Prenatal diagnosis of genetic disease in Canada: report of a collaborative study.

A study of 1223 amniocenteses carried out during 1020 pregnancies in 990 women showed that 2nd-trimester amniocentesis at about 16 weeks'' gestation is a safe, accurate and reliable procedure for the diagnosis of certain classes of genetic disease when it is monitored by ultrasound, performed by a trained obstetrician and carried out in a major health sciences centre. The percentage of fetal losses (4.7%) and neonatal deaths (0.5%) during the study was not greater than in control samples for women 35 years of age and older. The best results were obtained when needles of gauge 20 or 21 were used. The use of needles of gauge 19 or larger and more than two insertions during a single amniocentesis were associated with a significantly greater frequency of fetal loss than a second or even a third amniocentesis during the same pregnancy. For 39 fetuses (3.8%) a diagnosis of a genetic abnormality was made and 23 male fetuses were found to be potentially hemizygous for an X-linked gene. There were 51 therapeutic abortions as a result of the diagnosis. Sixty-six tests (5.4%) gave an inconclusive result and seven (0.6%) gave an erroneous diagnosis; five of the latter (two false-positives and three false-negatives) resulted from the alpha1-fetoprotein test for neural-tube defects and in two cases the sex was incorrectly determined. The frequency of all chromosome abnormalities was 1:20 when the mother''s age was 40 years or more and 1:60 when the mother''s age was between 35 and 39 years. When a mother had previously had a child with a chromosome abnormality the risk of recurrence of such an abnormality was 1:100 when the age of the mother was 35 years or more.     

Genetic heterogeneity in neural tube defects.

In 1985-1987, the authors attempted to ascertain all cases of confirmed neural tube defects (NTD) in California and Illinois, not only among live-born infants (postnatal) but also cases ascertained during pregnancy (prenatal). Mothers of both prenatal and postnatal NTD cases were interviewed within 5 months. Among postnatal NTD cases, 14.9% (45/303) had anomalies not ordinarily associated with NTD. The frequency of non-NTD related anomalies was 9.4% (5/53) in anencephaly, 0/3 in craniorachischisis, 22.9% (8/35) in encephalocele, 14.5% (27/186) in spina bifida, 20% (1/5) in multiple NTD cases and 19% (4/21) in other NTDs. However, relatively few postnatal NTD cases had known multiple malformation patterns; Meckel-Gruber syndrome was the most common, with 2 postnatal cases, and 3 additional prenatal cases. Maternal age, paternal age and birth order in postnatal cases were 26.7 +/- 5.4 SD, 28.9 +/- 5.8 and 2.8 +/- 1.8, respectively. These characteristics were similar in prenatal NTD cases (27.9 +/- 6.0, 30.1 +/- 6.3, 2.5 +/- 1.5, respectively). We also found no differences in parental ages among different types of NTD. Frequency of prior spontaneous abortion differed neither between postnatal NTD (9.3%) and postnatal controls (8.1%), nor between prenatal NTD (10.7%) and prenatal control (8.7%). Loss rates in the pregnancy immediately prior to the index NTD cases were not significantly higher than in control subjects. The high frequency of non-NTD associated malformations (14.9%) indicates the caution must be exercised before assuming that a given NTD case is polygenic-multifactorial in etiology, especially cases of encephalocele.     

Biomechanical basis of diazepam-induced neural tube defects in early chick embryos: a morphometric study

The biomechanical basis of diazepam (Valium/Roche)-induced neural tube defects in the chick was investigated using a combination of electron microscopy and morphometry. Embryos at stage 8 (four-somite stage) of development were explanted and grown for 6 hr in nutrient medium containing 400 micrograms/ml diazepam. Nearly 80% of these embryos exhibited neural tube defects that were most pronounced in the forming midbrain region and typified by a "relaxation" or "collapse" of neural folds. The hindbrain and spinal cord regions were less affected. Electron microscopy revealed that neuroepithelial cells in diazepam-treated embryos had smoother apical surfaces and broader apical widths than did controls. Morphometric measurements supported this observation and further showed that these effects were focused at sites within the wall of the forming neural tube that typically exhibit the greatest degree of bending and apical constriction (i.e., the floor and midlateral walls). Overall results indicate that neural tube defects associated with exposure to diazepam are due largely to a general inhibition of the contractile activity of apical microfilament bundles in neuroepithelial cells. These findings 1) emphasize the important contribution of microfilament-mediated apical constriction of neuroepithelial cells in providing the driving forces for bending of the neuroepithelium during neural tube formation and 2) suggest that agents or conditions that impair their contractile activity could play a role in the pathogenesis of certain types of neural tube defects.     

VANGL1 rare variants associated with neural tube defects affect convergent extension in zebrafish

In humans, rare non-synonymous variants in the planar cell polarity gene VANGL1 are associated with neural tube defects (NTDs). These variants were hypothesized to be pathogenic based mainly on genetic studies in a large cohort of NTD patients. In this study, we validate the potential pathogenic effect of these mutations in vivo by investigating their effect on convergent extension in zebrafish. Knocking down the expression of tri, the ortholog of Vangl2, using an antisense morpholino (MO), as shown previously, led to a defective convergent extension (CE) manifested by a shortened body axis and widened somites. Co-injection of the human VANGL1 with the tri-MO was able to partially rescue the tri-MO induced phenotype in zebrafish. In contrast, co-injection of two human VANGL1 variants, p.Val239Ile and p.Met328Thr, failed to rescue this phenotype. We next carried out overexpression studies where we measured the ability of the human VANGL1 alleles to induce a CE phenotype when injected at high doses in zebrafish embryos. While overexpressing the wild-type allele led to a severely defective CE, overexpression of either p.Val239Ile or p.Met328Thr variant failed to do so. Results from both tri-MO knockdown/rescue results and overexpression assays suggest that these two variants most likely represent “loss-of-function” alleles that affect protein function during embryonic development. Our study demonstrates a high degree of functional conservation of VANGL genes across evolution and provides a model system for studying potential variants identified in human NTDs.     

Neural tube defects without neural crest defects in splotch mice.

Homozygous Splotch mutant mice (Sp/Sp) die on day 14 of gestation with neural tube defects, curly tail, and malformations of neural crest derivatives. Sp1H mice, which have a radiation-induced allele of Splotch with a similar phenotype, were used for this study. The neural tube defects are always located in the lumbosacral region and in 50% of the cases also in the region of the hindbrain. In this report, rare cases of neural tube defects and tail defects among the offspring of crosses between Splotch (Sp1H) heterozygotes are presented, which are not associated with a neural crest defect. This suggests that the development of the neural tube and neural crest defects in this mutant is caused by independent mechanisms or is dependent on the dosage of the mutant gene, with different thresholds being pathogenetic in the neural tube and neural crest, respectively.     

Maternal zinc and fetal neural tube defects.

Among the factors implicated in the heterogeneous etiology of neural tube defects (NTDs) is the trace element zinc (Zn). In a case-control study, we collected midtrimester maternal toenail samples for multiple trace element analyses, including Zn, which were assayed by neutron activation analysis. We studied 17 women with NTD offspring and 1,787 controls. The crude OR for NTD comparing Zn values greater than normal range to normal Zn values was 3.2 (95% CI 1.1,9.7). These results were not materially affected when adjustment was made for folic acid supplementation. An overall increased risk for NTD associated with increasing toenail Zn was also evident. A matched subset of 17 cases and 73 controls yielded a crude OR of 3.1 (95% CI 0.9,10.3) when cases with elevated Zn (greater than or equal to 120 ppm) were compared to those with normal Zn. Matched analyses controlling for folic acid supplements, family history of NTD, assay batch, age of mother and year of delivery yielded an OR of 5.0 (95% CI 1.1,21.6). This study reveals an association between increased toenail Zn in the second trimester of pregnancy and the risk of having a child with an NTD. Whether Zn sequestration has resulted in relative Zn deficiency at the site of neural tube closure remains uncertain.     

Neural tube defects: a review of human and animal studies on the etiology of neural tube defects

Although neural tube defects are a common congenital anomaly, their etiology is not known. Human studies have emphasized the pathology and epidemiology of the defects and suggest that in the majority of cases the etiology is multifactorial. Factors which appear possibly to be important are genetic predisposition, maternal illness, and fetal drug exposure. Animal studies have utilized naturally occurring neural tube defects and teratologically induced lesions. No animal model has been convincingly established as the equivalent of human neural tube defects. However, animal models have allowed investigation of the mechanisms of suggested human teratogens and determination of the pathogenesis of naturally occurring animal defects. Their most important contribution has been in furthering the understanding of the normal mechanisms of neural tube closure. It may be through this understanding that the etiology of human neural tube defects will be determined.     

Epidemiology of neural tube defects in Germany

Summary A survey is made of the epidemiologic studies of neural tube defects (NTD) in Germany. A temporary increase is noted in the prevalence of NTD at birth for the time during and shortly after the Second World War, followed by a downward trend thereafter. Thus an earlier observation of Lenz (1965) could be confirmed. Falling rates of NTD were also reported from various other countries in recent years. No convincing etiological explanation is available so far. The current prevalence of NTD at birth can be estimated for Germany to be about 1.0–1.5 per thousand newborns with about an even distribution to anencephalus and spina bifida.     

Immunobiological methods in the prenatal diagnosis and evaluation of foetal neural tube defects

In cases of foetal neural tube defects (NTDs) macrophages are present in the amniotic fluid. These mononuclear cells were analysed with immunobiological methods: functional markers as Fc and C3b receptor-mediated phagocytosis and chemoluminescence have been studied. It was found that most of these pathognomic cells ingest haemolysin sensitized sheep red blood cells (sSRBCs) and zymosan (Mannozym) particles opsonized with fresh human serum. Amniotic fluid cell suspensions from pregnancies with and without foetal NTDs were stimulated by opsonized Mannozym; consistently higher chemoluminescence activities were found when open lesion was present. The evaluation of multiple functional markers is likely to provide a better basis for understanding the characteristics of amniotic fluid macrophages and may contribute to the prenatal diagnosis of NTDs.     

Computer modelling of neural tube defects

Neurulation, the curling of the neuroepithelium to form the neural tube, is an essential component of the development of animal embryos. Defects of neural tube formation, which occur with an overall frequency of one in 500 human births, are the cause of severe and distressing congenital abnormalities. However, despite the fact that there is increasing information from animal experiments about the mechanisms which effect neural tube formation, much less is known about the fundamental causes of neural tube defects (NTD). The use of computer models provides one way of gaining clues about the ways in which neurulation may be compromised. Here we employ one computer model to examine the robustness of different cellular mechanisms which are thought to contribute to neurulation. The model, modified from that of Odell et al (Odell, G.M., Oster, G., Alberch, P. and Burnside, B., (1981)) mimics neurulation by laterally propagating a wave of apical contraction along an active zone within a ring of cells. We link the results to experimental evidence gained from studies of embryos in which neurulation has been perturbed. The results indicate that alteration of one of the properties of non-neural tissue can delay or inhibit neurulation, supporting the idea, gained from observation of embryos bearing genes which predispose to NTD, that the tissue underlying the neuroepithelium may contribute to the elevation of the neural folds. The results also show that reduction of the contractile properties of a small proportion of the neuroepithelial cell population may have a profound effect on overall tissue profiling. The results suggest that the elevation of the neural folds, and hence successful neurulation, may be vulnerable to relatively minor deficiencies in cell properties.