Kimberly A. Greer

Neural Tube and Craniofacial Defects With Special Emphasis On Folate Pathway Genes

Neural tube and orofacial defects are common congenital malformations in humans. While etiologically heterogeneous, they are for the most part multifactorial in their pathogenesis, having both genetic and environmental components in their development. In recent years, there has been a great deal of epidemiologic evidence demonstrating that women who received multivitamins containing folic acid periconceptionally had significantly reduced occurrence and recurrence risks for producing infants with such malformations. This risk reduction is not observed in all populations, further suggestive of a genetic regulation of this phenomenon. Unfortunately, the mechanisms underlying the beneficial effects of folic acid are not well-understood. In this article, we review the relevant epidemiologic data on both neural tube defects and orofacial malformations, the fundamental embryological processes involved in closing the neural tube, and the development of the craniofacies, and propose a working hypothesis for susceptibility to these malformations. This hypothesis is based on the interworkings of cellular folate transport, focusing on the key elements involved in potocytosis. We propose that infants with mutations in the folate receptor alpha gene might be at increased risk for congenital anomalies due to a reduced binding affinity for 5-methyltetrahydrofolate, the physiologic form of folic acid. Various experimental approaches to test the working hypothesis are considered.

Lack of association between mutations in the folate receptor-α gene and spina bifida

Defects of neural tube closure are among the most common of all human malformations. Epidemiological and genetic studies indicate that most of these defects are multifactorial in origin with genetic and environmental causes. Although periconceptional supplementation of the maternal diet with folic acid has been shown to reduce the recurrence and occurrence of neural tube defects (NTDs) by up to 70%, the underlying mechanism remains unknown. Folic acid enters cells of certain tissues via a receptor-mediated process known as potocytosis. The folate receptor alpha (FR-alpha) gene codes for the protein responsible for binding folate, which is the first, and only, folate-dependent step in folate transport. The FR-alpha exons, which code for mature protein and the intron-exon boundaries, were examined for mutations in three separate studies. Initial screening was performed by single-stranded conformational polymorphism (SSCP) analysis in a subset of 1,688 samples obtained from a population-based case-control study of NTDs in California. In the second study, the DNA sequence of exons 5 and 6 was determined in a group of 50 NTD affected individuals. The final experiment involved using dideoxy fingerprinting (ddF) to screen a population-based case-control sample of 219 individuals who were stratified into four sub-groups on the basis of folate intake and pregnancy outcome. No polymorphism was detected in any of the four exons examined. It is unlikely that the beneficial effects of maternal folate supplementation in preventing NTDs acts through a mechanism involving pharmacological correction of a variant form of folate receptor alpha.

Preliminary evidence of phenytoin-induced alterations in embryonic gene expression in a mouse model

SWV mouse embryos collected on gestational days (GD) 9:12 and 10:00 following chronic in utero exposure to teratogenic concentrations of phenytoin were utilized for in situ transcription studies of gene expression. The substrate cDNA obtained from the frozen embryo sections was amplified into radiolabelled antisense RNA (RT/aRNA) and used as a probe to screen a panel of 20 cDNA clones representing genes that are important regulators of craniofacial and neural development. The magnitude of alteration in gene expression following phenytoin treatment was determined densitometrically by changes in the hybridization intensity of the aRNA probes to the cDNA clones immobilized to the slot blots. We found that both Wnt-1 and the calcium channel gene were developmentally regulated, as their level of expression decreased significantly between the two collection times. Phenytoin treatment produced a significant downregulation in the level of expression for 25% of the genes examined in the GD 9:12 embryos, including the growth factors TGF-beta and NT3, the proto-oncogene Wnt-1, the nicotinic receptor, and the voltage sensitive calcium channel gene. Additional changes in the coordinate expression of several of the growth and transcription factors were observed at both gestational timepoints. The application of RT/aRNA technology has extended our appreciation of the normal patterns of gene expression during craniofacial and neural development, and provided the first demonstration of multiple coordinate changes in transcription patterns following teratogenic insult.

Gene expression analysis in a canine model of X-linked Alport syndrome

Chronic kidney disease (CKD) often culminates in renal failure as a consequence of progressive interstitial fibrosis and is an important cause of illness and death in dogs. Identification of disease biomarkers and gene expression changes will yield valuable information regarding the specific biological pathways involved in disease progression. Toward these goals, gene expression changes in the renal cortex of dogs with X-linked Alport syndrome (XLAS) were examined using microarray technology. Extensive changes in inflammatory, metabolic, immune, and extracellular matrix biology were revealed in affected dogs. Statistical analysis showed 133 genes that were robustly induced or repressed in affected animals relative to age-matched littermates. Altered expression of numerous major histocompatibility complex (MHC) molecules suggests that the immune system plays a significant role in XLAS. Increased expression of COL4A1 and TIMP-1 at the end stage of disease supports the suggestion that expression increases in association with progression of fibrosis and confirms an observation of increased COL4A1 protein expression. Clusterin may function as one of the primary defenses of the renal cortex against progressive injury in dogs with XLAS, as demonstrated here by increased CLU gene expression. Cellular mechanisms that function during excess oxidative stress might also act to deter renal damage, as evidenced by alterations in gene expression of SOD1, ACO1, FDXR, and GPX1. This investigation provides a better understanding of interstitial fibrosis pathogenesis, and potential biomarkers for early detection, factors that are essential to discovering more effective treatments thereby reducing clinical illness and death due to CKD.

Analysis of gene expression in brain tissue from Greyhounds with meningoencephalitis

OBJECTIVE To elucidate the pathogenesis of Greyhound meningoencephalitis by evaluating gene expression in diseased brain tissue. ANIMALS Cadavers of 3 diseased (8- to 15-month-old) and 3 (10-month-old) control Greyhounds. PROCEDURES Samples of RNA were extracted from brain tissue of all dogs and evaluated by use of a canine-specific microarray. RESULTS A unique profile involving significant alterations in expression of 21 genes was evident in diseased dogs, compared with expression in control dogs. Most genes with up-regulated expression were related to immune function, with the remaining genes involved in ligand binding, signal transduction, transcriptional regulation, and formation and transportation of proteins including enzymes. Of notable involvement were genes encoding for major histocompatibility complexes, small inducible cytokine A5 precursor, myxovirus-resistant proteins, and components of the classical complement pathway, which are all genes common to pathways of viral infections and autoimmunity. CONCLUSIONS AND CLINICAL RELEVANCE Although results of microarray analysis did not clearly define a potential etiology of Greyhound meningoencephalitis, they did highlight a consistent gene alteration signature that would suggest a common etiology and pathogenesis for this condition.

Development of an in vitro model of excess intracellular reactive oxygen species

These investigations characterize an in vitro model for generating excess intracellular reactive oxygen species (ROS). This novel model may be useful in the identification and delineation of cellular mechanisms associated with aging due to the link between age and excess oxidative events. The human cell line, MCF7, was stably transfected using the pSV3.neo plasmid housing a gene encoding the Aequorea victoria green fluorescent protein (GFP). Transfected cells were analyzed for maintenance of GFP over time, showing stability of the GFP gene. These studies demonstrate that the presence of fluorescing GFP significantly increases intracellular ROS, creating oxidative stress in these cells. Antioxidant supplementation was evaluated to determine the effectiveness of intracellular H2O2 reduction. The results demonstrate that supplementation with a potent antioxidant, such as reduced glutathione, protects cells from oxidative damage by decreasing intracellular concentrations of H2O2. This model for intracellular generation of excess ROS establishes a clear method by which the utility of antioxidant supplementation to protect against intracellularly generated reactive oxygen species may be evaluated.