EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Congenital sideroblastic anemia is characterized by anemia and intramitochondrial iron accumulation in erythroid precursors that form ring sideroblasts. The most common recessive forms are caused by sequence variations in the ALAS2 and SLC25A38 genes. In patients with transfusion-dependent and pyridoxine-resistant severe congenital sideroblastic anemia, hematopoietic stem cell transplant is the only curative option. Herein, we described successful implemen-tations of allogeneic hematopoietic stem cell transplant in 4 Iranian children with congenital sideroblastic anemia. The patients had presented with clinical manifestations of anemia early in life, and the diagnoses of congenital sideroblastic anemia were established through blood tests and bone marrow aspiration. Congenital sideroblastic anemia was further confirmed by the identification of pathogenic variants in SLC25A38 in 2 patients. All 4 patients received allogeneic hematopoietic stem cell transplant with myeloablative conditioning regimen that included busulfan, cyclophosphamide, and rabbit antithymocyte globulin. A combination of cyclosporine A and methotrexate or mycophenolate mofetil was used for graft-versus-host disease prophylaxis. Bone marrow and peripheral blood from sibling or related donors with fully matched human leukocyte antigen profiles were applied. The outcomes of hematopoietic stem cell transplant in patients with congenital sideroblastic anemia were favorable. Three patients achieved full donor chimerism (>95%, 98%, and 100%), and the other patient showed mixed chimerism (75%). All patients remained transfusion independent. Hemato-poietic stem cell transplant is a curative treatment that can provide long-term survival for patients with congenital sideroblastic anemia, particularly when used in a timely manner. There remain ongoing challenges in various aspects of hematopoietic stem cell transplant in patients with congenital sideroblastic anemia, which remain to be elucidated.

Key words : Donor chimerism, Erythroid precursors, Graft-versus-host disease, Transplantation

Congenital sideroblastic anemia (CSA) is a rare heterogeneous category of inherited disorders of erythropoiesis characterized by the presence of ringed sideroblasts in the bone marrow due to pathological deposits of iron in the mitochondria of developing erythroblasts.^1,2 Defective iron utilization could disturb the intracellular reduction-oxidation reaction and induce apoptosis. Thus, the clinical markers of CSA are anemia (usually hypochromic microcytic anemia) and increased levels of serum iron and ferritin.³ Clinical presentation of patients with sideroblastic anemia includes the common symptoms of anemia such as fatigue, malaise, shortness of breath, palpitations, and headache. Pallor may be observed in physical examination.⁴

Congenital sideroblastic anemia is a disease of mitochondrial dysfunction related to defects in heme biosynthesis, iron-sulfur cluster biogenesis, gene-ralized mitochondrial protein synthesis, or the synthesis of specific mitochondrial proteins involved in oxidative phosphorylation.⁵ The most common inheritance pattern is X-linked recessive, caused by sequence variations in the 5?-aminolevulinate synthase 2 gene (ALAS2). The second most common form is caused by pathological variants in SLC25A38, which is responsible for the mitochondrial transport of glycine required for the initial step of heme biosynthesis.⁶

Patients with X-linked CSA (XL-CSA) are typically male patients under 40 years old. Anemia is categorized as hypochromic microcytic in variable degrees, accompanied by systemic iron overload. Sequence variations in the ALAS2 gene in patients with XL-CSA are heterogeneous and usually missense variants of conserved amino acids, resulting in inappropriate function. Today, more than 80 different sequence variations in ALAS2 have been reported in patients with XL-CSA.⁷

The other CSA genetic defects are found in the SLC25A38 gene, which encodes an erythroid-specific protein of the inner mitochondrial membrane. SLC25A38 is incorporated in the mitochondrial import of glycine, essential for the ALA synthesis. The molecular basis that contributes to the formation of ring sideroblasts is similar to that of XL-CSA. Patients typically present at birth or early childhood with severe, transfusion-dependent microcytic anemia and iron overload that clinically resembles thalassemia major.⁸ Other pathological variants underlying CSA have been identified in GLRX5, HSPA9, and ABCB7, which encode glutaredoxin 5 enzymes, member of the heat shock protein 70 gene family, and adenosine triphosphate-binding cassette B7, respectively.⁹ The nonhereditary form of sideroblastic anemia is known as acquired sideroblastic anemia, and it may be primary or secondary. Primary causes include clonal hematological disorders such as myelodysplastic syndrome with ring sideroblast and refractory anemia with ring sideroblasts. Secondary causes are related to drugs, toxins, copper deficiency, or chronic neoplastic disease.¹⁰

In up to two-thirds of cases, the XL-CSA anemia responds to the essential cofactor of ALAS in the form of pyridoxal 5?-phosphate by enhancing the function of some ALAS2 mutants. The hemoglobin (Hb) level normalizes in about one-third of responders. Uncommonly, severe anemia that is unresponsive to pyridoxine dictates blood transfusion with iron chelation therapy. Patients with severe anemia caused by SLC25A38 defects require lifelong transfusion therapy or, as an alternative, hemato-poietic stem cell transplant (HSCT).¹¹

Regarding the importance of transplant procedures in patients with CSA, our aim was to report the details of 4 patients with refractory CSA who underwent successful HSCT by myeloablative conditioning (MAC).

Informed consent for participation and publication was obtained from the parents of the patients. The present study was conducted according to the principles expressed in the Helsinki Declaration

The patient was a 3-year-old girl who was the second child of closely related parents (Figure 1 and Figure 2). She presented at 2 months of age with pallor and severe hypochromic microcytic anemia and later developed splenomegaly (115 cm), large head, and frontal bossing. Her initial blood count and iron indices included Hb 6.4 g/dL, mean corpuscular volume (MCV) 74 fL, mean corpuscular Hb (MCH) 25 pg, reticulocyte count 0.1%, and ferritin 923 ng/mL. In the bone marrow aspiration, the observations were normal cellularity, erythroid series increase, mild dyserythropoiesis, and 20% to 25% ring sideroblasts. The treatment with pyridoxin resulted in no response, and the patient remained dependent on blood transfusion and iron chelation therapy. Therefore, at 3 years of age, HSCT was planned, and she received allogeneic HSCT from her fully matched sibling donor. The conditioning regimen included busulfan (0.8 mg/kg intravenously [IV] every 6 h for 4 days), cyclophosphamide (50 mg/kg IV daily infusion for 4 days), and rabbit antithymocyte globulin (rATG) (1.25 mg/kg for 2 days) with graft-versus-host disease (GVHD) prophylaxis by cyclosporine A (3 mg/kg/day IV BD)and methotrexate (15 mg/m2 IV on first day and then 10 mg/m2 IV on days 3, 6, and 11). She received engraftment at 21 days after stem cell infusion. She later developed acute gastrointestinal (GI) GVHD (stage 3) and acute (stage 3) and chronic skin GVHD, cytomegalovirus reactivation, and posterior reversible encephalopathy syndrome. All of these complications were managed and resolved. Now, at 5 years after HSCT, she remains drug-free with >95% donor chimerism.

The patient was a 2.5-year-old girl born to first-degree consanguineous parents (Figure 3 and Figure 4). She was primarily found to have experienced severe anemia at 1.5 months old with Hb 7 g/dL, MCV 63 fL, MCH 19 pg, and reticulocyte count 0.5%. The Hb electrophoresis showed HbA 82%, HbA² 2.9%, and HbF 15.9%. At the time, she had no organomegaly but did have a large head and a thick diploic space of the frontal bone indicative of increased hematopoiesis rate. In bone marrow aspiration, the observations were erythroid hyperplasia, dyserythropoiesis, and ring sideroblasts (6%). Next generation sequencing (NGS) gene analysis revealed a homozygous variant in SLC25A38 that further confirmed the diagnosis of CSA. She was also refractory for pyridoxine and required regular blood transfusions and an iron chelator. Therefore, she underwent allogeneic HSCT at the age of 2.5 years from her fully matched mother with a conditioning regimen that consisted of busulfan (0.8 mg/kg IV evert 6 h for 4 days), cyclophosphamide (50 mg/kg IV daily infusion for 4 days), and low-dose rATG (1.25 mg/kg for 2 days) and GVHD prophylaxis by cyclosporine A (3 mg/kg/day IV BD) and mycophenolate mofetil (15 mg/kg/dose every 12 h). She received engraftment at 18 days after stem cell infusion, later complicated by skin GVHD (stage 3), but this resolved completely after treatment with corticosteroids. At 3 years after HSCT, she maintains stable health, receives no blood transfusions, and has 100% donor chimerism.

The patient was a 3.5-year-old boy born to closely related parents. He manifested with severe pallor, and at 2 months old he developed splenomegaly. His blood evaluation showed Hb 8 g/dL, MCV 78 fL, MCH 27 pg, reticulocyte count 0.1%, and ferritin 1685 ng/mL. In bone marrow aspiration, moderate erythroid hyperplasia and ring sideroblasts were evident, suggestive of sideroblastic anemia. He received blood transfusions and iron chelator and later showed transfusion-related complications (anti-immunoglobulin G C3d) that required intravenous immunoglobulin, corticosteroids, and 4 courses of rituximab. At the age of 3.9 years, he was a candidate for HSCT and received allogeneic HSCT from his fully matched sister. The conditioning regimen included busulfan (0.8 mg/kg IV every 6 h for 4 days), cyclophosphamide (50 mg/kg IV daily infusion for 4 days), and low-dose rATG (1.25 mg/kg for 2 days). Cyclosporine A (3 mg/kg/day IV BD) and methotrexate (15 mg/m2/day IV and then 10 mg/m2/day IV) were used for GVHD prophylaxis. He received the engraftment at 16 days after stem cell infusion. He exhibited cytomegalovirus reactivation (at 26 days after stem cell infusion) and posterior reversible encephalopathy syndrome, which improved gradually. In addition, for high ferritin level, he received oral iron chelator (deferasirox) 6 months after HSCT without complication. Now, 5 years after HSCT he is clinically stable without blood transfusion with a 75% donor chimerism.

The patient was a 2.6-year-old boy who was the second child of first-degree relative parents. He had initially presented with severe anemia (Hb 4 g/dL) at 2.5 months old and since then had received regular blood transfusions and iron chelation therapy. Bone marrow aspiration revealed cellular bone marrow, and staining results for ring sideroblasts were positive. He was diagnosed with hereditary sideroblastic anemia, which was later confirmed by a homozygote sequence variation in SLC25A38. He received allogeneic HSCT from a fully matched related donor (aunt) with a conditioning regimen of busulfan (0.8 mg/kg IV every 6 h for 4 days), cyclophosphamide (50 mg/kg IV daily infusion for 4 days), and rATG (1.25 mg/kg for 2 days) and GVHD prophylaxis by cyclosporine A (3 mg/kg/day IV BD) and methotrexate (15 mg/m2/day IV and then 10 mg/m2/day IV). He received engraftment at 12 days after stem cell infusion with 96% chimerism. During admission he developed bacteremia (Staphylococcus epidermidis), severe GI bleeding, diarrhea, and GI GVHD (stage 3), all of which were managed and improved. At 90 days after stem cell infusion, he showed mild GI GVHD with 98% chimerism after HSCT and was on GVHD treatment with low-dose prednisone and cyclosporine and supportive care. He remains transfusion independent.

Hematopoietic stem cell transplant plays a prominent role in the treatment of CSA. In this study, we described the clinical status of 4 patients with CSA and the transplant procedures, as well as the MAC regimens and GVHD prophylaxis (cyclosporine A and methotrexate). Pathogenic variants in SLC25A38 were identified in 2 of the 4 patients.

The first description of ringed sideroblasts in sideroblastic anemia dates back to the late 1950s, when a sequence variant of ALAS2 was first reported in a male patient with the sporadic form of sideroblastic anemia and ALAS2 sequence variations were confirmed in familial cases.¹²

Several studies have investigated the outcomes of HSCT for CSA. A study in Canada examined 7 patients with CSA. The median age of SLC25A38-related CSA diagnosis was 1.5 years (6 months to 22 years). For 2 of the 7 patients, the diagnosis of CSA was delayed until young adulthood. The remaining 5 patients were diagnosed within 6 months of presentation, prompted by positive family histories. The median age at first transfusion was 8 months (24 days to 4.4 years). In 4 of the 7 patients, the first red blood cell transfusion coincided with the initial presentation. In 6 of the 7 patients, a scheduled transfusion program was required with a median frequency of every 4 weeks from the time of the first transfusion. For all participants, iron chelation was recommended, and 6 patients started chelation therapy within 3 years of initiation of transfusion, at a median age of 3.2 years (15 months to 20 years). Three of the patients underwent allogeneic HSCT, at 5.5 years, 7.2 years, and 28 years of age, respectively. One of these patients received matched unrelated donor HSCT, with reduced-intensity conditioning that included total body radiation, cyclophosphamide, and fludarabine. Another patient received matched sibling donor HSCT, with a conditioning regimen that consisted of myeloablative doses of busulfan, fludarabine, and ATG. A third patient received a matched sibling donor HSCT with MAC with busulfan and fludarabine. One patient died in the immediate posttransplant period due to complications of sepsis. The other 2 patients remained transfusion independent, with full donor chimerism at 15 months and 16 years after HSCT; neither patient required immunosuppression, and both were free of GVHD.⁶

In another report, 31 patients from 24 families were diagnosed with rare pathological variants of SLC25A38 using whole exome sequencing. All of the patients required transfusions, most chronically, beginning in the neonatal period or infancy. Two, 12, 11, and 3 patients received their first transfusions in utero, during the neonatal period, at infancy, or between age 4 and 8 years, respectively. All patients who survived early childhood developed secondary iron overload and required chelation. One patient died at age 18 years from cardiomyopathy. Another patient died of central venous line?associated sepsis at age 3 years. The median age of patients alive at the time of the last follow?up was 11 years. Nine of 31 patients underwent allogeneic HSCT with a median follow?up of 7 years (6 months to 17 years). The outcome was positive as all transplant recipients remained alive. Eight of 9 patients had full engraftment and became transfusion independent. One patient had secondary graft failure at 18 months after HSCT. Also, the second transplant with the same donor failed to engraft. Four patients received MAC, and 4 others received reduced-intensity conditioning. Donors were matched unrelated donors for 4 patients, matched related donors for 4 patients, and 1 patient had a 1-antigen-mismatch sibling donor. Methotrexate and calcineurin inhibi-tors were the most common agents for GVHD prophylaxis. The GVHD was observed in 4 patients, and 1 patient developed chronic posttransplant autoimmune hemolytic anemia that required transfusion.¹

Another study reported the application of HSCT in a Hispanic girl who was noted to be pale since birth and was diagnosed with CSA and SLC25A38 gene variant. The patient had received blood transfusions every 4 to 6 weeks since infancy, and after age 15 months deferasirox was also administered. She received a transplant from a 6/6 matched sibling donor at age 4 years. The conditioning regimen consisted of busulfan, fludarabine, and an intermediate dose of alemtuzumab. Methotrexate and tacrolimus were used for GVHD prophylaxis. She tolerated the transplant very well without complications. Engraftment was on day 17, and a 100% donor chimerism was on day 30 after HSCT.¹³

In another case report, a 22-year-old man with CSA who was diagnosed at 8 months was studied. Due to the ineffectiveness of pyridoxal 5?-phosphate therapy, transplant was performed. The conditioning regimen was fludarabine, low-dose total body irradiation, and ATG. The transplant source was peripheral blood stem cells from his HLA-matched brother. Cyclosporine and mycophenolate mofetil were used for GVHD prophylaxis. Complete donor chimerism was observed at 131 days after stem cell infusion. Soon after the transplant, he became transfusion independent. Posttransplant complications were refractory lactic acidosis followed by fatal cardiovascular collapse that developed without evidence of infection.¹⁴

Positive outcomes were observed in a study of HSCT on 3 children with CSA (2 girls aged 1 year and 8 years, and 1 boy aged 2 years at the time of HSCT). All patients were unresponsive to the high dose of pyridoxine and were transfusion dependent. Only the 8-year-old girl had chelation therapy with deferoxamine. All donors were HLA-matched siblings. Conditioning consisted of cyclophosphamide, busulfan, and ATG. The agents for GVHD prophylaxis were cyclosporin A and methotrexate. The 3 patients received engraftment at 20 days, 13 days, and 19 days after HSCT, respectively. Acute skin GVHD (grade 2) ensued in only 1 patient, and this patient showed a positive response to treatment with steroids. Two patients developed mild self-limited veno-occlusive disease of the liver. All 3 patients remain alive and transfusion independent.¹⁵

Allogenic HSCT could be a curative treatment in patients with SLC25A38 gene variant of CSA. Further research studies and clinical trials are required to prove this statement.

Volume: 21 Issue: 1 January 2023

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