Alcohol (ethanol) exposure during pregnancy is a well-recognized cause of birth defects and central nervous system disturbances that lead to cognitive and behavioral problems across the lifespan. Although clusters of physical features, in particular those involving the craniofacies, and neurobehavioral symptoms define fetal alcohol spectrum disorders (FASDs), there is considerable variation in the consequences of prenatal alcohol exposure. This variation impedes the accurate diagnosis of FASDs and confounds our complete understanding of the damage that can be caused by alcohol exposure. While some of the individual differences in the consequences of alcohol exposure are due to variations in the timing of exposure, genetic variability is a strong modifier of the effects of ethanol exposure. Elucidating the genetic factors that confer risk and resilience has been a slow process, usually accomplished by comparing ethanol?s effects among various strains of animals, or by candidate gene approaches. Here, we propose a cross-species genetic analysis, utilizing state-of-the-art whole transcriptomic sequencing (RNA-Seq), high-throughput CRISPR/Cas9 gene editing techniques, and genetic screening to drive the discovery of novel candidate genes that modify susceptibility to early gestational ethanol exposure.
In Aim 1, RNA-Seq will be performed after ethanol or vehicle exposure in two closely related mouse strains that differ in their susceptibility to the teratogenic effects of ethanol. This experiment will reveal a number of genes that are differentially expressed in these ?at risk? and ?resilient? strains. Candidate genes are then refined and tested for significant associations with craniofacial and neuroanatomical dysmorphology, as well as neurobehavioral changes, using our zebrafish high-throughput screens, mouse MRI analysis (with Hammond) and mouse behavioral phenotyping. The dual species approach affords a highly conserved FASD model that is more relevant than studying either species alone.
In Aim 2, we will perform an unbiased forward genetic screen in zebrafish to identify mutations that suppress the teratogenicity of ethanol. The roles of these genes will be tested in mice to identify conserved mechanisms of ethanol teratogenesis. These conserved genetic mechanisms of ethanol teratogenesis can then be tested by CIFASD members Foroud, Hammond, Mattson in human populations with prenatal ethanol exposure who vary in their craniofacial and neurobehavioral manifestations. Likewise, human whole-exome sequencing experiments proposed by Dr. Foroud will generate numerous candidate genes that will be tested and confirmed in our animal models for the purpose of identifying conserved teratogenic mechanisms. These highly translational studies will significantly contribute to our understanding of the genetic factors underlying the susceptibility to prenatal ethanol exposure, which may be used to improve diagnosis, treatment, and prevention of FASD, as well as provide insight into the teratogenic mechanisms of FASD.
The goal of this work is to increase our understanding of the genetic predispositions underlying FASD and to uncover novel pathogenic mechanisms in order to aid in prevention and intervention efforts. In our first approach, we will use RNA-Seq in mice and genetic screens in zebrafish to discover novel candidate genes that modify susceptibility to early gestational ethanol exposure. Subsequent experiments will use gene editing in zebrafish and MRI and behavioral phenotyping in mice to further confirm candidate genes that may have an interaction with ethanol. In a second approach, we will perform a forward genetic screen to identify loci that modulate gene-ethanol interactions, with follow up analyses in mouse and human.
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