Erythropoiesis-hepcidin-iron axis in patients with X-linked sideroblastic anaemia: An explorative biomarker study.

X-linked sideroblastic anaemia (XLSA) arises from pathogenic variants in the 5-aminolevulinate synthase 2 (ALAS2) gene, encoding ALAS2. ALAS2 plays an important role in erythropoiesis as the first and rate-limiting step of heme synthesis in the mitochondria of erythroid precursor cells.1 Lower ALAS2-activity results in insufficient heme synthesis and microcytic anaemia.2 Consequently, iron accumulates in the mitochondria of erythroblasts and causes the formation of ring sideroblasts in the bone marrow. In addition, XLSA is associated with systemic iron overload due to ineffective erythropoiesis.1, 2 However, the exact underlying pathophysiology of this iron loading in XLSA is not fully understood. It is known that ineffective erythropoiesis in other iron-loading anaemias inhibits hepatic synthesis of hepcidin mediated by the erythroid regulator erythroferrone (ERFE), which is synthesized by erythroblasts upon erythropoietin (EPO) stimulation.3-6 Lower levels of hepcidin result in increased circulating iron levels and once these iron levels rise well near or above the maximum iron binding capacity of circulating transferrin, subsequent secondary parenchymal iron overload occurs. The toxic effect of this iron overload on erythropoiesis further worsens ineffective erythropoiesis, maintaining a vicious cycle between ineffective erythropoiesis and iron overload.7 In our previous ERFE-assay validation study, we showed increased ERFE levels in XLSA patients compared to controls.8 In the current study, we have enriched the ALAS2-patient population from our iron expertise centre and included several iron-related parameters to describe the role of the erythropoiesis–hepcidin–iron axis in the pathogenesis of XLSA. Understanding the relationship between this axis and iron loading will help to prevent iron-related toxicity. We performed a cross-sectional study on the relationship between biomarkers of iron metabolism and erythropoiesis in a small group of XLSA patients. All patients were treated in the Radboud university medical centre, Nijmegen, and signed for inclusion in the Iron Biobank (IB) of the Radboud Biobank between December 2016 and March 2020. At the time of inclusion in the IB, blood was collected for both direct laboratory measurements and storage of serum aliquots at −80°C until use (Supporting Information). Data were analysed using SPSS Statistics and reported as median and interquartile range (IQR) (Supporting Information). In total, 12 male patients were included. All patients were hemizygous for the c.1355G>A (p.Arg452His) ALAS2-variant (NM_000032.5), the most common ALAS2-variant in the Netherlands.2 Age at sample collection ranged from 7 to 72 years. Patients received different treatments, varying from no therapy to combined therapy with pyridoxine and phlebotomies (Table 1). All patients showed microcytic anaemia, but the degree of anaemia varied (Hb 12.6 g/dL [IQR 11.7–12.8], MCV 74 fL [IQR 70–75]; Table 1). Iron parameters differed among patients, with some patients showing levels outside the reference interval: median ferritin was 154 μg/L (IQR 101–317), transferrin saturation (TSAT) 46% (IQR 41–55), and hepcidin/ferritin-ratio was 14.9 pmol/μg (IQR 9.1–27.6). All patients showed EPO and sTfR levels within the reference interval, although levels varied between close to the lower limit and close to the upper limit of the interval. ERFE levels in XLSA patients ranged from 1.38 to 4.79 ng/mL indicating interindividual differences in severity of ineffective erythropoiesis (reference: healthy controls: median 1.44 ng/mL [IQR < 1.16–2.01]8). At presentation and before the patients were diagnosed with XLSA, a bone marrow biopsy was performed in four patients. In none of these patients erythroid hyperplasia was observed, in only one of these biopsies ring sideroblasts were found. Differences in erythropoietic biomarker levels between patients may be explained by concomitant (medical) conditions or the effect of treatment, as almost all of our XLSA patients already received treatment. Indeed, patient 6 received frequently phlebotomies for several years and showed the lowest ERFE level, associated with a low sTfR level (Table 1). It is conceivable that effective iron depletion by phlebotomies in this patient has resulted in more efficient erythropoiesis. In contrast, patient 5, who also was homozygous for the C282Y mutation in the HFE-gene, expressed the highest levels of both ERFE (4.79 ng/mL) and sTfR (1.62 mg/L), indicating relatively severe ineffective erythropoiesis. However, EPO level was relatively low in relation to these parameters. In addition, this specific patient had a severe parenchymal iron overload and a remarkable low setpoint of hepcidin relative to ferritin (TSAT 88%, ferritin 382 μg/L, hepcidin/ferritin ratio 1.3 pmol/μg) compared with other patients, which can be ascribed to HFE-hereditary haemochromatosis (HFE-HH) as coexisting disorder of hepcidin regulation. These combined findings of increased sTfR, ERFE and low hepcidin setpoint for body iron stores levels in this patient may be attributed to ineffective erythropoiesis associated with XLSA aggravated by the toxicity of iron overload on erythropoiesis. Although dysregulation of the EPO-ERFE-hepcidin axis has been shown for different iron-loading anaemias,6, 10 our findings for the various iron biomarkers suggest ineffective erythropoiesis in well-treated XLSA patients is less obvious. In line with our previous ERFE-assay study, our XLSA-population showed higher ERFE levels compared to the healthy control population (median 4.08 ng/mL vs. 1.44 ng/mL),8 but we did not observe higher levels of sTfR and EPO levels above the upper limit of the reference interval. Moreover, we did not find a clear correlation between different erythropoietic and iron biomarkers (data not shown due to a small number of patients). This could be explained by the fact that our treated group of XLSA patients is relatively homogeneous, with absent, or at most relatively mild, ineffective erythropoiesis. To our knowledge, this is the first study assessing the erythropoiesis–hepcidin–iron axis in XLSA patients. This study reveals an enhanced ERFE response with persistent microcytic anaemia in well-treated XLSA patients in a tertiary centre with varying levels of circulating and stored iron (defined by TSAT and ferritin, respectively), but without abnormality of other biomarkers involved in the erythropoiesis–hepcidin–iron axis (sTfR, EPO and hepcidin). Studies with larger sample sizes are needed to further explore the role and interplay of different biomarkers. Moreover, additional research is warranted to identify other erythroid factors involved in the erythropoiesis–hepcidin–iron axis. This knowledge will contribute to better identification and treatment of XLSA patients at risk for iron toxicity. Dorine W. Swinkels designed the research study. Rian Roelofs performed and interpreted measurements. Lieke E. Nijssen, Vera Hoving and Rian Roelofs collected and analysed the data. Lieke E. Nijssen and Vera Hoving wrote the original draft. Vera Hoving wrote the paper. Saskia E. M. Schols, Albertine E. Donker and Dorine W. Swinkels reviewed and edited the paper. Saskia E. M. Schols, Dorine W. Swinkels and Albertine E. Donker supervised the project. All authors have read and agreed to the published version of the manuscript. We would like to thank Siem Klaver and the clinicians of the Radboudumc expertise centre for iron disorders: Alexander Rennings, Paul Brons, Britta Laros and Marlijn Hoeks for their contribution to the Iron Biobank and Laura Diepeveen for sharing data and samples of the healthy controls and patients. We are grateful to Coby Laarakkers and Edwin van Kaauwen for the measurement of hepcidin and ALAS2-variants, respectively. The authors declare that they have no conflict of interest. This study was conducted in accordance with the Declaration of Helsinki, and approved by the Medical Research Ethics Committee Oost-Nederland (protocol number 2016-2362). Written informed consent was obtained from all individual patients for inclusion in the Iron Biobank and future use. Appendix S1. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.