Increased de novo copy number variants in the offspring of older males

Abstract

The offspring of older fathers have an increased risk of neurodevelopmental disorders, such as schizophrenia and autism. In light of the evidence implicating copy number variants (CNVs) with schizophrenia and autism, we used a mouse model to explore the hypothesis that the offspring of older males have an increased risk of de novo CNVs. C57BL/6J sires that were 3- and 12-16-months old were mated with 3-month-old dams to create control offspring and offspring of old sires, respectively. Applying genome-wide microarray screening technology, 7 distinct CNVs were identified in a set of 12 offspring and their parents. Competitive quantitative PCR confirmed these CNVs in the original set and also established their frequency in an independent set of 77 offspring and their parents. On the basis of the combined samples, six de novo CNVs were detected in the offspring of older sires, whereas none were detected in the control group. Two of the CNVs were associated with behavioral and/or neuroanatomical phenotypic features. One of the de novo CNVs involved Auts2 (autism susceptibility candidate 2), and other CNVs included genes linked to schizophrenia, autism and brain development. This is the first experimental demonstration that the offspring of older males have an increased risk of de novo CNVs. Our results support the hypothesis that the offspring of older fathers have an increased risk of neurodevelopmental disorders such as schizophrenia and autism by generation of de novo CNVs in the male germline.

Figure 1

Relative copy number (rCN) in the combined samples established by Sequenom. rCN in the combined samples was examined by Sequenom competitive quantitative PCR. The data from both sets of animals are shown with all values scaled to set a relative CN of 1 at 0. (a) Variation in CNV2 was established using three separate assays targeting the 5′ prime, central or 3′ prime region of this CNV, respectively. (b) The remaining CNVs were detected by one assay, each. CNV2–CNV6: Boxes denote positive calls for amplifications or deletions. No formal assignment of calls was performed for the highly variable loci CNV1 and CNV7. CNV, copy number variant.

Figure 2

Normalized values on CNV6 load—MRI and behavioral measures. HB center time=proportion of time spent in center zone of the hole board arena. Avoidance response and head dip are count variables. Tail flick and HB center time are timed outcomes (in seconds). Startle response is average amplitude of response to 120 decibels. MRI outcomes include volume of the striatum, hippocampus and ventricles, and width of the corpus callosum. Statistically significant group differences by CNV6 status are shown with an asterisk. CNV, copy number variant; HB, hole board; MRI, magnetic resonance imaging.

Figure 3

CNV2 details. (a) Array log(2) signal intensity ratios of the individual probes are plotted along mouse chromosome 5 and connected with a trend line based on triangular smoothening. A de novo deletion (CNV2) affecting the Auts2 gene was detected in one female offspring of an old sire. The two purple lines depict the result of a technical replicate with the animal containing the deletion. The black line visualizes the result obtained from a self-hybridization array, which was used to control for noise. The location of the CNV on the mouse genome is boxed. (b) CNV2 was aligned to the human genome using the UCSC genome browser (http://genome.ucsc.edu/) together with copy variants observed in the human and genomic regions associated with neurocognitive disease. HapMap: Case from the International Hapmap Project. SLEP, Sullivan Laboratory Evidence Project; CNV, copy number variant; DEL, deletion; AMP, amplification; DN, de novo.