Combined heterozygosity for hemoglobin Paksé, α-thalassemia and for hemoglobin E, β- thalassemia - first appearance in Europe

Abstract

Hemoglobinopathies are among the most common inherited diseases and they are believed to be one of the major etiologic factors contributing to anemia. Thalassemia is characterized by an altered hemoglobin (Hb) chain synthesis and may appear as alpha-(α-)thalassemia and/or beta-(β-)thalassemia. The clinical manifestations of thalassemia may range from asymptomatic to severe, with the potential to ultimately result in death. The search for an underlying cause was prompted by the discovery of an asymptomatic patient of Thai origin with microcytic anemia and no iron deficiency during a routine hematological examination. Genetic sequencing results revealed Hb Paksé, α2 CD 142 [A > T] (HBA2:c.429 A > T) and HbE β2 CD 26 [G > A] (HBB: c.79 G > A). It is essential that the identification and knowledge of Hb mutations facilitate the clinical recognition, genetic testing, and counseling of patients with thalassemia. The combination of two thalassemia Hb variants, one in the α-globin gene as Hb Paksé and one in β-globin gene as Hb E, in a single individual is occasionally described in East Asia. The first appearance of combined heterozygosity for Hb Paksé, and Hb E in Europe provides evidence that this is the result of a migration-caused occurrence.

Supplementary Information

The online version contains supplementary material available at 10.1007/s00277-025-06286-1.

Background

Hemoglobinopathies are classified into two categories: the hemoglobin (Hb) variants and the thalassemia syndromes. They represent some of the most prevalent inherited disorders globally, with a substantial impact on mortality and morbidity [1]. Thalassemia is an autosomal recessive disorder caused by a defect in the synthesis of Hb chains. According to the type of affected globin chain, it can be divided into two types: α-thalassemia and β-thalassemia. These Hb variants are caused by changes in the formation of the globin chains and are more common in regions with a high risk of malaria infection [2]. They are regarded as a significant contributor to anemia [3]. Homozygote, compound heterozygote or combinations of hemoglobinopathies may result in mild to severe phenotypes, including premature death. It is not necessary to provide treatment to asymptomatic, heterozygous carriers of α-thalassemia or individuals with the β-thalassemia trait [4]. Thalassemia is a prevalent condition, affecting up to 50% or more of the population in certain endemic areas of East Asia. Hb E (HBB: c79 G > A) is one of the more common Hb thalassemia variants [5]. Hb Paksé (HBA2:c.429 A > T), initially identified in Laos [6], is characterized as a rare form of thalassemia and has been observed to occur occasionally in co-inherited heterozygosity with a few other Hb mutations in East Asia. Due to travel and migration of individuals with hemoglobinopathies, there has been a notable increase in the number of individuals relocating to non-endemic Northern Europe [7]. Moreover, this manuscript offers insights by presenting the detected Hb E variant in combination with a heterozygous Hb Paksé, thereby enhancing comprehension of compound genetic variants and their associated phenotypes.

We present a case of an individual from northeastern Thailand, now residing in Austria, who carries two Hb thalassemia variants in combined heterozygosity: Hb Paksé in the α-globin gene and Hb E in the β-globin gene. This heterozygous combination of Hb Paksé and Hb E has been identified for the first time in Europe.

Case presentation

A 40-year-old female patient, originally from the province of Buriram in the northeastern region of Thailand, presented with elevated blood pressure (150/100 mmHg). A comprehensive physical examination, including a gynecological examination and abdominal ultrasound, did not reveal any abnormalities, including a normal-sized spleen.

The diagnosis of microcytic anemia was established using MS4Se (Melet Schloesing Laboratoires GmbH C&E, Vienna, Europe), which revealed a total Hb of 9.8 g/dL (normal 12.1–17.2), a hematocrit of 32.5% (range 36.1–50.1), a mean corpuscular volume of 73.7 fL (normal 82.2–97.4) and a mean corpuscular hemoglobin of 22.1.9 pg (normal 27-33.3). The red blood cell distribution width, red blood cell count, corpuscular hemoglobin concentration, white blood cells, platelet count, serum ion, ferritin, total transferrin, total ion and transferrin binding capacity were all within the normal range.

In order to ascertain the etiology of microcytic anemia, a systematic diagnostic approach was undertaken. The results of the acetate hemoglobin gel electrophoresis (Sebia, Hydragel 15 Haemoglobin(e), Lisses, France) demonstrated a band pattern that could be consistent with the presence of Hb E (Fig. 1). The high-performance liquid chromatography (HPLC) Variant II β-thalassemia Short Program (Bio-Rad, Hercules, CA, USA) detected an HbA2 fraction of 17.2% (normal 2.2–3.3) and a fetal Hb (Hb F) with 1.7% (normal < 1) (Fig. 2). Following the analysis of electrophoresis and HPLC, a tentative diagnosis of haemoglobinopathy was proposed. As neither Hb E nor Hb Paksé could be unambiguously diagnosed using the aforementioned methods, genetic testing was subsequently conducted using the α- and β- Globin StripsAssay Kit (ViennaLab Diagnostics GmbH, Vienna, Austria). The presence of a heterozygous Hb E (CD 26 [G > A]; (HBB: c.79 G > A) was identified in the β-Globin Strips Assay SEA, while a heterozygous Hb Paksé (α2 CD 142 [A > T] (HBA2:c.429 A > T) was observed in the α-Globin Strips Assay.

Fig. 1.

Acetate hemoglobin gel electrophoresis (Sebia, 15 HEMOGLOBIN(E)) of the patient (column 1) shows a band at the position where HbA migrates as well as in the A2 - C - E line. The positive control (column 8) shows the band pattern from top to bottom: A (A0 + A1), F, S - D, A2 - C - E and anhydrase

Fig. 2.

The graph below shows the chromatogram and above the analyte identification windows of the patient’s HPLC (BioRad Variant II, β-thalassemia Short Program). Analyte identification window: The HbA2 is 17.2% and the HbF is 1.7%. Chromatogram: The grey peak on the right (retention time 3.59 min) is identified as HbA2. The small grey peak on the left (retention time 1.04 min) is identified as HbF. All other peaks (percentage, retention time, peak area and peak name) are compatible with the measurement

Discussion

In general, hemoglobinopathies are inherited heterogeneous autosomal recessive single-gene disorders. In addition to the extremely rare forms of hemoglobin variants, such as methemoglobinemia caused by Hb M-Hyde Park (Hb M-Akita) [8], the more common variants result from mutations in the human α- or β globin genes [9]. Despite well-defined diagnostic methods for hemoglobin variants, a detailed understanding of the prevalence and trends of hemoglobinopathies remains lacking [1]. Overall, in Northern Europe, Hb variants are considered rare diseases. Hemoglobinopathies are prevalent in Southeast Asia, with an estimated prevalence of 10–30% for α-thalassemia and up to 50% for β- thalassemia Hb E in certain endemic regions. Hb E is a point mutation in codon 26 of the human Hb β globin gene as HBB: c.79 G > A. Hb Paksé, an α-thalassemia variant, is described as clinically silent and is located in the α globin gene as HBA2:c.429 A > T. The heterozygous Hb Paksé variant has been documented on a limited scale in Southeast Asia, with the initial identification occurring in the Paksé district of Laos [6].

The underlying defect in thalassemia is the inability to synthesize one or both of the hemoglobin chains. This results in imbalances of globin chains, which in turn cause hemolysis and/or impair erythropoiesis, leading to anemia, with mild to severe phenotypes [9]. In accordance with the aforementioned, the clinical presentation of α- and β-thalassemia exhibits a spectrum of severity from the severe forms, namely thalassemia major, to intermediate, mild and minima. The most severe form of α-thalassemia is the absence of all α-globin genes in Hb Bart’s, which is also is known as hydrops fetalis syndrome, and the fetus usually dies in-utero or soon after birth. In survivors this form is associated with a lifelong need for transfusions [2, 10]. Beta-thalassemia intermedia is a clinical condition in-between asymptomatic β-thalassemia minor and transfusion-dependent β-thalassemia major. Due to the clinical diversity of patients with thalassemia intermedia, individualized approaches to therapy are required. Treatment options are induction of fetal hemoglobin, occasional transfusions, ion chelation, and sometimes, stem cell transplantation. Genome editing with permanent DNA base changes by introducing single nucleotide variants into Hb DNA represents a new highly promising therapeutic approach in thalassemia [11].

Children with β-thalassemia major present symptoms within the first two years of life. They need regular blood transfusions and untreated thalassemia major eventually leads to early death. This underscores the significance of prenatal screening, prevention, and control programs. One newborn screening study demonstrated various thalassemia genes with 22 different genotypes in Thailand [5]. Individuals with thalassemia are advised to be referred for preconception genetic counseling. Those with α-thalassemia are advised to consider chorionic villus sampling which is used as a diagnostic tool for infants with Hb Bart’s, a condition that increases the risk of toxemia and postpartum bleeding [12].

It is important to note that the interactions between different Hb variants can result in a wide-ranging spectrum of clinical manifestations [13]. The following examples, which mainly originate from East Asia, demonstrate representative thalassemia Hb variants in various combinations. The Hb Paksé variants are associated with a range of health issues, as evidenced by the presented examples.

Hb Constant Spring (HBA2:c.427 T > C) (Hb CS) and Hb Paksé are both α-thalassemia Hb variants. The prevalence of Hb CS has been reported in northeastern Thailand and in Laos, of up to 10%. This compound heterozygosity may cause the development of mild hemolytic anemia and splenomegaly [14]. Although compound heterozygotes of Hb CS and Hb Paksé may also manifest clinically severe conditions, including jaundice, hepatosplenomegaly, and the need for regular blood transfusion [15].

Compound heterozygosity of Hb Adana (HBA2:c.179 G > A) with Hb Paksé is described in a child with severe anemia and hepatosplenomegaly. He required two monthly transfusions and was growth retarded. The patient’s father exhibited a normal red blood cell count, as determined by standard laboratory analysis. However, DNA studies revealed the presence of Hb Adana, confirming the diagnosis. His mother was found to be compound heterozygous for Hb CS and Hb Paksé [16]. This disease exhibits a high degree of heterogeneity, which may manifest as Hemoglobin H (Hb H) [17].

In general, a non-fatal α-thalassemia intermediate form is Hb H disease where Hb is built of four β Hb chains. Hb H disease belongs to the group of non-transfusion dependent thalassemia. In case that mutations like Hb H and Hb Paksé are inherited in a compound heterozygous state (Hb H/Hb Paksé), they may potentially result in the manifestation of a severe thalassemia phenotype. Fetuses with Hb H/Hb Paksé may be associated with hydrops fetalis, a condition that occurs during the mid-pregnancy period. During this critical period of fetal development, anemia may result in adverse effects on the growth and function of organs, potentially leading to the aforementioned condition. Consequently, prenatal diagnosis and intrauterine treatment of fetal Hb H/Hb Paksé is essential to prevent anemic hypoxia in developing organs and fetal diseases [18].

The combined heterozygote Hb variant with Hb E and Hb Paksé is clinically silent. The anemia observed in our patient is mild and is attributed to Hb E. However, Hb E is described as unstable, with reduced Hb synthesis and microcytosis. Eight distinct genotypes of Hb CS/Hb Paksé, encompassing an additional Hb E variant. Besides other variants heterozygous Hb Paksé, double heterozygous Hb E/Hb Paksé, triple heterozygous HbE/α-thalassemia/Hb Paksé, and a compound Hb CS/Hb Paksé were defined. In clinical practice, these are predominantly observed in patients with microcytic anemia, which may be transfusion-dependent, and occasionally accompanied by iron deficiency [19].

Hb Paksé and Hb C (HBB: p.6 Glu > Lys) leads to a condition known as Hb C/Hb Paksé, which causes primarily moderate anemia [20]. In contrast, Hb Paksé with Hb S (HBB: p.6 Glu > Val) manifests as heterozygosity and exhibits a range of complex clinical manifestations. The disease is characterized by mild to moderate anemia, jaundice and hepatosplenomegaly. Additionally, various complications are associated with vaso-occlusive crises and an increased risk of infections. Clinically these patients may present with bone pain, respiratory distress, stroke or multi-organ damage in severe cases [14]. It is possible for the clinically silent Hb D (HBB: b.121 (GH4)Glu > Gln) to manifest alongside Hb Paksé. This variant is characterized by hypochromic microcytic red cell morphology and is classified as Hb D/Hb Paksé [21]. Hb Lansing-Ramathibodi (HBA1:c.264 C > G) has been described in two members of a Thai family who were compound heterozygous with Hb Paksé, and had mildly elevated reticulocytes [22].

In general, thalassemia is most prevalent in Africa and Southeast Asia, as well as in the Mediterranean region. Nevertheless, as a consequence of increased mobility and migration, all forms of thalassemia are now spreading to Northern Europe [4, 6]. A precise prevalence, impact, and trends of hemoglobin variants remain uncertain, not only in Europe but also globally. It is essential to distinguish between thalassemia and iron deficiency anemia, particularly when microcytic anemia is present alongside a normal iron status [3, 21]. Although an early and accurate diagnosis enables the prompt initiation of treatment and ensures the delivery of comprehensive care, and prevents the implementation of inappropriate interventions [2], it is essential to acknowledge that these uncertainties persist.

The above-mentioned examples demonstrate the diverse clinical manifestations that can result from interactions between different Hb variants. Nevertheless, the identification of the wide spectrum of globin gene mutations is challenging. This underscores the need for medical personnel involved in the etiologic search for anemias to be aware of Hb variants and the availability of genetic sequencing. In non-endemic areas, it is imperative that healthcare professionals demonstrate clinical vigilance in order to identify hemoglobin variants. The estimated frequency of β-thalassemia in Austria is estimated to be 1/100,000 [7] although the Austrian Federal Statistical Office reports that there are approximately 5,000 individuals of Thai descent living in Austria. Furthermore, it is imperative to consider the potential impact of hemoglobin variants from other countries of immigration, as this may also contribute to an increase in thalassemia. Nevertheless, it is evident that the prevalence of thalassemia will continue to rise.

The combined occurrence of two heterozygous thalassemia Hb variants, one in the β-globin gene as Hb E and one in the α-globin gene as Hb Paksé, in the same individual is a rare phenomenon. To the best of our knowledge, this combined heterozygote Hb variant has only been described in East Asia. This is the first case to be reported in Europe, which provides evidence that this is the result of a migration-caused occurrence. However, it is of importance to recognise that as a consequence of ongoing migration, the number of heterozygous carriers of haemoglobinopathies is rising steadily in central Europe. This is resulting in a variety of combinations of Hb variants, which are occurring with increasing frequency. It is becoming increasingly evident that there is a necessity to gain a more profound understanding of the genotypic variants and the phenotypic clinical manifestations associated with Hb variant combinations.

Conclusions

Nevertheless, the identification and comprehension of Hb mutations are prognostically significant, thereby enabling the administration of appropriate treatment. It is therefore essential that clinical recognition, genetic testing, and counseling for patients with Hb variants, particularly in the case of thalassemia patients, are carried out.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

W.J. S., A. V-G. and H. M. drafted the manuscript– G. L., S. M. and D. E. performed laboratory work and data collection– W.J. S., G. L., A. V-G., S. M., H. M. and D. E. participated in data analysis and interpretation. All the authors approved this last version and the publication of the manuscript.

Funding

No funding has been received from any source for this work.

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

This is an observational, retrospective observation conducted on already available data and follows the ethical guidelines of the Declaration of Helsinki.

Competing interests

The authors declare no competing interests.