# A new unstable haemoglobin, Hb Alger, causes a transfusion-dependent anaemia in early childhood

Syrine Bouazizi, Mathieu Cerino, Suzy Lim, Camille Desgrouas, Catherine Badens, Sarah Szepetowski, Nathalie Bonello-palot

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Syrine Bouazizi, Mathieu Cerino, Suzy Lim, Camille Desgrouas, Catherine Badens, et al. A new unstable haemoglobin, Hb Alger, causes a transfusion-dependent anaemia in early childhood. 2025, pp.1716 - 1718. 10.1111/bjh.70040. hal-05525483

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# A new unstable haemoglobin, Hb Alger, causes a transfusion-dependent anaemia in early childhood

To the Editor,

Unstable haemoglobins are a group of pathological haemo-
globin variants resulting from genetic variants affecting
the genes encoding the globin chains (HBA1 and HBA2 for
α chains and HBB for β chains). These variants, generally
single nucleotide variations with autosomal dominant in-
heritance, alter the structure of haemoglobin, compromis-
ing its stability and biological function. These structural
abnormalities lead to reduced solubility of the haemoglobin
molecule and increased susceptibility to oxidation, resulting
in the formation of intracellular protein precipitates known
as Heinz bodies. These inclusions disrupt the integrity of the
erythrocyte membrane, causing increased rigidity, fragil-
ity and premature destruction of red blood cells by the re-
ticuloendothelial system, particularly in the spleen. In most
cases, these processes result in chronic haemolytic anaemia,
the severity of which varies depending on the degree of in-
stability of the abnormal haemoglobin.¹

Unstable haemoglobin can result from various genetic
defects, such as missense variants, premature stop codons,
deletions, insertions or frameshift variants, which can lead
to elongated or truncated globin chains.²

In this study, we report the case of a 5-year-old Algerian patient referred for diagnosis and management of transfusion-dependent anaemia. All protocols performed in this study complied with the ethics guidelines of the institutions involved.

The patient, from Algeria, was the third child of a non-
consanguineous couple. The mother had a history of three
miscarriages. At birth, the patient's height and weight were
within normal ranges. He developed neonatal jaundice re-
quiring phototherapy. At 2 months of age, he presented
with severe anaemia (haemoglobin 3 g/dL) and has required
monthly transfusions since then. At 18 months, his total bil-
irubin was 102.6 µmol/L, haptoglobin was <0.8 µmol/L and
Lactate Dehydrogenase was 5300 nkat/L. A bone marrow ex-
amination performed at 1 year showed erythroblastic hyper-
plasia and dyserythropoiesis, with no evidence of vitamin
B9 or B12 deficiency. Due to severe splenomegaly, a splenec-
tomy was performed at age of 4.

Upon his arrival in France at age of 5, the patient showed
signs of significant iron overload, prompting the initiation of

chelation therapy. He was treated with Exjade (450 mg/day) and Desferal (1400 mg, 5 days per week). The most recent fer-ritin level, measured in April 2025, was 673 pmol/L. Cardiac magnetic resonance imaging (MRI) revealed no abnormali-ties, while liver MRI showed significant iron accumulation.

Biochemical analysis of haemoglobin detected no anom-
alies, even when performed just prior to a transfusion, which
was surprising because a diagnosis of thalassaemia was first
suspected. Brilliant blue cresyl staining (methylene blue 1%)
revealed Heinz bodies immediately, and after 1 and 2 h of
incubation at 37°C (Figure 1). After informed consent was
obtained from all individuals included in this study, molecu-
lar analysis identified a de novo heterozygous HBB c.193G>T
(p.Gly65Cys) variant, which was not present in either par-
ent. This variant is absent from the Genome Aggregation
database and is predicted to be pathogenic by multiple bio-
informatics tools. Whole Exome Sequencing revealed no
additional anomalies. Structural modelling on the Mutation
Explorer webserver (https://proteininformatics.uni-leipzig.de/
mutation_explorer/) estimated that the total energy of wild-
type haemoglobin was –377 and was increased to +4 for the
mutant haemoglobin (p.Gly65Cys) (Figure 2).

The c.193G>T variant is in a region of the *HBB* gene known to be associated with haemoglobin instability. Notably, three other variants have been reported at codon 65: c.194G>A (p.Gly65Asp),³ c.194G>C (p.Gly65Ala)⁴ and c.194G>T (p.Gly65Val).⁵ These variants all affect the glycine residue at position 65 a highly conserved amino acid that plays a crucial role in maintaining the structural integrity

<figure><img src="image_2.png" /><figcaption>FIGURE 1 Visualisation of Heinz bodies in index case's blood film.</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>FIGURE 2 Mutation Explorer performed the mutation and calculated the energy of the structure (PDB: 7XGY) as the sum of energies computed for each residue. The red circle indicated the mutated residue and the difference colouring showed the mutant (mutated result structure) minus the wild type (WT) (parent structure) energy for each residue (with negative values in blue and positive values in red). It estimated that the total energy of wild-type haemoglobin was -377 and was increased to +4 for the mutant haemoglobin (p.Gly65Cys).</figcaption></figure>

<table><caption>TABLE 1 The different reported variants in codon 65 linked to unstable haemoglobin.</caption><thead><tr><td>Hb variants</td><td>Hb name</td><td>Clinical symptoms</td><td>Hb level (g/dL)</td><td>Transfusion regimen</td><td>Total energy of haemoglobin</td><td>References</td></tr></thead><tbody><tr><td>c.194G&gt;A (p.Gly65Asp)</td><td>Hb J-calabria</td><td>Completely asymptomatic</td><td>Hb ≈ 10</td><td>Rare</td><td>-25</td><td>3</td></tr><tr><td>c.194G&gt;T (p.Gly65Val)</td><td>Hb Calgary</td><td>Severe haemolytic anaemia and dyserythropoiesis</td><td>Hb = 7.6</td><td>Monthly</td><td>+398</td><td>5</td></tr><tr><td>c.194G&gt;C (p.Gly65Ala)</td><td>Hb Aubagne</td><td>Persistent anaemia during pregnancy</td><td>Hb = 8.5</td><td>Rare</td><td>-297</td><td>4</td></tr><tr><td>c.193G&gt;T (p.Gly65Cys)</td><td>Hb Alger</td><td>Severe anaemia since the age of 2 months, splenomegaly</td><td>Hb = 6</td><td>Monthly</td><td>+4</td><td>Our study</td></tr></tbody></table>

Abbreviation: Hb, haemoglobin.

of haemoglobin. A point is that de novo mutations occur most commonly at CpG dinucleotides, higher by a factor of ~18% than other dinucleotides, particularly at glycine and arginine, which account for ~40% of these nonsynonymous mutations, due to spontaneous deamination of methylated cytosine to thymine.⁶ In our case, substitution of this glycine alters the protein's conformation and induces instability, potentially leading to various clinical outcomes depending on the nature of the amino acid substitution.¹ This suggests a correlation between the biochemical properties of the substituted residue and the severity of the disease.⁷

The p.Gly65Asp and p.Gly65Ala variants are generally associated with moderate anaemia, rarely requiring transfusions.

The p.Gly65Asp variant, known as haemoglobin J-Calabria, is found in individuals with haemoglobin levels around 10 g/dL and minimal need for transfusions. The substitution introduces a negatively charged carboxyl group, which may interfere with electrostatic interactions in the haemoglobin molecule. However, the slight size difference

between aspartate and glycine likely limits the overall destabilising effect.³

The p.Gly65Ala variant, known as haemoglobin Aubagne, has been observed in a case of persistent anaemia during a second pregnancy, with haemoglobin levels around 8.5 g/dL. Transfusion needs are also minimal. In this case, glycine is replaced by alanine, a small yet hydrophobic residue. While less disruptive than aspartate, alanine may still affect protein packing and reduce haemoglobin stability.⁴

In contrast, the p.Gly65Val variant, known as haemoglobin Calgary, leads to severe haemolytic anaemia and dyserythropoiesis, with haemoglobin levels around 7.6 g/dL and monthly transfusion requirements. The replacement of glycine with valine, a larger, more hydrophobic amino acid, significantly disrupts the hydrophobic core and spatial structure of the haemoglobin molecule, contributing to its instability and the severity of clinical symptoms.⁷

In our case, glycine is replaced by cysteine, a larger, polar amino acid featuring a reactive thiol (–SH) group capable of
forming disulphide bonds. This chemical property can dra-
matically alter haemoglobin's three-dimensional structure,
potentially causing greater instability than previously re-
ported substitutions. Depending on its location and context
within the protein, cysteine may interfere with proper te-
tram er formation or promote aberrant disulphide linkages.
These disruptions can increase the molecule's susceptibility
to degradation or aggregation, thereby exacerbating haemo-
lysis and anaemia.⁸

Computational modelling revealed increased energy lev-
els for all four mutated haemoglobins compared to the wild-
type form, the most symptomatic variants exhibiting the
highest differences with the wild-type haemoglobin. Since
lower energy values indicate greater structural stability, this
significant energy increase suggests a substantial decrease in
haemoglobin stability (Table 1).

We have identified and characterised a novel variant in
the *HBB* gene, named Haemoglobin Alger, which results in
severe anaemia with both haemolytic and dyserythropoietic
features, necessitating a regular transfusion regimen in the
first year of life.

In this particular case, molecular analysis has thus
proved to be a crucial step in both diagnosis and therapeutic
management since it was instrumental not only in clarifying
the aetiology of the anaemia but also in enabling the con-
sideration of a curative treatment by haematopoietic stem
cell transplantation, the patient's father being a compatible
donor.

AUTHOR CONTRIBUTIONS

SB performed the research. SS and CB designed the research
study. MC and SL contributed essential reagents or tools. CD
estimated the total energy of the different haemoglobins.
NB-P analysed the data; SS and NB-P wrote the paper.

FUNDING INFORMATION

This research work was not supported.

KEYWORDS

haemoglobin instability, haemoglobinopathy, *HBB* gene,
paediatric anaemia

CONFLICT OF INTEREST STATEMENT

The authors report no competing interests.

Syrine Bouazizi¹ ID

Mathieu Cerino¹,²

Suzy Lim²

Camille Desgrouas³

Catherine Badens¹,²,³ ID

Sarah Szepetowski⁴

Nathalie Bonello-Palot¹

¹APHM, Molecular Genetics Laboratory, M2GM,
Biogenopole, Hôpital de la Timone Marseille,
Marseille, France

²APHM, Biochemistry Department, Biogenopole,
Hôpital de la Timone, Marseille, France

³Aix Marseille Univ, INSERM, C2VN, Marseille,
France

⁴AP-HM, Pediatric Hematology Department, Timone
Children's Hospital, Marseille, France

Correspondence

Syrine Bouazizi, APHM, Molecular Genetics
Laboratory, M2GM, Biogenopole, Hôpital de la
Timone Marseille, Marseille, France.

Email: syrine.bouazizi@ap-hm.fr

ORCID

Syrine Bouazizi ID https://orcid.org/0009-0001-7662-2784

Catherine Badens ID https://orcid.org/0000-0001-9024-310X

REFERENCES

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