X-linked sideroblastic anemia, or XLSA, arises from pathogenic variants in ALAS2, a gene carried on the X chromosome that provides the blueprint for erythroid-specific 5-aminolevulinate synthase, the first and rate-limiting enzyme in heme biosynthesis. When the enzyme falters, developing red blood cells cannot manufacture heme efficiently, and iron piles up inside their mitochondria instead of being incorporated into hemoglobin. Under the microscope, these iron-laden mitochondria ring the nucleus of immature erythroid cells, producing the ring sideroblasts that give the disease its name. Because the gene sits on the X chromosome, the condition typically follows an X-linked recessive pattern: hemizygous males, who possess only one X chromosome, are usually the ones who fall ill, while heterozygous females often remain asymptomatic or show only mild anemia thanks to their second, healthy copy.

The girl in this report had been flagged during a routine physical examination eight years before referral, when she was initially diagnosed with iron-deficiency anemia. Oral iron supplements failed to lift her hemoglobin, and two years before admission she began complaining of dizziness and fatigue. Testing at that point revealed macrocytic anemia accompanied by folate deficiency, and a bone marrow aspiration showed mild dyserythropoiesis, prompting a diagnosis of nutritional anemia. She was treated with high-dose oral folic acid, later combined with mecobalamin, a form of vitamin B12. The anemia proved stubbornly refractory: hemoglobin hovered around 80 g/L and then slid to roughly 60 g/L after another year of therapy, at which point she was referred for specialist evaluation.

On admission, her hemoglobin measured 59 g/L, with red cell indices pointing firmly toward macrocytosis: a mean corpuscular volume of 113.1 fL, well above the normal range, and a mean corpuscular hemoglobin of 36.9 pg. Reticulocyte analysis told an equally important story. Although the reticulocyte percentage was mildly elevated at 2.48 percent, the calculated reticulocyte production index, adjusted for her severe anemia, came out at approximately 0.40, a value far too low for the degree of anemia. That discrepancy signaled ineffective erythropoiesis: the bone marrow was working hard but producing few usable red cells. White blood cell and platelet counts, crucially, remained normal, arguing against a broad marrow failure syndrome.

Iron studies then delivered a decisive clue. Serum ferritin was markedly elevated at 871.7 micrograms per liter, and serum iron reached 44.12 micromoles per liter, with an unsaturated iron-binding capacity below the detectable limit, together painting a picture of substantial iron overload rather than iron deficiency. Nutritional markers showed borderline folate at 4.14 nanograms per milliliter, interpreted as persistent functional folate deficiency given her continuous supplementation, and a normal vitamin B12 level. Hemolysis markers added further complexity: plasma free hemoglobin was elevated, haptoglobin was nearly abolished, and both indirect and total bilirubin were raised, consistent with hemolytic activity or ineffective erythropoiesis. Erythropoietin, the kidney hormone that drives red cell production, was dramatically elevated at 213.62 mIU/mL, reflecting the marrow’s desperate attempt to compensate. A Coombs test was negative, ruling out autoimmune hemolysis, and the osmotic fragility test was unremarkable.

Morphology told its own story. The peripheral blood smear displayed striking anisocytosis and poikilocytosis, with red cells of wildly varying size and shape, including ovalocytes and teardrop cells. Bone marrow aspiration revealed relative erythroid hyperplasia dominated by intermediate and late erythroblasts, and the biopsy was markedly hypercellular at over 90 percent with erythroid predominance. Flow cytometry found no increase in myeloblasts and no monoclonal lymphoid population, helping exclude a clonal marrow cancer. Globin gene sequencing came back clean, ruling out thalassemia and related hemoglobin disorders, leaving the underlying cause still shrouded.

The breakthrough came from whole exome sequencing of bone marrow mononuclear cells, which uncovered a frameshift insertion in ALAS2: c.1277dupT, resulting in the protein change p.M426Ifs*66. Sanger sequencing confirmed the variant. A frameshift of this kind is catastrophic for protein architecture: the reading frame collapses, and the resulting truncated protein loses critical C-terminal functional domains. Structural modeling with AlphaFold 3 suggested the truncation disrupts the three-dimensional conformation of the enzyme’s active site, specifically displacing the Lys391 residue, the catalytic lysine that forms the essential Schiff base linkage with the pyridoxal 5′-phosphate cofactor. Without that interaction, the enzyme is predicted to be catalytically inert. The variant was therefore classified as highly pathogenic, providing molecular confirmation of XLSA. Notably, the sequencing also surfaced two missense variants in DHX34, predicted deleterious and potentially linked to familial myelodysplastic syndrome or acute myeloid leukemia risk, plus variants in FANCI and BRIP1 classified as uncertain in significance; without segregation or functional data, the team could not assign any of them a causal or modifying role in her blood disorder.

The diagnostic journey required careful exclusion of look-alike conditions. Myelodysplastic neoplasm with SF3B1 mutation, the commonest acquired cause of ring sideroblasts, was considered unlikely given the childhood onset, isolated anemia with preserved white cells and platelets, absence of excess myeloblasts, and the germline ALAS2 finding, though bone marrow iron staining was never performed, so ring sideroblasts could not be formally assessed. Congenital dyserythropoietic anemia, with its characteristic multinucleated erythroblasts and chromatin bridges, found no morphological support, and the features of Pearson syndrome, Diamond-Blackfan anemia, GATA1-related disorders, and telomere biology disorders were all absent. After the molecular diagnosis, the patient continued transfusions as needed for symptomatic anemia, with ferritin monitored but iron chelation not yet started. At last contact she remained transfusion-dependent and subsequently did not return for follow-up, leaving her long-term course unknown.

The case carries broader lessons for hematology. More than 100 pathogenic ALAS2 variants have now been described, scattered across nearly every exon with no clear mutational hotspots, and missense mutations account for roughly 90 percent of cases, making this frameshift a rare and severe class of defect. The authors propose that such profound loss of function may explain the unusually early onset in this female patient. Why she was macrocytic rather than microcytic remains the most intriguing question. Prior work suggests that in symptomatic heterozygous females, skewed X-chromosome inactivation leaves the mutant allele active in erythroid cells, and that circulating red cells derive almost exclusively from wild-type lineages, because ALAS2-deficient clones cannot complete terminal differentiation. Under hypoxic stress and soaring erythropoietin, those surviving wild-type cells may mature prematurely, dividing fewer times and emerging larger, a model the authors frame as hypothetical since X-inactivation studies could not be performed. The family declined further genetic testing, iron staining of stored marrow was never authorized, and transfusion began before pyridoxine response could be tested, though a complete loss-of-function variant was unlikely to respond to the vitamin B6 cofactor anyway. Clinically, the message is clear: when macrocytic anemia resists folate and B12, especially alongside iron overload and a poor reticulocyte response, inherited sideroblastic anemia deserves a place on the differential, and genomic sequencing can end years of diagnostic limbo.

Subject of Research: A novel ALAS2 frameshift mutation causing severe macrocytic X-linked sideroblastic anemia in a female patient

Article Title: Severe Macrocytic Anemia Associated With a Novel ALAS2 Mutation: A Case Report and Literature Review

Article References: Cai, H., Shen, K., Wang, J., Gu, J., Zhang, M., & Xiao, M. (2026). Severe Macrocytic Anemia Associated With a Novel ALAS2 Mutation: A Case Report and Literature Review. Clinical Case Reports, 14(9), Article e73529. https://doi.org/10.1002/ccr3.73529

Image Credits: AI Generated

DOI: 10.1002/ccr3.73529

Keywords: ALAS2, X-linked sideroblastic anemia, macrocytic anemia, heme biosynthesis, ring sideroblasts, whole exome sequencing, iron overload, frameshift mutation, X-chromosome inactivation, ineffective erythropoiesis, refractory anemia, genetic diagnosis

The research team, led by Ewa Karakulska-Prystupiuk and colleagues in Warsaw, analyzed 341 patients who received allogeneic stem cell transplants between 2014 and 2023. For each patient, the investigators recorded the lowest vitamin B12 and folate levels measured within two years after the transplant, applying deficiency thresholds derived from the NHANES population surveys. The results were striking: 49 percent of patients showed reduced vitamin B12 levels below 300 pg/mL, with 17 percent falling below 200 pg/mL, and folate deficiency, defined as a level under 3 ng/mL, affected 46 percent of the cohort.

Graft-versus-host disease occurs when donor immune cells recognize the recipient’s tissues as foreign and mount an attack. When this process becomes chronic and involves the gastrointestinal tract, the lining of the gut—the very tissue responsible for absorbing nutrients—is inflamed and damaged. The Polish team hypothesized that this mucosal injury, combined with reduced dietary intake and the effects of immunosuppressive drugs, would translate into measurable vitamin depletion, particularly for B12 and folate, both of which are absorbed through intestinal mechanisms vulnerable to inflammatory damage.

To test this, the investigators carved out a subgroup of 68 patients with confirmed chronic graft-versus-host disease, whose median age was 45.5 years. Among them, 42 had gastrointestinal involvement. When the researchers compared median vitamin levels between the two groups, the difference was unequivocal. Patients with gastrointestinal chronic graft-versus-host disease had a median vitamin B12 level of 258 pg/mL, versus 442 pg/mL in patients whose chronic disease spared the gut. Deficiency was documented in 88.6 percent of the gastrointestinal group, compared with just 11.4 percent of the others, a difference that reached high statistical significance with a p value below 0.001.

Perhaps even more telling was the dose-response relationship. The researchers found an inverse correlation between the severity of gastrointestinal graft-versus-host disease and vitamin B12 concentrations: the worse the gut involvement, the lower the circulating B12. This gradient supports a mechanistic interpretation in which progressive mucosal destruction progressively impairs the absorption machinery—including the ileal uptake pathway that normally captures vitamin B12 bound to intrinsic factor—rather than the deficiency arising from a single binary event.

Folate, the other B vitamin central to red blood cell production and DNA synthesis, followed a similar directional pattern. Levels were lower in the gastrointestinal group, consistent with the idea that an inflamed gut absorbs less of this nutrient as well, but the difference did not reach statistical significance. The authors suggest this may reflect folate’s more diffuse absorption along the small intestine, or the confounding influence of supplementation practices, though the precise explanation remains a question for future work.

The study then turned to anemia, one of the most persistent quality-of-life problems after transplantation. At 12 months post-transplant, hemoglobin concentrations were significantly lower in patients with gastrointestinal chronic graft-versus-host disease, 12.2 g/dL versus 13.25 g/dL in those without gut involvement, with a p value of 0.006. Since both vitamin B12 and folate deficiencies classically cause macrocytic anemia—oversized, underperforming red cells—one might expect the vitamin deficits to explain the anemia. They did not. Macrocytic anemia predominated in both patient groups, but it was not associated with the vitamin deficiencies, indicating that the red cell failure after transplantation stems from multiple converging causes rather than a single nutrient shortfall.

This dissociation is arguably the study’s most instructive result. Post-transplant anemia can arise from ongoing inflammatory suppression of the bone marrow, iron dysregulation, renal dysfunction, medications, and the residual effects of the conditioning regimen itself. The Warsaw data suggest that clinicians cannot assume that correcting B12 or folate levels will resolve anemia in these patients, and conversely, that a normal blood film does not rule out dangerous vitamin depletion. The two problems run on separate tracks, and each demands its own surveillance.

The practical implications are considerable. Vitamin B12 deficiency is not a benign laboratory curiosity; untreated, it can produce neurological injury, cognitive impairment, and worsening fatigue in a population already burdened by treatment toxicities. Given that roughly nine in ten patients with gastrointestinal chronic graft-versus-host disease in this cohort were deficient, the authors’ findings argue for routine, periodic measurement of B12 and folate in this subgroup, with a low threshold for supplementation even in the absence of anemia. Whether prophylactic supplementation should extend to all transplant recipients remains an open question the retrospective design cannot answer.

The study does carry the inherent limits of a single-center retrospective analysis, and vitamin levels were defined by the lowest recorded value rather than serial trends, which may capture transient dips. Still, the size of the cohort, the uniform threshold definitions, and the striking statistical separation between patient groups give the findings weight. As survival after allogeneic stem cell transplantation continues to improve, attention is steadily shifting from survival alone to the long-term functional health of survivors. Identifying a preventable, treatable deficiency affecting nearly half of all recipients—and nearly all of those with gut-directed graft-versus-host disease—is a concrete step in that direction, one that could translate into simple blood tests and inexpensive supplements protecting patients from an entirely avoidable secondary burden.

Subject of Research: Vitamin B12 and folate deficiency after allogeneic hematopoietic stem cell transplantation and its link to gastrointestinal chronic graft-versus-host disease and anemia.

Article Title: Vitamin B12 and folate deficiencies after allo-HSCT: association with gastrointestinal chronic graft-versus-host disease and anemia

Article References: Karakulska-Prystupiuk, E., Kumorek, A., Tomaszewska, A., Kacprzyk, P., Biliński, J., Jędrzejczak, W. W., & Basak, G. W. (2026). Vitamin B12 and folate deficiencies after allo-HSCT: association with gastrointestinal chronic graft-versus-host disease and anemia. Annals of Hematology. https://doi.org/10.1007/s00277-026-07276-7

Image Credits: AI Generated

DOI: 10.1007/s00277-026-07276-7

Keywords: allogeneic stem cell transplantation, vitamin B12 deficiency, folate deficiency, graft-versus-host disease, gastrointestinal GvHD, anemia, macrocytic anemia, hematology, bone marrow transplant, post-transplant care, malnutrition, Annals of Hematology