Complex mosaic structural variations in human fetal brains

Somatic mosaicism, manifesting as single nucleotide variants (SNVs), mobile element insertions, and structural changes in the DNA, is a common phenomenon in human brain cells, with potential functional consequences. Using a clonal approach, we previously detected 200–400 mosaic SNVs per cell in three human fetal brains (15–21 wk postconception). However, structural variation in the human fetal brain has not yet been investigated. Here, we discover and validate four mosaic structural variants (SVs) in the same brains and resolve their precise breakpoints. The SVs were of kilobase scale and complex, consisting of deletion(s) and rearranged genomic fragments, which sometimes originated from different chromosomes. Sequences at the breakpoints of these rearrangements had microhomologies, suggesting their origin from replication errors. One SV was found in two clones, and we timed its origin to ∼14 wk postconception. No large scale mosaic copy number variants (CNVs) were detectable in normal fetal human brains, suggesting that previously reported megabase-scale CNVs in neurons arise at later stages of development. By reanalysis of public single nuclei data from adult brain neurons, we detected an extrachromosomal circular DNA event. Our study reveals the existence of mosaic SVs in the developing human brain, likely arising from cell proliferation during mid-neurogenesis. Although relatively rare compared to SNVs and present in ∼10% of neurons, SVs in developing human brain affect a comparable number of bases in the genome (∼6200 vs. ∼4000 bp), implying that they may have similar functional consequences.

Somatic mosaicism, the presence of more than one genotype in the somatic cells of an individual, is a prominent phenomenon in the human central nervous system. Forms of mosaicism include aneuploidies and smaller copy number variants (CNVs), structural variants (SVs), mobile element insertions, indels, and single nucleotide variants (SNVs). The developing human brain exhibits high levels of aneuploidy compared to other tissues, generating genetic diversity in neurons (Pack et al. 2005; Yurov et al. 2007; Bushman and Chun 2013). Such aneuploidy was suggested to be a natural feature of neurons, rather than a distinctive feature of neurodegeneration. However, the frequency of aneuploidy in neurons has been debated, with a separate study suggesting that aneuploidies occur in only about 2.2% of mature adult neurons (Knouse et al. 2014). They hence infer that such aneuploidy could have adverse effects at the cellular and organismal levels. Additionally, analysis of single cells from normal and pathological human brains identified large, private, and likely clonal somatic CNVs in both normal and diseased brains (Gole et al. 2013; McConnell et al. 2013; Cai et al. 2014; Knouse et al. 2016; Chronister et al. 2019; Perez-Rodriguez et al. 2019), with 3%–25% of human cerebral cortical nuclei carrying megabase-scale CNVs (Chronister et al. 2019) and deletions being twice as common as duplications (McConnell et al. 2013). Given that CNVs often arise from nonhomologous recombination and replication errors, their likely time of origin is during brain development. However, when CNVs first arise in human brain development has not yet been investigated. The present work is the first to examine this question using clonal populations of neuronal progenitor cells (NPCs) obtained from fetal human brains.Detection of CNVs in single neurons is challenging, given the need to amplify DNA. Such amplification may introduce artifacts that could, in turn, be misinterpreted as CNVs. In order to address this technical limitation, Hazen et al. reprogrammed adult postmitotic neurons using somatic cell nuclear transfer (SCNT) of neuronal nuclei into enucleated oocytes (Hazen et al. 2016). These oocytes then made sufficient copies of the neuronal genome allowing for whole-genome sequencing (WGS), thus eliminating the need for amplification in vitro. Using this method, they identified a total of nine structural variants in six neurons from mice, three of which were complex rearrangements. However, it is not possible to extend such studies to humans, given the ethical issues involved, besides the technical challenges in obtaining and cloning adult neurons. To circumvent the need of single-cell DNA amplification or nuclear cloning, we examined clonal cell populations obtained from neural progenitor cells from the frontal region of the cerebral cortex (FR), parietal cortex (PA) and basal ganglia (BG) and describe here the discovery and analysis of mosaic SVs in these NPCs (Bae et al. 2018). These clones were sequenced at 30× coverage (much higher than most previous single-cell studies), allowing identification of SVs other than large deletions and duplications as well as precise breakpoint resolution.