Project II - Modeling meningomyelocele in frog using human alleles and folic acid exposure

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Abstract – Project II: Modeling meningomyelocele in frog using human alleles and FA exposure Neural tube defects (NTDs) are a relatively common birth defect with a complex etiology and gene-environment interactions (GXEs). Genetic factors play an important role, and these gene variants likely interact with one another in gene-gene interactions (GXGs). A major focus of our Program Project is the assessment of de novo mutations that can be assessed in patients with NTDs, and their modulation by environmental risk factors, such as folic acid (FA) accessibility. This complex etiology has made NTDs an extremely challenging syndrome to predict based on genetic testing and to treat clinically. This proposal is a part of a comprehensive Program Project to investigate the genetic basis of the NTD subtype known as meningomyelocele (MM), localized to the spinal neural tube, and occuring in approximately one in every 2,500 births. Project I in the application uses next generation sequencing to identify de novo gene variants that are associated with human MM patients, and assess for recurrence. Even for recurrent mutations, it is critical to functionally evaluate causality in an in vivo setting. This project exploits Xenopus as a high throughput and high content tetrapod experimental model to assess these variants, taking advantage of the fact that the morphogenetic process of neural tube formation and the underlying molecular pathways involved in neurulation are conserved between Xenopus and mammalian embryos. CrispR mutagenesis in F0 Xenopus embryos will be used to test whether genes that lie within deletions of LCR22C-D in the 22q11.2 interval and that substantially increase risk of MM in humans, cause NTDs as null mutations. Gene variants detected in the planar cell polarity pathway that may increase the risk of MM in humans will be tested using rescue experiments in Xenopus, taking advantage of quantitative assays for measuring planar axis formation. Finally, the assessment of MM gene variants will also exploit recent studies in Xenopus, indicating that folate deficiency also causes NTDs as in mammals. Interactions between genetic risk factors and folate deficiency on the incidence of NTDs will be rapidly assessed using Xenopus, as part of the overall goal to determine whether FA alters gene expression and thus the expressivity of critical gene mutants. In sum, project II will use Xenopus as model to prioritize MM gene variants identified in Project I that can be further pursued in experiments in the mouse embryo in project III. Aim 1. Test genes in the minimal 22q11.2 deletion interval for a role in NTDs using Xenopus. Aim 2. Test human MM alleles for impact on neural tube formation in Xenopus embryos. Aim 3. Test GXE by assessing the impact of FA on MM gene phenotypes in Xenopus.