X‐Linked Sideroblastic Anemia Caused by ALAS2 Intron 1 Mutation Successfully Treated by Allogenic Hematopoietic Stem Cell Transplant

To the Editor: Congenital sideroblastic anemia (CSA) is a rare and heterogeneous group of genetic disorders, with the most prevalent form being X-linked sideroblastic anemia (XLSA). XLSA is typically caused by mutations in the δ-aminolevulinic acid synthase 2 (ALAS2) gene on Xp11.21 [1, 2]. Here, we report a child with clinical features consistent with CSA in whom we identified a rare pathogenic variant at the ALAS2 intron 1 enhancer GATA (int-1-GATA) site by whole-genome sequencing (WGS). To the best of our knowledge, this is the first reported case of XLSA caused by an ALAS2 intron 1 variant and successfully treated with allogeneic hematopoietic stem cell transplantation (allo-HSCT). We report the case of a 13-year-old boy who presented with severe anemia and growth retardation, weighing 32 kg (−1.81 SDS) with a height of 133 cm (−3.44 SDS). Blood tests and bone marrow smears were performed at our hospital (Figure 1A and 1B). Detailed examination reports are shown in Table 1. These results supported a diagnosis of CSA. Notably, whole-exome sequencing (WES) did not identify any pathogenic variants at first, while the WGS identified a rare variant at the ALAS2 int-1-GATA site, establishing the diagnosis of XLSA (Table 1; Figure 1C). This finding was further confirmed by DNA sequencing of his mother (Figure 1D). Current treatment for XLSA is largely limited to pyridoxine supplementation and blood transfusions, which benefit only a subset of patients. At 6 years old, he began receiving vitamin B6 (six tablets daily) but remained transfusion-dependent. He received red blood cell transfusions every 2–4 weeks at a local hospital. Given the child's poor response to vitamin B6 and ongoing transfusion dependence, allo-HSCT was considered the only curative option. In the absence of a matched sibling donor, an HLA-matched unrelated donor was selected. A myeloablative conditioning regimen was administered (busulfan, 0.95 mg/kg for 3 days; fludarabine, 30 mg/ m2 for 4 days; thiotepa,160 mg for 1 day; antithymocyte globulin 9.3 mg/kg for 3 days and cyclophosphamide, 30 mg/kg for 2 days). Peripheral blood stem cells (CD34+ count: 5.14 × 106/kg; MNC: 11.63 × 108/kg) were transfused. Post-transplant, the patient received mycophenolate mofetil, methotrexate, and cyclosporine for graft-versus-host disease (GVHD) prophylaxis. Granulocyte colony-stimulating factor and recombinant thrombopoietin were administered to promote hematopoietic recovery. Neutrophil engraftment occurred on day 17 post-transplantation, and platelet engraftment on day 18. The patient's peripheral blood chimerism showed 99.86% of whole blood, consistent with complete chimerism. At 26 months post-transplant, the patient's hematopoietic function had fully recovered with no signs of chronic GVHD. CSA is a rare and heterogeneous group of disorders, with the most prevalent form being XLSA [3]. The majority of XLSA cases are caused by mutations in the ALAS2 gene on Xp11.21, which encodes 5-aminolevulinate synthase [4-6]. ALAS2 is the first enzyme in the heme biosynthesis pathway in erythroid precursors. To date, more than 80 ALAS2 variants have been reported in patients with XLSA [7]. These variants predominantly occur in the catalytic domain or the pyridoxal phosphate binding domain, although they may also involve the ALAS2 promoter, enhancer, or mitochondrial targeting sequence [8, 9]. As a key transcription factor that binds cis-regulatory elements of ALAS2, GATA-1 plays a central role in activating erythroid-specific genes, including the β- and γ-globin genes. Additionally, GATA-1 regulates essential cellular functions, including inhibition of apoptosis, suppression of cell proliferation, and promotion of erythroid progenitor cell development and maturation [10-14]. Zhang et al. generated mice lacking the int-1-GATA site (TAGATAAAGCCCC) and found that hemizygous deletion resulted in embryonic lethality due to severe anemia caused by the absence of ALAS2 expression. This finding underscores the indispensable role of this non-coding sequence in ALAS2 expression in vivo. Further analyses demonstrated that the int-1-GATA site interacts with the GATA site in intron 8 (int-8-GATA) and the proximal promoter, forming a long-range loop to enhance ALAS2 expression in erythroid cells. Moreover, compared with the int-8-GATA site, the int-1-GATA site is more essential for ALAS2 regulation, as shown by CRISPR/Cas9-mediated site-specific deletion studies [15]. Therefore, the int-1-GATA site could serve as a valuable diagnostic marker for XLSA. Different mutations in ALAS2 exhibit variable responses to treatment [1, 16]. Mutations affecting the pyridoxal phosphate binding site typically respond well to vitamin B6 therapy [17-19]. However, mutations in other regions, including the GATA-binding site, often exhibit poor responsiveness to vitamin B6 treatment and may lead to fatal organ failure due to iron overload [6, 8]. This explains the lack of response to vitamin B6 in our patient. In conclusion, several studies have reported successful HSCT in transfusion-dependent CSA cases, although genetic testing was not performed in most of these studies [20-27], however this is the first reported case of XLSA caused by an ALAS2 intron 1 mutation and successfully treated with allo-HSCT. This case underscores the importance of comprehensive genetic testing in the diagnosis of XLSA and suggests that the int-1-GATA site could serve as a valuable diagnostic marker for XLSA, particularly in patients with atypical mutations. It also highlights the potential of HSCT as a curative treatment that offers long-term survival for patients with transfusion-dependent XLSA, particularly those who do not respond to pyridoxal. Dao Wang was critical in diagnosing and treating this patient, supervised the study, and provided critical revision of the paper. Shurui Du analyzed the data and wrote the paper. Peihan An and Chi Li analyzed the data. Lei Zhang, Huanhuan Li, and Huixia Wei were engaged in diagnosing and treating this patient. All authors have read and agreed to the final version of the manuscript. We would like to thank our patient and every person involved in their treatment process. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The study involving humans were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University(2025-KY-1047). The parents of this child provided informed consent for publication of the case. Data sharing is not applicable to this article as no new data were created or analyzed in this study.