Figure 2.

Phosphatase activity assay of the identified SYNJ1 missense variants. The effect on enzymatic activity of SYNJ1 was tested for mutants resulting from missense variants. Wild-type or mutant FLAG-tagged SYNJ1 (NM_203446.2) was expressed in Expi293F cells, purified and tested for enzymatic activity against short chain phosphoinositides using a malachite-based assay (Maehama et al., 2000). The dephosphorylation of domain specific substrates was investigated: PI(4)P for the Sac1 domain, and PI(4,5)P₂ and PI(3,4,5)P₃ for the 5’PP domain. Because dephosphorylation of PI(4,5)P₂ by the 5’PP domain results in PI4P, which can then be dephosphorylated by the Sac1 domain, the phosphate released when using PI(4,5)P₂ as substrate reflects the action of both phosphatase domains. When PI(3,4,5)P₃ was used as a substrate, selective action of the 5’PP domain could be assessed. Graphics depict mean values of released inorganic free phosphate from three independent experiments (bars reflect standard error of mean). A clear reduction, but not a complete loss, of phosphate activity towards all tested substrates was found for the p.Tyr888Cys substitution identified in Family A. Compared to wild-type expressing cells, no differences in enzymatic activity was found for cells expressing the p.Met1020Ile/p.Tyr1057Ser substitutions. Unpaired t-test was used to analyse the difference between wild-type and mutants (***P = 0.0002–0.0003, ****P < 0.0001). ns = non-significant.