Linkage, association, and gene-expression analyses identify CNTNAP2 as an autism-susceptibility gene

Abstract

Autism is a genetically complex neurodevelopmental syndrome in which language deficits are a core feature. We describe results from two complimentary approaches used to identify risk variants on chromosome 7 that likely contribute to the etiology of autism. A two-stage association study tested 2758 SNPs across a 10 Mb 7q35 language-related autism QTL in AGRE (Autism Genetic Resource Exchange) trios and found significant association with Contactin Associated Protein-Like 2 (CNTNAP2), a strong a priori candidate. Male-only containing families were identified as primarily responsible for this association signal, consistent with the strong male affection bias in ASD and other language-based disorders. Gene-expression analyses in developing human brain further identified CNTNAP2 as enriched in circuits important for language development. Together, these results provide convergent evidence for involvement of CNTNAP2, a Neurexin family member, in autism, and demonstrate a connection between genetic risk for autism and specific brain structures.

Figure 1

Dense SNP Analysis of the 7q35 Autism-Related Language QTL Region Highlights Four Variants for Stage 2 Follow-Up The negative log of the significance level of each block or single-SNP transmission bias is plotted according to base-pair position. Nominally significant SNPs (p < 0.01, pLOD > 2) that were followed up in Stage 2 are boxed. Tics above the graph demarcate gene content.

Figure 2

Relative Location of Associated SNPs within CNTNAP2 Gene structure is depicted from 5′ to 3′ with exons represented by small blocks or lines along the gene, and the black arrowhead indicates gene direction. The SNPs that comprised the nominally significant (p < 0.01) block 254 from Stage 1 are represented by blue vertical lines over the bottom LD plot. The tag SNP, rs2710102, confirmed in the male-only trios (n = 187) in Stage 2 is circled in red. The LD plot was generated with the Haploview program.

Figure 3

Representational Oligonucleotide Microarray Analysis and Assessment of Mendelian Errors in SNPs Identify a Rare CNTNAP2 Deletion The two vertical lines in the graph show the endpoints of the deletion estimated from ROMA data by a Hidden Markov Model-based predictor. SNP genotyping data are superimposed on the graph at the bottom. The two green triangles represent SNPs that were informative and showed no Mendelian errors, indicating that the deleted region is somewhere between these two points. Purple diamonds denote SNPs that showed Mendelian errors consistent with a deletion. Orange squares represent SNPs that were uninformative. Chromosome 7 locations are from the UCSC Genome Browser May 2004 assembly.

Figure 4

CNTNAP2 Expression in Human Fetal Brain Demarcates Anterior Frontal and Temporal Perisylvian Regions, Striatum, and Thalamus (A) Autoradiograms of midline sagittal sections obtained from an 18-week and a 20-week fetal brain show striking enrichment of CNTNAP2 in frontal gray matter, corresponding roughly to a region between the orbital gyrus and superior frontal cortical anlagen. Strong labeling is also observed in basal ganglia, thalamus, and amygdala. (B) Autoradiograms of coronal sections from two 19-week fetal brains demonstrate high levels of CNTNAP2 in amygdala, caudate, cortical plate, dorsal thalamus, and putamen. Abbreviations: Amyg, amygdala; Caud, caudate; Ctx, cortex; Put, putamen; and Thal, thalamus.