Novel ALAS2 Mutation Unmasked as Hidden Cause of Severe Macrocytic Anemia in Teenage Girl

A puzzling case of severe anemia in a 15-year-old girl that defied years of conventional treatment has led researchers to identify a previously unknown mutation in the ALAS2 gene, the genetic culprit behind the most common form of hereditary sideroblastic anemia. The case, reported in Clinical Case Reports, is notable not only for the novelty of the mutation but also for the atypical way the disease presented. Instead of the small, pale red cells that usually characterize this disorder, the patient’s blood work showed the opposite: abnormally large red cells, or macrocytosis, a feature that repeatedly steered clinicians toward nutritional explanations and delayed the correct diagnosis by nearly a decade.

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