Human STEAP3 mutations with no phenotypic red cell changes

To the editor:

During recent years, variants in iron metabolism genes have been identified as molecular etiologic factors of hereditary microcytic anemias, such as iron-refractory iron deficiency anemia caused by TMPRSS6 mutations, hypotransferrinemia resulting from TF mutations, congenital sideroblastic anemia (CSA) due to mutations in SLC25A38, and so on.^1-3  Recently, the STEAP3 gene, also known as TSAP6, was identified as one of the potential candidate for CSA, because it encodes an endosomal ferrireductase required for efficient transferrin-dependent iron uptake in developing erythroblasts.^4-6 Steap3/Tsap6 null mice display moderately severe microcytic anemia due to reduced activity of ferrireductase and abnormal erythroid maturation.^5,7-9  Recently, the first, and thus far only, human STEAP3 mutation causing transfusion-dependent severe hypochromic anemia was reported in 1 family due to a combination of a mutant allele (p.Cys100Ter) and a hypomorphic allele.¹⁰ 

Our study investigated the prevalence of STEAP3 mutations in humans and their physiologic consequences. We used large cohorts of normal individuals and α-thalassemia subjects from China to study the phenotypic effect of STEAP3 mutations on normal and abnormal erythrocyte indices. Surprisingly, we found a relatively high prevalence of potentially harmful recessive alleles. However, although the identified STEAP3 mutations exhibited impaired ferrireductase activity in vitro, they had little or no effect on erythrocyte phenotypes.

Details regarding the study design and methods used are available in the supplemental Data available on the Blood Web site. In the present population-based study, using a molecular screening approach (supplemental Figure 1) and functional analysis, we found multiple mutations in STEAP3 with a surprisingly high prevalence of 5.3% in a total of 2338 Chinese healthy subjects, with slightly higher rates in southern China (6.5%) than in northern China (4.1%, P = .011; Figure 1A). A total of 179 mutant alleles involving 16 different loss-of-function STEAP3 mutations (14 missense mutations, 1 nonsense mutation, and 1 mutation in the 5′ untranslated region [UTR]) were identified in these 2 geographically distinct study populations (Figure 1B), each with their own characteristic spectrum of mutations. The 3 most common mutations, p.Cys261Ter, p.Arg290His, and p.Gly51Asp, accounted for 72.6% of the total (supplemental Table 1). As shown in Figure 1B and supplemental Figure 2, the affected residues within the key domains of the protein are highly conserved evolutionarily and may be critical for enzyme activity.

We systematically performed molecular and functional analysis of all variants on STEAP3 and identified 16 different loss-of-function STEAP3 mutations. As shown in Figure 2A, the various mutant STEAP3 proteins exhibited impaired ferrireductase activity (5 severe and 10 moderately reduced). Notably, the p.Arg59Cys and the p.His316Asn mutations completely abrogated iron reduction, as mutations at these 2 sites are implicated in NAD(P)H-binding motif (Ser⁵⁸ and Arg⁵⁹) or in heme-binding sites (His³¹⁶ and His⁴⁰⁹) of STEAP3.^5,6,11,12  Additionally, 2 other human STEAP3 mutants (p.Ala182Glu and p.Arg462Trp) also severely impaired protein function, demonstrating the functional importance of these 2 residues. Surprisingly, the mutant messenger RNA (mRNA) (p.Cys261Ter), while massively degraded, could still be detected by complementary DNA (cDNA) sequencing (Figure 2B). It is likely that either incomplete nonsense-mediated decay or other mechanisms resulted in extremely low ferrireductase activity (Figure 2A) and protein expression in vitro (Figure 2A,C). Subcellular localization of this mutant green fluorescent protein (GFP)-tagged truncated protein revealed abnormal aggregates in the endosomes and on the plasma membrane (Figure 2C). However, all 14 missense mutations had no effect on protein expression (Figure 2A) and exhibited normal cellular localization (data not shown). Figure 2D demonstrates that the luciferase activity was significantly reduced by >80% for the mutant (c.-25C>T), whereas the mRNA level remained relatively stable, suggesting that the mutation in the STEAP3 5′UTR disrupts posttranscriptional regulation. We further measured STEAP3 mRNA levels in human peripheral blood cells by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). As shown in Figure 2E, considerable variability and a wide range of STEAP3 mRNA expression were observed in the 162 normal individuals, and significantly lower expression levels of STEAP3 were noted in normal samples containing the C allele compared with the T allele at rs6721852 (P < .001), which is the expressed single nucleotide polymorphism (eSNP) in the STEAP3-C2orf76 intergenic region (http://www.gtexportal.org/home/gene/STEAP3).¹³  The expression levels were lower in all 48 subjects with STEAP3 mutations compared with the 162 normal samples (P < .001). These findings confirm a previous report that STEAP3 is expressed as a quantitative trait locus.^10,13,14 

Unexpectedly, we observed that there were no significant alterations in erythrocyte hematologic phenotypes in all subjects carrying those STEAP3 heterozygous mutations (79 in group A and 45 in group D; Figure 1A), including 33 individuals with p.Cys261Ter variants (29 in group A and 4 in group D; Figure 1A), as shown in supplemental Table 2. Hypochromic anemia was not found in subjects with STEAP3 mutations or normal individuals with low-level STEAP3 mRNA expression due to the hypomorphic C allele. In addition to this, we also confirmed no changes in the 2 iron metabolic indices, serum iron and serum ferritin, in the individuals with 8 representative heterozygous mutations in STEAP3 (supplemental Table 3). Interestingly, we identified a family (Figure 2F) in which the child carried compound heterozygous mutations (p.His316Asn, a null mutation from the father and p.Arg290His from the mother). There was decreased expression of STEAP3 expression in both the father and the child (Figure 2E). Importantly, the hematologic parameters and iron status were essentially normal despite the compound heterozygosity (Figure 2F). This finding is in marked contrast to a recent report that described a transfusion-dependent severe hypochromic anemia in a family due to a combination of a mutant allele (p.Cys100Ter) and a hypomorphic allele.¹⁰  Taking together, these findings suggest that only a very large reduction in enzyme activity or complete absence of activity could lead to the manifestation of the hypochromic erythrocyte phenotypes as suggested by Grandchamp et al.¹⁰  Homozygous null STEAP3 mutations such as the most common STEAP3 null variant, p.Cys261Ter, would be sufficient to cause moderately severe congenital hypochromic anemia arising from lack of heme synthesis. However, we were unable to identify such cases in our study. Another possibility is that other pathways of reducing iron in the transferrin-cycle endosome are present in erythroid cells, as evidenced by the previous findings that Steap3^−/− reticulocytes in mice still retain some ferrireductase activity.^5,15  The mRNA for Steap2 and Steap4, which express ferrireductase activity,¹⁶  are indeed consistently and significantly upregulated in bone marrow–derived macrophages and hepatocytes in the absence of Steap3.¹⁷ 

As α-thalassemia is a common inherited hemolytic disorder globally and can cause phenotypic alterations of erythrocytes,^18,19  we examined whether STEAP3 mutations have synergetic effects on hematologic indices and clinical severity in α-thalassemia. Unexpectedly, we were unable to document the adverse effects of heterozygous STEAP3 mutations on hematologic phenotypes of the α-thalassemia carriers (supplemental Figure 3). Furthermore, we identified STEAP3 mutations in 22 subjects among the 310 HbH disease patients studied (supplemental Table 4). Also, no adverse effects were noted as a consequence of STEAP3 mutation on the severity of HbH disease (odds ratio = 1.365, P = .499; supplemental Table 5).

Taken together, our findings imply that heterozygous STEAP3 mutations are relatively common in humans and that their deleterious effect in humans remains to be confirmed. The present study, to our knowledge, represents the first comprehensive mapping of genetic alterations in the human STEAP3 gene and shows that reduced ferrireductase activity resulting from STEAP3 heterozygous mutations in humans fails to have a significant effect on heme synthesis.