Our Research

 Finnell/Cabrera Birth Defects Laboratory

The Role of Planar Cell Polarity Genes in Complex Birth Defects


The planar cell polarity (PCP) genes have recently been associated with increased risks of neural tube and craniofacial malformations. In humans, polymorphisms in the PCP gene VANGL1 were found in several families affected by spina bifida and VANGL2 SNPs were found more frequently in anencephalic fetuses. The primary challenge remains to identify which gene variants under what circumstances increase NTD risk to developing embryos. PCP genes are also known to be mutated in several mouse models of NTDs, including the Loop tail (Lp) mouse, the Circle tail (Crc) mouse mutation, and the Celsr gene that is mutated in the Crash (Crsh) mouse. The Finnell Laboratory has developed a mouse line lacking the PCP gene, Fuzzy (Fuz), whose phenotype, including NTDs, suggesting an interaction with Wnt/Shh pathways. In addition to NTDs, the Wnt/Shh pathways have been implicated in craniofacial defects. The development of the vertebrate craniofacies requires complicated tissue-tissue interactions between all germ layers and coordinated movements in three dimensions. Small variations in programming the morphogenetic events can lead to a diverse range of congenital defects. A majority of all human birth defects are associated with some form of craniofacial malformation, creating tremendous medical and social burdens, as many require lifelong care. Fuz is essential for craniofacial development and normal closure of the cranial sutures. The Fuz knockout mouse presents with craniofacial deformities including: hypoplastic mandible, missing incisors, malformed molars, hyperplastic Meckel’s cartilage, premature closure of the sutures, anophthalmia and missing tongue. Given the critical function of canonical Wnt signaling in craniofacial development, we hypothesize that canonical Wnt signaling will be increased in the Fuz-/- mutant mice. The sonic hedgehog (Shh) signaling pathway has also been shown to work through primary cilia. Thus, we will also test the hypothesis that Fuz inactivation affects Shh signaling and the expression of Shh downstream genes during craniofacial development. We will determine how Fuz regulates target gene expression through both the Wnt and Shh signaling pathways and its impact on birth defects such as craniosynostosis.

Using Embryonic Stem Cells to Screen for Reproductive Toxicants


There are over 85,000 industrial chemicals presently registered with the federal government, the vast majority are inadequately studied with regards to their potential environmental and biological impacts. Over 30,000 of these industrial chemicals are sold at quantities exceeding 400 million tons per year; however, there also remains a considerable knowledge gap regarding the relationship between exposure to these industrial chemicals and possible adverse health effects. Many of these chemicals are released into the environment and can be absorbed via ingestion, inhalation, or dermal exposures. Additionally, these chemical can also be transported to the fetus after maternal exposure and can have adverse effects on fetal and neonatal health. This lack of information on industrial chemicals released into the environment leaves the federal government with an ever increasing number of chemicals that may contribute to immediate and long-term health effects in exposed animal and human populations, most of which need to be characterized and prioritized by their individual health risks. The Finnell Laboratory is working to develop a solution to this problem via high-throughput, high-information content biological screens on industrial chemicals. We hypothesize that high throughput screening (HTS) ESCs can recapitulate observations from in vivo models exposed to environmental pollutants. Further, in the absence of a priori toxicological data, we hypothesize that HTS of ESC can be used for prediction of in vivo impact, risk assessment, and prioritizing of chemicals for in vivo testing and epidemiological studies.

Role of Maternal Immune Factors and Risk for Complex Congenital Defects


There is increasing evidence that immune activation, and the subsequent maternal or fetal production of cytokines, may compensate for aberrant fetal development. Many of the known neural tube defect (NTD) risk factors, for example diabetes or obesity, have inflammatory features, suggesting that genes involved in the inflammatory response may represent increased risk factors for NTDs. These studies, combined with human studies demonstrating inflammatory protein promoter polymorphisms in mothers of babies affected by spina bifida, has led us to explore the potential role of the innate immune system in neural tube development. We hypothesize that: 1) Dietary folate stress results in the activation of the complement system in the developing embryo; 2) Complement, and in particular, the potent anaphylaxtoxin C5a, is involved in neural tube closure, especially under folate-deficient conditions; 3) Activation of the C5a receptor, CD88, is protective against the development of NTDs under conditions of maternal folate stress; and 4) C5a may also mediate neural tube closure in other folate-independent murine models of NTDs.

SUMOlyation and Congenital Heart Defects


Congenital heart defects (CHDs) are the most common of all human birth defects, occurring in 1% of newborns. We recently discovered a high incidence of CHDs in mouse embryos with inherited SUMO-1 and SUMO-3 haploid insufficiency mutations. DNA sequence analysis of human newborn screening blood spots identified SUMO-1 regulatory promoter mutations in a high percentage of patients with CHDs. A functional SUMO pathway is essential for proper cardiac development. We propose to test the hypothesis that genetic variants within the components and targets of the SUMO conjugation pathway are a significant cause of human CHDs using human DNA samples from newborn screening blood spots and samples from the National Birth Defects Prevention Study, as well as genetically modified mouse models.

CIC Gene Discovery


Cerebral folate deficiency (CFD) syndrome is characterized by very low concentrations of 5-methyltetrahydrofolate (5-MTHF) in the cerebrospinal fluid, while folate levels in the plasma and red blood cells are within normal limits. Previously, mutations in several folate pathway genes, including hFR (folate receptor alpha), DHFR (dihydrofolate reductase), and PCFT (proton coupled folate transporter) have been identified in patients with low concentrations of 5-MTHF in their cerebrospinal fluid. In an effort to identify causal mutations for CFD, we performed whole exome sequencing analysis of DNA samples collected from a CFD patient, her healthy siblings, and her biological parents. A de novo mutation in the Capicua gene (CIC), c.1057C>T (p.R353X), was identified in the patient. The results were confirmed using Sanger sequencing. In addition, a missense mutation predicted to be damaging, c.1738G>GT (p.G580GC) was identified in another CFD patient. The CIC protein is a HMG-box transcriptional repressor. The DNA binding domain located at amino acid residues 200-268 binds the octomer sequence T(G/C)AATG(A/G)A. The mutation identified in the CFD patient, p.R353X, yields a truncated protein which still contains the DNA binding domain (HMG box); therefore, it is still able to bind to its targets. CIC target binding octomer sequence has been found in the promoter regions of folate transport genes FOLR1, PCFT, RFC1, and DHFR, which is involved in folate metabolism. In the patient’s induced pluripotent stem (iPS) cell, the p.R353X mutation down regulated FOLR1, PCFT and RFC1 gene expression compared with H9 stem cells and an iPS cell line from an individual with the wildtype CIC genotype. Chromatin immunoprecipitation assays demonstrated that CIC bound to the FOLR1, PCFT and RFC1 promoters in vitro. In a dual-luciferase assay, the CIC protein repressed FOLR1 promoter transcription. Using CRISPR-Cas9 technology, we have made a p.R353X knock –in mouse strain. It will be used to study whether this variant could cause CFD in mice, the underlying mechanisms and possible therapy strategies.