Two Rare Blood Disorders in One Family: EPB41 Mutations and β-Thalassemia Unravel a Diagnostic Puzzle

In the world of medical genetics, some diagnoses arrive like a detective story. A single patient walks into a clinic with chronic anemia, and what initially appears to be one familiar blood disorder turns out to be the work of two entirely different genetic culprits operating in the same person. That is precisely what unfolded in a Chinese family recently described in a case report published in Annals of Hematology, where researchers identified the exceedingly rare co-inheritance of β-thalassemia and hereditary elliptocytosis, both traced to specific mutations in genes governing red blood cell structure and function.

The story begins with a proband—an individual who serves as the starting point for genetic investigation within a family—presenting with chronic hemolytic anemia, a condition in which red blood cells are destroyed faster than the body can replace them. In southern China, β-thalassemia is a common inherited disorder of hemoglobin synthesis, primarily caused by point mutations and small deletions or insertions in the β-globin gene, known as HBB. Hereditary elliptocytosis, by contrast, is a red blood cell membrane disorder characterized by elliptical rather than the normal biconcave-disc-shaped erythrocytes. The coexistence of these two conditions is exceedingly rare and presents a formidable diagnostic challenge because their hematological characteristics overlap in ways that can easily confound clinicians relying on standard blood work alone.

To disentangle the two conditions, the research team deployed a comprehensive molecular toolkit. Hematological and biochemical evaluations were performed alongside hemoglobin fraction analysis, which separates the different types of hemoglobin present in the blood to detect abnormalities in globin chain production. The investigators then turned to third-generation sequencing to analyze both α- and β-globin genes in the proband and available family members. Unlike earlier sequencing platforms, third-generation technology can read long stretches of DNA in a single pass, making it particularly valuable for detecting complex rearrangements and structural variants that shorter-read methods might miss. Whole-exome sequencing was subsequently performed to identify candidate variants across the protein-coding portion of the genome, followed by confirmation through Sanger sequencing, the gold-standard method for validating individual genetic variants.

The results revealed two heterozygous variants in the EPB41 gene, which encodes protein 4.1, a critical structural component of the red blood cell membrane skeleton. The first variant, designated NM_001376013.1(EPB41):c.906-1G>A, affects a splice site—the molecular signal that tells the cellular machinery where to cut and rejoin RNA during gene expression. The second, NM_001376013.1(EPB41):c.731 A>G, represents a single nucleotide substitution that alters the protein’s amino acid sequence. Both variants were confirmed by Sanger sequencing and demonstrated co-segregation with the elliptocytic phenotype in affected family members, meaning that the presence of these mutations tracked precisely with the appearance of elliptical red blood cells across the family pedigree. This pattern of inheritance is a hallmark of causality in genetic analysis and provides strong evidence that the EPB41 variants are responsible for the membrane disorder.

Both EPB41 variants are documented in population genetics databases including gnomAD, which aggregates genome sequences from tens of thousands of individuals worldwide, and dbSNP, a comprehensive database of single nucleotide polymorphisms. The c.731 A>G variant is additionally listed in ClinVar, the National Center for Biotechnology Information’s archive of clinically significant genetic variants, under Variation ID 2194273. The presence of these variants in public databases helps contextualize their rarity and provides a reference point for other clinicians who may encounter similar cases in the future.

Beyond the membrane disorder, the molecular investigation uncovered a frameshift mutation in the HBB gene, designated NM_000518.5:c.126_129delCTTT. Frameshift mutations occur when the deletion or insertion of nucleotides is not a multiple of three, disrupting the reading frame that determines how the genetic code is translated into protein. In this case, the deletion of four nucleotides—CTTT—shifts the frame and produces a nonfunctional β-globin protein, consistent with a diagnosis of β-thalassemia. The identification of this mutation alongside the EPB41 variants confirmed that the proband was carrying two independent genetic disorders simultaneously, each contributing to the overall clinical picture in distinct ways.

Pathogenicity assessment followed the American College of Medical Genetics and Genomics guidelines, a standardized framework that classifies genetic variants on a five-tier scale ranging from benign to pathogenic. The ACMG criteria incorporate multiple lines of evidence, including population frequency, computational predictions, functional data, and segregation patterns within families. By applying these rigorous standards, the researchers ensured that their classification of the EPB41 variants and the HBB frameshift was not merely speculative but grounded in established clinical genetics practice.

The integration of peripheral blood morphology—the microscopic examination of red blood cell shape and size—with family segregation analysis and molecular genetic findings ultimately supported a unified diagnosis of co-inherited hereditary elliptocytosis and β-thalassemia. This multi-pronged approach illustrates why no single diagnostic modality is sufficient when dealing with complex anemias. Blood smears can reveal the elliptocytic cells characteristic of the membrane disorder, while hemoglobin fraction analysis points toward the thalassemia component, but only genetic sequencing can definitively identify the underlying molecular lesions responsible for each condition.

The significance of this report extends beyond the individual family involved. By expanding the genotype–phenotype spectrum of EPB41-associated hereditary elliptocytosis, the study adds to the growing catalog of known disease-causing variants in this gene, which may help other clinicians recognize similar presentations in their own patients. The co-inheritance scenario also serves as a cautionary tale for diagnostic medicine: when a patient presents with anemia that does not fit neatly into a single diagnostic category, clinicians should consider the possibility that multiple genetic disorders are operating concurrently. The case underscores the diagnostic utility of combining traditional morphological assessment with modern molecular techniques, a synergy that is increasingly essential as the complexity of inherited blood disorders becomes better appreciated.

Funding for this work was partially provided by the Lishui Science and Technology Bureau Project under Grant No. 2023GYX49. The research was conducted in accordance with the ethical standards of institutional and national research committees and the 1964 Helsinki Declaration and its later amendments. Informed consent was obtained from all individual participants involved in the study, and the authors declared no competing interests. The article is published open access under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, ensuring that the findings are freely available to clinicians, researchers, and patients worldwide who may benefit from this detailed molecular characterization of a rare dual diagnosis.

Subject of Research: Co-inheritance of β-thalassemia and EPB41-associated hereditary elliptocytosis in a Chinese family

Article Title: Co-inherited β-thalassemia and hereditary elliptocytosis associated with EPB41 mutations in a Chinese family: a case report

Article References: Co-inherited β-thalassemia and hereditary elliptocytosis associated with EPB41 mutations in a Chinese family: a case report. (n.d.). https://doi.org/10.1007/s00277-026-07322-4

Image Credits: AI Generated

DOI: 10.1007/s00277-026-07322-4

Keywords: β-thalassemia, hereditary elliptocytosis, EPB41, HBB, genetic sequencing, red blood cell membrane, hemolytic anemia, whole-exome sequencing, ACMG guidelines, co-inheritance, case report, molecular diagnosis