Characterisation of the TBR1 interactome: variants associated with neurodevelopmental disorders disrupt novel protein interactions

TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein–protein interaction

De novo variants disturbing the transactivation capacity of POU3F3 cause a characteristic neurodevelopmental disorder

POU3F3, also referred to as Brain-1, is a well-known transcription factor involved in the development of the central nervous system, but it has not previously been associated with a neurodevelopmental disorder. Here, we report the identification of 19 individuals with heterozygous POU3F3 disruptions, most of which are de novo variants. All individuals had developmental delays and/or intellectual disability and impairments in speech and language skills. Thirteen individuals had characteristic low-set, prominent, and/or cupped ears. Brain abnormalities were observed in seven of eleven MRI reports. POU3F3 is an intronless gene, insensitive to nonsense-mediated decay, and 13 individuals carried protein-truncating variants. All truncating variants that we tested in cellular models led to aberrant subcellular localization of the encoded protein. Luciferase assays demonstrated negative effects of these alleles on transcriptional activation of a reporter with a FOXP2-derived binding motif. In addition to the loss-of-function variants, five individuals had missense variants that clustered at specific positions within the functional domains, and one small in-frame deletion was identified. Two missense variants showed reduced transactivation capacity in our assays, whereas one variant displayed gain-of-function effects, suggesting a distinct pathophysiological mechanism. In bioluminescence resonance energy transfer (BRET) interaction assays, all the truncated POU3F3 versions that we tested had significantly impaired dimerization capacities, whereas all missense variants showed unaffected dimerization with wild-type POU3F3. Taken together, our identification and functional cell-based analyses of pathogenic variants in POU3F3, coupled with a clinical characterization, implicate disruptions of this gene in a characteristic neurodevelopmental disorder

De novo BCL11A variants in neurodevelopmental disorder disrupt multiple aspects of protein function

The rare chromosome 2p16.1-p15 deletion syndrome (MIM 612513) is characterized by intellectual disability, dysmorphic features and microcephaly. A high proportion of affected individuals have autism, behavioral problems and language deficits. Skeletal and organ abnormalities have also been reported. Comparison of the chromosomal regions deleted in different cases suggests that haploinsufficiency of the zinc finger transcription factor BCL11A (MIM 606557) may underlie the neurological features of the syndrome. Although the role of BCL11A in brain development is poorly understood, the gene is expressed in the developing cortex, hippocampus, basal ganglia and cerebellum, and has recently been reported to play a role in the specification of subcortical projection neurons by repressing expression of TBR1 (MIM 604616), a gene recurrently mutated in cases of autism. In addition to its function in the brain, BCL11A has a key role in mediating the switch from the fetal to the adult form of hemoglobin, and persistence of fetal hemoglobin has been reported in several patients with BCL11A deletions. The first disorder-associated missense variants in BCL11A have recently been identified in three unrelated infants with developmental delay. The variants in these patients cluster within the N-terminal portion of the protein, and lie outside the zinc finger DNA-binding domains, in a region of unknown function. We therefore sought to characterize the effects of these variants on protein function in order to confirm their etiological role in disorder in these three patients, and to illuminate the molecular mechanism of disorder. We found that all three variants disrupt the localization of BCL11A within the nucleus and abolish protein dimerization and transcriptional regulatory activity. Our results therefore strongly support a causal role for BCL11A variants in the developmental delay in these patients, and add to the growing evidence that BCL11A is a recurrently mutated gene in neurodevelopmental disorder. Furthermore the characterization of these variants reveals a key role for the N-terminal region of BCL11A in mediating protein dimerization and regulation of transcription

Functional characterisation of FOXP1 mutations found in patients with intellectual disability

Intellectual disability (ID) is a neurodevelopmental disorder manifesting in children before the age of 18, and affects 1.5-2% of the population. It is characterised by a significant impairment of cognitive functioning and adaptive behaviours, and can be classified as mild (IQ 50-70), moderate (IQ >35), or severe (IQ >20). Although many of the exact molecular risk factors contributing to ID are still unknown, there is considerable evidence to support a genetic basis. Large de novo deletions and a nonsense variant disrupting the FOXP1 transcription factor gene have been described in patients with mild to moderate ID and severe speech and language deficits. Whole exome sequencing of 20 parent-child trios with sporadic autism reported a potentially causative de novo truncating mutation in FOXP1 in a severely affected child with evidence for regression, language delay and comorbidity for moderate ID. Functional analysis in cell systems showed that this FOXP1 protein variant mislocalised to the cytoplasm and lost its transcriptional repressor ability. Recently, sequencing of balanced chromosomal abnormalities in patients with autism or other neurodevelopmental disorders revealed disruption of FOXP1 in a subject with global developmental delay, including speech delay. These results are intriguing because FOXP1 is the closest paralogous human gene to FOXP2, a gene associated with rare forms of speech and language disorder. FOXP1 and FOXP2 are expressed in similar neural circuits and directly interact with each other, with the potential to co-regulate downstream targets, including those involved in language development (such as CNTNAP2). Previous sequencing of FOXP1 coding exons in 883 individuals with ID uncovered 8 non synonymous mutations, some of which are predicted to be damaging. In this study, we generated FOXP1 constructs carrying several of these mutations and performed cell based assays to identify their physiological significance. In our analysis we also included synthetic mutations targeting the FOX DNA binding domain in order to investigate nuclear translocation. FOXP1 variants were transfected into human embryonic kidney and neuroblastoma cell lines and the expressed proteins were analysed in terms of size, cellular localisation, transcription factor function and their ability to interact with wild type FOXP1 and FOXP2 proteins. Our findings highlight the importance of performing functional characterisation to help uncover the biological significance of variants identified by genomics approaches, thereby providing insight into pathways underlying complex neurodevelopmental disorders like ID and speech and language impairment

Functional characterization of a large series of NKX2-1 variants in Brain-Lung-Thyroid syndrome reveals diverse molecular mechanisms of disorder.

Brain-Lung-Thyroid syndrome (MIM 610978) is an autosomal dominant disorder resulting from disruption of the homeobox transcription factor gene NKX2-1 (MIM 600635). The disorder exhibits phenotypic variability, both between and within families, but is typically characterized by infant hypotonia that progresses to chorea and other movement abnormalities. Dysarthria (a motor speech disorder) and attentiveness disorder have recently been recognized as common neurological features of the syndrome. Most affected individuals also have thyroid dysfunction and/or lung problems such as neonatal respiratory distress and recurrent infections. Over 100 different lesions affecting the NKX2-1 gene have been reported in cases of Brain-Lung-Thyroid syndrome, including a number of missense variants. However there has been little functional investigation of the effects of these variants to elucidate the molecular mechanisms of disorder. Here we report the functional characterization of forty NKX2-1 variants, employing assay methodologies that could be extended for the high-throughput characterization of transcription factor gene variants identified by next-generation sequencing in disorders. By assessing the effects of NKX2-1 variants on protein expression, subcellular localization, protein-protein interactions and transcriptional regulatory activity, we uncovered diverse molecular-level effects for etiological variants, highlighting the importance of examining multiple aspects of protein function when characterizing putative disorder-related variants. In addition, we confirmed that NKX2-1 interacts with the forkhead transcription factor FOXP2 (MIM 605317) and show that NKX2-1 can also interact with the FOXP2 paralogs FOXP1 (MIM 605515) and FOXP4 (MIM 608924). The interaction between NKX2-1 and FOXP transcription factors may be of significance in brain and lung development. Heterozygous disruption of FOXP2 results in a severe speech/language disorder (MIM 602081). The presence of motor speech deficits as core features of both the NKX2-1 and FOXP2-related disorders points to roles for both genes in the development of speech-related motor circuitry. Strikingly, we find that disorder-related variants in the DNA-binding domains of both NKX2-1 and FOXP2 abrogate the interaction between these proteins