Associated malformations in infants and fetuses with upper or lower neural tube defects

A number of studies on sexual differences in the ilium have been reported. However, most of these studies have focused on the adult ilium. With regard to sexual differences in the fetal ilium, few studies have been carried out. Especially, there have few studies regarding sexual differences in the fetal ilium using dry bones. In the present study, sexual differences in the morphological characteristics of the greater sciatic notch were investigated using dry fetal iliac bones. We examined 212 fetuses (106 males and 106 females) measuring 20.0 cm or more in fetal length with free of gross malformations or deformities which were collected at Saga Medical School. The iliac bones were excised from these fetuses and dried as materials for study. Fetal length was measured in the fully extended position and was classified into six groups each for males and females in 5-cm increments, giving a total of twelve groups. The inner lateral surface of the greater sciatic notch was magnified 25x using projector, traced, and the tracings were inputted to a computer (7600/120: Apple Co. Ltd) using a flatbed scanner. The width and height of the greater sciatic notch were measured, as well as the area of the greater sciatic notch. No sexual differences were noted regarding the increase in the width of the greater sciatic notch in fetuses up to 39.9 cm in fetal length. In fetuses measuring between 40.0 anf 44.9 cm, a significant sexual difference was observed, with the increase in greater sciatic notch width markedly greater in females. With respect to the increase in the height of the greater sciatic notch, no significant differences were noted. No significant differences were observed in the total area of the greater sciatic notch in fetuses up to 39.9 cm in fetal length. On the other hand, a significant sexual difference was observed in fetuses measuring 40.0 cm or more, with the total area markedly increased in females. In addition, no significant differences were found in the area between the two sides of the greater sciatic notch in fetuses up to 39.9 cm in fetal length, although significant differences were observed in fetuses measuring 40.0 cm or more. In female fetuses 40.0 cm or more in fetal length, the greater sciatic notch was found to be wider than in males, and the peak of the greater sciatic notch was displaced in the anteroinferior direction, farther from the auricular surface of the ilium and nearer the pubic bone. Thus these female fetuses show morphological characteristics of the adult female pelvic bone. Based on the results obtained, it is concluded that sexual differences can be identified in fetuses from the 8th month of pregnancy (fetal length 40.0 cm or more).     

Relationship between altered axial curvature and neural tube closure in normal and mutant (curly tail) mouse embryos

In isolated smooth muscles of the rat stomach, the properties of electrical responses of the membrane elicited by transmural nerve stimulation were studied. The smooth muscle membranes were quiescent in the fundus and spontaneously active with slow waves and often action potentials superimposed on top of the slow wave in the antrum and pylorus. The maximum membrane potentials were larger in the antrum (-50 to -55 mV) than in the fundus (-40 to -45 mV). Transmural nerve stimulation elicited an excitatory junction potential (e.j.p.) which was followed by an inhibitory junction potential (i.j.p.) in the fundus, and an i.j.p. alone in the antrum. The e.j.p. was inhibited by atropine, indicating that this potential was cholinergic in nature. The amplitude of the e.j.p. was increased by apamin or nitroarginine, and the latter was more potent than the former. The i.j.p. was inhibited by apamin or nitroarginine, and was resistant to adrenergic and cholinergic blocking agents, and therefore this potential was non-adrenergic non-cholinergic (NANC) in nature. The inhibitory actions of nitroarginine on the i.j.p. were antagonized by L-arginine, suggesting the involvement of nitric oxide (NO) in this junctional transmission. The results indicate that smooth muscles of the rat stomach receive cholinergic excitatory and NANC inhibitory nerves, and that endogenous NO may either be partly responsible for the generation of the i.j.p. or may modulate the junctional transmissions.     

Pituitary gland and sella turcica in human trisomy 21 fetuses related to axial skeletal development

Fourteen members of family P. and four members of family N. were clinico-biochemically examined. Among twelve adult children (19-32 years old) of family P. five sons manifested angiokeratotic skin lesions and other clinical signs of Fabry disease. Three of the probands had additional symptoms not generally found in Fabry disease. Biochemical studies including an enzyme assay, analysis of storage products and alpha-galactosidase multiple forms, allowed us to confirm the diagnosis of Fabry disease in four affected brothers and to establish the heterozygous status of their mother. The data of biochemical investigation of patient N. with atypical variant of Fabry disease are also presented. The patient N. with strong skin lesions had a high residual alpha-galactosidase activity and unusual composition of alpha-galactosidase multiple forms.     

Reduced glucose consumption in the curly tail mouse does not initiate the pathogenesis leading to spinal neural tube defects

At embryonic stages of neural tube closure, the mouse embryo exhibits a high rate of glycolysis with glucose as the main energy source. In the curly tail mouse, often used as model system for study of human neural tube defects, a delay in closure of the posterior neuropore (PNP) is proposed to be indirectly caused by a proliferation defect in the caudal region. Because glucose is important for proliferation, we tested glucose uptake in curly tail and control embryos, and in a BALB/c-curly tail recombinant strain. The structure and expression of Glut-1, a glucose transporter molecule that is abundantly present during those embryonic stages and that has been mapped in the region of the major curly tail gene, were also studied; however, no strain differences could be demonstrated. Glucose uptake was determined by measuring glucose depletion from the medium in long-term embryo cultures that encompassed the stages of PNP closure and by measuring accumulation of 3H-deoxyglucose in short-term cultures at the stages of early and final PNP closure. Both approaches indicated a reduced glucose uptake by curly tail and recombinant embryos. Surprisingly, the uptake per cell appeared normal, accompanied by a significantly lower DNA content of the mutant embryos. Therefore, it is unlikely that reduced cell proliferation is caused by a reduction in glucose supply during the pathogenesis of the defects in curly tail embryos. The reduced DNA content as well as the reduced glucose uptake per embryo are likely downstream effects of the aberrant proliferation pattern.     

Blood folic acid and vitamin B12 in relation to neural tube defects

OBJECTIVE: To determine the relation between blood folic acid and serum vitamin B12 in neural tube defect pregnancies using data from the MRC Vitamin Study and a literature review of all studies. DESIGN: Stored blood samples collected as part of a randomised trial of vitamin supplementation in the prevention of neural tube defects were retrieved from affected pregnancies (cases) and unaffected pregnancies (controls). Four controls were matched with each case by centre, maternal age and duration of storage of the blood sample. The samples had been collected from women at entry to the trial, immediately before the women became pregnant, and at around 12 weeks of pregnancy. Our results were combined with those already published from other studies to obtain an overall assessment of blood folic acid and vitamin B12 in relation to neural tube defects. SETTING: Blood samples were collected as part of the MRC Vitamin Study. The collaborating centres were in the United Kingdom, Hungary, Israel, Australia, Canada and Russia. PARTICIPANTS: Twenty-seven women with neural tube defect pregnancies and 108 matched controls with unaffected pregnancies. RESULTS: Serum and red cell folic acid and serum vitamin B12 levels were lower in the cases than in controls at each of the three occasions when blood samples were collected, but no comparison was significant (P > 0.05). A systematic review of all studies from the literature showed that on average, during the 1st trimester of pregnancy, serum folic acid was 0.6 ng/ml lower in neural tube defect pregnancies (P < 0.01), red cell folic acid was 77 ng/ml lower (P < 0.001) and serum vitamin B12 was 38 ng/l lower (P < 0.001). A logistic regression showed no association between serum B12 and neural tube defects after allowing for serum folic acid. CONCLUSION: our results are consistent with other evidence that folic acid and vitamin B12 levels are lower in women with neural tube defect pregnancies and consistent with evidence from randomised trials which showed that folic acid is protective.     

The alpha-fetoprotein level analysis in open neural tube defects and chromosomal aberrations in the fetus

The effects of 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) and two Chinese traditional (Kampo) medicines, Dai-saiko-to and Saiko-ka-ryukotsu-borei-to on spontaneous soft tissue (heart and tongue) mineralization in DBA/2NCrj mice were studied. These agents were given orally for 4 weeks to DBA/2NCrj mice. After 2 weeks of administration in the heart, 0.006 and 0.03% (w/v) HEBP decreased calcium content by 90 and 30%, respectively, while 0.27 and 2.7 mg/ml Dai-saiko-to reduced calcium content by 30 and 45%, respectively. Saiko-ka-ryukotsu-borei-to (0.27 mg/ml) reduced both calcium and phosphorus content by 50 and 35%, respectively. However, their inhibitory effects on the heart were not observed after 4 weeks of administration. The compounds delayed the onset of increases of bulk calcium and phosphorus content. In the tongue, at 4 weeks, 0.006 and 0.03% (w/v) HEBP reduced calcium content by 30 and 45%, respectively, while two Kampo medicines (at both concentrations used) significantly reduced the content of calcium (by 27-79%) and phosphorus (by 24-32%). These results strongly suggest that two Kampo medicines as well as HEBP may be useful in preventing and curing soft tissue calcification.     

Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene

Transgenic mice were generated containing a cytomegaloviral promoter driven construct (CMV43) expressing the gap junction polylpeptide connexin 43. RNA and protein analysis confirmed that the transgene was being expressed. In situ hybridization analysis of embryo sections revealed that transgene expression was targeted to the dorsal neural tube and in subpopulations of neural crest cells. This expression pattern was identical to that seen in transgenic mice harboring other constructs driven by the cytomegaloviral promoter (Kothary, R., Barton, S. C., Franz, T., Norris, M. L., Hettle, S. and Surani, M. A. H. (1991) Mech. Develop. 35, 25-31; Koedood, M., Fitchel, A., Meier, P. and Mitchell, P. (1995) J. Virol. 69, 2194-2207), and corresponded to a subset of the endogenous Cx43 expression domains. Significantly, dye injection studies showed that transgene expression resulted in an increase in gap junctional communication. Though viable and fertile, these transgenic mice exhibited reduced postnatal viability. Examination of embryos at various stages of development revealed developmental perturbations consisting of cranial neural tube defects (NTD) and heart malformations. Interestingly, breeding of the CMV43 transgene into the Cx43 knockout mice extended postnatal viability of mice homozygote for the Cx43 knockout allele, indicating that the CMV43 trangsene may partially complement the Cx43 deletion. Both the Cx43 knockout and the CMV43 transgenic mice exhibit heart defects associated with malformations in the conotruncus, a region of the heart in which neural crest derivatives are known to have important roles during development. Together with our results indicating neural-crest-specific expression of the transgene in our CMV-based constructs, these observations strongly suggest a role for Cx43-mediated gap junctional communication in neural crest development. Furthermore, these observations indicate that the precise level of Cx43 function may be of critical importance in downstream events involving these migratory cell populations. As such, the CMV43 mouse may represent a powerful new model system for examining the role of extracardiac cell populations in cardiac morphogenesis and other developmental processes.     

Knowledge about folic acid and the prevention of neural tube defects in two general practice populations

Knowledge about the link between folic acid supplementation in pregnancy and the prevention of neural tube defects was assessed in women from two contrasting general practices using a questionnaire. The persisting lack of awareness, particularly in the more at-risk group from the inner city area, lends support to the argument in favour of the fortification of flour.     

PAX genes and human neural tube defects: an amino acid substitution in PAX1 in a patient with spina bifida

From studies in the mouse and from the clinical and molecular analysis of patients with type 1 Waardenburg syndrome, particular members of the PAX gene family are suspected factors in the aetiology of human neural tube defects (NTD). To investigate the role of PAX1, PAX3, PAX7, and PAX9, allelic association studies were performed in 79 sporadic and 38 familial NTD patients from the Dutch population. Sequence variation was studied by SSC analysis of the paired domain regions of the PAX1, PAX7, and PAX9 genes and of the complete PAX3 gene. In one patient with spina bifida, a mutation in the PAX1 gene was detected changing the conserved amino acid Gln to His at position 42 in the paired domain of the protein. The mutation was inherited through the maternal line from the unaffected grandmother and was not detected in 300 controls. In the PAX3 gene, variation was detected at several sites including a Thr/Lys amino acid substitution in exon 6. All alleles were present among patients and controls in about the same frequencies. However, an increased frequency of the rare allele of a silent polymorphism in exon 2 was found in NTD patients, but no significant association was observed (p = 0.06). No sequence variation was observed in the paired domain of the PAX7 and PAX9 genes. Our findings so far do not support a major role of the PAX genes examined in the aetiology of NTD. However, the detection of a mutation in PAX1 suggests that, in principle, this gene can act as a risk factor for human NTD.     

Disruption of the csk gene, encoding a negative regulator of Src family tyrosine kinases, leads to neural tube defects and embryonic lethality in mice

All Src family non-receptor tyrosine kinases are negatively regulated by phosphorylation at a carboxy-terminal tyrosine. To analyze the significance of this regulation during development, we have generated mice deficient in Csk, a kinase that phosphorylates this tyrosine, by gene targeting in embryonic stem cells. Homozygous mutant embryos exhibit a complex phenotype that includes defects in the neural tube and die between day 9 and day 10 of gestation. Cells derived from these embryos exhibit an order of magnitude increase in activity of Src and the related Fyn kinase. Phosphorylation at the carboxy-terminal tyrosine of Src was reduced but not eliminated and was accompanied by increased phosphorylation at another key tyrosine residue. These results demonstrate that Src family kinase activity is critically dependent on phosphorylation by Csk and suggest that the regulation of kinase activity may be essential during embryogenesis.     

Rbt (Rabo torcido), a new mouse skeletal mutation involved in anteroposterior patterning of the axial skeleton, maps close to the Ts (tail-short) locus and distal to the Sox9 locus on chromosome 11

Rbt (Rabo torcido) is a new semidominant mouse mutant with a variety of skeletal abnormalities. Heterozygous Rbt mutants display homeotic anteroposterior patterning problems along the axial skeleton that resemble Polycomb group and trithorax gene mutations. In addition, the Rbt mutant displays strong similarities to the phenotype observed in Ts (Tail-short), indicating also a homeotically transformed phenotype in these mice. We have mapped the Rbt locus to an interval of approximately 6 cM on mouse Chromosome (Chr) 11 between microsatellite markers D11Mit128 and D11Mit103. The Ts locus was mapped within a shorter interval of approximately 3 cM between D11Mit128 and D11Mit203. This indicates that Rbt and Ts may be allelic mutations. Sox9, the human homolog of which is responsible for the skeletal malformation syndrome campomelic dysplasia, was mapped proximal to D11Mit128. It is, therefore, unlikely that Ts and Rbt are mouse models for this human skeletal disorder.