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. 2013 Nov 1;342(6158):632-7.
doi: 10.1126/science.1243472.

Mosaic copy number variation in human neurons

Michael J McConnell  1 , Michael R Lindberg, Kristen J Brennand, Julia C Piper, Thierry Voet, Chris Cowing-Zitron, Svetlana Shumilina, Roger S Lasken, Joris R Vermeesch, Ira M Hall, Fred H Gage
Affiliations

Mosaic copy number variation in human neurons

Michael J McConnell et al. Science. .

Abstract

We used single-cell genomic approaches to map DNA copy number variation (CNV) in neurons obtained from human induced pluripotent stem cell (hiPSC) lines and postmortem human brains. We identified aneuploid neurons, as well as numerous subchromosomal CNVs in euploid neurons. Neurotypic hiPSC-derived neurons had larger CNVs than fibroblasts, and several large deletions were found in hiPSC-derived neurons but not in matched neural progenitor cells. Single-cell sequencing of endogenous human frontal cortex neurons revealed that 13 to 41% of neurons have at least one megabase-scale de novo CNV, that deletions are twice as common as duplications, and that a subset of neurons have highly aberrant genomes marked by multiple alterations. Our results show that mosaic CNV is abundant in human neurons.

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Figures

Figure 1
Figure 1. Mosaic copy number variation (CNV) is detected in human neurons
(A, B) Subchromosomal deletions (green arrow) and duplications (red arrow) are observed in hiPSC-derived neurons. (A) Neuron Dn_1 has a deletion on chromosome (chr) 4q (lower panel); neuron Dn_2 has no CNV on Chr4 (upper panel). Small gray dots show the predicted copy number at individual SNPs; red dots show every 30th SNP. (B) Neuron Cn_32 has a duplication on ChrXq (lower panel); neuron Cn_2 does not (upper panel). (C, D) Single cell sequencing reveals subchromosomal deletions (green arrow) and duplications (red arrow) in FCTX neurons. (C) FCTX079 has a deletion on Chr1p (lower panel); FCTX080 does not (upper panel). Blue dots show raw copy number predictions obtained by read-depth analysis (mean window size ~687 Kb; see methods) (D) Neuron FCTX197 has a duplication on Chr2p (lower panel) whereas FCTX185 does not (upper panel). Another likely duplication on Chr2q in FCTX197 (open arrow) was comprised of only four consecutive bins and therefore failed our five-bin confidence threshold.
Figure 2
Figure 2. Large CNVs are found in hiPSC-derived neurons
(A) Whole and subchromosomal duplications (red) and deletions (green) are summarized for 40 hiPSC-derived neurons (upper panel). The y-axis value represents the number of times each genomic interval was deleted (below in green) or duplicated (above in red). CNVs were detected in 9 of 21 C neurons (Cn), 2/6 D neurons (Dn) and 2/13 E neurons (En). In donor hiPSC-derived NPC populations (middle panel), CNVs were detected in 1/10 D NPCs (Dp) and 3/9 C NPCs (Cp). In donor fibroblast populations (lower panel), CNVs were detected in 7/20 D fibroblasts (Df) and 0/9 C fibroblasts (Cf). Note that chromosomes are not plotted to scale because data is summarized in 100-SNP bins. (B) Subchromosomal CNVs in fibroblasts were significantly smaller than in hiPSC-derived neurons (KS test, P < .001). No deletions were observed in NPCs. Deletions are denoted with blue markers; all other markers indicate duplications. Aneuploidies are not included in this plot. For completeness, subchromosomal CNVs from clonal fibroblasts (Fig. 3) were included in this plot, bringing the total n to 42 fibroblasts.
Figure 3
Figure 3. Large CNVs are found in cultured fibroblasts
(A) Single fibroblasts obtained by limiting dilution were expanded to a population of ~20 clonal fibroblasts after seven days in vitro (DIV). In one clonal population, a reciprocal chromosome missegregation event was detected. One fibroblast was trisomic for Chr2 (upper panel) and a sister was monosomic for Chr2 (lower panel). Chromosomes 1 - 3 are shown alongside the third euploid cell. (B, C) Two groups of Df (passage 7 and 8) were summarized in (Fig 2A); a parallel culture of the p7 group was sent for karyotyping and FISH. 20/20 metaphase chromosome spreads were euploid. (B) FISH was performed for a ChrX p arm telomere (green) and ChrX centromere (red). 13/200 nuclei were aneuploid. (C) FISH was performed for the Chr20 centromere (green) and Chr20 CNV (red). 26/200 nuclei had the CNV. (D) Single cell sequencing of 2 male fibroblasts with karyotypically defined trisomy 21. Genome-wide copy number profiles show that, in both cells, most of the genome is present at 2 copies, Chr21 is present at 3 copies, and ChrX is present at 1 copy. In addition, we identified a large deletion on Chr7q in FIBR030. DNA copy number (y-axis) was calculated by read-depth analysis of variably sized genomic windows containing 500 Kb of uniquely mappable sequence (blue), and CNVs were detected by circular binary segmentation (orange). Green and red arrows denote deletions and duplications, respectively, that were identified by segmentation and passed filtering criteria. Reported CNVs comprise five or more consecutive bins and exceed two MADs. Dotted gray lines show 1 and 2 median absolute deviations (MADs) from the median copy number of each dataset. See Figs. S6 and S7 for plots of additional cells.
Figure 4
Figure 4. Identification of CNVs in post-mortem neurons using single cell sequencing
(A) Genome-wide copy number profiles of five male (left panels) and five female (right) neurons from two individuals, #1583 and #1846, respectively. Data are plotted exactly as in Fig. 3D. Arrows denote deletions (green) and duplications (red) that were identified by copy number segmentation and passed filtering criteria. Note that single copy “losses” of ChrX in cells from male individual #1583 are not indicated by arrows, but were identified in 100% of cells. See Fig. S7 for plots of all cells. (B) Whole and subchromosomal duplications (red) and deletions (green) are summarized for the 110 FCTX neurons as in Fig. 2A. (C) Bar chart showing the number of individual neurons (y-axis) that exhibited a given number of CNVs (X-axis). See Fig. S11 for results at different CNV detection stringency thresholds.
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Comment in

  • Genetics. Our fallen genomes.
    Macosko EZ, McCarroll SA. Macosko EZ, et al. Science. 2013 Nov 1;342(6158):564-5. doi: 10.1126/science.1246942. Science. 2013. PMID: 24179207 No abstract available.

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