Phenotypic Effects of Loss of Function CNTNAP2 Variants

Background: CNTNAP2 is an autism spectrum disorder (ASD) candidate gene that encodes CASPR2, a large protein expressed in the nervous system. Heterozygous CNTNAP2 variants have been reported in individuals with ASD and speech-language disorders; however, these variants are frequently inherited, making it difficult to interpret their functional relevance. We have the unique opportunity to study Mennonite and Amish families that harbor a truncating, loss of function (LoF) mutation in CNTNAP2. Individuals who are homozygous for this variant present with cortical dysplasia-focal epilepsy syndrome (CDFE), characterized by epilepsy, intellectual disability, and ASD. No studies to date include a detailed assessment of the behavioral phenotype of CDFE in homozygotes nor the potential neurocognitive effects of this CNTNAP2 variant in their heterozygous carrier relatives.

Objectives: In the current study, we conducted behavioral assessments to phenotype individuals who are homozygous and heterozygous for CNTNAP2 LoF variants and compared results to their homozygous relatives without the variant. By advancing our understanding of the effects of rare LoF variants in CNTNAP2, we hope to elucidate an important mechanistic pathway in ASD.

Methods: We completed phenotypic batteries on four Old Order Amish and Mennonite families consisting of at least one child with CDFE. The phenotypic battery included developmental/cognitive assessment using the Mullen Scales of Early Learning (MSEL), Differential Ability Scales (DAS-2), or the Wechsler Abbreviated Scales of Intelligence (WASI-2). Speech assessments were also completed and included the Goldman-Fristoe Test of Articulation (GFTA-2) and the non-word repetition task of the Comprehensive Test of Phonological Processing (CTOPP-2). The non-word repetition task is robust measure of language impairment which reliably discriminates between individuals with and without a history of language impairment, including bilingual children from diverse language backgrounds. All children above a chronological and developmental age of 12 months were given the Autism Diagnostic Observation Schedule (ADOS-2) by a research reliable examiner.

Results: Children with CDFE scored in the moderate to profound range of intellectual disability. All verbal children with CDFE had significant phonological disorders as measured by the GFTA, providing new evidence for discrete language pathology in CDFE. Notably, carrier children and adults also performed significantly worse on the CTOPP non-word repetition task compared to non-carrier children (p = .02), providing additional evidence for a mild, intermediate language phenotype in heterozygous CNTNAP2 carriers. Several carrier siblings had a moderate level of ASD symptoms, as measured by the ADOS-2. Non-carrier siblings did not present with elevated ADOS-2 scores, and their social engagement was judged to be within the typical range.

Conclusions: Using a well-controlled experimental paradigm, we provide compelling evidence of mild language impairment and ASD symptomatology in individuals haploinsufficient for CNTNAP2, underscoring the fundamental role of this gene in the development of human language and social behavior.