A sequence variation in the promoter of the placental alkaline phosphatase gene (ALPP) is associated with allele-specific expression in human term placenta

Allele-specific mRNA expression of ALPP gene in term placenta from 12 subjects with heterozygous C/G genotype assayed by RFLP and GeneScan analysis as described in Materials and methods. An example of GeneScan analysis is given Fig. 1A (sample 9). In each case, the relative expression of C/G allele was calculated from GeneScan peak sizes with G + C alleles (Fig. 1B) or G allele (Fig. 1C) as 100% to adjust the amount between samples. Bars means ± SEM of the data. **p < 0.01, paired t-test.

2a- Schematic representation of human ALPP proximal promoter. The position of SNPs potentially affecting transcription factors biding sites within this part of the gene is indicated. The arrow depicts the transcription initiation site (+1). 2b- Expression of Luc reporter gene under the control of the ALPP proximal promoter with A or G at rs2014683. Plasmid constructs with the Luc gene under the control of the ALPP(G) (−899/+162) or ALPP(A) (−899/+162) promoter were tested for Luc activity by transfection into HTR-8/SVneo cells. The relative Luc activity was calculated with ALPP(A) promoter used as 100%. These data were derived from triplicate assays, representative of 5 independent experiments. Bars means ± SEM of the data. *p < 0.05, paired t-test.

rs2014683G>A dependent differential DNA binding of the ALPP promoter in HTR-8/Svneo cells. Nuclear extracts were prepared and incubated with DIG-labeled probes as described in Materials and methods. A: The figure is representative of five independent experiments. B: Densitometric analysis of EMSA experiments. Results are means ± SEM of five separate experiments. **p < 0.01.

In vitro alkaline phosphatase activity of Pl¹, Pl² and the rare D variant of ALPP gene after transfection in COS-7 cells. Alkaline phosphatase activity was normalized with β-galactosidase activity as described in Materials and methods. COS-7 cells only transfected with pcDNA3.1/His/LacZ were used as negative control of alkaline phosphatase activity. In each case, experiments were repeated at least four times independently. Bars means ± SEM of the data. (a) Pl² or D vs Pl¹, (b) Pl² vs D. **p < 0.01; ns: non-significant; one way ANOVA test.

Hypothetic model for counterbalancing of mRNA expression and enzymatic activity in the two common variants Pl¹ and Pl² and the rare D variant of ALPP gene. Arrows indicate relative increase or decrease of expression or activity, depending of the alleles harbored at the rs204683 locus in the promoter, the c.692G>C locus distinguishing Pl¹ and Pl² variants, and the c.1352G>A locus distinguishing D variant from others.