Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Single nucleotide polymorphisms (SNPs) are currently being identified and mapped at a remarkable pace, providing a rich genetic resource with vast potential for disease gene discovery, pharmacogenetics, and understanding the origins of modern humans. High-throughput, cost effective genotyping methods are essential in order to make the most advantageous and immediate use of these SNP data. We have incorporated the use of matrix-assisted laser desorption/ionization timeof-flight mass spectrometry (MALDI-TOF) in our laboratory as a tool for differentiating genotypes based on the mass of the variant DNA sequence, and have utilized this method for production scale SNP genotyping. We have combined a 4 ml PCR amplification reaction using 3 ng of genomic DNA with a secondary enzymatic reaction (mini-sequencing) containing oligonucleotide primers that anneal immediately upstream of the polymorphic site, dideoxynucleotides, and a thermostable polymerase used to extend the PCR product by a single base pair. Mass spectrometry (MS) analysis of mini-sequencing reactions was performed using a MALDI-TOF instrument (Voyager-DE, Perseptive Biosystems, Framingham, MA). We performed both single and multiplex PCR and mini-sequencing reactions, and genotyped seven different variant sites in a random sample of 989 individuals. Genotypes generated with MS methods were compared with genotypes produced using a 5¢ exonuclease fluorescence-based assay (Taqman, Applied Biosystems, Foster City, CA) and a gel-based genotyping protocol. Because multiple polymorphisms can be detected in a single reaction, the MS technique provides a cost-effective and efficient method for high-throughput genotyping. Hum Mutat 17:296-304, 2001.

Stroke is the leading cause of severe disability and the third leading cause of death, accounting for one of every 15 deaths in the USA. We investigated the association of polymorphisms in the soluble epoxide hydrolase gene (EPHX2) with incident ischemic stroke in African-Americans and Whites. Twelve single nucleotide polymorphisms (SNPs) spanning EPHX2 were genotyped in a case-cohort sample of 1336 participants from the Atherosclerosis Risk in Communities (ARIC) study. In each racial group, Cox proportional hazard models were constructed to assess the relationship between incident ischemic stroke and EPHX2 polymorphisms. A score test method was used to investigate the association of common haplotypes of the gene with risk of ischemic stroke. In African-Americans, two common EPHX2 haplotypes with significant and opposing relationships to ischemic stroke risk were identified. In Whites, two common haplotypes showed suggestive indication of an association with ischemic stroke risk but, as in African-Americans, these relationships were in opposite direction. These findings suggest that multiple variants exist within or near the EPHX2 gene, with greatly contrasting relationships to ischemic stroke incidence; some associated with a higher incidence and others with a lower incidence.

Background-Modulation of endogenous epoxide levels by soluble epoxide hydrolase (sEH) in the endothelium represents an important mechanism in the regulation of cardiovascular function. We examined the relationship between a common, functional polymorphism of the human sEH gene and coronary artery calcification (CAC) in young, largely asymptomatic African-American and non-Hispanic white subjects. Methods and Results-Multiple logistic regression and Tobit regression models were used to assess the relationship between the sEH Arg287Gln polymorphism and presence and quantity of CAC. Models adjusting for race (except in race-specific analyses), age, sex, smoking, body mass index, systolic blood pressure, LDL cholesterol, and HDL cholesterol were estimated. Allele and genotype frequency distributions were not significantly different between the 2 ethnic groups (Pϭ0.22; Pϭ0.17, respectively). The Arg287Gln polymorphism of the sEH gene was a significant predictor of CAC status in African-American participants, either alone or after adjusting for other risk factors. African-American subjects with at least 1 copy of the Gln287 allele had a 2-fold greater risk of having CAC compared with those not carrying this allele (95% CI, 1.1 to 2.9; Pϭ0.02). There was no relationship between Arg287Gln polymorphism and the probability of having CAC in white participants (OR, 0.8; 95% CI, 0.5 to 1.3; Pϭ0.49).Inferences from multivariable Tobit regression were similar to those obtained in the logistic regression models, indicating that the Arg287Gln polymorphism was a significant independent predictor of both presence and quantity of CAC in African-American but not white subjects. Conclusions-These data suggest an intriguing and possibly novel role for sEH in the pathogenesis of atherosclerosis, which deserves additional investigation.