Refinement and discovery of new hotspots of copy-number variation associated with autism spectrum disorder

Abstract

Rare copy-number variants (CNVs) have been implicated in autism and intellectual disability. These variants are large and affect many genes but lack clear specificity toward autism as opposed to developmental-delay phenotypes. We exploited the repeat architecture of the genome to target segmental duplication-mediated rearrangement hotspots (n = 120, median size 1.78 Mbp, range 240 kbp to 13 Mbp) and smaller hotspots flanked by repetitive sequence (n = 1,247, median size 79 kbp, range 3-96 kbp) in 2,588 autistic individuals from simplex and multiplex families and in 580 controls. Our analysis identified several recurrent large hotspot events, including association with 1q21 duplications, which are more likely to be identified in individuals with autism than in those with developmental delay (p = 0.01; OR = 2.7). Within larger hotspots, we also identified smaller atypical CNVs that implicated CHD1L and ACACA for the 1q21 and 17q12 deletions, respectively. Our analysis, however, suggested no overall increase in the burden of smaller hotspots in autistic individuals as compared to controls. By focusing on gene-disruptive events, we identified recurrent CNVs, including DPP10, PLCB1, TRPM1, NRXN1, FHIT, and HYDIN, that are enriched in autism. We found that as the size of deletions increases, nonverbal IQ significantly decreases, but there is no impact on autism severity; and as the size of duplications increases, autism severity significantly increases but nonverbal IQ is not affected. The absence of an increased burden of smaller CNVs in individuals with autism and the failure of most large hotspots to refine to single genes is consistent with a model where imbalance of multiple genes contributes to a disease state.

Figure 1

Recurrent Large CNVs in Individuals with Autism versus Developmental Delay Data from individuals with developmental delay (n = 31,518) were derived from recently published reports. CNVs (>400 kbp) enriched among children with autism (blue asterisk) as opposed to developmental delay (red asterisk) and vice versa.

Figure 2

Phenotypic Features of Deletions and Duplications as a Function of Size The scatter plot demonstrates that as the size of the deletion increases, nonverbal IQ significantly decreases, whereas as the size of duplication increases, autism severity significantly increases.

Figure 3

Properties of CNV Hotspots (A and B) Frequency of deletions (A) and duplications (B) at different size ranges is shown for SD-mediated hotspots and smaller (micro, mini, and AluY) hotspots. Although no enrichment for smaller non-SD hotspots is observed among autistic children, a significant enrichment for larger, SD-mediated CNVs is observed. An asterisk denotes a significant difference (p < 0.05) in cases compared to controls via a Mann-Whitney t test. (C and D) Sequence properties of genomic hotspots are shown. A comparison of size (C) and sequence identity (D) of repeats flanking “active” hotspots versus “inactive” genomic hotspots. The error bars indicate the minimum and maximum range of the data points.

Figure 4

Atypical Copy-Number Variants within Disease-Associated Regions Large recurrent CNVs were identified within the 1q21.1 (A) and 17q12 (B) microdeletion loci in autism cases. Private atypical deletion events reveal potential candidate genes CHD1L (A) and ACACA (B), highlighted by the dashed box. Blue (duplication) and red (deletion) histograms depict log₂ relative hybridization signals. SDs flanking the larger recurrent events are mapped at the top.

Figure 5

Autism Candidate Genes Enriched for Copy-Number Variants in Cases versus Controls Atypical deletions of TRPM1 (A) were observed in cases but not in controls within the 15q13.3 microdeletion locus. Targeted genotyping of autism candidate genes DPP10 (B), NRXN1 (C), PLCB1 (D), FHIT (E), and MBD5 (F) revealed multiple deletions (red) and duplications (blue) of genes observed in cases at a higher frequency than in controls. Blue (duplication) and red (deletion) histograms depict log₂ relative hybridization signals.

Figure 6

SD-Associated Copy-Number Variants of HYDIN A de novo deletion and duplication, as well as an inherited duplication, were identified in children with autism. The child with the inherited duplication shares the event with his affected identical twin, but not with his unaffected sibling. Though these events are large and encompass numerous genes, the de novo duplication directly breaks HYDIN. Blue (duplication) and red (deletion) histograms depict log₂ relative hybridization signals.