Contactin 4 as an autism susceptibility locus

Abstract

Structural and sequence variation have been described in several members of the contactin (CNTN) and contactin-associated protein (CNTNAP) gene families in association with neurodevelopmental disorders, including autism. Using array comparative genome hybridization (CGH), we identified a maternally inherited ∼535 kb deletion at 3p26.3 encompassing the 5' end of the contactin 4 gene (CNTN4) in a patient with autism. Based on this finding and previous reports implicating genomic rearrangements of CNTN4 in autism spectrum disorders (ASDs) and 3p- microdeletion syndrome, we undertook sequencing of the coding regions of the gene in a local ASD cohort in comparison with a set of controls. Unique missense variants were identified in 4 of 75 unrelated individuals with ASD, as well as in 1 of 107 controls. All of the amino acid substitutions were nonsynonomous, occurred at evolutionarily conserved positions, and were, thus, felt likely to be deleterious. However, these data did not reach statistical significance, nor did the variants segregate with disease within all of the ASD families. Finally, there was no detectable difference in binding of two of the variants to the interacting protein PTPRG in vitro. Thus, additional larger studies will be necessary to determine whether CNTN4 functions as an autism susceptibility locus in combination with other genetic and/or environmental factors.

Figure 1

Microarray and FISH characterization of 3p26.3 deletion in patient A154. (A) Microarray analysis using a bacterial artificial chromosome (BAC)-based microarray (SignatureChip v4.0, Signature Genomic Laboratories, Spokane, WA) was performed using previously published methods [Bejjani et al., 2005] and showed a single-copy loss of 3 BAC clones from the short arm of chromosome 3 at 3p26.3 (chr3:1,711,180-2,245,625, UCSC March 2006 hg18 assembly), approximately 535 kb in size. Probes are arranged with the most distal p-arm clones on the left and the most distal q-arm clones on the right. The blue line is a plot of the array CGH data from the first microarray experiment (reference Cy5/patient Cy3). The pink line is a plot of the array CGH data from the second microarray experiment in which the dyes have been reversed (patient Cy5/reference Cy3). (B) A zoomed-in image of the deletion shown in (A). The minimal deletion size is represented by the purple bar, and the blue bars represent genes. Results are visualized using Genoglyphix (Signature Genomic Laboratories, Spokane, WA). (C) Metaphase FISH analysis showing deletion of the region shown by microarray analysis in (A). BAC clone RP11-96G4 from 3p26.3 is labeled in red, and the chromosome 3 centromere probe is labeled in green as a control. Missing red signal and retained green signal indicates deletion at 3p26.3 on one homologue (arrow).

Figure 2

Schematic of domains of CNTN4 and approximate sites of variant amino acids. (A) The 6 Ig-like domains of CNTN4 are shown as open loops and the 4 fibronectin II-like domains as filled boxes. The sites of the variant amino acids are shown as gray (control) or black (ASD patients) arrows. (B) Surface representations of the 4 Ig-like domains of mouse CNTN4 (CNTN4^Ig1-4) with binding site of PTPRG shown in red. The altered amino acids in the ASD patients within the Ig-like domains are shown in blue. The variant Y630C is not shown since this mutation lies within the FNIII region of CNTN4, the structure of which has not yet been determined.