Prospective diagnostic analysis of copy number variants using SNP microarrays in individuals with autism spectrum disorders

Abstract

Copy number variants (CNVs) have repeatedly been found to cause or predispose to autism spectrum disorders (ASDs). For diagnostic purposes, we screened 194 individuals with ASDs for CNVs using Illumina SNP arrays. In several probands, we also analyzed candidate genes located in inherited deletions to unmask autosomal recessive variants. Three CNVs, a de novo triplication of chromosome 15q11-q12 of paternal origin, a deletion on chromosome 9p24 and a de novo 3q29 deletion, were identified as the cause of the disorder in one individual each. An autosomal recessive cause was considered possible in two patients: a homozygous 1p31.1 deletion encompassing PTGER3 and a deletion of the entire DOCK10 gene associated with a rare hemizygous missense variant. We also identified multiple private or recurrent CNVs, the majority of which were inherited from asymptomatic parents. Although highly penetrant CNVs or variants inherited in an autosomal recessive manner were detected in rare cases, our results mainly support the hypothesis that most CNVs contribute to ASDs in association with other CNVs or point variants located elsewhere in the genome. Identification of these genetic interactions in individuals with ASDs constitutes a formidable challenge.

Figure 1

Strategy used for the selection of rare, possibly deleterious CNVs. The criteria included the presence of coding regions in the CNV, a minor allele frequency (MAF) <1% in the DGV, a de novo occurrence of the CNV, a previous association with ASDs, the recurrence of rare CNVs in our cohort and the presence of genes of interest (ie expressed in the brain and with a function related to that of genes previously involved in ASDs). The CNVs highlighted in this study are indicated in italics for each subcategory.

Figure 2

Identification of three pathogenic CNVs: 15q11q12 triplication (family 772, patient 8082), 9p24 deletion (family 15, patient 8378) and 3q29 deletion (family 1315, patient 433). (a) SNP array profiles of the patient with the triplication (8082): the Y axis indicates the log R (above) and the B allele frequency (below); the X axis indicates the position on chromosome 15. The red line (log R ratio profile) corresponds to the median smoothing series (Genomestudio). (b) Confirmation of the triplication by FISH analysis with a probe specific to UBE3A (in red) on peripheral blood cell metaphases and nucleus. The arrows point the triplication (c) Details of the genes included in the triplication. (d) Microsatellite (D15S822) analysis showing two alleles inherited from the father in the proband. (e) Methylation indexes of genes included in the triplicated region showing abnormal methylation of UBE3A and SNRPN in the patient 8082 and comparison with a patient with Angelman syndrome. (f) SNP array profiles of the patient with the 9p24 deletion: the Y axis indicates the log R (above) and the B allele frequency (below); the X axis indicates the position on chromosome 9. (g) Confirmation of the deletion (shown by the arrow) by FISH analysis with a probe specific to the 9p subtelomeric region (green) on peripheral blood cell metaphases. (h) Details of the genes included in the deletion. (i) SNP array profiles of the patient with the 3q29 deletion: the Y axis indicates the log R (above) and the B allele frequency (below); the X axis indicates the position on chromosome 3. (j) Confirmation of the deletion (shown by the arrow) by FISH analysis with a probe specific to the 3q29 region (RP1-196F4, green) on peripheral blood cell metaphases and nuclei. (k) Details of the genes included in the deletion.

Figure 3

Identification of a deletion encompassing DOCK10 associated with the c.6460 G>A/p.Asp2154Asn in a male patient. (a) SNP array profiles of the patients with the deletion and details of the genes included the deletion: the Y axis indicates the log R (above) and the B allele frequency (below); the X axis indicates the position on chromosome 2. (b) Pedigree of family 625 and segregation analysis of the deletion and the c.6460G>A/p.Asp2154Asn variant. (c) Sequence electropherograms showing the c.6460G>A/p.Asp2154Asn variant in the hemizygous state in the proband of family 625 and in the heterozygous state in his father. (d) Alignment of the region flanking c.6460G>A/p.Asp2154Asn in orthologous proteins showing the conservation of the altered amino acids.