Contribution of Small Copy Number Variations (CNVs) to Autism Spectrum Disorder (ASD)

Background: Autism Spectrum Disorder (ASD) is characterized by significant and persistent deficits in communication/social interaction and by restricted and repetitive patterns of behavior, interests or activities. ASD has a complex genetic architecture, with involvement of rare, relatively large Copy Number Variations (CNVs) in nearly 10% of the cases. Despite the advancement of high-throughput genomic approaches, the detection of small CNVs (1 to 100 Kb) and large indels is still challenging and therefore little is known about their contribution to ASD. We used a customized Comparative Genomic Hybridization array (aCGH) in order to screen small potentially pathogenic CNVs in a sample of Brazilian subjects with idiopathic ASD.

Objectives: To assess the importance of small CNVs in the etiology of ASD.

Methods: All patients were diagnosed by psychiatrists using DSM-IV, ICD-10 and ASQ. Children's behavior was characterized by score questions of CARS. Patients with known autism-related syndromes, or exposed to teratogenic drugs /infectious agents during pregnancy were excluded from the study. All patients were negative for FMR1 expansion and for CNVs at 15q11.13, 16p11.2 (proximal region) and 22q13 through multiplex ligation probe amplification (MLPA) analysis (SALSA KIT P343-B1 AUTISM-1; MRC-Holland, Amsterdam). The customized aCGH included 269 ASD candidate genes, with a high density of probes targeting exons and exon-intron junctions. CNVs (≥ 1 kb) were only considered potentially pathogenic if they were located at coding regions and had a frequency ≤ 1% in our sample of 200 non-affected Brazilian subjects and in the Database of Genomic Variants (DGV).  All CNVs were validated through Affymetrix 500K SNP-array, MLPA, and/or qPCR.

Results: The customized aCGH was validated by analyzing 15 patients with CNVs from around 60 to 375 Kb at 3p26.3, 15q11-13, 16p11.2, and 22q13 previously detected by the Affymetrix 500K SNP array or by the commercial P343-B1 MLPA kit. All these CNVs were detected by the customized aCGH. Eighty patients with non-syndromic ASD and 20 with syndromic ASD were included in the study. We identified nine CNVs that fulfilled our abovementioned criteria of potential pathogenicity, ranging from 9.5 Kb to 25 Mb, in nine patients with idiopathic ASD. Four CNVs were considered small (< 100 kb). Two of them, detected in non-syndromic patients, affected single genes, namely a duplication at SLC17A6 and a deletion at GRM5. Other two distal 16p11.2 deletions, detected in one syndromic and in one non-syndromic patient, have between 38.9 and 61.8 kb, and involved three genes.

Conclusions: The use of an array designed to detect small CNVs, along with large ones, is a good strategy to improve the elucidation of ASD etiology, as we found a prevalence of 9% of CNVs in our cohort of idiopathic ASD patients, 44% of them smaller than 100 kb. To our knowledge, the two CNVs detected at 16p11.2 are the smallest distal 16p11.2 deletions described so far, and these findings may help to identify the causative ASD gene(s) in this region.