Gene-gene interaction in folate-related genes and risk of neural tube defects in a UK population.

OBJECTIVE: To investigate the contribution of polymorphic variation in genes involved in the folate-dependent homocysteine pathway in the aetiology of neural tube defects (NTD). DESIGN: Case-control association study. SUBJECTS: A total of 530 individuals from families affected by NTD, 645 maternal controls, and 602 healthy newborn controls from the northern UK. MAIN OUTCOME MEASURES: Seven polymorphisms in six genes coding for proteins in the folate-dependent homocysteine pathway (MTHFR 677C-->T, MTHFR 1298A-->C, MTRR 66A-->G, SHMT 1420C-->T, CbetaS 844ins68, GCPII 1561C-->T, RFC-1 80G-->A). The impact of each polymorphism and the effect of gene-gene interactions (epistasis) upon risk of NTD were assessed using logistic regression analysis. RESULTS: The MTHFR 677C-->T polymorphism was shown to represent a risk factor in NTD cases (CC v CT+TT odds ratio (OR) 2.03 [95% confidence interval (CI) 1.09, 3.79] p = 0.025) and the MTRR 66A-->G polymorphism was shown to exert a protective effect in NTD cases (AA v AG+GG OR 0.31 [95% CI 0.10, 0.94] p = 0.04). When statistical tests for interaction were conducted, three genotype combinations in cases (MTRR/GCPII; MTHFR 677/CbetaS; MTHFR 677/MTRR) and one combination in case mothers (CbetaS/RFC-1) were shown to elevate NTD risk. Maternal-fetal interaction was also detected when offspring carried the MTHFR 677C-->T variant and mothers carried the MTRR 66A-->G variant, resulting in a significantly elevated risk of NTD. CONCLUSION: Both independent genetic effects and gene-gene interaction were observed in relation to NTD risk. Multi-locus rather than single locus analysis might be preferable to gain an accurate assessment of genetic susceptibility to NTD.

A monoclonal antibody that enables specific immunohistological detection of prion protein in bovine spongiform encephalopathy cases.

The specificity of a monoclonal antibody (mAB) raised against recombinant bovine prion protein (PrP) for the immunohistological detection of PrP accumulation in the medulla oblongata of bovine spongiform encephalopathy (BSE) and ovine scrapie cases was investigated. mAB KG9 showed a diffuse low intensity reaction with the cytoplasm of neurones in normal cattle and sheep sections. In BSE sections the mAB detected widespread granular deposits of PrP associated with neurones and the neuropil. Although scrapie sections showed similar levels of granular deposits with another antibody to PrP these were not detected by KG9 which did however detect diffuse staining in neuronal cytoplasm. Possible explanations for the specificity of binding of KG9 are discussed.

Investigation of the susceptibility of equine autonomic neuronal cell lines, clonally derived from the same paravertebral ganglion, to toxic plasma from equine dysautonomia (grass sickness) cases.

In the autonomic nervous system (ANS) of equine grass sickness (GS) cases, some neurones show abnormal changes while neighbouring neurones are unaffected. To test whether noradrenergic neurones showed variable susceptibility to the GS toxin in culture, clonally-derived populations isolated from the same fetal thoracic sympathetic chain ganglion were challenged with plasma from GS cases previously shown to induce ANS damage when injected into normal horses. During the early stages of exposure to toxic plasma, cells within a clonal population showed variable susceptibility ranging from no obvious effect to characteristic patterns of pathology. However, after 3 days of exposure to toxic plasma all cells were killed. Dose response analysis on selected clonal populations showed no significant difference in TD(50) values.

Establishment and characterization of equine autonomic ganglion cell lines to enable direct testing of candidate toxins involved in equine dysautonomia (grass sickness).

To enable direct testing of a range of potential toxins or pathogens that might be involved in grass sickness, equine thoracic sympathetic chain ganglion cell lines were established from primary cell cultures by retroviral-mediated transduction of the temperature-sensitive mutant of the establishment oncogene encoding SV40 large T antigen. Morphological and behavioral features, temperature dependence, and immunocytochemical characteristics of the cell lines were investigated. The majority of cells were noradrenergic neurons in which dopamine-beta-hydroxylase, the enzyme that catalyzes norepinephrine synthesis, and neuropeptide Y coexisted. Cells treated with plasma from grass sickness cases that had previously been shown to induce autonomic nervous system damage when injected into normal horses showed significantly decreased mitochondrial function after 1 day. After 3 days exposure most cells showed severe degeneration in contrast to those treated with normal plasma. Liver and lung cell lines were also susceptible to plasma, suggesting that the toxin is not specifically neurotoxic.

Electrophysiological actions of phenytoin on N-methyl-D-aspartate receptor-mediated responses in rat hippocampus in vitro.

1. The effects of the anticonvulsant, phenytoin, have been examined on N-methyl-D-aspartate (NMDA) receptor-mediated population spikes in the CA1 region of the rat hippocampus in vitro. 2. The 'conventional' (AMPA receptor-mediated) CA1 population spike, evoked by electrical stimulation of the Schaffer collateral/commissural pathway, was abolished by 5 min treatment with 5 x 10(-6) M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), after which superfusion with a nominally Mg(2+)-free Krebs solution (containing 5 x 10(-6) M CNQX) led to the appearance of an epileptiform population spike which was fully developed by 30-40 min. 3. The epileptiform population spike was abolished by the non-competitive NMDA antagonist, dizocilpine (1 x 10(-6) M, 20-30 min) and inhibited by the competitive NMDA receptor antagonist, D-CPP (IC50 for reducing the amplitude of the first spike in the train = 8.3 x 10(-7) M), demonstrating that the response was mediated by activation of NMDA receptors and validating its use as an assay for antagonists acting at the NMDA receptor/channel complex. 4. Phenytoin (0.1, 0.3 and 1 x 10(-4) M applied cumulatively for 30 min at each concentration) failed to inhibit the NMDA receptor-mediated epileptiform population response (n = 7 slices). 5. Phenytoin (3 x 10-6 M to 1 x 10-4M) attenuated the effects of the sodium channel activator,veratridine (2 x 10-6 M), on the CAl population spike amplitude (recorded in normal Krebs solution),indicating that the previously observed lack of effect of phenytoin on the NMDA receptor-mediated response was not due to impaired access of phenytoin to the biophase.6. These data support the conclusion that antagonism of NMDA receptor-mediated events is not a pharmacological property of phenytoin and that such an action is therefore unlikely to contribute to the anticonvulsant activity of this drug.