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

How environmental factors shape patterns of biotic diversity in tropical ecosystems is an active field of research, but studies examining the possibility of ecological speciation in terrestrial tropical ecosystems are scarce. We use the isolated rainforest herpetofauna on the Montagne d'Ambre (Amber Mountain) massif in northern Madagascar as a model to explore elevational divergence at the level of populations and communities. Based on intensive sampling and DNA barcoding of amphibians and reptiles along a transect ranging from ca. 470–1470 m above sea level (a.s.l.), we assessed a main peak in species richness at an elevation of ca. 1000 m a.s.l. with 41 species. The proportion of local endemics was highest (about 1/3) at elevations >1100 m a.s.l. Two species of chameleons (Brookesia tuberculata, Calumma linotum) and two species of frogs (Mantidactylus bellyi, M. ambony) studied in depth by newly developed microsatellite markers showed genetic divergence up the slope of the mountain, some quite strong, others very weak, but in each case with genetic breaks between 1100 and 1270 m a.s.l. Genetic clusters were found in transect sections significantly differing in bioclimate and herpetological community composition. A decrease in body size was detected in several species with increasing elevation. The studied rainforest amphibians and reptiles show concordant population genetic differentiation across elevation along with morphological and niche differentiation. Whether this parapatric or microallopatric differentiation will suffice for the completion of speciation is, however, unclear, and available phylogeographic evidence rather suggests that a complex interplay between ecological and allopatric divergence processes is involved in generating the extraordinary species diversity of Madagascar's biota. Our study reveals concordant patterns of diversification among main elevational bands, but suggests that these adaptational processes are only part of the complex of processes leading to species formation, among which geographical isolation is probably also important.

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

A steady accumulation of experimental data argues that protein synthesis in neurons is not merely restricted to the somatic compartment, but also occurs in several discrete cellular micro-domains. Local protein synthesis is critical for the establishment of synaptic plasticity in mature dendrites and in directing the growth cones of immature axons, and has been associated with cognitive impairment in mice and humans. Although in recent years a number of important mechanisms governing this process have been described, it remains technically challenging to precisely monitor local protein synthesis in individual neuronal cell parts independent from the soma. This report presents the utility of employing microfluidic chambers for the isolation and treatment of single neuronal cellular compartments. Furthermore, it is demonstrated that a protein synthesis assay, based on fluorescent non-canonical amino acid tagging (FUNCAT), can be combined with this cell culture system to label nascent proteins within a discrete structural and functional domain of the neuron. Together, these techniques could be employed for the detection of protein synthesis within developing and mature neurites, offering an effective approach to elucidate novel mechanisms controlling synaptic maintenance and plasticity.

Abstract

We describe the calls of Stumpffia be Köhler, Vences, D’Cruze & Glaw, 2010. This is the first call description made for a species belonging to the large-bodied northern Madagascan radiation of Stumpffia Boettger, 1881. Stumpffia is a genus of small (~9–28 mm) microhylid frogs in the Madagascar-endemic subfamily Cophylinae Cope. Little is known about their reproductive strategies. Most species are assumed to lay their eggs in foam nests in the leaf litter of Madagascar’s humid and semi-humid forests (Glaw & Vences 1994; Klages et al. 2013). They exhibit some degree of parental care, with the males guarding the nest after eggs are laid (Klages et al. 2013). The bioacoustic repertoire of these frogs is thought to be limited, and there are two distinct call structures known for the genus: the advertisement call of the type species, S. psologlossa Boettger, 1881, is apparently unique in being a trill of notes repeated in short succession. All other species from which calls are known emit single, whistling or chirping notes (Vences & Glaw 1991; Vences et al. 2006).

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

Schizophrenia is a complex disorder that affects cognitive function and has been linked, both in patients and animal models, to dysfunction of the GABAergic system. However, the pathophysiological consequences of this dysfunction are not well understood. Here, we examined the GABAergic system in an animal model displaying schizophrenia-relevant features, the apomorphine-susceptible (APO-SUS) rat and its phenotypic counterpart, the apomorphine-unsusceptible (APO-UNSUS) rat at postnatal day 20-22. We found changes in the expression of the GABA-synthesizing enzyme GAD67 specifically in the prelimbic-but not the infralimbic region of the medial prefrontal cortex (mPFC), indicative of reduced inhibitory function in this region in APO-SUS rats. While we did not observe changes in basal synaptic transmission onto LII/III pyramidal cells in the mPFC of APO-SUS compared to APO-UNSUS rats, we report reduced paired-pulse ratios at longer inter-stimulus intervals. The GABA(B) receptor antagonist CGP 55845 abolished this reduction, indicating that the decreased paired-pulse ratio was caused by increased GABA(B) signaling. Consistently, we find an increased expression of the GABA(B1) receptor subunit in APO-SUS rats. Our data provide physiological evidence for increased presynaptic GABAB signaling in the mPFC of APO-SUS rats, further supporting an important role for the GABAergic system in the pathophysiology of schizophrenia.