. As the amount of tissue available to the molecular pathology laboratory is often limited and the DNA is frequently very fragmented, this chapter will be concerned with polymerase chain reaction (PCR)-based methodologies. It will also focus on those methodologies that are used or are likely to be utilized by diagnostic laboratories.
methylation in eukaryotes occurs as a result of the addition of methyl groups to cytosine to form 5-methylcytosine. In humans, DNA methylation occurs almost exclusively at cytosines located within CpG dinucleotides. The CpG dinu-cleotide (the p denotes physical linkage via a phosphodiester bond) is the unit of DNA methylation. It pairs with a CpG din-ucleotide on the opposite DNA strand. This one-to-one correspondence of CpGs enables the replication of methylation patterns immediately following DNA replication during which the methylation of CpGs on the parental strands acts as a template for the corresponding CpGs on the newly synthesized daughter strands.
CpG dinucleotides, which are comparatively rare in DNA, are generally methylated. The observed frequency of CpG
dinucleotides in most regions of the DNA is much lower than the expected frequency that would be predicted from the product of the frequencies of the cytosine residues and the guanine residues in those regions (1). This is a result of the accelerated loss of methylated cytosine residues over evolutionary time. When spontaneous cytosine deamination occurs, the resultant uracil is excised by uracil glycosylase in the first step of base excision repair. On the other hand, 5-methylcytosine is deaminated to thymine. The resultant T : G mismatch is recognized by thymine DNA glycosylases, which then excise the thymine. The repair rate is slightly less efficient for 5-methylcytosines than for cytosines probably because uracils, which are not normally part of DNA, are more readily recognized than mismatched thymines. The mutability of 5-methylcytosine underlies much somatic variation and pathogenic mutations in the coding regions of genes (2,3). The CpG dinucleotide becomes either a TpG or a CpA, depending on which strand the deamination has occurred.
CpG islands are cytosine- and guanine- rich segments of the genome with minimal CpG suppression (1,4). They are often associated with the promoter region of genes, and the CpG dinucleotides within the island are generally unmethylated. Methylation of a CpG island in the promoter region of a gene acts to turn off (silence) gene transcription by recruiting histone deacetylases and inducing the formation of inactive chromatin (5,6). In the remainder of this chapter, when we refer to methy-lation, we will specifically refer to promoter region CpG island methylation.
Methylated DNA influences gene expression via a set of proteins carrying a common methylated CpG- binding domain (MBD). The MBD was first described in the transcriptional repressor MeCP2. Like MeCP2, MBD1, MBD2, and MBD3 are involved in recruiting histone deacetylases to methyl CpG-enriched promoter regions to repress transcription (reviewed in ref. 7). MBD4 does not affect transcription but is a thymine DNA glycosylase that binds to CpG : TpG mismatches when the cytosine is methylated (8).
Methylation patterns are maintained during replication by the DNA methyltransferase DNMT1, which recognizes the methyl groups on the parental strand and methylates the daughter strand accordingly. The DNA methyltransferases DNMT3A and DNMT3B show de novo methylation activity. All three of
From: Molecular Diagnostics: For the Clinical Laboratorian, Second Edition Edited by: W. B. Coleman and G. J. Tsongalis © Humana Press Inc., Totowa, NJ
these enzymes have been shown to be essential for normal embryonic development (9,10).
Two more DNA methyltransferase family members have been described. The role of DNMT2 remains unclear. It is not necessary for either de novo or maintenance methylation but might be involved in regulatory pathways as it shows strong binding to DNA (10,11). DNMT3L has been shown to be required for the establishment of maternal methylation imprints at imprinting centers (12). It lacks the catalytic domain common to other DNA methyltransferases but strongly enhances de novo methylation by DNMT3A (13).
DNA methylation was postulated to play a central role in the control of gene expression during development (14,15). Although it is no longer considered central, its precise contribution remains poorly understood. Methylation patterns are determined during early embryogenesis by mechanisms that are also poorly understood. Genomewide demethylation after fertilization is followed by de novo methylation in pregastrulation (16). The patterns are then faithfully transmitted by DNMT1-mediated maintenance methylation. The instructions for de novo methylation must be encoded within the chromatin, as patterns of methylation are highly conserved between individuals (17).
DNA methylation can vary between the two alleles of a gene. DNA methylation is commonly found on the inactive allele when there is differential activity of the two alleles of a gene, as seen in imprinting and X-inactivation. This differential methylation can be used for diagnostic purposes as in the diagnosis of Prader-Willi and Angelman syndromes or the determination of clonality by the X-inactivation method, as will be discussed in Section 3.3.
1.3. DNA METHYLATION IN HUMAN PATHOLOGY DNA methylation pathways have recently been shown to be directly or indirectly involved in the development of several genetic diseases (reviewed in ref. 18). Inherited deficiencies affecting the methylation machinery underpin three genetic disorders: ICF, ATR-X, and Rett syndromes. It is of interest that all of these syndromes involve mental retardation as part of their clinical spectrum. It indicates that methylation plays a crucial but as yet unknown role in the development or function of the brain.
Mutations in DNMT3B lead to the ICF (immunodeficiency, centromeric region instability, facial abnormalities) syndrome (19,20). Clinically, this disease is characterized by facial dys-morphism, mental retardation, and immune deficiency associated particularly with a decrease in IgA. The heterochromatic regions of chromosomes 1, 9, and 16 are decondensed and show frequent chromatid and chromosome breaks and interchanges (21,22). The centromeric abnormalities arise from lack of methylation of satellite II and III DNA, which is found at the centromeres of chromosomes 1, 9, and 16 (23).
Patients with ATR-X (X-linked a-thalassemia/mental retardation) syndrome have characteristic developmental abnormalities, including severe mental retardation, facial dysmorphism, urogenital abnormalities, and a-thalassaemia. The ATRX gene product is a member of the chromatin remodeling SWI/SNF family and is associated with the pericentromeric heterochro-matin. Mutations in the ATRX gene also underlie several other
X-linked mental retardation syndromes (24). Patients with the ATR-X syndrome show altered methylation of highly repeated sequences in the genome, although why this occurs and the relationship of these changes to the clinical phenotype remains unknown (25).
Mutations in the MBD family gene MECP2 lead to Rett syndrome, which is characterized by mental retardation, other neurological abnormalities, and mild skeletal abnormalities (26). MECP2 is X-linked and the disease is normally limited to girls. The mutations are normally lethal in males, although males carrying some of the less deleterious changes can survive past childbirth.
In the Fragile X syndrome, methylation plays a role in the development of the disease, although the methylation machinery is normal. The expansion of a CCG repeat in the 5' untranslated region past a critical number of copies results in de novo methylation of the repeats and inactivation of the adjacent FMR1 gene (27,28). This same phenomenon is also seen in mental impairment as a result of expansion of CCG repeats at the FRAXE fragile site. The de novo methylation of the repeats leads to inactivation of the FMR2 and FMR3 genes (29,30).
1.4. DNA METHYLATION CHANGES IN CANCER Early in cancer development, the distribution of methylation goes awry. Overall methylation is decreased but de novo methylation of some promoter-associated CpG islands occurs (reviewed in refs. 31 and 32). Inactivation by methylation of genes such as tumor suppressor genes, DNA repair genes, and pro-apoptotic genes offers a strong selective advantage to the tumor. Thus, the altered methylation seen during the development of cancer is the origin of much variation that forms the basis for selection of gene expression patterns enhancing the development and progression of malignancy.
A tumor suppressor gene generally requires two "hits" to lose its function; that is, both alleles must be inactivated. Traditionally, one of these hits has been considered to be mutation and the other has been considered to be unmasking of the mutation by physical loss of the second allele (often observed as loss of heterozygosity). Methylation of the tumor-suppressor-gene-promoter region can act as an alternative, either as a first hit or as a second hit. Mutation of one allele can be followed by methylation of the second allele. Methylation of one allele can be followed by physical loss of the second allele or methylation of the second allele.
Methylation has been shown to cause transcriptional silencing of numerous tumor suppressor genes. The pathogenic nature of methylation is illustrated by the observation that for many tumor suppressor genes, methylation of that gene is largely limited to the same type of tumor as that which mutational inactivation occurs; for example, the RB gene is methylated in sporadic retinoblastoma (33-35), the VHL (von Hippel-Lindau) gene is methylated in renal cancer (36), and the MLH1 gene is methylated in colorectal cancer (37,38). TheBRCA1 gene, which is methylated in breast and ovarian cancer, is not inactivated in colon cancer or leukemia (39,40). The gene expression profile of BRCA1-methylated breast cancers is closely related to that of cancers that arose from a BRCA1 mutation, further supporting the direct role of BRCA1 methylation in the development of breast cancer (41).
Some tumor suppressor genes with promoter region CpG islands do not become methylated in cancer (e.g., the BRCA2 gene in breast cancer) (42). The reason why some genes with promoter region CpG islands undergo inactivation by methyla-tion and others do not remains unknown. It has been estimated that about 1% of islands can undergo methylation in cancer (32). How much of this methylation is pathogenic remains unknown. In breast cancer, higher-grade tumors have methy-lation of more CpG islands than the less aggressive lower-grade tumors (43). In colorectal cancer, tumors have been classified as showing a CpG island methylator phenotype (CIMP) if they show methylation of two or more of the five loci; p16, MINT1, MINT2, MINT31, and MLH1 (44).
Methylation abnormalities can be reversed by several drugs, notably the inhibitors 5-azacytidine and 5-aza-2'-deoxycytidine. These drugs have been used extensively in vitro to study methylation and are currently undergoing clinical trials as therapeutic agents for several cancers, in particular for the myelodysplastic syndromes, where both drugs have had some success (45-48).
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