T he potential importance of DNA methylation for specifying epigenetic inheritance in eukaryotic cells was recognized soon after the discovery of the role that methylation plays in the modification and restriction of bacterial and bacteriophage DNA (1-5). In eukaryotic cells, inheritance of the methylated state usually involves 5-methylcytosine and predominantly depends on enzymatic recognition of CpG and CNG motifs. Base-pairing rules (6) ensure that these motifs are symmetrically located on complementary strands of DNA (for example, CpG͞CpG dyads), thus providing the opportunity for the inheritance of cytosine methylation after DNA replication (7). In mammals, maintaining a methylated state of CpG cytosines is an important component of X-chromosome inactivation and genomic imprinting (8-10). The failure to maintain a methylated or an unmethylated state of key cytosines can lead to ''epimutations''; such changes may alter cell and developmental pathways, resulting in new phenotypes (11-14) including disease (15-17). The mechanisms and fidelity of epigenetic inheritance are thus of crucial biological and medical importance.A central issue in epigenetics concerns the mechanism by which a locus maintains a stable epigenetic state through many cell divisions. It appears that epigenetic mechanisms that use 5-methylcytosine within CpG dinucleotides have moderate to high fidelities of maintaining a methylated state of cytosine, after a transitory hemimethylation state during DNA replication (9, 18-23). Hemimethylated sites are also transitional states in developmental processes; active demethylation or de novo methylation may sometimes be involved in gene reactivation or inactivation (24-26). In a study to assess the dynamics of DNA methylation, Riggs and colleagues (9, 27), estimated the fidelity of maintenance methylation (E m ) within partially methylated CpG islands to be Ͼ0.99 per methylated cytosine per cell division; de novo methylation efficiency (E d ) for unmethylated cytosines was estimated to be 0.05 per site per generation. This study, carried out with clones of tissue-culture cells in which methylation was perturbed with 5-azacytidine, also provides a useful mathematical model of the kinetics of DNA methylation (9).Current inferences on epigenetic fidelities and transitional methylation states are based on data for single methylation sites or on patterns of methylation derived from populations of complementary strands. A major experimental limitation has been the difficulty in obtaining methylation patterns from the two complementary strands of an individual DNA molecule. If such a method were available, patterns of methylation fidelity, and detection of both gain and loss of methylation, could be studied relatively directly.We have developed ''hairpin-bisulfite PCR'' for this purpose of analyzing patterns of cytosine methylation on complementary strands of individual DNA molecules. This method uses a hairpin linker, targeted and ligated to restriction-enzyme-cleaved genomic DNA, to maintain attachment o...

Huntington's disease (HD) is an autosomal dominant disorder with a variable age of onset that is influenced by the sex of the affected parent. The recent recognition that HD is caused by an expanded triplet repeat suggests the possibility that the onset age may be predicted by the length of the repeat. This hypothesis was tested by assaying the length of the repeat in 114 individuals who were clinically diagnosed with HD and had a known onset age. Every individual had an expanded allele. The range was from 36 to 82 repeats (mean = 48.4 +/- 9.51) and larger than the normal range (6 to 31). The size of the expanded allele was correlated with the age of onset (r = -0.65 p < .0001). Despite the highly significant correlation, the repeat size explains less than half of the variance in onset age. Furthermore, the age of onset cannot be predicted from the size of the triplet repeat, particularly if the number of repeats is in the smaller end of the expanded range. If the repeat is < or = 50 triplets, the amount of variation in the age of onset explained by the length of the triplet repeat is less than 10%. As an illustration, the onset age of individuals with 39 repeats ranges from 30 to 65 years old. The sex of the affected parent had no effect in our sample beyond the effect of the length of the repeat. Affected offspring of affected fathers had longer repeats and a larger variance in allele size than offspring of affected mothers, perhaps reflecting greater instability in paternal transmission.

There are currently 13 diseases known to be caused by unstable triplet repeat mutations; however, there are some instances (as with FRAXF and FRA16) when these mutations appear to be asymptomatic. In a search for polymorphic CTG repeats as candidate genes for bipolar disorder, we screened a genomic human chromosome 18-specific library and identified a 1.6 kb clone (7,6A) with a CTG24 repeat that maps to 18q21.1. The CTG repeat locus, termed CTG18.1, is located within an intron of human SEF2-1, a gene encoding a basic hellx-loop-hellx DNA binding protein involved in transcriptional regulation. The CTGn repeat is highly polymorphic and very enlarged alleles, consistent with expansions of up to CTG2100, were identified. PCR and Southern blot analysis in pedigrees ascertained for a Johns Hopkins University bipolar disorder linkage study and in CEPH reference pedigrees revealed a tripartite distribution of CTG18.1 alleles with stable alleles (CTG10-CTG37), moderately enlarged and unstable alleles (CTG53-CTG250), and very enlarged, unstable alleles (CTG800-CTG2100). Moderately enlarged alleles were not associated with an abnormal phenotype and have a combined enlarged allele frequency of 3% in the CEPH and bipolar populations. Very enlarged alleles, detectable only by Southern blot analysis of genomic digests, have thus far been found in only three individuals from our bipolar pedigrees, and to date, have not been found in any of the CEPH reference pedigrees. These enlarged alleles may arise, at least in part, via somatic mutation.