Molecular Characterization of Non-Transfusion Dependent Beta-Thalassemia (NTDT) in Spain
Authors: Ropero Gradilla Paloma , Menor Gómez María , Morado Arias Marta , Salido Fiérrez Eduardo José , Montañés García María Ángeles , Recasens Flores Valle , Tenorio Núñez María , Senent Peris María Leonor , De La Iglesia Íñigo Silvia , Lo Riso Laura , López Rubio Montserrat , Ricard Andrés Pilar , Zamanilllo Herreros Irene , Orbe Jaramillo Irene , Esperanza Marco Josefa , Abío Calvete Mariola , Vara Míriam , Fernandez Garcia Sergio , Rodríguez Servan Isabel , Molina Arrebola María Angustias , Linares Garcia Mariano , Vagace Valero José Manuel , Hinojosa Orantos Cristina , Hidalgo Campos Manuela Ines , Hernández García M.ª del Carmen , López López Rosa María , Ugalde Fernández Nazaret , Ferre Bermejo Oscar , Herrera Robles Kenneth , Popa Dusacovschi Oana , Groiss Buiza Jorge , Palma Vallellano Antonio José , Vidan Estevez Julia Maria , Herrera Esther , Ríos Rull Pablo , Luciana Peri Valeria , Rodríguez Ana , Ferrer Sara , Gómez Álvarez Miguel , Martínez Nieto Jorge , Fernandez Fernando Ataulfo Gonzalez , Villegas Ana , Benavente Cuesta Celina , Sehh Grupo Español de Eritropatología 
Published: December 2024

Introduction
In recent years, the clinical classification of beta thalassemia has been based on transfusion needs. Non-transfusion dependent beta-thalassemias (NTDT), previously referred to as thalassemia intermedia, do not require regular transfusions or may not need them at throughout the patient's life, only receiving transfusions in specific clinical situations such as pregnancy, infections, or surgery, etc. These patients represent a very heterogeneous and complex group with various associated Hb abnormalities.
Objective
Molecular characterization and registry of the cases of non-transfusion dependent thalassemia detected in Spain in the last 20 months.
Material and Methods
Between November 2022 and July 2024, 54 individuals from different Spanish regions, including native and migrant populations, were studied. Conventional hemocytometric analysis, determination of HbA2 and HbF by ion exchange HPLC 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
Twenty-two women (40.7%) and 32 men (59.3%) comprised the sample, with a mean age of 41.7 years. Twenty-two percent were splenectomized at a mean age of 11 years. Of the patients, 85.2% did not receive transfusions, 14.8% (8) received a transfusion, 2 sporadically and the other 6 needed at least 1.5 units per year. All patients presented with variable moderate anemia, with hemoglobin levels between 7.9 and 12.1 g/dL, accompanied by microcytosis (MCV (fL) = 67.5 ± 10.5); (MCH (pg) = 19.9 ± 3.2) and increased RDW (%) (22.6 ± 4.6). Hb A2 (%) levels were also elevated (4.8 ± 0.9) and Hb F levels ranged from 0.6% to 25%. Reticulocytes were moderately elevated.
Ferritin was at a mean value of 304.9 ng/mL. Fifteen patients received chelation monotherapy the rest the treatment was unknown.
The most common HBB gene genotypes were CD39 (C&gt;T) (50% of patients) and IVS-1-nt6 (T&gt;C) (11%), with the predominant alpha genotype being ααα/αα (65% of patients). Six patients had 2 mutations in the HBB gene, 2 were carriers of Hb Lepore Baltimore and IVS-1-nt1 (G&gt;A), and another with an IVS-1-nt110 (G&gt;A); the first of them also had an associated -α3.7/αα. Three patients presented with homozygous Spanish δβ-thalassemia, and 2 patients were carriers of an Hb C associated with a β0-thalassemia [CD39 and IVS-1-nt1 (G&gt;A)]. Finally, 2 patients were carriers of heterozygous beta-thalassemia [IVS-1-nt6 (T&gt;C) and IVS-1-nt110 (G&gt;A)] and 6 alpha genes (ααα/ααα and αααα/αα, respectively).
Conclusions
NTDT patients in Spain represent a very heterogeneous group both genetically and clinically. This is evidenced by the diversity of mutations and combinations of mutations found, as well as the variability in clinical presentations, such as hemoglobin levels and transfusion requirements.
Splenectomized patients tend to have higher ferritin levels, especially those with the CD39 genotype (514 ng/mL) compared to those who have not been splenectomized (140.5 ng/mL).
This study contributes to understanding the complex molecular foundations of NTDT in Spain, emphasizing the need for personalized management strategies based on genetic profiles and clinical phenotypes.
FUNDING: Agios Pharmaceuticals
CONFLICT OF INTEREST: The authors declare that they have no conflict of interest