Hemoglobinopathies represent the most prevalent monogenic disorders, characterized by anomalies in globin gene expression leading to the synthesis of structurally abnormal globin chains or an insufficient production of globin chains, causing thalassemia.1 Often, the discovery of hemoglobin (Hb) variants occurs serendipitously during the course of thalassemia and hemoglobinopathy control and prevention efforts. Advancements in Hb separation techniques, allow for finer distinctions between rarer variants and Hb A or Hb A2, which along with improvements in molecular diagnostic tools, have led to the identification of an increasing number of Hb variants. The HbVar database has recorded approximately 1420 human Hb variants to date.2 The clinical impact of Hb variants can range from clinically silent to symptomatic expression, contingent upon the specific mutation site. Within the group of pathogenic Hb variants, clinically relevant outcomes arise due to mutations that alter the intersubunit contacts of α1β1 or α1β2. These modifications confer instability upon the α-globin chain, making it prone to denaturation, proteolytic degradation, and membrane binding, triggering hemolysis.3,4 Mutations can disrupt structural elements proximal to the heme group, destabilizing heme interactions and directly affecting oxygen binding.5 While many Hb variants exhibit limited clinical significance and are linked to normal red blood cell (RBC) parameters, particularly in heterozygotes, there exists variants that induce mild to severe α+-thalassemia phenotypes and serous clinical symptoms. These variants are often associated with deletional α-thalassemia, Hb Constant Spring, and Hb Paksé, which are prevalent in Southeast Asia.6,7 Moreover, rare unstable variants, such as Hb-Quong Sze and Hb-Suan-Dok, which follow analogous mechanisms, contribute to mild to severe Hb H disease within different populations.7-15

However, a notable challenge arises in diagnosing unstable Hb variants, as they can evade detection due to their asymptomatic or milder phenotypes. Moreover, they might be misdiagnosed since their electrophoretic mobility matches that of normal structurally intact Hb. Employing a combination of methods, such as Hb high-performance liquid chromatography (HPLC), Hb electrophoresis, Heinz body assessment, and tests evaluating heat and isopropanol stability, emerges as a strategy to reveal these elusive variants. While most abnormal Hbs constitute non-pathological variants, a subset of them can cause clinical symptoms associated with hemolytic anemia. Consequently, precise identification and differential diagnosis of these variants is essential. In this study, the clinical and molecular characterization of a rare unstable Hb variant in a Thai patient, who presented with an unexplained moderate anemia, is described.

This case identified and presented evidence supporting the pathogenicity of a rare α-thalassemic Hb variant, namely Hb Jax [HBA2:c.44G>C] in a Thai patient with unexplained chronic anemia but without any history of hepatosplenomegaly. This variant is defined by a transition from TGG>TCG at codon 14 of the α2-globin gene, leading to the substitution of tryptophan with serine. The mobility behavior of Hb Jax (αJax2βA2) in HPLC is expectedly distinct from that of Hb A. Notably, Hb Jax elutes more slowly than Hb A. This divergence is critical because it ensures the complete separation of Hb Jax from Hb A using the HPLC-Variant-II system. This capability permits the accurate estimation of Hb Jax levels. Furthermore, the derivative Hb Jax (Hb A2-Jax), resulting from the assembly of two αJax and two δ chains into tetramers, is also entirely separated from Hb A2 and quantifiable via this system. Conversely, when subjected to analysis by the Premier-Resolution HPLC system, Hb Jax demonstrates a marginally faster elution than Hb A. Consequently, this system only partially separates Hb Jax from Hb A, making it challenging to accurately estimate their respective quantities when compared with the HPLC variant-II system.

Additionally, an eluted fraction of Hb A2-Jax was indiscernible, suggesting the possibility of co-elution with Hb A2. While the chromatograms of the patient displayed excellent resolution between normal Hb and the mutant Hb, the chromatogram of the heterozygote did not exhibit any noticeable peak, indicating that co-presence with other α-thalassemia might be necessary for visibility via chromatographic techniques. One plausible explanation for this divergence lies in the substantial formation of the Hb Jax tetramer in conjunction with α0-thalassemia. In this scenario, the limitation of α-globin production leads to a more pronounced expression of the αJax chain, owing to an affected mutation on the α2-globin gene, compared with the αA chain derived from the α1-globin gene. This accelerated expression facilitates the rapid incorporation of excess unbound β-chains into the newly synthesized αJax2βA2 tetramer. The use of the CE technique for Hb analysis resulted in indiscernible anomalous Hb peaks in both heterozygotes and compound heterozygotes with α0-thalassemia. This was suggestive of Hb Jax and its derivative co-migrating with Hb A and Hb A2. This phenomenon might be attributed to the substitution of the amino acid serine, an uncharged amino acid similar to tryptophan. Consequently, the net charge within the Hb tetramers remains unaltered. It is evident that reliance solely on the CE technique for Hb separation can lead to the misidentification of Hb Jax and other variants with physicochemical similarities.

The hydrophobic tryptophan residue, occupying position 14, is situated within a helical A segment of the α-chain. This location rests on the internal surface of the tetramer and does not engage in intersubunit contacts, including α1β1 or α1β2, nor does it interact directly with the heme group.5,23,24 Despite its apparent seclusion, this region holds a pivotal role as a highly conserved hydrophobic residue.24-26 The substitution of this vital position in Hb Jax with a polar serine residue, commonly found on the molecule's external surface, introduces a heightened degree of hydrophilicity to this region. Consequently, a noticeable transformation occurs in the physicochemical attributes of the Hb Jax tetramer, ultimately influencing its molecular stability. Bioinformatic predictions support this observation, proposing that Hb Jax's physiochemical disparities between tryptophan and serine render it potentially damaging and deleterious.27 Furthermore, a reduced molecular stability of this variant was successfully demonstrated using isopropanol stability testing. This is evidenced by the gradual increase in flocculent precipitate over time, indicating the minor instability of Hb Jax.

An intriguing feature of Hb Jax is the absence of detectable Hb H (β4 tetramers) despite employing CE and HPLC techniques for Hb separation. This is remarkable because the patient possesses only two α-globin genes, one of which carries the TGG>TCG mutation (αJax). However, her phenotype and elevated reticulocyte count are notably similar to individuals with Hb H.28-30 Based on this observation, we infer that the increased imbalance in co-inheritance with the deletion defects did not lead to a substantial overproduction of β-globin chains, thus preventing the significant formation of β4 tetramers in peripheral RBCs. This suggests that the Hb Jax mutation only marginally impacts the synthesis of the αJax chain. However, in Hb Bart's, the accumulation of excess γ-globin chains produced in this patient was consistently observed through CE and HPLC techniques. Notably, the quantified percentage of Hb Jax using HPLC in the patient is less than 25% of the total circulating Hb, which is lower than the expression level of the functional α2 globin gene.31 Chemical evidence for supporting the variant's instability was established by the positive results from the test for inclusion bodies with brilliant cresyl blue and the positive outcome of the isopropanol tests for unstable Hb. These findings align with the low levels of the variant, leading us to conclude that the significant reduction in Hb Jax levels in this context results from denaturation and proteolytic degradation of Hb Jax tetramers rather than a reduction in synthesis due to mRNA instability. Consequently, we consider Hb Jax to be a slightly unstable Hb variant equivalent to α+-thalassemia.

The phenotypic expression in the heterozygosity of Hb Jax observed in the daughter of the patient is greatly compatible with individuals displaying a typical α+-thalassemia trait. This combination presents iron deficiency and more abnormalities in RBC parameters compared with those observed in individuals with the α+-thalassemia trait alone. The exact phenotype influenced by the mutant gene will be elucidated after their ferritin levels are improved.32 Although we have not provided information about another individual with pure heterozygous Hb Jax, it is reasonable to posit that the phenotypic expression of a person with a pure heterozygote of this variant would be highly similar to that of an individual with the α+-thalassemia trait. The severe clinical phenotype observed in the patient can be attributed directly to the co-inheritance of two α-globin gene deletions in trans, as there is no evidence suggesting the presence of iron deficiency or other causes of microcytic anemia in the patient. The frequency of Hb Jax in the Thai population remains unknown through routine Hb separation alone due to the technological limitations for Hb analysis that fails to virtually detect the Hb Jax fraction in heterozygotes. DNA level analysis is required to accurately determine the prevalence of this variant, particularly in a population with a high prevalence of α-thalassemia determinants. Such investigations would prove valuable for genetic counseling. Additionally, our patient had moderate anemia, marked microcytosis, and hypochromia. The analysis of common α+-thalassemia genes yielded negative results, underscoring the necessity to investigate this mutation in the α-globin gene, which may interact with other forms of thalassemia or hemoglobinopathies.

A mutation at codon 14 of α2-globin has been reported in Hb Evanston [HBA2:c.[43T>A (HBA1) or 43T>C (HBA1)].33 The relatively unstable Hb Evanston is associated with defective tetramer stability and globin chain synthesis as the αEvanston gene exists on a chromosome cis to a -α3.7gene. When linked with thalassemia, this variant contributes to an α-thalassemia-like phenotype, potentially leading to Hb H when co-inherited with another α-thalassemia defect,34 as observed in our patient. The Hb Evanston is virtually undetectable in heterozygous individuals using CE and HPLC techniques.35 In contrast, its combination with an additional α-thalassemia deletion gene allows for more readily detectable quantities of Hb Evanston, approximately 1–2% in association with the leftward deletion in trans and 6–10% in association with two α-globin gene deletions.34,35 The findings in the Hb Evanston and Hb Jax emphasize that the Hb variant tetramer resulting from the affected α2-globin gene mutation on codon 14 is relatively unstable, generating the α-thalassemia-like phenotype.

Analyzing the α-globin haplotype provides a better understanding of the origins and spread of this variant within the population. The α-haplotype associated with this variant is similar to that found in Hb Prato, documented in a Thai patient,15 indicating a shared evolutionary origin stemming from this ancestry.

Hb Jax is a rarely identified α-globin variant resulting from the substitution of tryptophan with serine at codon 14 of the α2-globin gene. It displays susceptibility to tetramer instability. In a heterozygous state, it manifests as marginally abnormal hematologic findings similar to α+-thalassemia. However, when combined with an α0-thalassemia gene in trans, it assumes a clinically significant role, resembling an Hb H-like phenotype. Notably, the mutation remains cryptic on a wild-type allele but becomes visible through chromatographic techniques when associated with other α-thalassemic defects. These findings represent the first description of this variant in published literature. The developed allele-specific PCR approach improved the accuracy and speed of diagnosing this variant, with potential applications for comprehensively studying its prevalence in regions with high incidence of common α-thalassemia determinants.

SP performed study concept and design, development of methodology and writing, review and revision of the paper; KJ, PK, PN were involved in analysis and interpretation of data; SS was involved in analysis and interpretation of data, and statistical analysis. All authors read and approved the final paper.

This work was supported by the Thailand Science Research and Innovation Fund and the University of Phayao, Thailand (Grant number FF65-RIM135).

The data presented in this study are available upon request from the corresponding author.