Abstract
Preeclampsia is a serious disorder affecting nearly 3% of all in the Western world. It is associated with hypertension and proteinuria, and several lines of evidence suggest that the renin-angiotensin system (RAS) may be involved in the development of hypertension at different stages of a preeclamptic pregnancy. In this study, we developed rapid genotyping assays on the LightCycler® instrument to allow the detection of genetic variants in the renin gene (REN) that may predispose to preeclampsia. The method is based on real-time PCR and allele-specific hybridization probes, followed by fluorescent melting curve analysis to expose a change in melting temperature (Tm). Ninety-two mother-father-child triads (n=276) from preeclamptic pregnancies were genotyped for three haplotype-tagging single nucleotide polymorphisms (htSNPs) inREN. All three htSNPs (rs5705, rs1464816 and rs3795575) were successfully genotyped. Furthermore, two unexpected nucleotide substitutions (rs11571084 and rs61757041) were identified within the selected hybridization probe area of rs1464816 and rs3795575 due to aberrant melting peaks. In conclusion, genotyping on the LightCycler® instrument proved to be rapid and highly reproducible. The ability to uncover additional nucleotide substitutions is particularly important in that it allows the identification of potentially etiological variants that might otherwise be overlooked by other genotyping methods.
Highlights
Preeclampsia is a heritable complication affecting nearly 3% of all pregnancies in Western populations [1]
Wee et al / Identification of unexpected nucleotide substitutions within the selected hybridization probe area analysis of the gene coding for renin gene (REN) might contribute to an improved understanding of the underlying causes of preeclampsia
When genotypes from the LightCycler analysis were compared with the results of DNA sequencing in a random set of 30 patient samples, identical genotypes were obtained for each haplotype-tagging single nucleotide polymorphisms (htSNPs)
Summary
Preeclampsia is a heritable complication affecting nearly 3% of all pregnancies in Western populations [1]. Duced placental perfusion, oxidative stress, activation of thrombosis and the renin-angiotensin system (RAS) are probably involved at different stages of a preeclamptic pregnancy [2,3,4,5]. Studies have shown that genetic factors contribute to the development of preeclampsia, but the exact mechanism is still unknown despite extensive studies [6,7,8]. The presence of renin (REN) in the placenta indicates a local synthesis and suggests that REN may be involved in the regulation of maternal blood pressure [9,10]. Wee et al / Identification of unexpected nucleotide substitutions within the selected hybridization probe area analysis of the gene coding for REN might contribute to an improved understanding of the underlying causes of preeclampsia
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