Genetic basis of susceptibility to environmentally induced neural tube defects.

Neural tube defects (NTDs) are among the most common of all human congenital defects, with multifactorial etiologies comprising both environmental and genetic components. Several murine model systems have been developed in an effort to elucidate genetic factors regulating expression of NTDs. Strain-dependent differences in susceptibility to teratogenic insults and altered patterns of gene expression observed within the neuroepithelium of affected embryos support the hypothesis that subtle genetic changes can result in NTDs. Since several affected genes are folate-regulated, transgenic knockout mice lacking a functional folate receptor were developed. Nullizygous embryos died in utero with significant morphological defects, supporting the critical role of folic acid in early embryogenesis. While epidemiological studies have not established an association between polymorphisms in the human folate receptor gene and NTDs, it is known that folate supplementation reduces infant NTD risk. Continued efforts are therefore necessary to reveal the mechanism by which folate works and the nature of the gene(s) responsible for human NTDs.

A rapid and sensitive reporter gene that uses green fluorescent protein expression to detect chemicals with estrogenic activity.

A reporter gene sequence was constructed within a eukaryotic expression vector. The altered plasmid contained 2 sequential estrogen response elements (ERE) coupled to a human phosphoglycerate kinase (PGK) promoter inserted upstream from a cDNA sequence encoding enhanced green fluorescent protein (GFP) with a 3'-polyadenylation signal. The plasmid was linearized and transfected into MCF-7 cells, a human breast cancer-derived line that expresses the estrogen receptor (ER). No selectable marker was present in the plasmid, requiring stably transfected cells to be selected by fluorescence-activated cell sorting based on GFP expression after the cells were treated with 10(-9) M 17beta-estradiol (E2). Stably transfected MCF-7 cells (MCF7-ERE) exhibited 2000-3000 times more fluorescence at 488 nm excitation and 512 nm emission than non-transfected cells. MCF7-ERE cells exhibited a linear increase in GFP expression induced over a range of 10(-12) M E2, a concentration giving 2 times the background expression, to maximal expression at 3 x 0(-10) M E2. From the maximal level, GFP expression plateaued, and then declined when E2 was increased to the highest concentration tested, 10(-7) M. 4-Hydroxytamoxifen (TFN-OH) treatment of cells produced a dose-dependent inhibition of E2-induced GFP expression, indicating the interaction of ER in the regulation of GFP gene expression. A series of estrogenic chemicals were evaluated for their capacity to induce GFP expression in MCF7-ERE cells, showing induced expression of GFP at concentrations 2-4 log units higher than the E2 concentration giving maximal GFP expression. The ERE-PGK-GFP reporter gene system is capable of rapid GFP expression in the presence of low concentrations of E2, and of quantifying estrogenicity of chemicals compared with a standard curve of the natural ligand, 17beta-estradiol.

Mice lacking the folic acid-binding protein Folbp1 are defective in early embryonic development.

Periconceptional folic acid supplementation reduces the occurrence of several human congenital malformations, including craniofacial, heart and neural tube defects. Although the underlying mechanism is unknown, there may be a maternal-to-fetal folate-transport defect or an inherent fetal biochemical disorder that is neutralized by supplementation. Previous experiments have identified a folate-binding protein (Folbp1) that functions as a membrane receptor to mediate the high-affinity internalization and delivery of folate to the cytoplasm of the cell. In vitro, this receptor facilitates the accumulation of cellular folate a thousand-fold relative to the media, suggesting that it may be essential in cytoplasmic folate delivery in vivo. The importance of an adequate intracellular folate pool for normal embryogenesis has long been recognized in humans and experimental animals. To determine whether Folbp1 is involved in maternal-to-fetal folate transport, we inactivated Folbp1 in mice. We also produced mice lacking Folbp2, another member of the folate receptor family that is GPI anchored but binds folate poorly. Folbp2-/- embryos developed normally, but Folbp1-/- embryos had severe morphogenetic abnormalities and died in utero by embryonic day (E) 10. Supplementing pregnant Folbp1+/- dams with folinic acid reversed this phenotype in nullizygous pups. Our results suggest that Folbp1 has a critical role in folate homeostasis during development, and that functional defects in the human homologue (FOLR1) of Folbp1 may contribute to similar defects in humans.

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.