Satellite 2 methylation patterns in normal and ICF syndrome cells and association of hypomethylation with advanced replication

Mutation in the DNMT3B DNA methyltransferase gene is a common cause of ICF (immunodeficiency, centromeric heterochromatin, facial anomalies) immunodef iciency syndrome and leads to hypomethylation of satellites 2 and 3 in peri centric heterochromatin. This hypomethylation is associated with centromeri c decondensation and chromosomal rearrangements, suggesting that these sate llite repeats have an important structural role. In addition, the satellite regions may have functional roles in modifying gene expression. The extent of satellite hypomethylation in ICF cells is unknown because methylation s tatus has only been determined with restriction enzymes that cut infrequent ly at these loci. We have therefore developed a bisulfite conversion-based method to determine the detailed cytosine methylation patterns at satellite 2 sequences in a quantitative manner for normal and ICF samples. From our sequence analysis of unmodified DNA, the internal repeat region analyzed fo r methylation contains an average of 17 CpG sites. The average level of met hylation in normal lymphoblasts and fibroblasts is 69% compared with 20% in such cells from ICF patients with DNMT3B mutations and 29% in normal sperm . Although the mean satellite 2 methylation values for these groups do not overlap, there is considerable overlap at the level of individual DNA stran ds. Our analysis has also revealed a pattern of methylation specificity, su ggesting that some CpGs in the repeat are more prone to methylation than ot her sites. Variation in satellite 2 methylation among lymphoblasts from dif ferent ICF patients has prompted us to determine the frequency of cytogenet ic abnormalities in these cells. Although our data suggest that some degree of hypomethylation is necessary for pericentromeric decondensation, factor s other than DNA methylation appear to play a major role in this phenomenon . Another such factor may be altered replication timing because we have dis covered that the hypomethylation of satellite 2 in ICF cultures is associat ed with advanced replication.