Figure 2. Loss of EPHX1 hydrolysis activity due to the p.Thr333Pro and p.Gly430Arg variants.

(A) Schematic representation of the EPHX1 protein, showing its sub-cellular localization and function. Epoxide hydrolases open three membered cyclic ethers, known as epoxides, by the addition of water to yield 1,2-diols. The location of the amino acids affected by the missense variants identified in this study are indicated by stars. (B) Schematic representation of the variants used in functional tests. Residues of the catalytic triad are shown above the protein structure. Variants used in functional assays are depicted below. Variants identified in patients are displayed in red. (C) Model of the 3D structure of EPHX1, based on the quaternary structure of the closely homologous EH enzyme from the Aspergillus niger fungus (Zou et al., 2000). On the left panel, the location of the two variants identified in patients are indicated by a star. On the right panel, the two variants found in patients are depicted in red and the three key residues of the catalytic site in blue. (D) c-SO (cis-stilbene oxide) hydrolysis assay performed in HEK 293 cells transiently expressing Flag-tagged wild-type (WT) and mutated forms of human EPHX1, as indicated. Results are expressed as means ± SEM of three independent biological experiments, each of them being performed in duplicates. # indicates that hydrolysis activity of EPHX1 carrying the p.Thr333Pro and p.Gly430Arg de novo variants was abolished, compared with WT and other variants. (E) Western blot analysis aimed at controlling the expression of WT and mutant forms of EPHX1 in protein extracts used in c-SO hydrolysis assays presented in (D), using antibodies as indicated. Numbers on the left correspond to molecular weight markers (kDa). Western blot images are representative of two independent experiments.

Figure 2—figure supplement 1. Oxylipin profiling in patient 1.

A panel of epoxy fatty acids (EpFAs) dervived from arachidonic acid, linoleic acid, eicosapentaenoic acid, and docosahexaenoic acid was investigated, together with their corresponding diols. Nine measurements were performed in plasma samples from patient 1 collected on different days and 11 sex- and age-matched control women with normal BMI. Ratios between EpFA products and diol substrates were calculated showing increased levels of 7,8 epoxydocosapentaenoic acid (EpDPE) and decreased levels of 7,8 dihydroxydocosapentaenoic acid (7,8 DIHDPE) in patient 1, as compared to controls. Results are expressed as means ± SEM.

Figure 2—figure supplement 2. cis-stilbene oxide (c–SO) hydrolysis activity of co-expressed WT and mutant EPHX1 isoforms.

HEK 293 cells co-expressing WT EPHX1 (hEPHX1^WT) and hEPHX1^Thr333Pro (A), or hEPHX1^Gly430Arg (B), or hEPHX1^Glu404Asp (C) in varying ratios did not reveal a dominant negative effect of pathogenic variants. WT and mutant EPHX1 isoforms were also expressed alone, the total amount of transfected DNA being kept constant using an empty vector. Results are expressed as means ± SEM of four independent biological experiments.