Somatic retrotransposition alters the genetic landscape of the human brain

Abstract

Retrotransposons are mobile genetic elements that use a germline 'copy-and-paste' mechanism to spread throughout metazoan genomes. At least 50 per cent of the human genome is derived from retrotransposons, with three active families (L1, Alu and SVA) associated with insertional mutagenesis and disease. Epigenetic and post-transcriptional suppression block retrotransposition in somatic cells, excluding early embryo development and some malignancies. Recent reports of L1 expression and copy number variation in the human brain suggest that L1 mobilization may also occur during later development. However, the corresponding integration sites have not been mapped. Here we apply a high-throughput method to identify numerous L1, Alu and SVA germline mutations, as well as 7,743 putative somatic L1 insertions, in the hippocampus and caudate nucleus of three individuals. Surprisingly, we also found 13,692 somatic Alu insertions and 1,350 SVA insertions. Our results demonstrate that retrotransposons mobilize to protein-coding genes differentially expressed and active in the brain. Thus, somatic genome mosaicism driven by retrotransposition may reshape the genetic circuitry that underpins normal and abnormal neurobiological processes.

Figure 1

Overall RC-seq methodology (a) Retrotransposon capture: sheared genomic DNA is hybridized to custom tiling arrays probing full-length retrotransposons (nucleotides highlighted with light blue background). (b) Sequencing: post-hybridization, DNA fragments are eluted and analyzed with an Illumina sequencer, producing ~2.5×10 paired-end reads per library that are subsequently aligned to the reference genome. (c) Reads mapping as a pair to a single locus indicate known retrotransposon insertions. (d) Unpaired reads where one end maps to a single locus and the other end maps to a distal retrotransposon indicate novel retrotransposition events.

Figure 2. Multiplex qPCR confirms L1 copy number variation in the human brain

The relative abundance of L1 open reading frame 2 (ORF2) versus α-satellite repeats (SATA) was quantified using an existing TaqMan based approach. Genomic DNA from five brain regions was assayed in three donors (A, B and C). Hi, hippocampus; Pu, putamen; TG, middle temporal gyrus; Ca, caudate nucleus; FG, middle frontal gyrus. Values are normalized to caudate nucleus for each donor. Error bars equal one s.e.m. *p < 0.001 for repeated measures one way analysis of variance (ANOVA) within each donor, followed by pairwise least significant difference (LSD) post hoc tests with Bonferroni correction.

Figure 3. Characterization of non-reference genome insertions

(a) Proportions of novel insertions found for each family. (b) Annotation of novel L1 insertions across all brain libraries. The vast majority of insertions detected by fewer than three reads could not be annotated and were considered putative somatic events. Note: logarithmic scale.

Figure 4. Discovery of somatic insertions in HDAC1 and RAI1

(a) Alignment of an RC-seq read from donor C caudate nucleus indicated an antisense L1 insertion in intron 9 of HDAC1. Nested PCR primers were designed to span the L1 5′ terminus, with an initial reaction combining outside retrotransposon (ORP) and insertion site (OIP) primers and a second reaction using inside retrotransposon (IRP) and insertion site (IIP) primers. (b) Amplification of the nested PCR target, confirmed for specificity by capillary sequencing, was achieved in caudate nucleus but not in hippocampus. Sequencing indicated that the L1 mobilized from chromosome 9 and was accompanied by 5′ transduction. (c) Alignment of an RC-seq read pair from donor A caudate nucleus indicated a sense Alu insertion in exon 3, and the CDS, of RAI1. (d) As for (b) amplification of the nested PCR target was achieved in caudate nucleus but not in hippocampus. Sequencing indicated that the Alu mobilized from chromosome 4. Note: L1 and Alu elements in (a) and (c) are not drawn to scale.