Epistatic interaction between variations in the angiotensin I converting enzyme and angiotensin II type 1 receptor genes in relation to extent of coronary atherosclerosis.

OBJECTIVE: To test the hypothesis that gene-gene interaction of the renin-angiotensin system is associated with an effect on the extent of coronary atherosclerosis. SETTING AND RESULTS: A cohort of 1162 patients with coronary artery disease were genotyped for genetic polymorphisms in the renin-angiotensin system. Patients carrying the D allele of the angiotensin I converting enzyme (ACE) gene had greater coronary extent scores (defined as the number of coronary segments with 5% to 75% stenosis) than those not carrying this allele (p = 0.006 in non-parametric analysis and p = 0.019 in parametric analysis). This association remained significant after adjusting for age, body mass index, hypertension, and diabetes, which were also significantly associated with coronary extent scores. There was a significant interaction (p = 0.033) between genotypes of ACE and angiotensin II type 1 receptor (AGTR1). The association between the ACE gene D allele and increased coronary extent scores was significant (p = 0.008 in non-parametric and p = 0.027 in parametric analysis) in those carrying the +1166 C allele of the AGTR1 gene, but was absent in those not carrying the AGTR1 gene +1166 C allele. CONCLUSION: These findings suggest that variation in the ACE and AGTR1 genes and their interaction may not only contribute to susceptibility of coronary artery disease as previously found but also modify the disease process, thus contributing to interindividual differences in severity of the disease.

Effects of medial and dorsal cortex lesions on spatial memory in lizards.

In mammals and birds, the hippocampus is a major learning and memory center that plays a prominent role in spatial memory, the use of distal cues to guide navigation. The role of reptilian hippocampal homologues, the medial and dorsal cortex, in spatial memory has not been thoroughly investigated. The medial and dorsal cortex of reptiles is known to play a role in learning both tasks that are hippocampally dependent and tasks that are not hippocampally dependent in mammals and birds. In order to examine the specific role of the medial and dorsal cortex in spatial memory, we trained medial cortex, dorsal cortex, and sham lesioned Cnemidophorus inornatus lizards to locate the one heated rock of four identical rocks spaced evenly around the perimeter of a circular, sand filled, arena in a cool room. We used probe trials to examine the strategies used by lizards to locate the goal. Medial cortex lesions and dorsal cortex lesions slowed acquisition and altered the strategies used to locate the goal. However, none of the lizards adopted a spatial strategy to locate the goal suggesting that the dorsal cortex and medial cortex are involved in using non-spatial strategies for navigation.

Anticholinergic effects in frogs in a Morris water maze analog.

We determined the effect of two doses of the centrally acting anticholinergic drug, atropine sulfate (AS), on the performance of female Northern Leopard frogs (Rana pipiens) in a visual cue analog of the Morris water task. Untreated frogs learned the visually cued task, while frogs treated with 150 mg/kg AS were significantly slower than controls in learning to escape warm water by finding a visible platform, and there was a dose-dependent response, with frogs treated with 50 mg/kg AS performing midway between the higher dose and control frogs. These results suggest that the general role of the cholinergic system in learning is important in amphibians, and that this role is evolutionarily conserved across vertebrate species.

The hippocampus is not necessary for a place response but may be necessary for pliancy.

Rats with kainate-colchicine hippocampal lesions (HL) and controls (C) were initially trained in the Morris water maze with procedures that deterred their prepotent thigmotaxic response. Training began with an escape platform that occupied nearly the entire pool. The area to which the rats could escape was made smaller by substituting smaller platforms as training progressed. In contrast to standard procedures, HL rats and C rats showed comparable performance during acquisition and preferentially searched the goal quadrant on probe trials during which the platform was removed. In a follow-up experiment, the platform was moved to a random position along the wall, which required a switch to a thigmotaxic response for most effective escape. HL rats that were thigmotaxic before place training did not switch to a thigmotaxic response as readily as did controls, behavior consistent with the view that hippocampal damage reduces pliancy.

Corneal arcus, case finding and definition of individual clinical risk in heterozygous familial hypercholesterolaemia.

Premature corneal arcus may identify individuals with hyperlipidaemia and increased cardiovascular risk. We have attempted to quantitate relationships through determination of graded prevalence of corneal arcus with age for 81 males and 73 females suffering from heterozygous familial hypercholesterolaemia (HFH) at presentation, and for 280 male and 353 female unselected patients (age range 16-76 years) attending a country general practice. Some degree of arcus affected 50% of HFH patients by age 31-35 years, and 50% of practice patients by age 41-45 years. Complete full ring arcus affected 50% of the familial hypercholesterolaemia (FH) group by age 50 years, with only 5% similarly affected in the non-FH group. Arcus grade with age was advanced by some 5 years in males versus females. Premature arcus potentially alerting to HFH can be broadly defined for males and females combined, as heavy full ring by age 50 years, or any degree of arcus by age 30-35 years. Arcus grade was not related to the presence of coronary disease. Accelerated development of corneal arcus with age is an indicator of HFH, but premature arcus is not an additional marker of premature coronary disease for individual cases of HFH.

Identification of a common low density lipoprotein receptor mutation (R329X) in the south of England: complete linkage disequilibrium with an allele of microsatellite D19S394.

Familial hypercholesterolaemia is commonly caused by mutations in the low density lipoprotein receptor (LDLR) gene and more than 300 different mutations have been described worldwide. Some mutations occur at relatively higher frequency in certain populations, reflecting both chance and demography, most evident in founder populations. As part of a study of kindreds of 78 probands from Southampton and south west Hampshire, we identified the same mutation (R329X) in 9/78 (11.5%) probands. In all (100%) of these probands, length allele 259nt of the 17 allele microsatellite D19S394, sited approximately 250 kilobases telomeric and 5' to the LDLR gene, was observed, although in the general population this allele has a prevalence of only 16.1%. A simple diagnostic assay for R329X was constructed in conjunction with more detailed family studies. Both the R329X and linked D19S394 allele also cosegregated with the FH phenotype within each kindred. Although R329X involves a CpG site, it is highly likely that the families are identical by descent for R329X, we surmise with a common ancestor within 500 to 1000 years, although the mutation is not restricted to this geographical area. This relationship illustrates that the linkage disequilibrium of gene LDLR with marker D19S394 will enable rapid recognition using D19S394 genotype of possible common FH mutation(s) within a cohort of FH patients from a particular locality or ethnic group.

Development of a microsatellite-based approach to co-segregation analysis of familial hypercholesterolaemic kindreds.

Co-segregation studies based on a selection of intragenic restriction fragment length polymorphisms of the low density lipoprotein receptor (LDLR) gene have been used extensively both for research and diagnostic studies of familial hypercholesterolaemia (FH) families, because direct mutation screening remains complex. Here we describe the development and application of a more efficient approach to co-segregation studies based on highly informative dinucleotide and tetranucleotide repeats flanking the LDLR gene. A series of microsatellites (D19S391, D19S394, D19S221 and D19S179) were selected for study on the basis of linkage analysis in the CEPH families using intragenic polymorphisms for a TA repeat (exon 18) in the LDLR gene, and earlier data for a Pvu II polymorphism (intron 15). A physical map of the region of chromosome 19 also contributed to this selection. One marker in particular, D19S394, sited 150 kilobases telomeric to the gene, was extremely useful, displaying 90% heterozygosity, robust PCR of tetranucleotide repeats without stutter bands, and no recombination with the LDLR gene (theta = 0, LOD 68). Use of this marker in the families of twenty-three FH probands from Hampshire demonstrated co-segregation of the hyperlipidaemia phenotype with the LDLR gene region, except in one family with defective apolipoprotein B-100, and a family turning out to display familial combined hyperlipidaemia. This approach should facilitate the search for any families where FH does not co-segregate with the LDLR gene, and will enhance the repertoire of molecular diagnostic tools available for FH.

Anticholinergic effects on acquisition of place learning in the Morris water task: spatial mapping deficit or inability to inhibit nonplace strategies?

The role of central cholinergic blockage in spatial learning was examined by testing atropine sulfate-treated (50 mg/kg) rats and saline-injected controls in the Morris water task using training procedures designed to promote the use of a spatial search strategy. First, constraints used in early trials deterred thigmotaxis. Second, an originally oversized hidden platform that nearly occupied the entire pool was effectively "shrunk" into the southwest quadrant of the pool by substituting smaller platforms over trials, a procedure intended to focus attention on the hidden platform in relation to extramaze cues. Task acquisition did not differ between groups, and on the probe trial both groups increased distance and latency and swam preferentially in the previously correct quadrant. Impairments caused by atropine sulfate may be the result of deficits in ability to inhibit nonefficient escape strategies.

Postural asymmetries and language lateralization in humans (Homo sapiens).

Is hemispheric specialization for speech more closely related to left hemisphere specialization for manual skill and sequencing, as is usually supposed, or to control of asymmetries in whole body posture, as recent findings of right-handedness in nonhuman primates suggest? This question can be evaluated in the 10% of humans who have mixed handedness and footedness. Footedness entails postural asymmetry, and persons with mixed limb preferences often prefer the hand ipsilateral to the preferred foot in asymmetrical actions for which whole body postural adjustments are obligatory (e.g., throwing). The dichotic listening test , and indicator of language laterality, was administered to 4 groups of 48 persons with the 4 possible combinations of hand and foot preference. As in 2 past studies, language lateralization was somewhat more strongly related to postural asymmetries than to asymmetries in manual skill and sequencing.

Utilities for high throughput use of the single strand conformational polymorphism method: screening of 791 patients with familial hypercholesterolaemia for mutations in exon 3 of the low density lipoprotein receptor gene.

We have modified several aspects of the single strand conformational polymorphism (SSCP) method to increase the speed with which the technique can be used for mutation detection. The methods attain high resolution of small mobility differences using long (30 cm) gels and use a modified polymerase reaction to maximise detection sensitivity using a minimised quantity of 32P. By using custom cut "sharktooth" combs (4.5 mm between teeth) as the slot formers, commercially available multichannel pipettes (9 mm tip to tip) can be used to load eight or 12 samples at a time from standard microtitre plates. PCR products that have been prepared and radiolabelled using simplified protocols are loaded on to the gel, and after a precalculated time of electrophoresis another set of samples can be loaded, either with combs moved across 2.25 mm or onto the same gel tracks. The run conditions are calculated so that there is no overlap between the bands produced by the two loadings, thus doubling the amount of information that can be gained from one gel. A computer program has been developed to solve equations to determine suitable timings for repetitive loadings. Finally, a modification of the gel pouring system is described so that two gels can be poured between three standard glass plates, with both gels run simultaneously. Of the order of 1000 PCR reactions can be prepared and analysed in 24 man hours using five 40 cm x 30 cm gel tanks. The application of these techniques is described to detect SSCPs in exon 3 of the low density lipoprotein receptor (LDLR) gene in 791 patients with familial hypercholesterolaemia (FH). Eight different SSCP patterns were seen, one of which was caused by the previously described E80K mutation, which was present in 11 patients (1.4%). In total, 32 patients (4%) were identified with exon 3 mutations.