Characterization 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.

Heterozygous variants that disturb the transcriptional repressor activity of FOXP4 cause a developmental disorder with speech/language delays and multiple congenital abnormalities.

PURPOSE: Heterozygous pathogenic variants in various FOXP genes cause specific developmental disorders. The phenotype associated with heterozygous variants in FOXP4 has not been previously described. METHODS: We assembled a cohort of eight individuals with heterozygous and mostly de novo variants in FOXP4: seven individuals with six different missense variants and one individual with a frameshift variant. We collected clinical data to delineate the phenotypic spectrum, and used in silico analyses and functional cell-based assays to assess pathogenicity of the variants. RESULTS: We collected clinical data for six individuals: five individuals with a missense variant in the forkhead box DNA-binding domain of FOXP4, and one individual with a truncating variant. Overlapping features included speech and language delays, growth abnormalities, congenital diaphragmatic hernia, cervical spine abnormalities, and ptosis. Luciferase assays showed loss-of-function effects for all these variants, and aberrant subcellular localization patterns were seen in a subset. The remaining two missense variants were located outside the functional domains of FOXP4, and showed transcriptional repressor capacities and localization patterns similar to the wild-type protein. CONCLUSION: Collectively, our findings show that heterozygous loss-of-function variants in FOXP4 are associated with an autosomal dominant neurodevelopmental disorder with speech/language delays, growth defects, and variable congenital abnormalities.

Mutations of the Transcriptional Corepressor ZMYM2 Cause Syndromic Urinary Tract Malformations.

Congenital anomalies of the kidney and urinary tract (CAKUT) constitute one of the most frequent birth defects and represent the most common cause of chronic kidney disease in the first three decades of life. Despite the discovery of dozens of monogenic causes of CAKUT, most pathogenic pathways remain elusive. We performed whole-exome sequencing (WES) in 551 individuals with CAKUT and identified a heterozygous de novo stop-gain variant in ZMYM2 in two different families with CAKUT. Through collaboration, we identified in total 14 different heterozygous loss-of-function mutations in ZMYM2 in 15 unrelated families. Most mutations occurred de novo, indicating possible interference with reproductive function. Human disease features are replicated in X. tropicalis larvae with morpholino knockdowns, in which expression of truncated ZMYM2 proteins, based on individual mutations, failed to rescue renal and craniofacial defects. Moreover, heterozygous Zmym2-deficient mice recapitulated features of CAKUT with high penetrance. The ZMYM2 protein is a component of a transcriptional corepressor complex recently linked to the silencing of developmentally regulated endogenous retrovirus elements. Using protein-protein interaction assays, we show that ZMYM2 interacts with additional epigenetic silencing complexes, as well as confirming that it binds to FOXP1, a transcription factor that has also been linked to CAKUT. In summary, our findings establish that loss-of-function mutations of ZMYM2, and potentially that of other proteins in its interactome, as causes of human CAKUT, offering new routes for studying the pathogenesis of the disorder.

Investigating genetic links between grapheme-colour synaesthesia and neuropsychiatric traits.

Synaesthesia is a neurological phenomenon affecting perception, where triggering stimuli (e.g. letters and numbers) elicit unusual secondary sensory experiences (e.g. colours). Family-based studies point to a role for genetic factors in the development of this trait. However, the contributions of common genomic variation to synaesthesia have not yet been investigated. Here, we present the SynGenes cohort, the largest genotyped collection of unrelated people with grapheme-colour synaesthesia (n = 723). Synaesthesia has been associated with a range of other neuropsychological traits, including enhanced memory and mental imagery, as well as greater sensory sensitivity. Motivated by the prior literature on putative trait overlaps, we investigated polygenic scores derived from published genome-wide scans of schizophrenia and autism spectrum disorder (ASD), comparing our SynGenes cohort to 2181 non-synaesthetic controls. We found a very slight association between schizophrenia polygenic scores and synaesthesia (Nagelkerke's R2 = 0.0047, empirical p = 0.0027) and no significant association for scores related to ASD (Nagelkerke's R2 = 0.00092, empirical p = 0.54) or body mass index (R2 = 0.00058, empirical p = 0.60), included as a negative control. As sample sizes for studying common genomic variation continue to increase, genetic investigations of the kind reported here may yield novel insights into the shared biology between synaesthesia and other traits, to complement findings from neuropsychology and brain imaging. This article is part of a discussion meeting issue 'Bridging senses: novel insights from synaesthesia'.

Toward Robust Functional Neuroimaging Genetics of Cognition.

A commonly held assumption in cognitive neuroscience is that, because measures of human brain function are closer to underlying biology than distal indices of behavior/cognition, they hold more promise for uncovering genetic pathways. Supporting this view is an influential fMRI-based study of sentence reading/listening by Pinel et al. (2012), who reported that common DNA variants in specific candidate genes were associated with altered neural activation in language-related regions of healthy individuals that carried them. In particular, different single-nucleotide polymorphisms (SNPs) of FOXP2 correlated with variation in task-based activation in left inferior frontal and precentral gyri, whereas a SNP at the KIAA0319/TTRAP/THEM2 locus was associated with variable functional asymmetry of the superior temporal sulcus. Here, we directly test each claim using a closely matched neuroimaging genetics approach in independent cohorts comprising 427 participants, four times larger than the original study of 94 participants. Despite demonstrating power to detect associations with substantially smaller effect sizes than those of the original report, we do not replicate any of the reported associations. Moreover, formal Bayesian analyses reveal substantial to strong evidence in support of the null hypothesis (no effect). We highlight key aspects of the original investigation, common to functional neuroimaging genetics studies, which could have yielded elevated false-positive rates. Genetic accounts of individual differences in cognitive functional neuroimaging are likely to be as complex as behavioral/cognitive tests, involving many common genetic variants, each of tiny effect. Reliable identification of true biological signals requires large sample sizes, power calculations, and validation in independent cohorts with equivalent paradigms.SIGNIFICANCE STATEMENT A pervasive idea in neuroscience is that neuroimaging-based measures of brain function, being closer to underlying neurobiology, are more amenable for uncovering links to genetics. This is a core assumption of prominent studies that associate common DNA variants with altered activations in task-based fMRI, despite using samples (10-100 people) that lack power for detecting the tiny effect sizes typical of genetically complex traits. Here, we test central findings from one of the most influential prior studies. Using matching paradigms and substantially larger samples, coupled to power calculations and formal Bayesian statistics, our data strongly refute the original findings. We demonstrate that neuroimaging genetics with task-based fMRI should be subject to the same rigorous standards as studies of other complex traits.

A set of regulatory genes co-expressed in embryonic human brain is implicated in disrupted speech development.

Genetic investigations of people with impaired development of spoken language provide windows into key aspects of human biology. Over 15 years after FOXP2 was identified, most speech and language impairments remain unexplained at the molecular level. We sequenced whole genomes of nineteen unrelated individuals diagnosed with childhood apraxia of speech, a rare disorder enriched for causative mutations of large effect. Where DNA was available from unaffected parents, we discovered de novo mutations, implicating genes, including CHD3, SETD1A and WDR5. In other probands, we identified novel loss-of-function variants affecting KAT6A, SETBP1, ZFHX4, TNRC6B and MKL2, regulatory genes with links to neurodevelopment. Several of the new candidates interact with each other or with known speech-related genes. Moreover, they show significant clustering within a single co-expression module of genes highly expressed during early human brain development. This study highlights gene regulatory pathways in the developing brain that may contribute to acquisition of proficient speech.

Rare variants in axonogenesis genes connect three families with sound-color synesthesia.

Synesthesia is a rare nonpathological phenomenon where stimulation of one sense automatically provokes a secondary perception in another. Hypothesized to result from differences in cortical wiring during development, synesthetes show atypical structural and functional neural connectivity, but the underlying molecular mechanisms are unknown. The trait also appears to be more common among people with autism spectrum disorder and savant abilities. Previous linkage studies searching for shared loci of large effect size across multiple families have had limited success. To address the critical lack of candidate genes, we applied whole-exome sequencing to three families with sound-color (auditory-visual) synesthesia affecting multiple relatives across three or more generations. We identified rare genetic variants that fully cosegregate with synesthesia in each family, uncovering 37 genes of interest. Consistent with reports indicating genetic heterogeneity, no variants were shared across families. Gene ontology analyses highlighted six genes-COL4A1, ITGA2, MYO10, ROBO3, SLC9A6, and SLIT2-associated with axonogenesis and expressed during early childhood when synesthetic associations are formed. These results are consistent with neuroimaging-based hypotheses about the role of hyperconnectivity in the etiology of synesthesia and offer a potential entry point into the neurobiology that organizes our sensory experiences.

Proteomic analysis of FOXP proteins reveals interactions between cortical transcription factors associated with neurodevelopmental disorders.

FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical associations with other transcription factors might also modulate the functions of FOXP proteins. However, few FOXP-interacting transcription factors have been identified so far. Therefore, we sought to discover additional transcription factors that interact with the brain-expressed FOXP proteins, FOXP1, FOXP2 and FOXP4, through affinity-purifications of protein complexes followed by mass spectrometry. We identified seven novel FOXP-interacting transcription factors (NR2F1, NR2F2, SATB1, SATB2, SOX5, YY1 and ZMYM2), five of which have well-estabslished roles in cortical development. Accordingly, we found that these transcription factors are co-expressed with FoxP2 in the deep layers of the cerebral cortex and also in the Purkinje cells of the cerebellum, suggesting that they may cooperate with the FoxPs to regulate neural gene expression in vivo. Moreover, we demonstrated that etiological mutations of FOXP1 and FOXP2, known to cause neurodevelopmental disorders, severely disrupted the interactions with FOXP-interacting transcription factors. Additionally, we pinpointed specific regions within FOXP2 sequence involved in mediating these interactions. Thus, by expanding the FOXP interactome we have uncovered part of a broader neural transcription factor network involved in cortical development, providing novel molecular insights into the transcriptional architecture underlying brain development and neurodevelopmental disorders.

A Common CYFIP1 Variant at the 15q11.2 Disease Locus Is Associated with Structural Variation at the Language-Related Left Supramarginal Gyrus.

Copy number variants (CNVs) at the Breakpoint 1 to Breakpoint 2 region at 15q11.2 (BP1-2) are associated with language-related difficulties and increased risk for developmental disorders in which language is compromised. Towards underlying mechanisms, we investigated relationships between single nucleotide polymorphisms (SNPs) across the region and quantitative measures of human brain structure obtained by magnetic resonance imaging of healthy subjects. We report an association between rs4778298, a common variant at CYFIP1, and inter-individual variation in surface area across the left supramarginal gyrus (lh.SMG), a cortical structure implicated in speech and language in independent discovery (n = 100) and validation cohorts (n = 2621). In silico analyses determined that this same variant, and others nearby, is also associated with differences in levels of CYFIP1 mRNA in human brain. One of these nearby polymorphisms is predicted to disrupt a consensus binding site for FOXP2, a transcription factor implicated in speech and language. Consistent with a model where FOXP2 regulates CYFIP1 levels and in turn influences lh.SMG surface area, analysis of publically available expression data identified a relationship between expression of FOXP2 and CYFIP1 mRNA in human brain. We propose that altered CYFIP1 dosage, through aberrant patterning of the lh.SMG, may contribute to language-related difficulties associated with BP1-2 CNVs. More generally, this approach may be useful in clarifying the contribution of individual genes at CNV risk loci.

Identification and functional characterization of de novo FOXP1 variants provides novel insights into the etiology of neurodevelopmental disorder.

De novo disruptions of the neural transcription factor FOXP1 are a recently discovered, rare cause of sporadic intellectual disability (ID). We report three new cases of FOXP1-related disorder identified through clinical whole-exome sequencing. Detailed phenotypic assessment confirmed that global developmental delay, autistic features, speech/language deficits, hypotonia and mild dysmorphic features are core features of the disorder. We expand the phenotypic spectrum to include sensory integration disorder and hypertelorism. Notably, the etiological variants in these cases include two missense variants within the DNA-binding domain of FOXP1. Only one such variant has been reported previously. The third patient carries a stop-gain variant. We performed functional characterization of the three missense variants alongside our stop-gain and two previously described truncating/frameshift variants. All variants severely disrupted multiple aspects of protein function. Strikingly, the missense variants had similarly severe effects on protein function as the truncating/frameshift variants. Our findings indicate that a loss of transcriptional repression activity of FOXP1 underlies the neurodevelopmental phenotype in FOXP1-related disorder. Interestingly, the three novel variants retained the ability to interact with wild-type FOXP1, suggesting these variants could exert a dominant-negative effect by interfering with the normal FOXP1 protein. These variants also retained the ability to interact with FOXP2, a paralogous transcription factor disrupted in rare cases of speech and language disorder. Thus, speech/language deficits in these individuals might be worsened through deleterious effects on FOXP2 function. Our findings highlight that de novo FOXP1 variants are a cause of sporadic ID and emphasize the importance of this transcription factor in neurodevelopment.

A de novo FOXP1 variant in a patient with autism, intellectual disability and severe speech and language impairment.

FOXP1 (forkhead box protein P1) is a transcription factor involved in the development of several tissues, including the brain. An emerging phenotype of patients with protein-disrupting FOXP1 variants includes global developmental delay, intellectual disability and mild to severe speech/language deficits. We report on a female child with a history of severe hypotonia, autism spectrum disorder and mild intellectual disability with severe speech/language impairment. Clinical exome sequencing identified a heterozygous de novo FOXP1 variant c.1267_1268delGT (p.V423Hfs*37). Functional analyses using cellular models show that the variant disrupts multiple aspects of FOXP1 activity, including subcellular localization and transcriptional repression properties. Our findings highlight the importance of performing functional characterization to help uncover the biological significance of variants identified by genomics approaches, thereby providing insight into pathways underlying complex neurodevelopmental disorders. Moreover, our data support the hypothesis that de novo variants represent significant causal factors in severe sporadic disorders and extend the phenotype seen in individuals with FOXP1 haploinsufficiency.

Prevalence of Borrelia burgdorferi infection in a series of 98 primary cutaneous lymphomas.

Borrelia burgdorferi has been variably associated with different forms of primary cutaneous lymphoma. Differences in prevalence rates among reported studies could be a result of geographic variability or heterogeneity in the molecular approaches that have been employed. In the present study, we investigated the prevalence of Borrelia burgdorferi sensu lato DNA in diagnostic tissue samples from fresh cutaneous biopsies of 98 primary cutaneous lymphomas and 19 normal skin controls. Three different polymerase chain reaction (PCR) protocols targeting the hbb, flagellin, and Osp-A genes were used. Direct sequencing of both sense and antisense strands of purified PCR products confirmed the specificity of the amplified fragments. Sequence specificity was assessed using the Basic Local Alignment Search Tool, and MultAlin software was used to investigate the heterogeneity of target gene sequences across the different samples. Borrelia DNA was not detected in 19 controls, 23 cases of follicular lymphoma, 31 cases of extranodal marginal zone lymphoma, or 30 cases of mycosis fungoides. A single case of 14 diffuse large B-cell lymphoma cases was positive for B. burgdorferi. This study does not support a pathogenic role of B. burgdorferi in primary cutaneous B- and T-cell lymphomas from areas nonendemic for this microorganism and the consequent rationale for the adoption of antibiotic therapy in these patients.