Genetic and molecular functional characterization of variants within TNFSF13B, a positional candidate preeclampsia susceptibility gene on 13q

Abstract

Background: Preeclampsia is a serious pregnancy complication, demonstrating a complex pattern of inheritance. The elucidation of genetic liability to preeclampsia remains a major challenge in obstetric medicine. We have adopted a positional cloning approach to identify maternal genetic components, with linkages previously demonstrated to chromosomes 2q, 5q and 13q in an Australian/New Zealand familial cohort. The current study aimed to identify potential functional and structural variants in the positional candidate gene TNFSF13B under the 13q linkage peak and assess their association status with maternal preeclampsia genetic susceptibility.

Methodology/principal findings: The proximal promoter and coding regions of the positional candidate gene TNFSF13B residing within the 13q linkage region was sequenced using 48 proband or founder individuals from Australian/New Zealand families. Ten sequence variants (nine SNPs and one single base insertion) were identified and seven SNPs were successfully genotyped in the total Australian/New Zealand family cohort (74 families/480 individuals). Borderline association to preeclampsia (p = 0.0153) was observed for three rare SNPs (rs16972194, rs16972197 and rs56124946) in strong linkage disequilibrium with each other. Functional evaluation by electrophoretic mobility shift assays showed differential nuclear factor binding to the minor allele of the rs16972194 SNP, residing upstream of the translation start site, making this a putative functional variant. The observed genetic associations were not replicated in a Norwegian case/control cohort (The Nord-Trøndelag Health Study (HUNT2), 851 preeclamptic and 1,440 non-preeclamptic women).

Conclusion/significance: TNFSF13B has previously been suggested to contribute to the normal immunological adaption crucial for a successful pregnancy. Our observations support TNFSF13B as a potential novel preeclampsia susceptibility gene. We discuss a possible role for TNFSF13B in preeclampsia pathogenesis, and propose the rs16972194 variant as a candidate for further functional evaluation.

Figure 1. Schematic representation of the TNFSF13B gene and variants detected in a sub-set of founding or proband preeclamptic women from the Aust/NZ study population.

Solid blocks; untranslated exons, open blocks; translated exons.

Figure 2. Linkage disequilibrium (LD) pattern for the successfully genotyped TNFSF13B SNPs in the Aust/NZ study population.

LD is measured by the squared value of the pair wise correlation (rho) amongst intra-genic genotypes and the strength of correlation is depicted in the colored bar to the right of the LD plot. The intensity of red color increases with the strength of SNP allele correlation from white (0) indicating no correlation (i.e. no LD) to red (1.0) indicating a perfect correlation (i.e. complete LD).

Figure 3. Electrophoretic mobility shift assays for the TNFSF13B SNPs associated with preeclampsia in the Aust/NZ families.

Panel A: Lanes 1 and 6; No nuclear extract, Lanes 2 and 7; nuclear extract only, Lanes 3 and 8, Nuclear extract with unspecific competitor, Lanes 4 and 10; Nuclear extract with specific competitor (unlabelled double stranded oligo for the major allele), Lanes 5 and 9; Nuclear extract with specific competitor (unlabelled double stranded oligo for the minor allele). Panel B: Major shifts without competitor. Lane 1; no nuclear extract, Lane 2; HeLa nuclear extract, Lane 3; T47D nuclear extract. SS; specific shift, US; unspecific shift.