Molecular characterisation of the gene, LGALS13, and its putative involvement in pre-eclampsia
Thesis (MSc (Genetics))--University of Stellenbosch, 2009.
Pre-eclampsia is one of the most common hypertensive disorders of pregnancy in South Africa. Presently, the only cure for pre-eclampsia is delivery, which brings with it, additional complications. As an alternative, clinical management of this disorder relies on timely diagnosis. The predictive biomarker, Placental Protein 13 (PP13), is currently used for the early diagnosis of pre-eclampsia, in an ELISA-based diagnostic kit, developed by Diagnostic Technologies Limited (DTL)1. A decrease in serum PP13 levels has been reported during the first trimester of pregnancy in women who later develop pre-eclampsia. The function of PP13 has not been fully elucidated and it is also not known whether the reduction in PP13 levels is a cause or an effect of the disease. The use of PP13 as a predictive biomarker for pre-eclampsia therefore warrants a comprehensive study of this peptide and the encoding gene, LGALS13. The aim of this study was firstly to characterise LGALS13 using a range of in silico tools. PP13 was found to be most homologous to the predicted protein product of a neighbouring “putative” gene, LOC148003. A gene conversion event between these two genes most likely underlies the so-called “hotspot mutation” in LGALS13. Data also demonstrates that the DelT mutation disrupts functionally and structurally important features of the gene and peptide sequences. Through the analysis of the putative promoter region of LGALS13, the presence of a Stimulatory protein-1 (Sp1) binding sequence element was predicted, which has implications for regulation of LGALS13. Secondly, the study aimed to establish a study cohort for the investigation of the effect that the LGALS13 genotype has on the expression of its mRNA and protein products. Serum, plasma and whole blood samples were collected and prepared from 316 pregnant women. Placental tissue samples were obtained from a selected group of these subjects for RNA extraction. Once the sampling on the two remaining targeted deliveries has occurred, the collection of samples will be batched and sent to DTL in Israel, for PP13 measurement. DNA was extracted from the whole blood samples obtained, and all study participants were genotyped for seven sequence variants within the LGALS13 gene using (i) Multiphor Single Stranded Conformational Polymorphism and Heteroduplex (SSCP/HD) analysis, (ii) restriction enzyme analysis and (iii) DNA sequencing. The genotype data sets will be compared with PP13 levels when they become available, and also with clinical parameters, once the deliveries have all occurred and the database is complete. This study demonstrated the power of an in silico approach to direct the focus of future experimental work. The newly established study cohort will be used for prospective studies aiming at a better understanding of the role which LGALS13 and PP13 play in the early prediction of preeclampsia.