Discussion
Main findings. This study demonstrates that there are measurable
differences in patterns of DNA methylation at initial diagnosis of PCOS,
between women who do, or do not, later develop diabetes. Genome-wide
methylation array screening identified 273 differentially methylated
sites in at least five of six case-control pairs. 19 CpG sites were
differentially methylated in the same direction, separating best the
women with PCOS and those that developed diabetes, and thus are the best
targets for further investigation as potential at-diagnosis prognostic
biomarkers.
Strengths. To identify potential clinically useful prognostic DNA
methylation biomarkers, we used peripheral blood samples taken from
women at their initial diagnosis of PCOS. These samples had accompanying
long-term clinical follow-up data (up to 12 years), which enabled direct
comparison of ‘at-diagnosis’ methylation patterns in women with PCOS who
did or did not later develop diabetes. We reasoned that the best
potential methylation biomarkers would be detectable at this early stage
where dietary, exercise, and weight management interventions are most
likely to be effective[5].
We used well-established methods of genome-wide methylation assessment
(HumanMethylation450 arrays) to identify differences between women with
PCOS, and women with PCOS who later developed diabetes. Similar to
previous array-based prognostic marker screening studies, and to reduce
the likelihood of false positive targets, we used a ≥0.2 β-value
difference to define differential methylation[9].
Limitations . Given the number of samples evaluated and the
variation in criteria used to define PCOS (and therefore variable
population prevalence), we cannot estimate prognostic or predictive
values. Furthermore, plotted Pyrosequencing™ data raise the possibility
that single nucleotide polymorphisms (SNPs) may be implicated in
methylation patterns observed in some of our candidates. Although known
SNPs were excluded during array filtering, polymorphic CpG sequences (in
all Infinium methylation arrays) necessitate caution in interpretation
of results[11].
Interpretation. Although there are no striking visual differences
between cases and controls in the heatmap of the differentially
methylated CpG sites, clustering reassuringly separately grouped the
women with PCOS who developed diabetes from the women that did not. The
273 differentially methylated CpG sites and the 19 putative biomarker
candidates identified is broadly in keeping with numbers in previous
methylation biomarker studies in benign tissue types[10], but is
difficult to interpret in the context of lack of similar prior studies
in PCOS. Notwithstanding, methylation differences were substantiated by
Pyrosequencing™ in our candidates, confirming ‘proof of principle’ that
DNA methylation holds the exciting potential as a prognostic biomarker
in PCOS.
Whilst it cannot be implied from our data, it is possible that
differential methylation at one or more of the sites identified may be
contributory to diabetes development in women with PCOS. Further studies
in larger patient cohorts are required to confirm our initial findings,
evaluate the possible importance of SNPs in the differentially
methylated sites identified, and to facilitate the mechanistic
investigation of DNA methylation changes in women with PCOS who develop
diabetes.