A functional genetic link between distinct developmental language disorders

Abstract

Background: Rare mutations affecting the FOXP2 transcription factor cause a monogenic speech and language disorder. We hypothesized that neural pathways downstream of FOXP2 influence more common phenotypes, such as specific language impairment.

Methods: We performed genomic screening for regions bound by FOXP2 using chromatin immunoprecipitation, which led us to focus on one particular gene that was a strong candidate for involvement in language impairments. We then tested for associations between single-nucleotide polymorphisms (SNPs) in this gene and language deficits in a well-characterized set of 184 families affected with specific language impairment.

Results: We found that FOXP2 binds to and dramatically down-regulates CNTNAP2, a gene that encodes a neurexin and is expressed in the developing human cortex. On analyzing CNTNAP2 polymorphisms in children with typical specific language impairment, we detected significant quantitative associations with nonsense-word repetition, a heritable behavioral marker of this disorder (peak association, P=5.0x10(-5) at SNP rs17236239). Intriguingly, this region coincides with one associated with language delays in children with autism.

Conclusions: The FOXP2-CNTNAP2 pathway provides a mechanistic link between clinically distinct syndromes involving disrupted language.

Figure 1 (facing page). Identification of CNTNAP2 as a Direct Neural Target Bound by Human FOXP2

In Panel A, a 300-bp clone was identified through shotgun cloning of gene fragments identified by FOXP2-chromatin immunoprecipitation and localized to intron 1 of the human CNTNAP2 gene in 7q35. Semiquantitative PCR analysis indicated consistent enrichment of this region in multiple independent experiments in a neuronlike cell line immunoprecipitated with an N-terminal FOXP2 antibody (lane 2), as compared with a control sample without the antibody (lane 3) and input DNA samples (lane 1). Lane 4 shows the water control sample. Two FOXP2 consensus binding sites were identified (highlighted in red). In Panel B, electrophoretic mobility shift assays (EMSAs) using nuclear extracts from transfected HEK293T cells assessed the ability of FOXP2 protein to bind to the 5′ consensus binding site (highlighted in red). Efficient binding to the CNTNAP probe was observed when FOXP2 was present but not when either un-transfected cells or cells expressing a mutant form of FOXP2 (R553H) were used. Binding to the labeled probe was efficiently reduced by competition with an un-labeled probe (CNTNAP) but not by a mutant form of the probe (CNTNAP-M) or an irrelevant binding site (NFK). The arrow shows the position of the shift caused by FOXP2 binding to the CNTNAP probe.

Figure 2. Analyses of the Effects of FOXP2 on Neural Expression of CNTNAP2

Panel A shows the regulation of CNTNAP2 expression by FOXP2 in human neuronlike cells. The expression of messenger RNA (mRNA) was assessed with quantitative reverse-transcriptase PCR in SH-SY5Y cells. The cells were stably transfected either with a construct expressing FOXP2 (FOXP2-positive cells) or with an empty control vector that does not contain a gene insert (control cells). Levels of CNTNAP2 mRNA in these cells were inversely proportional to that of FOXP2. Findings were consistent for three sets of primer pairs recognizing distinct combinations of CNTNAP2 exons (primers A to C). Expression changes are given as the mean log₂ expression ratios in FOXP2-positive cells, as compared with empty controls, normalized for equal expression of the internal control, GAPDH. The I bars represent standard errors. The P values were calculated with the use of two-tailed unpaired t-tests. Panel B shows nonoverlapping mRNA expression for CNTNAP2 and FOXP2 in human fetal cortex. Adjacent sections from human fetal brain (at 18 to 22 weeks’ gestation) were processed for in situ hybridization, dipped in film emulsion, and visualized by dark-field microscopy. Within the cerebral cortex, the highest levels of CNTNAP2 mRNA are observed between bands of FOXP2 expression, putatively within layers II and III of the cortical plate (subpanel a). In contrast, FOXP2 is present at high levels in the molecular zone, deep layers of the cortical plate, and subplate (subpanel b). Cortical lamination is highlighted in a bright-field image of a section stained with cresyl violet (subpanel c). Nonoverlapping expression patterns for CNTNAP2 and FOXP2 are schematized in subpanel d. CP denotes cortical plate, MZ molecular zone, and SP subplate.

Figure 3. Association between CNTNAP2 and Language Deficits in Families with Specific Language Impairment

Panel A shows the results of analyses with a quantitative transmission disequilibrium test (QTDT) of SNPs from the CNTNAP2 locus and their association with measures of an ability to repeat nonsense words (orange), to express language (green), and to understand language (blue). The circles show the positions of individual SNPs, and the black diamonds at the top of the graph indicate the relative positions of exons, according to the National Center for Biotechnology Information genetic sequence (build 35). The orange square denotes the position of the FOXP2-bound fragment from the shotgun cloning of gene fragments isolated by chromatin-immunoprecipitation screening. (Details regarding SNP locations, allele frequencies, QTDT results, and effect sizes are available in Table S3 in the Supplementary Appendix.) Panel B shows the effect of the multimarker haplotype ht1 on scores evaluating the ability to repeat nonsense words on the Children’s Test of Nonword Repetition. Scores on the scale range from 46 to 141, with a mean of 100 and a standard deviation of 15 in the general population. Lower scores indicate poorer performance. Children (gray) were divided into three groups on the basis of the numbers of copies of the putative risk allele ht1 that they carried. The mean score on nonsense-word repetition dropped by about 6 points as a consequence of carrying one or more copies of ht1. Similar results were seen with a larger sample that also included parents with available phenotypic data (black). The I bars represent standard errors. QTDT analyses of the multimarker haplotypes yielded a P value of 6.0×10^-4 for the association between ht1 and measures of nonsense-word repetition (Table S4 in the Supplementary Appendix).