Effects of mutations on STEAP3 function. (A) Transient transfection of HeLa cells with GFP-tagged STEAP3 expression constructs of wild-type (WT) and mutant cDNA to determine relative ferrireductase activity by iron reductase assay. Five mutants completely abrogated this activity, whereas 10 expressed moderately decreased activity compared with the wild-type STEAP3. Similarly transfection efficiency and stable STEAP3 protein expression were noted for all mutant STEAP3s except for 2 nonsense mutants (p.Cys261Ter and p.Cys100Ter) by flow cytometry analysis. (B) Sequence analysis of genomic DNA and cDNA from a case with the STEAP3 nonsense mutation (c.783C>A, p.Cys261Ter) indicated that the mutant allele was massively degraded but still could be detected at the cDNA level. (C) Subcellular localization of GFP-tagged wild-type STEAP3 and the 2 mutants in COS7 cells in images taken at ×4 and ×180 magnification. Confocal microscopy confirmed the endosomal and the plasma membrane localization of the wild-type protein, whereas the p.Cys261Ter mutant formed unusual aggregates, and the p.Cys100Ter mutant localized in both the nucleus and the cytoplasm of COS7 cells. Only a small number of cells exhibited these changes (bottom center/right figures), and most of cultured cells were not detected due to lack of fluorescence. (D) The Renilla luciferase gene was used as a reporter driven by the STEAP3 5′UTR with firefly luciferase as the endogenous control. The −25C>T mutant decreased STEAP3 expression at the protein level, whereas the mRNA levels remained relatively stable compared with the wild type. (E) Quantitative RT-PCR analysis of the STEAP3 mRNA from peripheral blood cells. A cohort of 210 samples randomly selected from southern China was used for mRNA quantification, which included the healthy group (n = 162), consisting of 3 su-groups according to the eSNP allele at rs6721852 (n = 79 in T/T, n = 69 in T/C, and n = 14 in C/C) and the STEAP3 mutant group (n = 48). Three members from a family with compound heterozygous mutations (p.His316Asn and p.Arg290His) are shown in the last column. (F) Study of a family with compound heterozygous STEAP3 mutations. The hematologic parameters (Hb, mean cell volume [MCV], and mean cell hemoglobin [MCH]), serum iron, and serum ferritin are indicated in the table. The p.His316Asn is a severe mutation, whereas the p.Arg290His is a moderate mutation. Data in A, D, and E represent mean ± standard deviation of 3 independent experiments.