Figure 5. Failure of mutant LRP5 in activation of the Norrin/β-catenin pathway. SuperTopFlash (STF) cells cotransfected with LRP5-pRK5 and FZD4 constructs were treated with Norrin and assayed for luciferase reporter activity. Each assay was performed in triplicate at the same time and repeated three times. The results are an average of three measurements. Both of the novel LRP5 mutants failed to induce the luciferase reporter activity in STF cells in response to Norrin (87% reduction for p.A422T, 97% reduction for p.L540P). The additional LRP5 mutation in Patient 1 (p.Q816P) from the unaffected mother had similar luciferase reporter activity compared with the wild type, while the second mutation of Patient 2 (p.T852M), which was not detected in either parent, exhibited a 94.9% reduction. The luciferase intensities of the two combinations (p.A422T and p.Q816P, p.L540P and p.T852M) decreased significantly compared to the wild type, but no significant differences were observed compared with the mere mutation (p.A422T or p.L540P; p>0.05). The mutant p.R570Q (positive control) exhibited a 96% reduction of its wild-type activity, which was similar to the results of a previous report. Asterisks indicate significant differences between the mutant and wild type as judged by the Student two-tailed t test (p<0.05).