Excess Genome-wide Burden of Rare and De Novo Genic CNVs
To explore the contribution of CNV to ASD, we expanded our previous study (stage 1)6 with an additional 1,604 families (stage 2), bringing the total to 9,050 individuals from 2,845 ASD-affected families. We used an analytical pipeline of Illumina 1M arrays6,30 to detect rare CNV in families and applied a series of QC filters, including validation of all de novo events by at least one method (Tables S1A–S1C). In total, 1,359 stage 2 families passed QC, and 2,446 families were used in the combined analyses of both stages (Tables S2A and S2B). Of these, 2,147 families were European, and 299 were of other ancestries.21 We used the same pipeline to analyze 2,640 control individuals of European ancestry26,27,29 who were genotyped with the same array platforms. Ancestry was inferred by analysis of SNP genotype data (Table S1B). The rate, size, and number of genes affected by rare (<1% frequency) CNVs were assessed. Consistent with our previous data, we observed that compared to control subjects, affected subjects had an increased burden in the number of genes affected by rare CNVs (1.41-fold increase, empirical p = 1 × 10−5; Table 1). This enrichment was apparent for both deletions and duplications and remained after we controlled for potential case-control differences (Table 1). Similar findings were obtained when each stage was considered separately (Tables S3A–S3C).
Array- and exome-based studies have revealed a substantial contribution of de novo variation to ASD risk,19 prompting us to assess this further. After screening 2,096 trios (of all ancestries), we found 102 rare de novo CNVs in 99 affected subjects (three of whom had two events; Table S4). Overall, 4.7% of trios had at least one de novo CNV, whereas control subjects had a frequency of 1%–2%.4,31,38 The average length of de novo events in our affected subjects (1.17 Mb) was larger than that of de novo CNVs in unaffected siblings from the Simons Simplex Collection (0.67 Mb, p = 0.01)4 and in control trios (0.55 Mb, p = 0.01).31 The average size of de novo CNVs was also larger than the size of all rare CNVs in our affected (188 kb) and control (159 kb) subjects. De novo CNVs affected 3.8-fold more genes in affected subjects than in control subjects4,31 (2.6-fold for deletions and 6.1-fold for duplications). Even after controlling for the difference in CNV size by proportionally scaling the number of intersected genes in each group, we observed a 1.77-fold difference (1.2-fold for deletions and 2.8-fold for duplications, p = 0.02). Furthermore, de novo CNVs in simplex families intersected 4.0-fold more genes than did CNVs in controls4,31 (1.8-fold after size correction, p = 0.01). There were no significant differences between subjects from simplex families and those from multiplex families in the frequency (5% and 4.2%, respectively) or gene content (n = 18.7 and 18.8, respectively) of de novo CNVs. Similarly, no significant difference was found between males and females in the size (1.17 and 1.2 Mb, respectively) or gene content (n = 18 and 17.3, respectively) of de novo CNVs. For 85 of 102 de novo events, it was possible to determine the parent of origin, and roughly equal numbers of events originated on the paternal allele (n = 45) and the maternal allele (n = 40) (Tables S5A–S5H). Taken together, our data indicate that there is an increased burden of de novo events in ASD-affected subjects. The clinical relevance of de novo CNVs in ASD is confirmed by the fact that among 102 such events identified, half (n = 46) are considered etiologically relevant, including 40 loci known to be involved in ASD and ID (see below).
We replicated previous observations, such as a de novo deletion intersecting PTCHD1AS in a male (adding to the evidence that both PTCHD1 and PTCHD1AS contribute to ASD risk14) and de novo events involving the miRNA miR137 (MIM 614304) in 1p21.2–p21.3 in two subjects. Microdeletions of miR137 have been reported in ASD,39 ID,40 and schizophrenia.41 Examples of ASD candidate genes identified by small de novo CNVs include SETD5, DTNA (MIM 601239), and LSAMP (MIM 603241) (Supplemental Data section “Highlighted Genes,” Figures S9, S10, and S14).