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 clinical puzzle began with a routine coagulation workup. The patient’s activated partial thromboplastin time, or APTT, one of the most commonly ordered tests in hospital laboratories, was found to be significantly prolonged. APTT measures the efficiency of the intrinsic pathway of coagulation, the branch of the clotting system that depends on a chain of protein factors circulating in plasma. When one link in that chain is missing or defective, the test tube reaction slows dramatically and the reported time lengthens. In most circumstances, a markedly prolonged APTT signals an elevated risk of bleeding and triggers a cascade of further testing, transfusion planning, and, in surgical contexts, potential postponement of procedures. For this patient, however, the laboratory abnormality stood in stark contradiction to the clinical picture, a phenomenon hematologists call genotype-phenotype dissociation.

The Chinese team, led by Fengjiao Wang and corresponding author Lihong Yang of the Department of Laboratory Medicine, pursued the discrepancy systematically. Mixing studies and factor assays pointed away from the classic deficiency states and toward a deficit of high-molecular-weight kininogen, a large plasma protein known as HMWK that serves as a cofactor in the intrinsic pathway. HMWK does not itself possess procoagulant enzymatic activity; instead, it acts as a molecular scaffold, binding to negatively charged surfaces and positioning factor XI and prekallikrein for activation by factor XIIa. Without HMWK, this assembly fails, and the in vitro clotting reaction measured by APTT grinds to a halt. Crucially, however, the in vivo relevance of this pathway to hemostasis has long been questioned, and individuals with complete HMWK deficiency characteristically bleed no more than healthy people, because the extrinsic and tissue factor pathways compensate for the intrinsic defect in real life.

Genetic sequencing provided the definitive answer. The analysis identified a novel homozygous deletion, designated c.628_629delAA, located in exon 5 of the KNG1 gene, which encodes the kininogen precursor protein. The deletion of two adenine bases is a classic frameshift mutation: it shifts the reading frame of the genetic code from that point onward, scrambling every downstream codon. In this case, the frameshift generates the amino acid substitution p.Asn210Phe at the new reading position and then, just fourteen codons later, introduces a premature termination codon, producing the notation fs*15. The result is a truncated protein that lacks the functional domains essential for HMWK’s role in the contact phase of coagulation. Applying the standards of the American College of Medical Genetics and Genomics, the team classified the variant as pathogenic, given its nature as a loss-of-function frameshift in a gene whose biallelic loss produces the observed biochemical phenotype.

The inheritance pattern followed classical autosomal recessive genetics. Because the patient inherited two copies of the mutant allele, one from each parent, essentially no functional HMWK was produced, and APTT values soared. Family screening revealed that heterozygous relatives carrying a single mutant copy had mildly reduced HMWK activity, yet their APTT values remained entirely within the normal range. This dose-effect relationship demonstrates that a single functional KNG1 allele produces enough kininogen to sustain a normal laboratory coagulation profile, while two defective alleles are required to unmask the abnormality. The finding has direct implications for genetic counseling: carriers are asymptomatic and undetectable by routine coagulation testing, so only molecular analysis can identify them, and only molecular analysis of both parents can predict recurrence risk in future pregnancies.

The rarity of the condition adds to its scientific value. Hereditary HMWK deficiency, historically known as Fitzgerald trait after the first described patient, has been reported in only a small number of families worldwide, and each new case contributes to a mutational catalog that remains strikingly short. The novel c.628_629delAA deletion identified in this Chinese patient expands that catalog and illustrates the kind of variant most likely to cause the condition: loss-of-function mutations that truncate the protein before its critical binding domains. Previous reported cases have involved diverse mutations scattered across the gene, consistent with the expectation that many different disruptions of KNG1 can produce the same laboratory phenotype. The Wenzhou case also highlights that the condition may be substantially underdiagnosed, since many affected individuals, being clinically healthy, may never come to medical attention unless an APTT happens to be checked before surgery or during an unrelated evaluation.

The clinical stakes of correct diagnosis are considerable. A prolonged APTT in a preoperative patient conventionally prompts either cancellation of surgery, empirical correction attempts with fresh frozen plasma, or extensive hematologic investigation for hemophilia, von Willebrand disease, lupus anticoagulants, or acquired inhibitors. Each of these pathways carries cost, delay, and in the case of plasma transfusion, genuine risk of transfusion reactions or alloimmunization. Recognizing that isolated APTT prolongation with normal prothrombin time, normal bleeding history, and reduced HMWK activity indicates a benign entity allows clinicians to proceed with appropriate reassurance. The authors of the report emphasize that systematic genetic screening of both patients and their family members is clinically important, not merely for scientific completeness, but because definitive molecular characterization resolves diagnostic uncertainty that biochemical testing alone cannot.

The report also contributes to a broader biological conversation about why the contact pathway exists at all. Decades of research have shown that individuals deficient in factor XII or HMWK do not bleed abnormally, even though their APTT values can be extraordinarily prolonged. This paradox has reshaped thinking in thrombosis research: if the intrinsic pathway is dispensable for preventing bleeding, it may nevertheless be essential for pathological clot formation, and inhibiting contact-phase proteins has become an attractive antithrombotic strategy that could theoretically prevent clots without increasing bleeding risk. Rare patients like this one serve as natural experiments that validate such approaches, demonstrating at the level of human physiology that a lifetime of absent HMWK activity is compatible with normal hemostasis. Epidemiological observations have even suggested that severe HMWK deficiency may protect against venous thromboembolism, adding an intriguing twist to the story of a mutation that leaves its carrier laboratory-abnormal but clinically well.

From a laboratory medicine perspective, the case underscores the importance of a disciplined diagnostic algorithm for unexplained APTT prolongation. The standard sequence begins with mixing studies, which distinguish factor deficiencies, where pooled normal plasma corrects the prolongation, from inhibitors, where it does not. Correction then leads to sequential factor assays through the intrinsic pathway: factors VIII, IX, XI, and XII, followed by the less commonly tested contact proteins, prekallikrein and HMWK. Because HMWK deficiency is so rare, it is often the last possibility considered, and many laboratories lack routine access to kininogen activity assays. The Wenzhou group’s workflow, moving from coagulation phenotyping to targeted sequencing of the KNG1 gene, offers a template other centers can follow. The measurement of profoundly reduced HMWK activity in the proband, alongside mildly reduced activity in heterozygous relatives, illustrates how genotype and biochemical phenotype track together in this condition with unusual precision.

The report, which was peer reviewed and published with a permanent DOI as an early-access article subject to final editorial production, was approved by the ethics committee of The First Affiliated Hospital of Wenzhou Medical University, and the authors declared no competing interests. The work was supported by the Key Laboratory of Clinical Laboratory Diagnosis and Translational Research of Zhejiang Province and by municipal science and technology funds from Wenzhou. Beyond its immediate value to hematologists and laboratory physicians, the case stands as a vivid reminder that the genome continues to hold explanations for laboratory curiosities that have puzzled clinicians for generations, and that the tool most likely to resolve them, systematic sequencing of patients and their families, has now become fast and affordable enough to be applied whenever a blood test and a bedside picture refuse to agree. For the patient at the center of the report, the conclusion is the most reassuring one possible in coagulation medicine: the frightening numbers on the laboratory report were never a threat to life.