Paternal age effects on sperm FOXK1 and KCNA7 methylation and transmission into the next generation

Abstract

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.

Figure 1.

Correlation between DMPK, FOXK1, KCNA7, and NCOR2 methylation and paternal age in sperm cohort 1 (upper panel) and cord blood (lower panel). Each dot represents average methylation of several target CpGs in an individual sperm or cord blood sample, measured by bisulfite pyrosequencing. Regression lines suggest a negative correlation with paternal age.

Figure 2.

Distribution of single sperm (allele) methylation of FOXK1 (left diagram) and KCNA7 (right). Red dots represent sperm samples of older (40–55 years) and blue dots of younger (25–35 years) donors. The FOXK1 assay targets 5 and the KCNA7 assay 12 CpG sites. The Y axis indicates the percentage of reads (individual sperm) with 0, 1, 2, 3, 4 or 5 methylated CpGs for FOXK1 and 0–12 methylated CpGs for KCNA7, respectively. Methylation measurements were performed using deep bisulfite sequencing. Average methylation values for the measured CpG sites are provided in Supplementary Material, Table S10.

Figure 3.

Boxplots showing the distribution of FOXK1 methylation in 74 male ASD patients (red boxes) and 41 age and sex-matched controls (blue boxes) in three different age groups (2–5, 5–10, and 10–20 years). The median is represented by a horizontal black line. The bottom of the box indicates the 25th, the top the 75th percentile. Outliers are shown as open circles.

Figure 4.

Luciferase activity of pCpGL vector containing either methylated or unmethylated FOXK1 promoter, normalized to activity of an internal control vector (Firefly/Renilla ratio). pCpGL and control vector were co-transfected in a 500:1 ratio in three different cell lines, SH-SY5Y, U2OS, and HELA. Empty vector and non-transfected cells served as negative controls. All measurements were done in duplicates.