Congenital abnormalities are defined as malformations which are present birth due to factors which have affected the fetus' development. These malformations can be found anywhere on the baby but the most common organs affected are the heart, brain and limbs (Thein et al 1992). Congenital malformations affecting the heart are seen to be the most deadly; around 50% of deaths due to congenital malformations are due to malformations of the heart (Yang et al 1997). The causes of congenital abnormalities can be classified into 4 groups - genetic, environmental, multifactorial (combination of genetic and environmental factors) and unknown. Up to 60% of congenital abnormalities are still caused by unknown factors. Only around 10% of malformations have been recognized as having an environmental basis. Environmental factors are classed as teratogens and several groups of exist; infection (rubella virus, herpes), radiation (nuclear bombs, radiation therapy), metabolic (diabetes, folic-acid deficiency) and drugs (ecstasy, isotretinoin, alcohol). Different environmental teratogens will cause different effects through different mechanisms. The fetus will also have 'most-susceptible-periods' to each teratogen depending on the organs it effects. As a general rule the most susceptible period for the fetus is during the first eight weeks of development as the most important developmental processes are taking place at this time (Finnell et al. 2002). This report will focus on a common environmental teratogen, folic-acid deficiency, and will investigate the mechanisms with which it effects fetal development.
Folic-acid deficiency
Folic acid deficiency has been shown to have a high correlation with incidence of neural tube defects (NTD) (Kirke et al 1993) with supplementation of folic acid to pregnant mothers reducing the risk of NTD development in the fetus by up to 70 %(DeWals et al 2007). This has led to the accepted view of folic acid having a role in normal neural development. Deficiency in folic acid can result in malformations such as anencephaly (failure of the cerebrum, cerebellum and cranial vault to develop with only rudimentary brain stem structures developing), lubosacral myelomeningocele (failure of the lumbar bones to form causing the lumbar part of the spinal cord and associated meninges to poke through) or craniorachischisis where none of the nervous system develops. To understand how these abnormalities in development come about we need to look at normal neural tube formation
Normal neural tube formation
Formation of neural plate via signals originating from the notochord is the first step of neural tube formation. This plate of neuroepithelial cells will shape itself so that it extends over the anterior-posterior axis of the embryo. Neurulation (see figure-1) of the neural plate occurs around a median hinge point. Conformational changes in the neuroepithelial cells, which become narrower at the apex and wider at the base, are the main mechanism for this folding movement. Elevation of the neural folds is thought to be accomplished through mainly extrinsic forces generated by the ectoderm lateral to the neural plate. Fusion of the neural folds will first occur near the middle of the forming neural tube where the first somites appear. This fusion will then proceed to continue both in the anterior and posterior directions until a hollow, open-ended tube is formed. The open ends at each end of the neural tube are called the anterior and posterior neuropore. These will close off forming a closed tube. This neural tube gives rise to the CNS.
Figure 1. - lateral folding of the neural plate in Ambystoma mexicanum (process is close to that of mammals)
Lateral folding of the neural plate Anterior neuropore
Neural plate Median hinge point Primary fusion point Posterior neuropore
NTDs will form as a result of defects in neural tube closure with different resulting defects depending on the position of where the defect occurs (e.g.
anencephaly - anterior neuropore fails to close;
lubosacral myelomeningocele - posterior neuropore fails to close;
craniorachischisis - failure of the whole neural plate to fuse). Thus the main cause of neural tube defects comes from a common cause - failure of normal neural tube folding and closure.
Folic acid in normal neural tube closure
Folic acid is a water-soluble B vitamin and has a range of functions within the body including gene expression, cell function and cell differentiation (Vanderput et al 2001). As folic acid cannot be synthesized by humans, sufficient intake is needed. Deficiencies may arise through a variety of reasons such as; insufficient intake through diet, malabsorption, altered metabolism, increased elimination. Genetic factors are thought to be the main reason for folic acid deficiencies (poor absorption and altered metabolism) arise rather than a poor diet. The mechanisms in which folic acid may affect neural tube closure can be traced back to two major pathways; the synthesis of nucleic acids (guanine and adenosine) which are needed for cell proliferation and the role folic acid plays in methylation reactions. Reduction of folic acid will reduce the efficiency of these pathways and consequently effect neural tube formation.
Folic acid in Methylation reactions
The AdoMet (adenosylmethionine) enzyme is responsible for many methyltransfer reactions in the body. AdoMet requires folic acid for synthesis as methionine is produced either directly from folic acid derivatives (via methionine synthase) or via transfer of a methyl group from folic acid derived enzymes onto homocystein to form methionine (MTHFR, MS, CBS). MAT (methionine adenosyl transferase) will then transfer ATP to methionine to form AdoMet. AdoMet acts in the body to methylate DNA and cytoskeletal proteins, both of which are important in neural tube folding and closure. As folic acid is involved in the production of AdoMet, when folic acid levels in the body are reduced, AdoMet would be produced at lower levels leading to a reduced ability to undertake methylation reactions.
Epigenetic regulation of DNA is essential for normal development to take place as it allows selective silencing of genes within particular cell types. A type of epigenetic regulation is DNA methylation where the promoter region for a gene is methylated, interfering with the binding of transcription factors and thus suppressing gene expression. AdoMet is one such molecule which may transfer a methyl group to DNA and thus regulate gene expression (Klein 1997). An example of the importance of epigenetics in neural tube closure is in the selective expression of specific cadherins, glycoproteins responsible for cell-cell adhesion. During neural plate formation the neuroectoderm will express only N-cadherins while during neural plate folding, the neuroectoderm cells will express E-cadherins on the dorsal surface and N-cadherins on the ventral surface. These specific surface cadherins allow the movement of cells so that neural tube formation may be accomplished and also facilitates fusion of the two folds at the ventral surface (Halbleib & Nelson 2006). The change from just N-cadherins to N and E cadherins is under epigenetic control; i.e. AdoMet will act to suppress specific genes via methylation as to produce a specific result (N and E cadherin expression in neuroectoderm cells) which allows neural folding and closure.
With a reduced ability to regulate gene expression via DNA methylation (due to folic acid deficiency), proper gene expression may not be able to be maintained. In the neuroectoderm cadherin-expression example, this might mean that E-cadherin is not expressed when it should be. This could lead to restriction of cell movement needed for neural plate folding due to the difference in properties between E-cadherin and N-cadherin. Also as lateral plate fusion is partially dependant on cadherin expression, fusion may not occur also due to incorrect cadherins expressed on cells. These would lead to failure of normal neural tube formation and thus NTDs.
Cytoskeletal proteins such as microtubules and microfilaments are present in the neural plate ectodermal cells at the apices. These filaments will cause narrowing around the apex of the neuroectodermal cells. This apical narrowing combined with cell proliferation and also the positioning of nuclei to the base of cells to broaden the base will generate intrinsic forces which act to fold the neural plate around to form a tubular structure. The methylation of 3 cytoskeletal filaments produces the observed narrowing at cell apices; actin, α and β tubulin, neurofilament-L (Bjrklund & Gordon 2006). AdoMet will act to generate an intrinsic force via methylation of cytoskeletal proteins which allows neural plate folding.
Generation of intrinsic folding forces via cytoskeletal proteins could not take place also if folic acid availability (and thus AdoMet) was limited. Without intrinsic folding forces caused by methylation of cytoskeletal filaments which narrow the apical ends of neuroepithelial cells, neural tube formation may not occur normally because of the reduction of force being generated to fold over the neural plate. Without enough force to fold the neural plate, the neural plate could not meet dorsally and fusion would not occur leading to NTDs.
Homocysteine levels
After AdoMet donates a methyl group, AdoHyc (adenosylhomocysteine) is formed. As this is a toxic substance it is quickly acted upon by SAH (S-adenosylhomocysteine hydrolase) which cleaves AdoHyc to form adenosine and homocysteine. Usually homocysteine levels are quickly reduced via folic acid derived enzymes which either remethylate homocysteine into methionine (MTHFR and MS) or transsulfate homocysteine into cysteine or turine (CBS). A deficiency in folic acid will cause reduction in the amount of homocysteine that may be readily transformed at any time due to a reduction in the amount various folic acid derived enzymes. (MTHFR, MS, CBS).
Homocysteine has been shown to be teratogenic at high levels with studies showing that a high level of homocysteine in the mother is correlated with neural tube defects seen in offspring (Rosenquist et al. 1996).
The teratogenic effects of homocysteine may be linked to its growth factor effects on cells; as a growth factor it alters the expression of genes to the process of neural tube formation and closure (Finkelstein 1998). This view of homocysteine is consistent with the fact that low levels are essential for normal growth of embryos but at high concentrations, just like other growth factors (eg. Pax-2), homocysteine will effects the normal expression of genes and through this mechanism, adversely affect normal development of the neural tube.
Synthesis of nucleic acids
Folic acid is needed as a substrate for the synthesis of guanine and adenosine. As such, cell proliferation requires folic acid for DNA synthesis (mitosis) to occur. As neural tube closure relies on cell proliferation to produce intrinsic as well as extrinsic forces, a reduced capacity for the embry to undergo mitosis will affect the amount of force generated to produce folding of the neural plate and concequently effect neural tube formation.
Conclusion
Folic acid deficiency has a probable genetic basis where folic acid is not properly transported into cells or metabolized leading to a reduction of folic acid levels in mother and fetus. As folic acid is essential for methylation reations (epigenetic gene regulation, contraction of neuroepithelial microfilaments) and also for cell proliferation (important in generating intrinsic and extrinsic forces), a reduction in the levels of folic acid available to the fetus will reduce the ability of the fetus to carry out these essential processes, leading to the development of neural tube defects. Yet such devastating effects can be easily eliminated in most cases by simple supplementation of folic acid to the mother before and during pregnancy. Informing the general public about the ease of which NTD may be prevented should be undertaken as to reduce the number of lives ruined by the range of devastating conditions caused by a simple deficiency in folic acid.
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