Pubertal development in healthy children is mirrored by DNA methylation patterns in peripheral blood

Kristian Almstrup, Marie Lindhardt Johansen, Alexander S Busch, Casper P Hagen, John E Nielsen, Jørgen Holm Petersen, Anders Juul, Kristian Almstrup, Marie Lindhardt Johansen, Alexander S Busch, Casper P Hagen, John E Nielsen, Jørgen Holm Petersen, Anders Juul

Abstract

Puberty marks numerous physiological processes which are initiated by central activation of the hypothalamic-pituitary-gonadal axis, followed by development of secondary sexual characteristics. To a large extent, pubertal timing is heritable, but current knowledge of genetic polymorphisms only explains few months in the large inter-individual variation in the timing of puberty. We have analysed longitudinal genome-wide changes in DNA methylation in peripheral blood samples (n = 102) obtained from 51 healthy children before and after pubertal onset. We show that changes in single methylation sites are tightly associated with physiological pubertal transition and altered reproductive hormone levels. These methylation sites cluster in and around genes enriched for biological functions related to pubertal development. Importantly, we identified that methylation of the genomic region containing the promoter of TRIP6 was co-ordinately regulated as a function of pubertal development. In accordance, immunohistochemistry identified TRIP6 in adult, but not pre-pubertal, testicular Leydig cells and circulating TRIP6 levels doubled during puberty. Using elastic net prediction models, methylation patterns predicted pubertal development more accurately than chronological age. We demonstrate for the first time that pubertal attainment of secondary sexual characteristics is mirrored by changes in DNA methylation patterns in peripheral blood. Thus, modulations of the epigenome seem involved in regulation of the individual pubertal timing.

Figures

Figure 1. Identification of CpGs associated with…
Figure 1. Identification of CpGs associated with pubertal age.
(a) From the COPENHAGEN puberty study of children followed longitudinally, selected pre- and post-pubertal samples from 31 boys and 20 girls were included in the study. Time relative to onset of puberty as measured by a testicular volume of 4 ml or breast tanner stage B2 or above was calculated and defined as the pubertal age. (b) Genomic inflation of the genome-wide DNA methylation patterns correlating with pubertal age were corrected by applying an improved surrogate variant analysis (SmartSVA) resulting in a genomic inflation factor of 1.2 and a qq-plot leaving 457 significant CpG sites at a FDR of 0.05 (red). (c) Manhattan plot of the significant CpG sites reveal a distribution across all autosomes. (d) Unsupervised hierarchical centroid clustering of CpGs associated with pubertal age. The resulting dendrograms were colour coded according to their height and divided samples into two major groups that nearly uniquely represented pre- and post-pubertal boys and girls. (e) Gene ontology analysis of all significant CpGs revealed several ontologies (p-value < 0.05) that could be related to pubertal development. Biological process gene ontologies were plotted in a sematic space, using REVIGO, that groups related ontologies together.
Figure 2. Identification of CpGs associated with…
Figure 2. Identification of CpGs associated with changes in circulating reproductive hormones in boys.
(a) Venn diagram of CpGs associated with Testosterone (n = 999), FSH (n = 492), LH (n = 403), AMH (n = 282), and Inhibin B (n = 218) at a FDR cut-off of 0.05 in boys after correcting for genomic inflation and age. (b) Binary tree obtained from a sequence of union operations showing how related the hormone CpG clusters are to each other. (c) Venn diagram of the CpGs associated with pubertal age and circulating reproductive hormones (Supplementary Tables 1, 5 and 6).
Figure 3. Identification of differentially methylated regions…
Figure 3. Identification of differentially methylated regions associated with pubertal age.
(a) The most significant differentially methylated genomic region (nine probes with a mean p-value of 1.3e–31) was found on chromosome 7 (chr7:100463206-100464771) that contains both gene body and 3′ UTR of SLC12A9 and transcription start site of TRIP6. The CpGs were located in several putative transcription factor bindings sites upstream of TRIP6. TX Factor ChIP, DNase1 clusters tracks were obtained from the ENCODE database and the CpG Islands from the UCSC database. (b) Immunohistochemical staining for TRIP6 in normal adult testicular tissue showing intense and specific staining in Leydig cells (arrow heads) producing testosterone. (c) TRIP6 staining of pre-pubertal (7.9 years old) testicular tissue. TRIP6 staining was absent from Leydig cells (arrow heads) in pre-pubertal testis. Bar equals 20 μm. Negative controls are shown in Supplementary Figure 4 together with staining of ovarian tissue. (d) Circulating levels of TRIP6 were determined by ELISA in boys and girls pre-pubertally, around pubertal onset and post-pubertally. The box plots show the distribution of the measurements (the band inside the box depicts the median) at each time point and the connected lines are drawn from the mean of each group. **Denotes a p-value below 0.01.
Figure 4. Prediction models of pubertal and…
Figure 4. Prediction models of pubertal and biological aging.
(a) Scatter plot of chronological age and pubertal age. Using an elastic net prediction model and a 10-fold cross validation, the pubertal age was predicted from DNA methylation patterns in peripheral blood from (b) boys and (c) girls. A residual error of 0.39 and 0.74 year, equalling five and nine months was observed in boys and girls, respectively. (d) Prediction of biological aging from our data using the same algorithm as applied for pubertal age revealed a residual error of 0.94 year (11 months). The dashed red line is the identity line.

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