Children of older fathers carry an increased risk for developing autism and other disorders. To elucidate the underlying mechanisms, we investigated the correlation of sperm DNA methylation with paternal age and its impact on the epigenome of the offspring. Methylation levels of nine candidate genes and LINE-1 repeats were quantified by bisulfite pyrosequencing in sperm DNA of 162 donors and 191 cord blood samples of resulting children (conceived by IVF/ICSI with the same sperm samples). Four genes showed a significant negative correlation between sperm methylation and paternal age. For FOXK1 and KCNA7, the age effect on the sperm epigenome was replicated in an independent cohort of 188 sperm samples. For FOXK1, paternal age also significantly correlated with foetal cord blood (FCB) methylation. Deep bisulfite sequencing and allele-specific pyrosequencing allowed us to distinguish between maternal and paternal alleles in FCB samples with an informative SNP. FCB methylation of the paternal FOXK1 allele was negatively correlated with paternal age, whereas maternal allele was unaffected by maternal age. Since FOXK1 duplication has been associated with autism, we studied blood FOXK1 methylation in 74 children with autism and 41 age-matched controls. The FOXK1 promoter showed a trend for accelerated demethylation in the autism group. Dual luciferase reporter assay revealed that FOXK1 methylation influences gene expression. Collectively, our study demonstrates that age-related DNA methylation changes in sperm can be transmitted to the next generation and may contribute to the increased disease risk in offspring of older fathers.

The prevalence of metabolic disorders, in particular obesity has dramatically increased worldwide. Genetic variants explain only a minor part of the obesity epidemic induced by physical inactivity and over-nutrition. Epidemiological studies in humans and animal models indicate that epigenetic changes associated with adverse parental and/or intrauterine factors may contribute to the missing heritability of metabolic disorders. Possible adverse paternal effects are likely transmitted by sperm to the next-generation. To investigate this hypothesis, we have systematically analyzed the effects of male body mass index (BMI) on sperm epigenome and its association with next-generation fetal cord blood (FCB) DNA methylation. Methylation levels of maternally imprinted (PEG1, PEG4, PEG5, and PEG10), paternally imprinted (H19-IG DMR, IGF2-DMR0, and MEG3-IG DMR) regions, and obesity-related non-imprinted HIF3A gene were quantified by bisulphite pyrosequencing in sperm samples of 294 human donors undergoing in vitro fertilization or intracytoplasmic sperm injection, and in 113 FCBs of the resulting offspring. Multivariable regression analyses revealed that MEG3 intergenic differentially methylated region (IG DMR) showed positive correlation between sperm methylation and donor’s BMI. A gender-specific correlation between paternal BMI and FCB methylation was observed for MEG3-IG DMR, HIF3A, and IGF2-DMR0. The former two genes displayed same directional nominal association (as sperm) between paternal BMI and FCB methylation in male offspring. Hypomethylation of IGF2-DMR0 with increased paternal BMI was observed in FCBs from female offsprings. Our results suggest that male obesity is nominally associated with modification of sperm DNA methylome in humans, which may affect the epigenome of the next-generation. Nevertheless, it is important to note that none of the associated p-values survived multiple testing adjustments. Future work should test the effect of associated methylation aberrations in the offspring as DNA methylation was shown to control expression and/or imprint establishment across the studied genes.

Hutchinson–Gilford Progeria Syndrome (HGPS) is an extremely rare genetic disorder caused by mutations in the LMNA gene and characterized by premature and accelerated aging beginning in childhood. In this study, we performed the first genome‐wide methylation analysis on blood DNA of 15 patients with progeroid laminopathies using Infinium Methylation EPIC arrays including 8 patients with classical HGPS. We could observe DNA methylation alterations at 61 CpG sites as well as 32 significant regions following a 5 Kb tiling analysis. Differentially methylated probes were enriched for phosphatidylinositol biosynthetic process, phospholipid biosynthetic process, sarcoplasm, sarcoplasmic reticulum, phosphatase regulator activity, glycerolipid biosynthetic process, glycerophospholipid biosynthetic process, and phosphatidylinositol metabolic process. Differential methylation analysis at the level of promoters and CpG islands revealed no significant methylation changes in blood DNA of progeroid laminopathy patients. Nevertheless, we could observe significant methylation differences in classic HGPS when specifically looking at probes overlapping solo‐WCGW partially methylated domains. Comparing aberrantly methylated sites in progeroid laminopathies, classic Werner syndrome, and Down syndrome revealed a common significantly hypermethylated region in close vicinity to the transcription start site of a long non‐coding RNA located anti‐sense to the Catenin Beta Interacting Protein 1 gene (CTNNBIP1). By characterizing epigenetically altered sites, we identify possible pathways/mechanisms that might have a role in the accelerated aging of progeroid laminopathies.

In somatic cells/tissues, methylation of ribosomal DNA (rDNA) increases with age and age‐related pathologies, which has a direct impact on the regulation of nucleolar activity and cellular metabolism. Here, we used bisulfite pyrosequencing and show that methylation of the rDNA transcription unit including upstream control element (UCE), core promoter, 18S rDNA, and 28S rDNA in human sperm also significantly increases with donor's age. This positive correlation between sperm rDNA methylation and biological age is evolutionarily conserved among mammals with widely different life spans such as humans, marmoset, bovine, and mouse. Similar to the tandemly repeated rDNA, methylation of human α‐satellite and interspersed LINE1 repeats, marmoset α‐satellite, bovine alpha‐ and testis satellite I, mouse minor and major satellite, and LINE1‐T repeats increases in the aging male germline, probably related to their sperm histone packaging. Deep bisulfite sequencing of single rDNA molecules in human sperm revealed that methylation does not only depend on donor's age, but also depend on the region and sequence context (A vs. G alleles). Both average rDNA methylation of all analyzed DNA molecules and the number of fully (>50%) methylated alleles, which are thought to be epigenetically silenced, increase with donor's age. All analyzed CpGs in the sperm rDNA transcription unit show comparable age‐related methylation changes. Unlike other epigenetic aging markers, the rDNA clock appears to operate in similar ways in germline and soma in different mammalian species. We propose that sperm rDNA methylation, directly or indirectly, influences nucleolar formation and developmental potential in the early embryo.

Aim: To examine the effects of genetic variation, parental age and BMI on parental allele-specific methylation of imprinted genes in fetal cord blood samples. Methodology: We have developed SNP genotyping and deep bisulphite sequencing assays for six imprinted genes to determine parental allele-specific methylation patterns in diploid somatic tissues. Results: Multivariate linear regression analyses revealed a negative correlation of paternal age with paternal MEG3 allele methylation in fetal cord blood. Methylation of the maternal PEG3 allele showed a positive correlation with maternal age. Paternal BMI was positively correlated with paternal MEST allele methylation. In addition to parental origin, allele-specific methylation of most imprinted genes was largely dependent on the underlying SNP haplotype. Conclusion: Our study supports the idea that parental factors can have an impact, although of small effect size, on the epigenome of the next generation, providing an additional layer of complexity to phe...