Rapid genotyping of the human renin (REN) gene by the
LightCycler® instrument: Identification of unexpected nucleotide
substitutions within the selected hybridization probe area
Affiliations: Department of Medical Biochemistry, Stavanger
University Hospital, Stavanger, Norway | Division of Epidemiology, Norwegian Institute of
Public Health, Oslo, Norway | Craniofacial Research, Musculoskeletal Disorders,
Murdoch Childrens Research Institute, Royal Children's Hospital, Parkville,
Australia | Department of Public Health and Primary Health Care,
University of Bergen, Bergen, Norway
Note: [] Corresponding author: Line Wee, Department of Medical
Biochemistry, Stavanger University Hospital, P.O. Box 8100, N-4068 Stavanger,
Norway. Tel.: +47 51 519528; Fax: + 47 51 519907; E-mail: weln@sus.no
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 (T_{m}). Ninety-two
mother-father-child triads (n=276) from preeclamptic
pregnancies were genotyped for three haplotype-tagging single nucleotide
polymorphisms (htSNPs) in REN. 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.
