Molecular Characterization of Hemoglobin H Disease in Spain

Introduction: Alpha-thalassemia is an autosomal recessive inherited disorder in which there is a defect in the synthesis of the α globin chain. Double heterozygosity for α⁺-thalassemia and α⁰-thalassemia results in HbH disease, which is associated with anemia, microcytosis, chronic hemolysis, and sometimes splenomegaly.

The molecular basis of α-thalassemia is usually due in 90% of cases to deletions and less frequently to point mutations affecting the expression of one or more of the α duplicated genes.

Objective: Molecular characterization and registry of the cases of HbH disease detected in Spain in the last 20 months.

Material and Methods: Between November 2022 and July 2024, 29 individuals from different Spanish regions, including native and migrant populations, were studied. Conventional hemocytometric analysis, determination of HbA2, HbF, and HbH by ion exchange HPLC, and quantification of HbH inclusion bodies were performed. Molecular diagnosis included multiplex PCR with hybridization using the α-globin StripAssay® method, Multiplex Ligation dependent Probe Amplification (MLPA), and automated Sanger sequencing.

Results: Seventeen women (58.6%) and 12 men (41.4%) comprised the sample, with a mean age of 37.1 years. Of the total, 11 (37.9%) were Spanish, and 18 (62.1%) were immigrants, 94.4% of the latter being Asian, mainly Chinese (72.2%), 4 Filipinos (13.8%), and 1 Maghrebi (5.5%). No splenectomies were recorded. All patients present with variable moderate anemia, with hemoglobin levels between 7.6 and 13.4 g/dL, accompanied by microcytosis (MCV = 61.52 ± 7.79) and hypochromia (MCH = 18.59 ± 2.70).

Reticulocytes are increased as a compensatory response to anemia, with inclusion bodies detected in 93.2% of the patients. The diagnosis is confirmed by the presence of HbH (1-12%).

Of the patients, 69% did not receive transfusions, 27.6% received them occasionally due to pregnancy, infection, or surgery, and only one (3.4%) needed them in the last year.

The most common genotype is -α³.⁷/–SEA, present in 8 patients (29.6% of the total), followed by -α³.⁷/–FIL, with 6 patients (22.2% of the total), and finally the genotypes -α³.⁷/ααPolyA and ααPolyA/ααPolyA in 2 patients each (7.4% of the total). There is a notable diversity of alpha genotypes, with several unique genotypes present in only one patient each. The distribution of genotypes according to ethnicity varies. Among Spanish, no clearly dominant genotype exists, although the most common are -α³.⁷/–SEA and -α³.⁷/ααPolyA. There is significant genotype diversity, including less frequent combinations such as ααHbAgrinio/ααHbAgrinio and -α³.⁷/– del13.5kb. Among Asians, the -α³.⁷/–SEA genotype is the most predominant (8 patients), followed by -α³.⁷/–FIL (6 patients). The distribution is less diverse compared to the Spanish group, with only 4 distinct genotypes identified in this group.

Conclusions: The results show a predominance of diagnoses in females, possibly due to the combination of anemia inherent to HbH disease and menstrual losses, exacerbating the clinical picture and leading to a greater search for medical attention in this group. In addition, most patients are immigrants, especially Asians, due to the high incidence of α-thalassemia in this area.

Genotyping reveals that the most common mutation, regardless of ethnicity, is the 3.7Kb deletion. In Asians, this deletion is inherited along with α^0-thalassemia (SEA or FIL), while in the Spanish population, there is a greater variety of mutations, including both deletions and point mutations. These genetic findings provide a deeper understanding of the etiology of the disease in different populations.

FUNDING: Agios Pharmaceuticals

CONFLICT OF INTEREST: The authors declare that they have no conflict of interest

Villegas:Agios: Consultancy.