Unlocking Rare Diagnoses: The Essential Role of Molecular Techniques in Hemoglobin Agrinio. A Case Report

ABSTRACT

Alpha thalassemia is characterized by the reduced or absent synthesis of alpha globin chains, most commonly due to deletional mutations. Hb Agrinio [α29(B10) Leu→Pro, CTG>CCG (α2)] is an infrequent nondeletional alpha thalassemia (α‐Thal) that has been described in less than 20 individuals in the world. We presented the case of a girl with anemia since birth and high transfusion needs, with a recent diagnosis of homozygous Hb Agrinio.

Abbreviations

α Thal

alpha thalassemia

G6PD

glucose‐6‐phosphate dehydrogenase

Hb

hemoglobin

HPLC

high‐performance liquid chromatography

HSCT

hematopoietic stem cell transplant

ICU

intensive care unit

US

ultrasound

1. Introduction

Hemoglobin Agrinio is a hyper‐unstable structural variant of the alpha chain, where the substitution of leucine with proline at the 29th amino acid of the B helix of the alpha‐2 chain [α29 (B10) Leu→Pro, CTG>CCG (α2)] conditions its post‐translational precipitation in erythroid precursors. Few cases have been described, and although conclusive data on the associated thalassemic phenotype and long‐term repercussions are limited, it is believed to behave like thalassemia intermedia, with moderate transfusion dependence [1, 2, 3, 4].

2. Methods

Hematological data were obtained using electrical impedance in an automated cell counter (UniCel DxH 800 Coulter). Hemoglobin analysis was performed via capillary electrophoresis (Minicap CDT and Sebia Capillarys 2 Flex Piercing) and quantified by ion‐exchange high‐performance liquid chromatography (HPLC) (VARIANT; Bio‐Rad). Glucose‐6‐phosphate dehydrogenase (G6PD) enzyme activity was measured using a spectrophotometric assay, while the direct antiglobulin test was conducted via the column agglutination method.

Genomic DNA was automatically extracted (Biorobot EZ1; Qiagen), and common mutations were screened using α‐globin StripAssay. Molecular characterization was performed through automatic sequencing (ABI PRISM 3100 Genetic Analyzer). The α2‐globin gene was amplified using specific primers and sequenced with the ABI PRISM BigDye Kit V1.1. following the protocol previously described [1]. All hematological data and clinical findings were collected with prior informed consent from the patient.

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3. Case Description

Neonatal with hemolytic anemia at birth was admitted to the intensive care unit (ICU) with 67 g/L of hemoglobin (Hb), respiratory distress, and significant paleness. Prenatal testing only showed a mild pericardial effusion and cardiomegaly at the third trimester ultrasound (US). Instrument‐assisted vaginal delivery proceeded without incidents.

In the complementary tests carried out during her admission, the following stand out: absence of organomegaly in US, moderate pulmonary hypertension 48 h after birth, with subsequent correction, but persistence of a patent foramen ovale. Toxic, secondary infectious diseases and Rh/AB0 incompatibility were ruled out.

At 16 days of life, she was discharged with an Hb of 103 g/L, after the transfusion of packed red blood cells, with supportive oral iron therapy as home treatment.

One month later, she was readmitted for study after finding an Hb of 62 g/L and clinical worsening. The neonatal screening was inconclusive, and a confirmatory test showed the presence of 2.4% of Hb Bart (Figure 1).

FIGURE 1.

On the left: Electropherogram of hemoglobin obtained by capillary electrophoresis using the Sebia system. The main peak corresponds to Hb A (77.1%), followed by a suspected Hb F or variant (19.0%), and Hb Bart (2.4%). A small Hb A2 fraction is also present (1.5%). On the right: DNA sequencing chromatograms showing the Hb Agrinio variant. The mutation (CTG>CAG) is identified in homozygosis in the proband and in heterozygosis in other family members.

At 2 months of life, she needed a new hospital admission because of rejection of feeds. In the analysis: Hb 67 g/L, VCM 70.3 fL with normal iron metabolism. Peripheral blood smear showed striking anisopoikilocytosis, presence of abundant erythroblasts, variability in terms of hemoglobinization with the often presence of very gross basophilic stippling.

Given the suspicion of possible congenital hemolytic anemia despite the normality of the complementary tests carried out, a sample was sent for genetic sequencing to the reference center (Hospital Clínico San Carlos). After their analysis, they concluded that the patient was a homozygous carrier of the CTG>CCG mutation in codon 29 of the first exon of the alpha 2 gene, which determines the change from leucine to proline at position 10 of the B helix of the alpha chain (Figure 1).

At the age of 2 years, the patient was slightly underweight and presented with microcephaly but exhibited no significant symptoms of anemia, despite a persistent moderate transfusion dependence.

At 2 years and 2 months of age, the patient underwent an allogeneic hematopoietic stem cell transplant (HSCT) using bone marrow from an HLA‐identical sibling donor. The conditioning regimen included thiotepa, fludarabine, treosulfan, and thymoglobulin. Neutrophil engraftment occurred on day +16, and platelet engraftment on day +14, with full donor chimerism established within the first month. The patient developed grade 2 cutaneous graft‐versus‐host disease, which resolved completely.

Currently, at +2 years post‐HSCT, the patient remains disease‐free, off immunosuppression, and without any significant toxicities.

4. Discussion

Hemoglobin (Hb) Agrinio represents a rare structural hemoglobinopathy that underscores the vital role of molecular diagnostic techniques. In this case, dependence on these techniques is not merely a convenience but a necessity, particularly for identifying rare conditions that traditional diagnostic methods may miss. Progress in molecular tools such as high‐throughput sequencing has allowed clinicians to detect mutations with precision, paving the way for diagnoses that were unattainable in the past.

The diagnosis of Hb Agrinio, while lacking a specific treatment, could become a cornerstone for broader family health management: it allows for comprehensive family screening (Figure 2) (which is particularly significant in regions with a high risk of consanguinity, where genetic disorders may be more prevalent) and prenatal counselling becomes possible (helping parents understand their risks and make informed decisions). Identifying carriers provides critical information, enabling affected families to make informed reproductive decisions. Prenatal advice becomes a particularly valuable intervention, potentially reducing the occurrence of severe hemoglobinopathies in future generations [3, 4].

FIGURE 2.

Pedigree of the family with Hb Agrinio. The proband (index case) is homozygous for the Hb Agrinio variant. Heterozygous carriers are indicated, and consanguinity (first cousins) is noted. Some individuals were not available for genetic testing (? = no data).

Moreover, the diagnosis has significant therapeutic implications, even in the absence of direct treatment for the condition itself. HSCT emerges as the only available curative option, highlighting the importance of accurate diagnostic and genetic information. The integration of molecular diagnostics and human leukocyte antigen (HLA) typing enables the selection of the most suitable stem cell donor, a critical factor in the success of HSCT. This comprehensive approach helps minimize transplant‐related complications and improves post‐transplant outcomes, demonstrating how the diagnosis of a rare condition can directly influence therapeutic strategies and patient care.

Equally important is the role of international data sharing to advance the understanding and management of rare disorders such as Hb Agrinio and indispensable for generating strong scientific evidence. In contrast to the series reported by Szepetowski et al. [2], our patient survived the neonatal period despite presenting with severe anemia and transfusion dependence from birth. The absence of antenatal findings such as hydrops fetalis and low birth weight and the ability to reach 2 years of age and undergo a successful allogeneic HSCT define a mild phenotype, suggesting a broader variability in disease expression. This discrepancy may suggest that additional genetic or environmental modifiers may influence disease severity.

Our report expands the clinical spectrum of homozygous Hb Agrinio and highlights the need for individualized and evidence‐based assessment and follow‐up.

Author Contributions

M.M.C. conceived the study, participated in the initial follow‐up, and drafted the manuscript. A.G.M. and S.F.P.M. contributed to data collection. M.G.M. and E.C. were responsible for the transplant. C.R.V. managed the clinical follow‐up from a pediatric perspective, both before and after transplant. P.R. performed the molecular studies. Á.M. and V.R. contributed to patient follow‐up, transfusion support, and coordination between centers for transplant and genetic studies. All authors critically revised the manuscript, approved the final version, and agree to be accountable for all aspects of the work.

Ethics Statement

Ethical approval was not required for this case report in accordance with the policies of the institutions. Written informed consent for publication was obtained from the patient's parents.

Conflicts of Interest

The authors declare no conflicts of interest.

Moreno Carbonell M., Martínez A. G., Mariño S. F. P., et al., “Unlocking Rare Diagnoses: The Essential Role of Molecular Techniques in Hemoglobin Agrinio. A Case Report,” European Journal of Haematology 117, no. 3 (2026): 701–704, 10.1111/ejh.70050.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.