AIMS appears as a sensitive approach to screen for the differential methylation of DNA in cancer cells. Moreover, its application to a large series of samples appears feasible because of its technical simplicity and low requirement for starting material. The method is specially powerful in the detection of hypermethylated sequences, which are often associated with the inactivation of tumor suppressor genes (12
). Hypomethylations are also detectable although contaminating cells maintaining the methylation status may limit the sensitivity to ~10%. To be amplified, a sequence must fulfill two requirements: (i) to contain two closely spaced methylated Sma
I sites and (ii) to show homology to the nucleotides extended at the 3′ end of the primer. The restricted amplification nature of AIMS renders fingerprints of adjustable complexity that are readily interpretable. Complementary representations may be obtained in experiments using different primers or combinations of primers.
The distribution of AIMS bands along chromosomes clearly shows that most of them originate from a subset of R bands, including T bands, that correspond to the CpG island and gene-rich regions (20
), which are also identified as the H3 isochore (21
). This is reinforced by the striking coincidence of chromosome hybridization patterns of AIMS products with that reported for H3 isochore (22
). While this concordance does not indicate the nature of the amplified sequences, it can be concluded that the AIMS screening for methylated regions is specially intensive in the chromosomal areas with a higher functional interest. This bias in genome screening jointly with the high sensitivity of AIMS to detect hypermethylations provide the most important advantages of this technique.
The isolation and characterization of bands showing differential display between two samples is readily achievable. In a preliminary setting, we have identified 11 bands showing a differential methylation status between normal and tumor tissue. Novel bands appearing in the tumor tissue are indicative of sequence hypermethylation. Bisulfite genomic sequencing of some of these sequences confirmed this postulate. Recurrent hypermethylation of specific sequences might be considered an indicator of the putative inactivation of tumor suppressor genes (11
). Nevertheless, AIMS raw data should be interpreted cautiously. Age-related methylation (23
) is also likely to be observed and, due to the high sensitivity of this approach, hypermethylations affecting a small subset of cells may also arise. Additionally, some apparent normal–tumor changes may reflect clonal expansions of heterogeneous methylation profiles in normal colonic mucosa (24
). Spurious amplification of bands due to incomplete digestion with Sma
I cannot be totally excluded, but the robustness of the competitive amplification and the characteristic patterns of the artifacts (smears for poor digestions and bands appearing sporadically; data not shown) can be easily distinguished from recurrent changes. Nevertheless, as for most screening approaches, functional interpretation of the methylation changes revealed by AIMS requires confirmatory and specific investigations. While analysis of a larger series of samples is needed before attempting a thorough study of each one of the loci showing differential methylation, the preliminary results unveil the power of AIMS to track novel targets of abnormal methylation in cancer.
The fingerprints analyzed show clear normal–tumor changes and also little intensity variations between paired samples. In this setting, we have taken into consideration only the definite variations and, therefore, we are probably underestimating the extent of aberrant methylation in cancer cells. According to our data, hypomethylations are slightly more frequent than hypermethylations in most primary tumors. Hypomethylation is considered as a general trend in most colorectal cancers (25
), which is accompanied by specific hypermethylation of a limited number of loci (reviewed in 11). Nevertheless, the skewness of our approach toward complex sequences may skip a high proportion of hypomethylations occurring at repetitive elements, which are the 5mC-richest regions in mammalian genome (27
In addition to the discovery of novel targets, due to its arbitrary nature AIMS allows the assessment of global methylation profiles. The small number of cases analyzed precludes further interpretations, but the arbitrary nature of the approach offering a genome-wide representation of the methylome may be essential to investigate the nature and extent of the postulated CpG island methylator phenotype (28
In conclusion, we have developed and evaluated a novel method to screen for differential DNA methylation. The approach is feasible for the analysis of large series of samples. Moreover, a high number of sequence tags may be generated in a few experiments. Represented tags arise from the chromosomal regions that are richest in CpG islands and genes. AIMS appears to be a powerful tool for identifying new genes critical to carcinogenesis.