Pathology
Spina bifida is typically considered a primary failure of neurulation. Failure of neurulation and, thereby, loss of neural tube closure, prevents the mesoderm adjacent to the notochord from forming muscle and bone (ie, via somitic mesoderm), which normally forms around the tube to protect it.
Therefore, the mechanisms involved in this process are suspect in the pathology of this disorder. Although this is the most popularly accepted theory, there are other proposed mechanisms. Dias and colleagues have discussed the idea that several forms of myelomeningoceles are not failures of neurulation, but a failure of Henson's node to lay down the notochord correctly—in other words, a failure in gastrulation (10,11) that causes significant errors in induction of the neural tube. Further research is necessary to elucidate and verify currently proposed theories.The mechanisms by which the neural tube is formed and closed are varied. Morphogenic changes in cell populations such as wedging result in the shaping of the neural plate into a tubelike structure early in neurulation (12-14). Several mechanisms are proposed for closure of the neural tube, such as interaction between various glycoproteins and cell adhesion molecules (CAM), multiple roles for various signaling protein/receptor interactions, the interlinking of numerous cell filopodia, and formation of intercellular junctions. The current view suggests that the process likely involves all of these and perhaps others not yet visualized.
Failures of induction of NTD by the notochord can result in incomplete CNS development and/or overgrowth of CNS precursors. Indeed, NTDs are described not only as failures of neural tube closures, but as failure to properly induce the development of mesenchymal and neuroectodermal structures. Neural induction involves numerous soluble, diffusible factors produced by a variety of genes (eg, sonic hedgehog), specific cells-surface signaling molecules important for appropriate migration of cells within the developing neural tube, and direct cell-cell signaling (eg, CAMs).
Genetic Influences
Genetic mutations can certainly have a significant impact on all of the previously mentioned processes and have been both demonstrated experimentally in rodents and documented clinically in humans. Alterations in genes that affect metabolism, nucleotide synthesis, cell programming, and cell-cell signaling can all affect aspects of neural development, ranging from the signaling aspects of the induction of neural tube formation initiated by the notochord to alterations in programmed cell death. This affects overall CNS development.
Induction of the neural plate is controlled by a variety of genes. Sonic hedgehog (SHH) is a vertebrate gene expressed by cells within the notochord that—in conjunction with the Patched (PTC) gene—produce proteins that are involved in the induction of the floor plate during embryogenesis, the proliferation of neuronal subtypes such as motor neurons, and the beginnings of somite development. Early work in Drosophila and then in avian systems have described how the proteins produced by these genes induce the expression of various signaling proteins on the surfaces of cells, allowing for the sequential transmission of signals regulating the cells' fate.
PTC is a gene that functions downstream of SHH. Its function is hypothesized to serve as a negative feedback to SHH, thereby regulating the induction of numerous cell types in the developing neural tube. Failure of this system and its feedback loops and/or overexpression of one portion of the process could easily be involved in neural tube development failure. Although not currently implicated by empirical data, much research is currently in place to elucidate the impact of this system on human NTDs.
Genes associated with folate metabolism and methyltransferase reactions associated with methionine and homocysteine metabolism are both of major interest. Folate serves as a cofactor for enzymes that participate in nucleotide synthesis as well as being important in methylation processes.
Evaluations of folate levels of mothers with NTDs shortly after birth have produced equivocal results, suggesting that absolute folate deficiency is rare. Indeed, disturbances in the metabolic pathways that utilize folate may predispose to NTDs. This could conceivably be corrected by supplementation with folate. Metabolism of folate and homocysteine is interdependent, and the risks associated with alterations in their metabolism are thought to be connected. Indeed, elevated homocysteine levels in pregnant women are a known risk factor for NTDs. Mutations/polymorphisms in the enzyme 5,10- methylenetetrahydrofolate reductase (MTHFR) have been associated with diminished plasma folate levels, with commensurate elevated homocysteine levels. These alterations have been identified in patients with spina bifida as well as their mothers and fathers. In addition, using cultures of fibroblasts from NTD-affected patients, homozygosity for defects in the MTHFR gene have been shown to have a 7.2-fold increased risk for neural tube defects. The prevalence of these defects appears to vary by race. Homozygosity for the C677T MTHFR mutation is a known risk factor for upperlevel spina bifida lesions in Hispanics. The MTHFD1 1958G>A polymorphism is also associated with NTDs in those of Irish descent.It is well known that in early stages of nervous system development, more cells are produced than needed and that the process of apoptosis and autophagy are coordinated during development to yield a well-defined and functioning nervous system. Apoptosis, the most studied of these processes, is modulated by various members of the Bcl2 gene family, the caspase family of cysteine proteases, and other genes which produce proteins that are necessary intermediators. Expression of these genes at different times and in different combinations essentially controls the development of specific populations of cells within the CNS. Several mouse models have shown that altering the expression of these genes (ie, knockout experiments) results in neural tube defects similar to that identified in humans.
Such evidence strongly suggests their involvement in human neural tube pathology. Autophagy, an autode- generative cell process, has a significant impact on the recycling of cellular components in the cytoplasm as a result of cellular organelle damage. This process can also be affected by nutritional stresses. A variety of genes that affect this process have been investigated using mouse models. Loss of Beclin 1 and Ambra 1 expression has been noted to result in overgrowth of the developing CNS. Therefore, identifying the human equivalent of these and other similar genes could yield information as to cause of various NTDs and provide information for new therapeutic targets.Environmental Influences
The external environment has a significant impact on embryonic development and the incidence of NTDs. This has been documented in several ways. Hyperthermia during early pregnancy—the first 28 days during which neurulation occurs—has been shown to increase the incidence of NTDs. Specifically, maternal febrile events as well as sauna/hot tub use has increased the risk of NTDs (15—19).
Parental occupation has been demonstrated to have a definitive influence on the risk for neural tube defects. Increases in risk for NTDs have been noted for occupations involving exposure to solvents (eg, painters, industrial process workers, etc.). The health care profession has also been seen to impart an increased NTD risk. Also, agricultural workers, along with those involved in the transportation industry, have been noted to have an increased risk for NTDs. The exact etiology behind these changes in risk can only by hypothesized at this time.
Nutritional influences have a broad impact and interact in many ways with environmental as well as genetic influences. A primary example is folate metabolism. As indicated previously, folate is a cofactor for the enzymatic process involved in purine and pyrimidine synthesis and is also important in facilitating the transfer of methyl groups during the metabolism of methionine and homocysteine.
Taken together, alterations in these folate-sensitive processes can have an impact on cellular proliferation. Lowered intake of foods containing folate in the diet is associated with an increase in the risk for NTDs. Also, as one can imagine, disorders of absorption of folate in the intestine can significantly affect folate levels and potentially affect NTD risk. However, studies of folate receptor/ carrier densities in the intestines of women with NTD offspring or their progeny do not have abnormally low receptor levels. A significant number of studies, both in the United States as well as Europe, have shown that supplementation can alter this risk. Indeed, mandatory supplementation of folate in grain products in the United States has caused a steadily declining incidence of NTDs since it was initiated in 1996. Since the introduction of this program, it has been estimated that the number of pregnancies affected by NTDs has declined from approximately 4,000 to 3,000 per year. In fact, studies have shown that the risk for recurrence of NTDs can be decreased approximately 50% by taking recommended folate supplementation.The risk for NTDs varies for couples, depending on whether there is a prior history of such defects. U.S. couples with a prior history of NTD births have an increased risk for recurrence (2%-5%) (2). Because of this, the U.S. Public Health Service and the CDC have two separate recommendations for supplementation based on prior NTD histories. Elevated supplementation is appropriate for couples with a prior NTD birth. Limited studies have also identified zinc as a nutritional entity that can also elevate NTD risk. It was discovered that women with the genetic disorder of zinc metabolism acrodermatitis enteropathica are at high risk for NTDs and that supplementation can lower those risks.
Maternal obesity and associated diabetes have been found to be associated with increases in risk for NTDs. Specifically, women with a pre-pregnancy body mass index (BMI) suggestive of obesity (>29 kg/m) are more inclined to give birth to children with NTDs.
This holds true for women with diabetes, although the etiology of this association may be linked to alterations in glucose metabolism during organogenesis. It is notable that experimentally manipulated glycosylation in rodents results in birth defects not unlike those seen born to mothers with diabetes. Risks for NTD-affected births has been estimated at 2% here in the United States and as high as 7% in England. These risks include spina bifida as well as other significant NTDs such as anencephaly. The NTD recurrence risk for mothers with diabetes in the United States is around 4%, which is similar to that found for mothers without diabetes.Teratogenic influences from the environment— such as the consumption of prescribed drugs—have been associated with neural tube defects, particularly myelomeningocele. Valproic acid taken for seizures during pregnancy has been shown to increase the incidence of neural tube defects. Mechanistically, it appears to work by disrupting folate metabolism, thereby inhibiting neural tube closure. Alterations in folate-dependent methylation of regulatory proteins is theorized to be the cause. Regardless, administration of folate during pregnancy counteracts valproic acid- associated neural tube defects.
The rising use of highly active antiretroviral therapy (HAART) in the treatment of human immunodeficiency (HIV) disease has increased the incidence of women exposed to these drugs entering and during pregnancy. A variety of case reports as well as animal studies have suggested an association between antiretroviral drug use and NTDs (20). Drug-induced interference with DNA synthesis during development would likely have an impact on gastrulation and neu- rulation. Other drugs are also associated with NTDs, such as isotretinoin (Accutane), which is used for acne treatment; etretinate (Tegison), which is a psoriasis treatment; and anticancer agents such as methotrexate. Indeed, even fetal alcohol syndrome has an association with increased risk for abnormal CNS development, including NTDs.
Some chromosomal disorders that have multivariate etiologies and presentation are known to have an association with increases in risk for NTDs. Trisomy 21 (Down's syndrome) and trisomy 13 (Patau syndrome) are notable examples. Although the incidence is relatively small, studies have shown that various NTDs, including spina bifida but not anencephaly, have been found upon autopsy of definitively karyotyped infants. Interestingly, trisomy 21 has been shown to be associated with genetic polymorphisms involved in homocysteine/methionine methylation (see the previous discussion on folate metabolism) and has a noted familial clustering with NTDs.