Publications

Abstract

Genome-wide association screens aim to identify common genetic variants contributing to the phenotypic variability of complex traits, such as human height or brain morphology. The identified genetic variants are mostly within noncoding genomic regions and the biology of the genotype–phenotype association typically remains unclear. In this article, we propose a complementary targeted strategy to reveal the genetic underpinnings of variability in subcortical brain volumes, by specifically selecting genomic loci that are experimentally validated forebrain enhancers, active in early embryonic development. We hypothesized that genetic variation within these enhancers may affect the development and ultimately the structure of subcortical brain regions in adults. We tested whether variants in forebrain enhancer regions showed an overall enrichment of association with volumetric variation in subcortical structures of >13,000 healthy adults. We observed significant enrichment of genomic loci that affect the volume of the hippocampus within forebrain enhancers (empirical P = 0.0015), a finding which robustly passed the adjusted threshold for testing of multiple brain phenotypes (cutoff of P < 0.0083 at an alpha of 0.05). In analyses of individual single nucleotide polymorphisms (SNPs), we identified an association upstream of the ID2 gene with rs7588305 and variation in hippocampal volume. This SNP-based association survived multiple-testing correction for the number of SNPs analyzed but not for the number of subcortical structures. Targeting known regulatory regions offers a way to understand the underlying biology that connects genotypes to phenotypes, particularly in the context of neuroimaging genetics. This biology-driven approach generates testable hypotheses regarding the functional biology of identified associations.

Abstract

Background Heterozygous mutations in CNTNAP2 have been identified in patients with a range of complex phenotypes including intellectual disability, autism and schizophrenia. However heterozygous CNTNAP2 mutations are also found in the normal population. Conversely, homozygous mutations are rare in patient populations and have not been found in any unaffected individuals. Case presentation We describe a consanguineous family carrying a deletion in CNTNAP2 predicted to abolish function of its protein product, CASPR2. Homozygous family members display epilepsy, facial dysmorphisms, severe intellectual disability and impaired language. We compared these patients with previously reported individuals carrying homozygous mutations in CNTNAP2 and identified a highly recognisable phenotype. Conclusions We propose that CASPR2 loss produces a syndrome involving early-onset refractory epilepsy, intellectual disability, language impairment and autistic features that can be recognized as CASPR2 deficiency disorder. Further screening for homozygous patients meeting these criteria, together with detailed phenotypic and molecular investigations will be crucial for understanding the contribution of CNTNAP2 to normal and disrupted development.

Abstract

Forkhead-box protein P2 is a transcription factor that has been associated with intriguing aspects of cognitive function in humans, non-human mammals, and song-learning birds. Heterozygous mutations of the human FOXP2 gene cause a monogenic speech and language disorder. Reduced functional dosage of the mouse version (Foxp2) causes deficient cortico-striatal synaptic plasticity and impairs motor-skill learning. Moreover, the songbird orthologue appears critically important for vocal learning. Across diverse vertebrate species, this well-conserved transcription factor is highly expressed in the developing and adult central nervous system. Very little is known about the mechanisms regulated by Foxp2 during brain development. We used an integrated functional genomics strategy to robustly define Foxp2-dependent pathways, both direct and indirect targets, in the embryonic brain. Specifically, we performed genome-wide in vivo ChIP–chip screens for Foxp2-binding and thereby identified a set of 264 high-confidence neural targets under strict, empirically derived significance thresholds. The findings, coupled to expression profiling and in situ hybridization of brain tissue from wild-type and mutant mouse embryos, strongly highlighted gene networks linked to neurite development. We followed up our genomics data with functional experiments, showing that Foxp2 impacts on neurite outgrowth in primary neurons and in neuronal cell models. Our data indicate that Foxp2 modulates neuronal network formation, by directly and indirectly regulating mRNAs involved in the development and plasticity of neuronal connections

Abstract

Mutations of the human FOXP2 gene have been shown to cause severe difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech (developmental verbal dyspraxia or DVD). Affected individuals are impaired in multiple aspects of expressive and receptive linguistic processing and ­display abnormal grey matter volume and functional activation patterns in cortical and subcortical brain regions. The protein encoded by FOXP2 belongs to a divergent subgroup of forkhead-box transcription factors, with a distinctive DNA-binding domain and motifs that mediate hetero- and homodimerization. This chapter describes the successful use of FOXP2 as a unique molecular window into neurogenetic pathways that are important for speech and language development, adopting several complementary strategies. These include direct functional investigations of FOXP2 splice variants and the effects of etiological mutations. FOXP2’s role as a transcription factor also enabled the development of functional genomic routes for dissecting neurogenetic mechanisms that may be relevant for speech and language. By identifying downstream target genes regulated by FOXP2, it was possible to identify common regulatory themes in modulating synaptic plasticity, neurodevelopment, and axon guidance. These targets represent novel entrypoints into in vivo pathways that may be disturbed in speech and language disorders. The identification of FOXP2 target genes has also led to the discovery of a shared neurogenetic pathway between clinically distinct language disorders; the rare Mendelian form of DVD and a complex and more common form of language ­disorder known as Specific Language Impairment.

Abstract

It is a challenge to identify the molecular networks contributing to the neural basis of human speech. Mutations in transcription factor FOXP2 cause difficulties mastering fluent speech (developmental verbal dyspraxia, DVD), while mutations of sushi-repeat protein SRPX2 lead to epilepsy of the rolandic (sylvian) speech areas, with DVD or with bilateral perisylvian polymicrogyria. Pathophysiological mechanisms driven by SRPX2 involve modified interaction with the plasminogen activator receptor (uPAR). Independent chromatin-immunoprecipitation microarray screening has identified the uPAR gene promoter as a potential target site bound by FOXP2. Here, we directly tested for the existence of a transcriptional regulatory network between human FOXP2 and the SRPX2/uPAR complex. In silico searches followed by gel retardation assays identified specific efficient FOXP2 binding sites in each of the promoter regions of SRPX2 and uPAR. In FOXP2-transfected cells, significant decreases were observed in the amounts of both SRPX2 (43.6%) and uPAR (38.6%) native transcripts. Luciferase reporter assays demonstrated that FOXP2 expression yielded marked inhibition of SRPX2 (80.2%) and uPAR (77.5%) promoter activity. A mutant FOXP2 that causes DVD (p.R553H) failed to bind to SRPX2 and uPAR target sites, and showed impaired down-regulation of SRPX2 and uPAR promoter activity. In a patient with polymicrogyria of the left rolandic operculum, a novel FOXP2 mutation (p.M406T) was found in the leucine-zipper (dimerization) domain. p.M406T partially impaired FOXP2 regulation of SRPX2 promoter activity, while that of the uPAR promoter remained unchanged. Together with recently described FOXP2-CNTNPA2 and SRPX2/uPAR links, the FOXP2-SRPX2/uPAR network provides exciting insights into molecular pathways underlying speech-related disorders.