Session 2 Abstracts

Transgenerational Epigenetic Markers of Autism

Minoo Rassoulzadegan

Erciyes University Medical Faculty, Medical Genetics Department, Kayseri, Turkey. Université Côte d’Azur, CNRS, Inserm, France

Autism spectrum disorder (ASD) is a group of developmental pathologies that impair social communication and cause repetitive behaviors. The suggested roles of noncoding RNAs in pathology led us to perform a comparative analysis of the microRNAs expressed in the serum of human ASD patients and their relatives. The analysis of a cohort of 45 children with ASD revealed that six microRNAs (miR-19a-3p, miR-361-5p, miR-3613-3p, miR-150-5p, miR-126-3p, and miR-499a-5p) were expressed at low to very low levels compared to those in healthy controls. A similar but less pronounced decrease was registered in the clinically unaffected parents of the sick children and in their siblings but never in any genetically unrelated control. Results consistent with these observations were obtained in the blood, hypothalamus and sperm of two of the established mouse models of ASD: valproic acid-treated animals and Cc2d1a+/- heterozygotes (a gene of the ASD constellation encoding a transcriptional repressor of serotonin receptors). In both instances, the same characteristic miRNA profile was evidenced in the affected individuals and inherited together with disease symptoms in the progeny of crosses with healthy animals. The consistent association of these genetic regulatory changes with the disease provides a starting point for evaluating the changes in the activity of the target genes and, thus, the underlying mechanism(s). From the applied societal and medical perspectives, once properly confirmed in large cohorts, these observations provide tools for the very early identification of affected children and progenitors.

Assisted Reproductive Technologies: Epigenetic Impacts and Long-term Outcomes

Marisa S. Bartolomei, Lisa A. Vrooman, Eric Rhon-Calderon and Laren Narapareddy

Department of Cell & Developmental Biology, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA  19104

Assisted reproductive technologies (ART) are increasingly used to treat human infertility. ART, however, can induce abnormal placentation, affect birth weight, and increase the risk of imprinting disorders and other pregnancy complications. Moreover, children conceived using in vitro fertilization (IVF) appear to be at increased risk for a number of abnormal physiological outcomes. Nevertheless, it remains to be determined whether these adverse outcomes result from the underlying infertility or the procedures used in ART. To investigate the question of whether ART itself is associated with abnormal outcomes, we have employed a normally fertile mouse model of IVF. In this model we have tested procedures used in ART determine which procedures are associated with adverse outcomes. We have shown that even the most basic ART procedures, e.g., embryo transfer, can compromise fetal growth at mid-gestation with the most severe phenotypes, including fetal weight, abnormal placental morphology, and placenta DNA hypomethylation, being linked to embryo culture. We have also followed offspring longitudinally and shown that abnormal metabolic phenotypes appear in the adult mouse and these phenotypes are sexually dimorphic. Finally, this model is being used to determine if observed phenotypes can be transmitted across generations. 

The Legacy of Paternal Exposure to Toxicants: More Than Just Gene Mutations

Francesco Marchetti

Environmental Health Science Research Bureau, Health Canada

Over the past 70 years, many environmental exposures have been demonstrated to induce heritable genetic changes in laboratory animals. Despite that, we still lack conclusive proof of an environmentally related increase in heritable genetic changes in the children of exposed parents. Characterization of the impact of environmental agents on the human germline has been hampered by the lack of efficient approaches to characterize the entire spectrum of genomic changes that can affect health. The advent of next generation sequencing (NGS) approaches, together with great gains in bioinformatics pipelines are providing an unparalleled opportunity to investigate genomic changes over the entire genome. In addition, approaches are available to detect a wide range of genomic alterations, spanning single base substitutions to structural variants affecting large regions of the genome that are increasingly being recognized as causal factors of human genetic diseases. We have been using NGS tools in both laboratory animals and human families to gain a better understanding of the exogenous and endogenous factors that influence the burden of genetic abnormalities in the next generation. NGS studies in rodents demonstrate that paternal exposure to germ cell mutagens induce genome-wide mutations in the offspring and that different mutagenic agents can differentially affect various types of genetic changes. Studies in human families consistently show a parental age-related increase in inherited mutations. However, the rate of de novo mutations varies considerably among families, pointing to the potential impact of the environment. Overall, these studies support the notion that alterations outside of coding regions are involved in determining adverse health effects and that a comprehensive evaluation of the environmental impact on genome integrity in the offspring necessitate analyses of a variety of genomic changes.

Multiparametric Analysis of the Spermatogonial Stem Cell Epigenome

John R. McCarrey, Keren Cheng

Department of Biology, University of Texas at San Antonio, San Antonio, TX, USA

Intergenerational inheritance of environmentally-induced epigenetic defects normally requires introduction of disruptions of the epigenome (epimutations) into the germ line. In both sexes, the germ lines first appear as primordial germ cells (PGCs), but then display sexually dimorphic development during gametogenesis. In males PGCs give rise to prospermatogonia that then form undifferentiated spermatogonia, a subset of which become spermatogonial stem cells (SSCs). SSCs both self-renew and give rise to progenitor spermatogonia that initiate spermatogenic differentiation in the mammalian testis. SSCs are the only self-renewing germline cell type in either sex and, upon replication, must either maintain SSC fate or commit to entry into the spermatogenic differentiation pathway as progenitor spermatogonia. We have used both bulk and single-cell RNA-seq to describe differential gene expression associated with each of these cell fates. In addition, we have conducted the first multiparametric integrative analysis of any mammalian germ cell epigenomes comparable to that done for >100 somatic cell types by the ENCODE Project. Differentially expressed genes distinguishing SSC- and progenitor-enriched spermatogonia showed distinct histone modification patterns, particularly related to enrichment of H3K27ac and H3K27me3. Motif analysis predicted transcription factors that may regulate spermatogonial subtype-specific fate, and immunohistochemistry and gene-specific chromatin immunoprecipitation analyses confirmed subtype-specific differences in target gene binding of a subset of these factors. Taken together, these results show that SSCs and progenitors display distinct epigenetic profiling consistent with these spermatogonial subtypes being differentially programmed to either self-renew and maintain regenerative capacity as SSCs, or lose regenerative capacity and initiate lineage commitment as progenitors.

Epigenetic Variation in Human Sperm Under Lifestyle Influences

Romain Barrès

Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, DK-2200 N Copenhagen, Denmark

Accumulating evidence supports that paternal nutritional stress before conception is associated with altered phenotype in the next generation offspring. We and others have identified epigenetic changes in gametes from animals fed a high fat diet, but the relative contribution of DNA methylation, histone modification and small RNA expression in the modulation of phenotype in the offspring is incompletely understood. In this presentation, results supporting a role of DNA methylation variation in sperm at genes controlling the development of the brain in paternal inheritance will be presented.