PMC1164411_01
Crypt methylation before and neo-adjuvant therapy. There are eight tags per crypt with each tag arranged in a 5' to 3' horizontal order of CpG sites. CSX (eight CpG sites) and BGN (nine CpG sites) tags were sampled from eight of the same crypts. Crypt methylation patterns are summarized by percent methylation and numbers of unique tags per crypt. Methylation patterns were not significantly different before and after neo-adjuvant therapy, with averages summarize in the Table. The seemingly random methylation patterns are consistent with a model [4] in which stochastic methylation errors start to accumulate from birth.
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F2
Crypt patterns (5' to 3' order of methylation) before and after neo-adjuvant therapy were complex and seemingly random, consistent with stochastic epigenetic errors (Figure 2). Methylation tag values differed between crypts sampled before and after neo-adjuvant therapy, but these differences were not significant and did not follow a trend (Figure 2). In addition to numbers of divisions since birth, methylation patterns (Figure 2) may also record stem cell dynamics.
Crypt methylation tags are consistent with a clock-like stochastic accumulation of methylation errors from birth. Such a model using previously published crypt parameters [4] can be simulated on a computer to yield expected individual (dotted lines include 95% of simulated crypt outcomes) and average (solid grey line) crypt values. Little difference is expected between the initial (black X 's are individual crypt values and black triangles are average values) and final specimens (red bars are individual crypt values and red circles are average values) because by this model, methylation errors slowly accumulate from birth, and few additional changes are expected to accumulate during the 62 days between specimens.
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F3
The model can be simulated on a computer (using previously published human colon crypt parameters) to estimate average tag values and intervals that include 95% of simulated individual crypt outcomes (Figure 3). Observed crypt tag values were generally consistent with the predictions of the model (Figure 3). This AFAP patient appeared to have normal numbers of crypt stem cells because his crypt diversity (3.22 and 2.33 average unique CSX crypt tags) was consistent with a normal colon (Figure 3).