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

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.

Abstract

Mutations of the epidermal growth factor receptor (EGFR) gene are found at a relatively high frequency in glioma, with the most common being the de2-7 EGFR (or EGFRvIII). This mutation arises from an in-frame deletion of exons 2–7, which removes 267 amino acids from the extracellular domain of the receptor. Despite being unable to bind ligand, the de2-7 EGFR is constitutively active at a low level. Transfection of human glioma cells with the de2-7 EGFR has little effect in vitro, but when grown as tumor xenografts this mutated receptor imparts a dramatic growth advantage. We have now mapped the phosphorylation pattern of de2-7 EGFR, both in vivo and in vitro, using a panel of antibodies unique to the different phosphorylated tyrosine residues. Phosphorylation of de2-7 EGFR was detected constitutively at all tyrosine sites surveyed both in vitro and in vivo, including tyrosine 845, a known target in the wild-type EGFR for src kinase. There was a substantial upregulation of phosphorylation at every tyrosine residue of the de2-7 EGFR when cells were grown in vivo compared to the receptor isolated from cells cultured in vitro. Upregulation of phosphorylation could be mimicked in vitro by the addition of specifi c components of the ECM such as collagen via an integrin-dependent mechanism. Since this increase in in vivo phosphorylation enhances de2-7 EGFR signaling, this observation explains why the growth enhancement mediated by de2-7 EGFR is largely restricted to the in vivo environment. In a second set of experiments we analyzed the interaction between EGFRvIII and ErbB2. Co-expression of these proteins in NR6 cells, a mouse fi broblast line devoid of ErbB family members, dramatically enhanced in vivo tumorigenicity of these cells compared to cells expressing either protein alone. Detailed analysis of these xenografts demonstrated that EGFRvIII could heterodimerize and transphosphorylate the ErbB2. Since both EGFRvIII and ErbB2 are commonly expressed at gliomas, this data suggests that the co-expression of these two proteins may enhance glioma tumorigenicity.

Abstract

FOXP2, the first gene to have been implicated in a developmental communication disorder, offers a unique entry point into neuromolecular mechanisms influencing human speech and language acquisition. In multiple members of the well-studied KE family, a heterozygous missense mutation in FOXP2 causes problems in sequencing muscle movements required for articulating speech (developmental verbal dyspraxia), accompanied by wider deficits in linguistic and grammatical processing. Chromosomal rearrangements involving this locus have also been identified. Analyses of FOXP2 coding sequence in typical forms of specific language impairment (SLI), autism, and dyslexia have not uncovered any etiological variants. However, no previous study has performed mutation screening of children with a primary diagnosis of verbal dyspraxia, the most overt feature of the disorder in affected members of the KE family. Here, we report investigations of the entire coding region of FOXP2, including alternatively spliced exons, in 49 probands affected with verbal dyspraxia. We detected variants that alter FOXP2 protein sequence in three probands. One such variant is a heterozygous nonsense mutation that yields a dramatically truncated protein product and cosegregates with speech and language difficulties in the proband, his affected sibling, and their mother. Our discovery of the first nonsense mutation in FOXP2 now opens the door for detailed investigations of neurodevelopment in people carrying different etiological variants of the gene. This endeavor will be crucial for gaining insight into the role of FOXP2 in human cognition.