Prioritization of Copy Number Variation Loci Associated with Autism from AutDB–An Integrative Multi-Study Genetic Database

<div><p>Copy number variants (CNVs) are thought to play an important role in the predisposition to autism spectrum disorder (ASD). However, their relatively low frequency and widespread genomic distribution complicates their accurate characterization and utilization for clinical genetics purposes. Here we present a comprehensive analysis of multi-study, genome-wide CNV data from AutDB (<a href="http://mindspec.org/autdb.html" target="_blank">http://mindspec.org/autdb.html</a>), a genetic database that accommodates detailed annotations of published scientific reports of CNVs identified in ASD individuals. Overall, we evaluated 4,926 CNVs in 2,373 ASD subjects from 48 scientific reports, encompassing ∼2.12×10⁹ bp of genomic data. Remarkable variation was seen in CNV size, with duplications being significantly larger than deletions, (<i>P</i>  =  3×10⁻¹⁰⁵; Wilcoxon rank sum test). Examination of the CNV burden across the genome revealed 11 loci with a significant excess of CNVs among ASD subjects (<i>P</i><7×10⁻⁷). Altogether, these loci covered 15,610 kb of the genome and contained 166 genes. Remarkable variation was seen both in locus size (20 - 4950 kb), and gene content, with seven multigenic (≥3 genes) and four monogenic loci. CNV data from control populations was used to further refine the boundaries of these ASD susceptibility loci. Interestingly, our analysis indicates that 15q11.2-13.3, a genomic region prone to chromosomal rearrangements of various sizes, contains three distinct ASD susceptibility CNV loci that vary in their genomic boundaries, CNV types, inheritance patterns, and overlap with CNVs from control populations. In summary, our analysis of AutDB CNV data provides valuable insights into the genomic characteristics of ASD susceptibility CNV loci and could therefore be utilized in various clinical settings and facilitate future genetic research of this disorder.</p></div

ASD susceptibility CNV loci on human chromosome 15

<p>The number of individuals with duplications (green) and deletions (red) are plotted for both ASD cases (continuous lines) and controls (broken lines, on a log₂ scale) along human chromosome 15q11.2-13.3. RefSeq genes overlapping with these regions are depicted in blue rectangles. Genes that have been associated with ASD according to AutDB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066707#pone.0066707-Basu1" target="_blank">[18]</a> are colored in orange. The variation in CNV burden along the 15q11.2 – 13.3 region, suggests three distinct ASD susceptibility loci: Locus 1 within breakpoints (BP) 1-2, Locus 2 within BPs 2-3, and Locus 3 within BPs 4-5.</p

Whole-genome distribution of CNV burden.

<p>A Manhattan plot showing CNV burden among ASD subjects in 10 kb regions continuously distributed across the human genome. A dashed horizontal line indicate the burden score of 6.5 (0.995 quantile) that was used a threshold to determine the top ranked ASD susceptibility CNV loci.</p

Top ASD susceptibility CNV loci.

a<p>The boundaries of the ASD susceptibility CNV loci were determined as the midpoint of the 10kb region with a θ>6.5.</p>b<p>RefSeq genes including both protein-coding and non-coding RNA genes, and excluding Pseudogenes. For loci with >3 genes, only the number of RefSeq genes is given.</p>c<p>CNV type. Inh  =  Inherited, DN  =  De-Novo, NR  =  Not Reported.</p>d<p>CNV burden score.</p>e<p>P-value calculated based on a Poisson distribution of CNV burden scores.</p>f<p>4400 individuals with no ASD diagnosis from three large genome-wide studies.</p

ASD susceptibility CNV loci on human chromosome 22.

<p>The number of individuals with duplications (green) and deletions (red) are plotted for both ASD cases (continuous lines) and controls (broken lines, on a log₂ scale) along human chromosome 22q11.21-13.33. RefSeq genes overlapping with these regions are depicted in blue rectangles. Genes that have been associated with ASD according to AutDB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066707#pone.0066707-Basu1" target="_blank">[18]</a> are colored in orange. (<b>A</b>) The CNV locus on 22q11.21 contains primarily copy number gains (duplications). A black arrow indicates the peak in duplications count due to the CNV data from Glessner <i>et. al</i>. (<b>B</b>) The CNV locus on 22q13.32-13.33 contains primarily copy number losses (deletions).</p

ASD susceptibility CNV locus on human chromosome 16.

<p>The number of individuals with duplications (green) and deletions (red) are plotted along human chromosome 16p11.2. RefSeq Genes overlapping with this region are depicted in blue rectangles. Genes that have been associated with ASD according to AutDB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0066707#pone.0066707-Basu1" target="_blank">[18]</a> are colored in orange.</p

Physical locations of the top ranked 11 ASD susceptibility CNV loci on Human G-banded ideogram.

<p>CNV loci length and width are proportional to their genomic size and burden score respectively. Green, red, and blue are for CNV loci containing primarily copy number gains (Duplications), copy number losses (Deletions), or both Duplications and Deletions, respectively.</p

Expression and anatomical properties of Clique I.

<p>(A) Maximal-intensity projection of the sum of normalized expressions of genes in this clique highlight regions in the cerebellum. (B) The expression fittings in these regions are higher than expected by chance (P = 0.00002, based on 100,000 random permutations). The brain regions of the ABA at 200 micron resolutions (one dot per region on the figure) are grouped into the following main regions: COR (cerebral cortex), OLF (olfactory areas), Hi (hippocampal region), RHi (Retrohippocampal region), STR (striatum), PAL (pallidum), THA (thalamus), HYP (hypoyhalamus), MID,(midbrain), PON (pons), MED (medulla), CER (cerebellum).</p

Expression and anatomical properties of Clique II.

<p>(A) Maximal-intensity projection of the sum of expressions of genes in this clique highlights the cerebellar cortex. (B) The fitting score in the cerebellar cortex is highest among all other brain regions and higher than expected by chance (<i>P</i> = 0.018, based on 100,000 random permutations). Some other regions in the cerebral cortex also show a slight deviation from expression values expected by chance (P<0.05). The brain regions of the ABA at 200 micron resolutions (one dot per region on the figure) are grouped into the following main regions: COR (cerebral cortex), OLF (olfactory areas), Hi (hippocampal region), RHi (Retrohippocampal region), STR (striatum), PAL (pallidum), THA (thalamus), HYP (hypoyhalamus), MID,(midbrain), PON (pons), MED (medulla), CER (cerebellum).</p

Cell type properties of Cliques I & II.

<p>(A) Sections of the sum of expressions of genes in cliques I (left) and II (right) are depicted through the most frontal coronal section of the cerebellum that intersects Nucleus X. The boundaries of the cerebellar cortex and of nucleus X are outlined. (B) The corresponding coronal section of the mouse brain in the Allen Mouse Brain Atlas, Allen Institute for Brain Science <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003128#pcbi.1003128-Allen1" target="_blank">[57]</a>.</p