Variants of the CNTNAP2 5′ promoter as risk factors for autism spectrum disorders: a genetic and functional approach

Abstract

Contactin-associated protein-like 2 gene (CNTNAP2), a member of the Neurexin gene superfamily, is one of the best-replicated risk genes for autism spectrum disorders (ASD). ASD are predominately genetically determined neurodevelopmental disorders characterized by impairments of language development, social interaction and communication, as well as stereotyped behavior and interests. Although CNTNAP2 expression levels were proposed to alter ASD risk, no study to date has focused on its 5' promoter. Here, we directly sequenced the CNTNAP2 5' promoter region of 236 German families with one child with ASD and detected four novel variants. Furthermore, we genotyped the three most frequent variants (rs150447075, rs34712024, rs71781329) in an additional sample of 356 families and found nominal association of rs34712024G with ASD and rs71781329GCG[7] with language development. The four novel and the three known minor alleles of the identified variants were predicted to alter transcription factor binding sites (TFBS). At the functional level, the respective sequences spanning these seven variants were bound by nuclear factors. In a luciferase promoter assay, the respective minor alleles showed cell line-specific and differentiation stage-dependent effects at the level of promoter activation. The novel potential rare risk-variant M2, a G>A mutation -215 base pairs 5' of the transcriptional start site, significantly reduced promoter efficiency in HEK293T and in undifferentiated and differentiated neuroblastoid SH-SY5Y cells. This variant was transmitted to a patient with autistic disorder. The under-transmitted, protective minor G allele of the common variant rs34712024, in contrast, increased transcriptional activity. These results lead to the conclusion that the pathomechanism of CNTNAP2 promoter variants on ASD risk is mediated by their effect on TFBSs, and thus confirm the hypothesis that a reduced CNTNAP2 level during neuronal development increases liability for ASD.

Identification of Human Neuronal Protein Complexes Reveals Biochemical Activities and Convergent Mechanisms of Action in Autism Spectrum Disorders

“…Large-scale profiling of physical protein interactions in specific tissues or induced pluripotent stem cell (iPSC)-derived neurons is currently challenging due to the large number of cells required. We therefore differentiated SH-SY5Y cells into neuron-like cells (Supplemental Experimental Procedures), which have been frequently used to model neurological and neuropsychiatric diseases including ASD(Chiocchetti et al, 2014). We confirmed the neuronal characteristics of the differentiated cells by visual inspection of neuronal morphology (Fig.…”

Intragenic CNTNAP2 Deletions: A Bridge Too Far

“…After genotyping a large sample of families with a single child with ASD according to ADI-R, ADOS-G, and DSM-IV criteria, 7 variants in the 5 ′ core promoter region 600 bp upstream from the transcription start site of CNTNAP2 were identified [Chiocchetti et al, 2015]. Nominal association with ASD was found for rs34712024 [G] and with language development for rs71781329GCG [7].…”

“…The 7 variants were located in transcription factor binding sites, and by luciferase assays, the respective minor alleles showed effects on CNTNAP2 promoter activation. The authors hypothesized that reduced CNTNAP2 transcription during neuronal development increases liability for ASD [Chiocchetti et al, 2015].…”

A common variant of the CNTNAP2 gene is associated with structural variation in the left superior occipital gyrus

“…Among these targets, the gene encoding contactin-associated protein-like 2, CNTNAP2, is of particular interest (Vernes et al, 2008). Common polymorphisms of CNTNAP2 have been associated with quantitative variation in language-related phenotypes in different neurodevelopmental disorders, including specific language impairment (Newbury et al, 2011;Vernes et al, 2008), autism spectrum disorder (ASD) (Alarcon et al, 2008;Chiocchetti et al, 2014) and dyslexia (Peter et al, 2011); but see (Newbury et al, 2011). Recent large-scale studies suggest that common CNTNAP2 variants are not associated with a qualitative diagnosis of ASD (i.e.…”