Publications

Abstract

Contactin-associated protein-like 2 (CNTNAP2) gene encodes for CASPR2, a presynaptic type 1 transmembrane protein, involved in cell–cell adhesion and synaptic interactions. Biallelic CNTNAP2 loss has been associated with “Pitt-Hopkins-like syndrome-1” (MIM#610042), while the pathogenic role of heterozygous variants remains controversial. We report 22 novel patients harboring mono- (n = 2) and bi-allelic (n = 20) CNTNAP2 variants and carried out a literature review to characterize the genotype–phenotype correlation. Patients (M:F 14:8) were aged between 3 and 19 years and affected by global developmental delay (GDD) (n = 21), moderate to profound intellectual disability (n = 17) and epilepsy (n = 21). Seizures mainly started in the first two years of life (median 22.5 months). Antiseizure medications were successful in controlling the seizures in about two-thirds of the patients. Autism spectrum disorder (ASD) and/or other neuropsychiatric comorbidities were present in nine patients (40.9%). Nonspecific midline brain anomalies were noted in most patients while focal signal abnormalities in the temporal lobes were noted in three subjects. Genotype–phenotype correlation was performed by also including 50 previously published patients (15 mono- and 35 bi-allelic variants). Overall, GDD (p < 0.0001), epilepsy (p < 0.0001), hyporeflexia (p = 0.012), ASD (p = 0.009), language impairment (p = 0.020) and severe cognitive impairment (p = 0.031) were significantly associated with the presence of biallelic versus monoallelic variants. We have defined the main features associated with biallelic CNTNAP2 variants, as severe cognitive impairment, epilepsy and behavioral abnormalities. We propose CASPR2-deficiency neurodevelopmental disorder as an exclusively recessive disease while the contribution of heterozygous variants is less likely to follow an autosomal dominant inheritance pattern.

Abstract

Aging, often considered a result of random cellular damage, can be accurately estimated using DNA methylation profiles, the foundation of pan-tissue epigenetic clocks. Here, we demonstrate the development of universal pan-mammalian clocks, using 11,754 methylation arrays from our Mammalian Methylation Consortium, which encompass 59 tissue types across 185 mammalian species. These predictive models estimate mammalian tissue age with high accuracy (r > 0.96). Age deviations correlate with human mortality risk, mouse somatotropic axis mutations and caloric restriction. We identified specific cytosines with methylation levels that change with age across numerous species. These sites, highly enriched in polycomb repressive complex 2-binding locations, are near genes implicated in mammalian development, cancer, obesity and longevity. Our findings offer new evidence suggesting that aging is evolutionarily conserved and intertwined with developmental processes across all mammals.

Abstract

INTRODUCTION
Comparative epigenomics is an emerging field that combines epigenetic signatures with phylogenetic relationships to elucidate species characteristics such as maximum life span. For this study, we generated cytosine DNA methylation (DNAm) profiles (n = 15,456) from 348 mammalian species using a methylation array platform that targets highly conserved cytosines.
RATIONALE
Nature has evolved mammalian species of greatly differing life spans. To resolve the relationship of DNAm with maximum life span and phylogeny, we performed a large-scale cross-species unsupervised analysis. Comparative studies in many species enables the identification of epigenetic correlates of maximum life span and other traits.
RESULTS
We first tested whether DNAm levels in highly conserved cytosines captured phylogenetic relationships among species. We constructed phyloepigenetic trees that paralleled the traditional phylogeny. To avoid potential confounding by different tissue types, we generated tissue-specific phyloepigenetic trees. The high phyloepigenetic-phylogenetic congruence is due to differences in methylation levels and is not confounded by sequence conservation.
We then interrogated the extent to which DNA methylation associates with specific biological traits. We used an unsupervised weighted correlation network analysis (WGCNA) to identify clusters of highly correlated CpGs (comethylation modules). WGCNA identified 55 distinct comethylation modules, of which 30 were significantly associated with traits including maximum life span, adult weight, age, sex, human mortality risk, or perturbations that modulate murine life span.
Both the epigenome-wide association analysis (EWAS) and eigengene-based analysis identified methylation signatures of maximum life span, and most of these were independent of aging, presumably set at birth, and could be stable predictors of life span at any point in life. Several CpGs that are more highly methylated in long-lived species are located near HOXL subclass homeoboxes and other genes that play a role in morphogenesis and development. Some of these life span–related CpGs are located next to genes that are also implicated in our analysis of upstream regulators (e.g., ASCL1 and SMAD6). CpGs with methylation levels that are inversely related to life span are enriched in transcriptional start site (TSS1) and promoter flanking (PromF4, PromF5) associated chromatin states. Genes located in chromatin state TSS1 are constitutively active and enriched for nucleic acid metabolic processes. This suggests that long-living species evolved mechanisms that maintain low methylation levels in these chromatin states that would favor higher expression levels of genes essential for an organism’s survival.
The upstream regulator analysis of the EWAS of life span identified the pluripotency transcription factors OCT4, SOX2, and NANOG. Other factors, such as POLII, CTCF, RAD21, YY1, and TAF1, showed the strongest enrichment for negatively life span–related CpGs.
CONCLUSION
The phyloepigenetic trees indicate that divergence of DNA methylation profiles closely parallels that of genetics through evolution. Our results demonstrate that DNA methylation is subjected to evolutionary pressures and selection. The publicly available data from our Mammalian Methylation Consortium are a rich source of information for different fields such as evolutionary biology, developmental biology, and aging.

Abstract

Horizontal transfer of transposable elements (TEs) is an important mechanism contributing to genetic diversity and innovation. Bats (order Chiroptera) have repeatedly been shown to experience horizontal transfer of TEs at what appears to be a high rate compared with other mammals. We investigated the occurrence of horizontally transferred (HT) DNA transposons involving bats. We found over 200 putative HT elements within bats; 16 transposons were shared across distantly related mammalian clades, and 2 other elements were shared with a fish and two lizard species. Our results indicate that bats are a hotspot for horizontal transfer of DNA transposons. These events broadly coincide with the diversification of several bat clades, supporting the hypothesis that DNA transposon invasions have contributed to genetic diversification of bats.

Abstract

The past few years have seen a surge of interest in using machine learning (ML) methods for studying the behavior of nonhuman animals (hereafter “animals”) (1). A topic that has attracted particular attention is the decoding of animal communication systems using deep learning and other approaches (2). Now is the time to tackle challenges concerning data availability, model validation, and research ethics, and to embrace opportunities for building collaborations across disciplines and initiatives.

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.

Abstract

Purpose: Factors affecting the efficacy of therapeutic monoclonal antibodies (mAb) directed to the epidermal growth factor receptor (EGFR) remain relatively unknown, especially in glioma. Experimental Design: We examined the efficacy of two EGFR-specific mAbs (mAbs 806 and 528) against U87MG-derived glioma xenografts expressing EGFR variants. Using this approach allowed us to change the form of the EGFR while keeping the genetic background constant. These variants included the de2-7 EGFR (or EGFRvIII), a constitutively active mutation of the EGFR expressed in glioma. Results: The efficacy of the mAbs correlated with EGFR number; however, the most important factor was receptor activation. Whereas U87MG xenografts expressing the de2-7 EGFR responded to therapy, those exhibiting a dead kinase de2-7 EGFR were refractory. A modified de2-7 EGFR that was kinase active but autophosphorylation deficient also responded, suggesting that these mAbs function in de2-7 EGFR–expressing xenografts by blocking transphosphorylation. Because de2-7 EGFR–expressing U87MG xenografts coexpress the wild-type EGFR, efficacy of the mAbs was also tested against NR6 xenografts that expressed the de2-7 EGFR in isolation. Whereas mAb 806 displayed antitumor activity against NR6 xenografts, mAb 528 therapy was ineffective, suggesting that mAb 528 mediates its antitumor activity by disrupting interactions between the de2-7 and wild-type EGFR. Finally, genetic disruption of Src in U87MG xenografts expressing the de2-7 EGFR dramatically enhanced mAb 806 efficacy. Conclusions: The effective use of EGFR-specific antibodies in glioma will depend on identifying tumors with activated EGFR. The combination of EGFR and Src inhibitors may be an effective strategy for the treatment of glioma.

Abstract

Mutations in FOXP2, a member of the forkhead family of transcription factor genes, are the only known cause of developmental speech and language disorders in humans. To date, there are no known targets of human FOXP2 in the nervous system. The identification of FOXP2 targets in the developing human brain, therefore, provides a unique tool with which to explore the development of human language and speech. Here, we define FOXP2 targets in human basal ganglia (BG) and inferior frontal cortex (IFC) by use of chromatin immunoprecipitation followed by microarray analysis (ChIP-chip) and validate the functional regulation of targets in vitro. ChIP-chip identified 285 FOXP2 targets in fetal human brain; statistically significant overlap of targets in BG and IFC indicates a core set of 34 transcriptional targets of FOXP2. We identified targets specific to IFC or BG that were not observed in lung, suggesting important regional and tissue differences in FOXP2 activity. Many target genes are known to play critical roles in specific aspects of central nervous system patterning or development, such as neurite outgrowth, as well as plasticity. Subsets of the FOXP2 transcriptional targets are either under positive selection in humans or differentially expressed between human and chimpanzee brain. This is the first ChIP-chip study to use human brain tissue, making the FOXP2-target genes identified in these studies important to understanding the pathways regulating speech and language in the developing human brain. These data provide the first insight into the functional network of genes directly regulated by FOXP2 in human brain and by evolutionary comparisons, highlighting genes likely to be involved in the development of human higher-order cognitive processes.

Abstract

We previously discovered that mutations of the human FOXP2 gene cause a monogenic communication disorder, primarily characterized by difficulties in learning to make coordinated sequences of articulatory gestures that underlie speech. Affected people have deficits in expressive and receptive linguistic processing and display structural and/or functional abnormalities in cortical and subcortical brain regions. FOXP2 provides a unique window into neural processes involved in speech and language. In particular, its role as a transcription factor gene offers powerful functional genomic routes for dissecting critical neurogenetic mechanisms. Here, we employ chromatin immunoprecipitation coupled with promoter microarrays (ChIP-chip) to successfully identify genomic sites that are directly bound by FOXP2 protein in native chromatin of human neuron-like cells. We focus on a subset of downstream targets identified by this approach, showing that altered FOXP2 levels yield significant changes in expression in our cell-based models and that FOXP2 binds in a specific manner to consensus sites within the relevant promoters. Moreover, we demonstrate significant quantitative differences in target expression in embryonic brains of mutant mice, mediated by specific in vivo Foxp2-chromatin interactions. This work represents the first identification and in vivo verification of neural targets regulated by FOXP2. Our data indicate that FOXP2 has dual functionality, acting to either repress or activate gene expression at occupied promoters. The identified targets suggest roles in modulating synaptic plasticity, neurodevelopment, neurotransmission, and axon guidance and represent novel entry points into in vivo pathways that may be disturbed in speech and language disorders.