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Article

Haemoglobinopathies: Integrated Biochemical and Molecular Diagnosis in 5243 Patients

by
Domenico Dell’Edera
*,
Brunilde Persia
,
Francesco La Rocca
and
Carmela Centoducati
Unit of Medical Genetics and Immunogenetics, Madonna Delle Grazie Hospital, 75100 Matera, Italy
*
Author to whom correspondence should be addressed.
Hemato 2025, 6(4), 36; https://doi.org/10.3390/hemato6040036
Submission received: 31 July 2025 / Revised: 22 September 2025 / Accepted: 11 October 2025 / Published: 14 October 2025
(This article belongs to the Section Non Neoplastic Blood Disorders)

Abstract

Background: Haemoglobinopathies are among the most common monogenic disorders worldwide. Early identification of asymptomatic carriers through reliable screening and molecular diagnostics is crucial for prevention programmes, especially in high-prevalence regions such as Southern Italy. Methods: A total of 5243 individuals were analysed between 2013 and 2024 using both biochemical and genetic parameters. First-level screening included full blood count, iron status, and high-performance liquid chromatography (HPLC) for haemoglobin variant quantification. Molecular analyses were performed using next-generation sequencing (NGS) for the HBA1, HBA2, and HBB genes. Results: We identified 267 individuals (11.2%) as carriers of α-thalassaemia and 473 individuals (16.7%) as carriers of β-thalassaemia. Among them, 5 were compound heterozygotes and 3 homozygous for the α-3.7 deletion. A rare case of HbG Philadelphia in association with a triplicated α-gene was also observed. The most common β-globin mutations included c.118C>T039, 44%), IVS-I-110 (17.7%), IVS-I-6 (12.7%), and IVS-I-1 (12.3%). Among α-globin mutations, the most prevalent were 3.7 (48%), α2 IVS1 -5nt (15.4%), -20.5 Kb (14.2%), and triplicated α (11%). In total, 18.7% of individuals were found to carry either α- or β-thalassaemia traits. Conclusion: Our findings highlight the limitations of traditional diagnostic methods—such as the osmotic fragility test—and the importance of integrating haematological, biochemical, and molecular data to accurately identify thalassaemia carriers. The variability of genotype–phenotype correlations, especially in the context of immigration and genetic diversity, underscores the need for comprehensive molecular analysis. We propose a three-step diagnostic algorithm combining first-level screening, iron status assessment, and NGS-based sequencing for inconclusive cases.

1. Introduction

The term “haemoglobinopathies” refers to all qualitative and quantitative defects of the globin genes. These disorders are inherited in an autosomal recessive manner. “Asymptomatic carriers” of the genetic defect have a high prevalence in geographical areas where malaria is endemic, as this condition provides an evolutionary advantage by hindering infestation by Plasmodium falciparum [1,2].
The marked haematological, molecular, and clinical heterogeneity of haemoglobinopathies increasingly requires experience, expertise, and the use of advanced laboratory diagnostic technologies. The variations and combinations observed today are also the result of migratory movements over recent decades, which have brought a significant number of individuals to Italy and Europe from regions where haemoglobin disorders are particularly prevalent [3,4].
So-called “first-level” laboratory tests still represent the fundamental step in the diagnostic approach to haemoglobinopathies. However, confirmatory testing and molecular characterisation remain essential, with a diagnostic strategy based on the integrated use of haematological, biochemical, and molecular techniques [5]. Laboratories involved in the diagnosis of haemoglobinopathies aim to support precision medicine by providing tools that contribute to increasingly personalised healthcare [6].
Within the spectrum of haemoglobinopathies, it is important to distinguish between conditions resulting from a quantitative defect in globin chain production (thalassaemia syndromes) and those due to qualitative abnormalities in the globin chains (haemoglobin variants, such as sickle cell anaemia).
Regarding thalassaemia syndromes, since adult haemoglobin consists of approximately 97% haemoglobin A (HbA: two α-chains and two β-chains), the phenotypic expression will depend on whether there is a deficiency of β-chains (β-thalassaemias) or α-chains (α-thalassaemias).
The synthesis of β-chains is controlled by two genes (one on each allele) located on the short arm of chromosome 11 (11p15.5), whereas α-chain synthesis is regulated by four genes (two on each allele), situated on the short arm of chromosome 16 (16p13.3).
From this, it follows that clinically significant phenotypes are more likely to occur in β-thalassaemia forms. The pathophysiology of β-thalassaemia primarily depends on the degree of imbalance between α- and β-globin chains. The greater the deficiency of β-chains, the greater the accumulation of unpaired α-chains, resulting in ineffective erythropoiesis. This process may be further exacerbated—or in some cases mitigated—by coexisting genetic or acquired modifiers [7].
Clinically relevant forms of α-thalassaemia arise when at least three α-globin genes are affected. When only two α-globin genes are involved—either by point mutations or deletions—the resulting phenotype is mild and typically presents as microcytic anaemia of low severity, usually requiring no treatment other than intermittent folic acid supplementation [8,9]. Due to the associated biochemical profile, such cases are frequently misdiagnosed as iron deficiency anaemia.
A clinically significant condition is haemoglobin H disease (HbH), [10] which occurs in individuals with only one functional α-globin gene (genotype –/-α). This form is characterised by haemoglobin levels ranging from 7 to 10 g/dL and may present with jaundice and splenomegaly. Microcytosis is typically more pronounced (MCV between 50 and 65 fL), HbA2 levels are reduced (1–2%), and HbH may account for up to 8–10% of total haemoglobin. The presence of HbH inclusion bodies is characteristic and can be demonstrated using specific staining techniques.
In the complete absence of α-chains, the foetus produces only Hb Bart’s (haemoglobin composed of four γ-chains), a form of haemoglobin with extremely high oxygen affinity but entirely ineffective at oxygen delivery to tissues. This results in severe intrauterine hypoxia, rapidly progressing to hydrops fetalis, cardiac failure, and, in the vast majority of cases, intrauterine or early neonatal death.
The aim of the present study is to analyse, in a cohort of 5243 patients, the correlation between biochemical parameters and specific genotypic mutations identified by molecular biology techniques in the α- and β-globin genes. This approach is particularly valuable in cases where first-level screening is insufficient for an accurate diagnosis, especially in the presence of rare variants or complex genotypic combinations.

2. Materials and Methods

2.1. Patients

A total of 5243 individuals were analysed for the presence of pathogenic variants in the α- and β-globin genes between 2013 and 2024.

2.2. First-Level Screening

The following tests were performed on the 5243 recruited individuals, using venous blood samples collected in EDTA-K3 tubes (Becton Dickinson, Berkshire, UK):
Peripheral blood smear to evaluate erythrocyte morphology.
Measurement of red cell indices using the automated haematology analyser Sysmex XE 2100 (Dasit, Cornaredo, Milan, Italy): red blood cell count (RBC), haemoglobin concentration (Hb), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC).
High-performance liquid chromatography (HPLC) for the quantification of haemoglobin subtypes using the Tosoh HPLC G7 system (Tosoh Bioscience S.r.l., Turin, Italy) [11].
Serum iron and ferritin levels were measured using the Elecsys 2010 analyser (Roche Diagnostics GmbH, Indianapolis, IN, USA).

2.3. Second-Level Molecular Analysis

2.3.1. Genomic DNA Extraction

Genomic DNA (gDNA) was extracted from peripheral blood samples. A total of 200 µL of whole blood was used for each extraction, performed with the MagCore HF 16 Plus automated nucleic acid extractor (Diatechline, Jesi, Italy).

2.3.2. Molecular Analysis of Globin Genes

Molecular analysis of the HBB, HBA1, and HBA2 genes—including major structural rearrangements within the α-globin gene cluster—was performed using the Devyser Thalassemia NGS kit (Devyser, Hägersten, Sweden). This test enables the simultaneous identification of point mutations, small insertions/deletions, and copy number variations (CNVs) in the HBB, HBA1, and HBA2 genes, including the principal structural rearrangements of the α-globin cluster. DNA samples were sequenced using MiSeq DX (Illumina, San Diego, CA, USA). A bioinformatics workflow Devyser Amplicon Suit v3.8 was used for data analysis.

3. Results

Analysis of the α-globin genes revealed that 267 individuals (11.2%) were asymptomatic carriers of the α-thalassaemia trait, including 190 females (70.6%) and 77 males (29.4%) (Table 1).
Among these, 5 individuals (0.2%) were found to be compound heterozygotes for α-globin gene mutations, and 3 individuals were homozygous for the α-3.7 deletion (Table 2).
Of particular interest is the detection of the α2 cd68 (C>G) variant, known as HbG-Philadelphia, in heterozygosity in a female patient of Nigerian origin. The same patient was also heterozygous for the −ααα3.7deletion, resulting in the loss of a single α-globin gene (genotype: −α3.7/αα). As for the β-globin gene analysis, 473 individuals (approximately 16.7% of the cohort) were identified as asymptomatic carriers of the β-thalassaemia trait, including 302 females (63.8%) and 171 males (36.2%). Among these, 17 individuals carried so-called “silent” mutations in the β-globin gene, specifically the −87 and −101 variants (Table 3).
Finally, 47 individuals were found to carry structural haemoglobin variants associated with amino acid substitutions in the β-globin chain: 41 with HbS, 3 with HbC, 1 with HbD, 1 with HbE, and 1 with HbO-Arab (Table 4).
These cases represent approximately 1.7% of the total population studied. In addition, 25 individuals were identified as compound carriers of both an α-globin and a β-globin gene mutation (Table 5).

4. Discussion

The World Health Organization (WHO) recommends the implementation of screening programmes within healthcare systems of countries with a high incidence of haemoglobinopathies [12].
Diagnostic strategies for identifying carriers are primarily based on first-level screening. Although this approach is more economically sustainable, it has lower sensitivity in the presence of mild or silent β-globin gene mutations (often associated with point mutations in the promoter region), or α-thalassaemia involving the deletion of a single α-globin gene.
Moreover, common clinical conditions such as iron deficiency anaemia, megaloblastic anaemia, and hereditary persistence of foetal haemoglobin (HPFH) can mask typical microcytic features, complicating interpretation. Pre-analytical issues, such as inadequate sample storage, further contribute to diagnostic inaccuracies.
HbA2 levels can be influenced by numerous factors, including mutations in the KLF1 gene, δ- or δβ0-haemoglobinopathies, α-thalassaemia (including triplicated α-globin genes), iron deficiency, hyperthyroidism, antiretroviral therapy, and megaloblastic anaemia [13].
An HbA2 value ≥3.5% is commonly considered indicative of β-thalassaemia carrier status, although cut-off values may vary between laboratories. Borderline results—between the upper limit of the normal range and the diagnostic threshold—require confirmation through molecular techniques such as PCR or β-globin gene sequencing.
In our study, the most frequent α-globin gene mutations were single-gene deletions associated with the α+-thalassaemia phenotype (α2-TAL), which frequently mimic the biochemical pattern of iron deficiency anaemia. In individuals with two deleted α-globin genes (α1-TAL phenotype), biochemical findings may also be ambiguous, potentially leading to a misdiagnosis of iron deficiency.
A notable case involved the HbG Philadelphia variant, which in our study showed an Hb fraction of 32.6% on HPLC—consistent with cis inheritance of the -ααα 3.7 Kb deletion. While simple heterozygosity for HbG typically causes no significant haematological changes, its coexistence with a triplicated α-gene arrangement may lead to microcytosis and hypochromia. Although many β-thalassaemia mutations are clinically silent, their identification is crucial for accurate genotype–phenotype correlation—especially when biochemical profiles appear nearly normal. Such cases may simulate mild iron deficiency anaemia and mislead diagnosis. The most common β-globin mutations identified in our β-thalassaemia carrier cohort were as follows:
  • NM_000518.5(HBB):c.118C>T (β039), detected in 44% of cases, typical of the Mediterranean region;
  • NM_000518.5(HBB):c.93-21G>A (IVS-I-110), in 17.7%, common in Eastern Europe;
  • NM_000518.5(HBB):c.92+6T>C (IVS-I-6), in 12.7%, prevalent in Eastern Europe and China;
  • NM_000518.5(HBB):c.92+1G>A (IVS-I-1), in 12.3%, also typical of the Mediterranean basin.
  • Among α-globin mutations, the most frequent were as follows:
  • 3·7 (NG_000006.1:g.34164_37967del3804), found in 48% of carriers, widespread in the Mediterranean and Middle East;
  • α2 IVS1 -5nt (HbA2: c.95+2_95+6delTGAGG), in 15.4%, common in Morocco and the Middle East;
  • -20.5 Kb (NG_000006.1:g.15164_37864del22701), in 14.2%;
  • Triplicated α (NG_000001:g.34247_38050dup), in 11% of cases.
Given the over 400 known β-thalassaemia mutations and the influence of genetic modifiers and comorbidities, genotype–phenotype correlations remain highly variable. No single biochemical or morphological marker is universally reliable [14,15,16].
Our findings confirm that the osmotic fragility test (Simmel test) is outdated and unsuitable as a first-line diagnostic method for thalassaemia carriers. In contrast, excluding external causes, the measurement of HbA2 is fundamental to suspecting thalassaemia carrier state and/or haemoglobin variants.
This result must be supported by a complete blood count and iron status assessment. In the past, molecular analysis of globin genes was targeted at the native population and focused on a limited number of mutations. Today, due to increasing immigration, the region studied is home to individuals from diverse ethnic backgrounds. Therefore, in cases with ambiguous haematological and biochemical profiles, full sequencing of the globin genes should be performed to avoid missing pathogenic variants not covered by standard molecular screening panels. Currently, in the Basilicata Region, a health education programme is in place for both healthcare professionals and the general population. This initiative is justified by the 14% prevalence of thalassaemia carrier state carriers in Basilicata, higher than the average of 11% observed in Italy’s most affected regions—Sicily, the Po Delta, and Sardinia [16]. A previous study of 2990 individuals in Basilicata [17] found a β-thalassaemia trait prevalence of 9.36%. In contrast, in the present study, 18.7% of individuals were found to be thalassaemia trait carriers (11.2% α and 16.7% β).
Based on these findings, our centre proposes the following diagnostic algorithm:
First-level testing, including a complete blood count with RDW index, and quantification of HbA, HbA2, HbF, and pathological haemoglobin variants (HbS, HbC, HbE, HbD) by HPLC.
(1)
Serum iron and ferritin analysis;
(2)
NGS-based sequencing of the α-, β-, γ- and δ-globin genes in cases of isolated HbA2 elevation, suspected silent β-thalassaemia, or persistent diagnostic uncertainty.
This study highlights the diagnostic complexity of identifying asymptomatic carriers of α- and β-thalassaemia, underlining the limitations of traditional methods such as the osmotic fragility test, and the need for an integrated approach.
While the analysis of HbA2 levels—together with a complete blood count and iron status—is a critical step in suspecting β-thalassaemia trait, isolated interpretation of this parameter may be misleading in the presence of interfering clinical conditions or rare genetic variants.

Author Contributions

Conceptualization and methodology, D.D.; validation, D.D.; writing—original draft preparation F.L.R.; writing—review and editing C.C. and B.P.; supervision, D.D.; project administration, D.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

The study was approved by the Clinical Trial Center of Azienda Sanitaria di Matera (n. 1022—7 April 2023) and all patients provided informed consent according to the Declaration of Helsinki.

Informed Consent Statement

Informed consent was obtained from all subjects involved in the study. All those who underwent this study received genetic counselling associated with the test.

Data Availability Statement

The authors confirm that the data supporting the findings of this study are available within the article.

Acknowledgments

We thank the Volunteer Organizations (ODV) Gian Franco Lupo and AnimaMundi.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

CNVCopy number variation
EDTAethylenediaminetetraacetic acid
gDNAGenomic DNA
HbAHaemoglobin A1, two α-chains and two β-chains
HbA2Haemoglobin A2
HbFFoetalhaemoglobin
HBBHaemoglobin Subunit Beta
HbHHaemoglobin H
Hb Bart’sHaemoglobin Bart’s
HCTHaematocrit
HGVSHuman Genome Variation Society
HPLCHigh-performance liquid chromatography
HPFHPersistence of foetal haemoglobin
MCVMean corpuscular volume
MCHMean corpuscular haemoglobin
MCHCMean corpuscular haemoglobin concentration
NGSNext-generation sequencing
PLTPlatelet count
RBCRed blood cell count
RDWRed cell distribution width
WHOWorld Health Organization

References

  1. Cyrklaff, M.; Sanchez, C.P.; Kilian, N.; Bisseye, C.; Simpore, J.; Frischknecht, F.; Lanzer, M. Hemoglobins S and C interfere with actin remodeling in Plasmodium falciparum-infected erythrocytes. Science. 2011, 334, 1283–1286. [Google Scholar] [CrossRef] [PubMed]
  2. Luzzatto, L. Sickle cell anaemia and malaria. Mediterr. J. Hematol. Infect. Dis. 2012, 4, e2012065. [Google Scholar] [CrossRef] [PubMed]
  3. Henderson, S.; Timbs, A.; McCarthy, J.; Gallienne, A.; Van Mourik, M.; Masters, G.; May, A.; Khalil, M.S.; Schuh, A.; Old, J. Incidence of haemoglobinopathies in various populations—The impact of immigration. Clin. Biochem. 2009, 42, 1745–1756. [Google Scholar] [CrossRef] [PubMed]
  4. Angastiniotis, M.; Vives Corrons, J.L.; Soteriades, E.S.; Eleftheriou, A. The impact of migrations on the health services for rare diseases in Europe: The example of haemoglobin disorders. Sci. World J. 2013, 2013, 727905. [Google Scholar] [CrossRef] [PubMed]
  5. Traeger-Synodinos, J.; Harteveld, C.L.; Old, J.M.; Petrou, M.; Galanello, R.; Giordano, P.; Angastioniotis, M.; De la Salle, B.; Henderson, S.; May, A. EMQN Best Practice Guidelines for molecular and haematology methods for carrier identification and prenatal diagnosis of the haemoglobinopathies. Eur. J. Hum. Genet. 2015, 23, 426–437. [Google Scholar] [CrossRef] [PubMed]
  6. Dell’Edera, D.; Epifania, A.A.; Milazzo, G.N.; Leo, M.; Santacesaria, C.; Allegretti, A.; Mazzone, E.; Panetta, P.; Iammarino, G.; Lupo, M.G.; et al. Identification of patients with defects in the globin genes. J. Prenat. Med. 2013, 7, 47–50. [Google Scholar] [PubMed] [PubMed Central]
  7. Dell’Edera, D.; Centoducati, C.; Allegretti, A.; La Rocca, F.; Persia, B. Genotype–Phenotype Correlation in a Couple in Which the Wife Is a Carrier of the Beta-Thalassemia Trait and the Husband Is a Carrier of a Mutation in the ALAS2 Gene: Both Gene Defects Are Associated with Non-Iron-Deficiency Microcytic Anemia. Thalass. Rep. 2024, 14, 118–121. [Google Scholar] [CrossRef]
  8. Songdej, D.; Fucharoen, S. Alpha-Thalassemia: Diversity of Clinical Phenotypes and Update on the Treatment. Thalass. Rep. 2022, 12, 157–172. [Google Scholar] [CrossRef]
  9. Vijian, D.; Wan Ab Rahman, W.S.; Ponnuraj, K.T.; Zulkafli, Z.; Bahar, R.; Yasin, N.; Hassan, S.; Esa, E. Gene Mutation Spectrum among Alpha- Thalassaemia Patients in Northeast Peninsular Malaysia. Diagnostics 2023, 13, 894. [Google Scholar] [CrossRef] [PubMed]
  10. Chui, D.H. Alpha-thalassemia: Hb H disease and Hb Barts hydrops fetalis. Ann. N. Y Acad. Sci. 2005, 1054, 25–32. [Google Scholar] [CrossRef] [PubMed]
  11. Kalleas, C.; Tentes, I.; Margaritis, D.; Anagnostopoulos, K.; Toli, A.; Pendilas, D.; Bourikas, G.; Tsatalas, C.; Kortsaris, A.H. Effect of HbS in the determination of HbA2 with the TOSOH HLC-723G7 analyzer and the HELENA Beta-Thal Quik column kit. Clin. Biochem. 2007, 40, 242–247. [Google Scholar] [CrossRef] [PubMed]
  12. Tuo, Y.; Li, Y.; Li, Y.; Ma, J.; Yang, X.; Wu, S.; Jin, J.; He, Z. Global, regional, and national burden of thalassemia, 1990–2021: A systematic analysis for the global burden of disease study 2021. eClinicalMedicine 2024, 72, 102619. [Google Scholar] [CrossRef] [PubMed]
  13. Giambona, A.; Passarello, C.; Renda, D.; Maggio, A. The significance of the hemoglobin A2 value in screening for hemoglobinopathies. Clin. Biochem. 2009, 42, 1786–1796. [Google Scholar] [CrossRef] [PubMed]
  14. Baig, M.A.; Swamy, K.B.; Baksh, A.D.; Bahashwan, A.; Moshrif, Y.; Al Sawat, A.; Al Mutairi, N.; Alharbi, N. Evaluation of role of HPLC (Merits & Pitfalls), in the diagnosis of various hemoglobinopathies &thalassemic syndromes. Indian J. Pathol. Microbiol. 2021, 64, 518–523. [Google Scholar] [CrossRef] [PubMed]
  15. Baird, D.C.; Batten, S.H.; Sparks, S.K. Alpha- and Beta-thalassemia: Rapid Evidence Review. Am. Fam. Physician 2022, 105, 272–280. [Google Scholar] [PubMed]
  16. Mansoor, N.; Meraj, F.; Shaikh, A.; Jabbar, N. Spectrum of hemoglobinopathies with hematological and biochemical profile: A five year experience from a tertiary care hospital. Pak. J. Med. Sci. 2022, 38, 2143–2149. [Google Scholar] [CrossRef] [PubMed]
  17. Dell’Edera, D.; Epifania, A.A.; Malvasi, A.; Pacella, E.; Tinelli, A.; Capalbo, A.; Lioi, M.B.; Di Renzo, G. Incidence of β-thalassemia carrier on 1495 couples in preconceptional period. J. Matern. Fetal Neonatal Med. 2013, 26, 445–448. [Google Scholar] [CrossRef] [PubMed]
Table 1. Comparison between pathogenic variant and erythrocyte biochemical data in asymptomatic carriers of the alpha-thalassaemia trait. From the analysis of the α globin genes we found that 267 subjects are healthy carriers of the α-thalassemia trait (11.2%), of which 190 are female (70.6%) and 77 are male (29.4%).
Table 1. Comparison between pathogenic variant and erythrocyte biochemical data in asymptomatic carriers of the alpha-thalassaemia trait. From the analysis of the α globin genes we found that 267 subjects are healthy carriers of the α-thalassemia trait (11.2%), of which 190 are female (70.6%) and 77 are male (29.4%).
Mutation
HGVS Nomenclature
Carriers of the α-thalassaemia Trait
Nomenclature IthanetRBC 106/μLHb g/dLHCT %MCV fLMCH pgMCHC g/dLHBA2%HBF%
1NG_000006.1: g.34164_37967del3804
(n = 129)
-3.7 (tipo I) heterozygotes5.10 ± 0.5813.1 ± 1.5339.6 ± 4.6677.8 ± 5.2725.7 ± 2.0333.0 ± 1.092.5 ± 0.841.3 ± 0.53
2HbA2: c.95+2_95+6delTGAGG
(n = 41)
α2 IVS1 -5nt5.2 ± 0.7112.4 ± 1.9638.1 ± 5.0674.0 ± 4.3123.8 ± 2.0132.3 ± 1.622.5 ± 1.110.8 ± 0.22
3NG_000006.1: g.15164_37864del22701
(n = 38)
-20.5 Kb double gene del5.4 ± 0.7011.6 ± 1.2036.0 ± 3.9065.9 ± 6.6021.7 ± 3.1032.3 ± 0.912.3 ± 0.400.7 ± 0.30
4NG_000001: g.34247_38050dup
(n = 29)
anti-3.7
(ααα−3.7)
4.12 ± 0.5212.7 ± 1.7837.5 ± 4.0381.7 ± 5.2428.3 ± 3.3233.7 ± 1.682.7 ± 0.631.7 ± 2.31
5HBA2: c.*+94A>G
(n = 10)
α 2poly-A15.30 ± 0.4512.4 ± 0.1138.1 ± 0.2372.6 ± 5.2823.6 ± 1.8732.4 ± 0.902.5 ± 0.230.6 ± 0.20
6NC_000016.10: g.169818_174075del
(n = 10)
-4.25.02 ± 0.2012.0 ± 0.7540.3 ± 6.4078.6 ± 6.3724.1 ± 1.9530.5 ± 5.222.3 ± 0.430.3 ± 0.25
7HbA2: c.427T>C
(n = 5)
α 2 cd 142 Hb Constant Spring5.62 ± 0.4313.0 ± 1.0740.7 ± 3.9371.8 ± 5.6023.2 ± 1.3832.3 ± 0.782.2 ± 0.210.4 ± 0.28
8HbA2: c.2T>C
(n = 4)
α 2 init cd5.60 ± 0.7713.3 ± 2.7341.5 ± 4.8278.6 ± 2.9626.6 ± 0.5032.5 ± 1.762.3 ± 0.500.7 ± 0.42
9NG_000006.1: g.24664_41064del 16401
(n = 1)
--MED double gene del5.5012.137.068.922.432.03.50.1
Table 2. Comparison between pathogenic variants and biochemical data in compound heterozygotes or homozygotes for the HBA gene, 5 individuals (0.2%) were found to be compound heterozygotes for α-globin gene mutations, and 3 individuals were homozygous for the α-3.7 deletion.
Table 2. Comparison between pathogenic variants and biochemical data in compound heterozygotes or homozygotes for the HBA gene, 5 individuals (0.2%) were found to be compound heterozygotes for α-globin gene mutations, and 3 individuals were homozygous for the α-3.7 deletion.
Mutation
HGVS Nomenclature
Compound Heterozygotes and Homozygotes for the α-globin Genes
Nomenclature IthanetRBC 106/μLHb
g/dL
HCT
%
MCV
fL
MCH
pg
MCHC g/dLHBA2
%
HBF
%
HB variants
1NG_000001:g.34247_38050 dup/HbA2:c.427T>C
(n = 3)
Anti-3.7 (ααα-3.7)/α2 cd 142 Hb Constant Spring5.60 ± 1.3213.2 ± 1.6745.0 ± 0.8275.2 ± 1.5624.5 ± 1.0632.6 ± 0.782.5 ± 0.140.4 ± 0.14----
2NG_000006.1:g.34164_37967del3804/HBA2: c.*+94A>G
(n = 1)
-3.7 (tipo I)/α 2 Poly A-1 (HBA2:c.*+94A>G)5.7011.437.560.023.133.32.40.3----
3HBA1:c.91G>C/NG_000006.1:g.34164_37967del3804
(n = 1)
α2 cd68 (C>G)/-3.7 (tipo I)
HbG
4.77.424.953.115.829.7--------32.6
4NG_000006.1:g.34164_37967del3804
(n = 3)
-3.7 (tipo I) Omozigote5.42 ± 0.3610.9 ± 0.8134.5 ± 1.6364.2 ± 3.6420.3 ± 0.5231.6 ± 1.562.7 ± 0.151.0 ± 0.10----
Table 3. Comparison between pathogenic variant and erythrocyte biochemical data in asymptomatic carriers of the β-thalassaemia trait. A total of 473 healthy carriers of the β-thalassemia trait were identified (approximately 16.7% of the sample), of which 302 were female (63.8%) and 171 were male (36.2%). Among these, 17 subjects presented so-called “silent” mutations in the β-globin gene (specifically the variants -87 and -101).
Table 3. Comparison between pathogenic variant and erythrocyte biochemical data in asymptomatic carriers of the β-thalassaemia trait. A total of 473 healthy carriers of the β-thalassemia trait were identified (approximately 16.7% of the sample), of which 302 were female (63.8%) and 171 were male (36.2%). Among these, 17 subjects presented so-called “silent” mutations in the β-globin gene (specifically the variants -87 and -101).
Mutation
HGVS Nomenclature
Carriers of the β-thalassaemia Trait
Nomenclature IthanetRBC 106/μLHb g/dLHCT %MCV fLMCH pgMCHC g/dLHBA2%HBF%
1NM_000518.5(HBB):c.118C>T
(n = 210)
CODON 39 [C>T]5.76 ± 0.7411.2 ± 1.3935.0 ± 4.8061.1 ± 5.1019.6 ± 1.7432.2 ± 1.725.4 ± 1.071.8 ± 2.63
2NM_000518.5(HBB):c.93-21G>A
(n = 84)
IVS 1.110 [G>A]5.70 ± 0.6311.8 ± 1.2636.4 ± 3.8363.8 ± 3.0120.7 ± 1.4232.2 ± 1.125.0 ± 0.671.2 ± 1.23
3NM_000518.5(HBB):c.92+6T>C
(n = 60)
IVS 1.6 [T>C]5.76 ± 1.1213.1 ± 3.9139.0 ± 5.7967.2 ± 7.722.6 ± 2.0732.8 ± 0.973.8 ± 0.541.0 ± 0.38
4NM_000518.5(HBB):c.92+1G>A
(n = 58)
IVS 1.1 [G>A]5.83 ± 0.7011.1 ± 1.2035.6 ± 3.6064.2 ± 6.4019.5 ± 0.830.7 ± 2.605.5 ± 0.41.3 ± 0.5
5NM_000518.5(HBB):c.316-106C>G
(n = 22)
IVS 2.745 [C>G]5.60 ± 0.7110.9 ± 1.6033.8 ± 4.5061.0 ± 4.2319.5 ± 1.9031.9 ± 1.535.1 ± 0.501.7 ± 1.5
6NM_000518.5(HBB):c.315+1G>A
(n = 15)
IVS 2.1 [G>A]6.21 ± 0.6112.1 ± 0.9137.6 ± 3.5661.2 ± 5.3619.8 ± 1.6332.4 ± 2.285.5 ± 0.881.6 ± 0.82
7NM_000518.5(HBB):c.-151C>T
(n = 14) (silente)
-101 C>T (c.151C>T)4.63 ± 0.4712.9 ± 1.3238.3 ± 3.8583.5 ± 2.6228.8 ± 0.9633.6 ± 0.893.7 ± 0.501.4 ± 0.98
8NM_000518.5(HBB):c.-137C>G
(n= 3) (silente)
-87 [C>G]5.04 ± 0.8611.8 ± 1.5036, ± 4.5372.2 ± 5.3723.6 ± 1.3032.7 ± 0.835.1 ± 1.062.55 ± 0.64
9NG_000007.3:g63632_7104 del
(n = 3)
Hb Lepore BW5.40 ± 0.211.6 ± 0.6032.6 ± 1.0064.4 ± 0.9022.1 ± 1.0033.3 ± 0.42.7 ± 0.105.0 ± 0.60
10NM_000518.5(HBB):c.92+2T>A
(n = 2)
IVS 1.2 [T>A]5.39 ± 0.1010.5 ± 0.4932.0 ± 0.2659.8 ± 2.4019.6 ± 0.7733.0 ± 0.855.6 ± 0.141.55 ± 0.35
11NM_000518.5(HBB):c.47G>A
(n = 2)
CODON 15 [TGG>TGA]5.31 ± 0.3310.4 ± 0.6432.9 ± 2.2661.9 ± 0.4919.7 ± 0.0031.7 ± 0.216.20 ± 0.281.05 ± 0.35
Table 4. Haemoglobin variants identified in the present cohort. A total of 47 subjects were identified as carriers of structural variants of haemoglobin associated with amino acid substitutions in the β-globin chain: 41 carriers of HbS, 3 of HbC, 1 of HbD, 1 of HbE and 1 of HbO-Arab.
Table 4. Haemoglobin variants identified in the present cohort. A total of 47 subjects were identified as carriers of structural variants of haemoglobin associated with amino acid substitutions in the β-globin chain: 41 carriers of HbS, 3 of HbC, 1 of HbD, 1 of HbE and 1 of HbO-Arab.
Haemoglobin Variants HGVS NomenclatureNomenclature IthanetRBC 106/μLHb g/dLHCT%MCVfLMCHpgMCHCg/dLHBA2%HBF%
1HBB:c.20A>T
(n = 41)
CODON 6 [A>T]
HbS
4.49 ± 0.7012.2 ± 2.4035.0 ± 3.9379.0 ± 6.7027.2 ± 3.6934.5 ± 1.193.3 ± 1.101.2 ± 1.03
2HBB:c.19G>A
(n = 3)
CODON 6 [G>A]
HbC
4.22 ± 1.219.7 ± 1.5734.5 ± 2.3569.7 ± 3.0424.0 ± 3.3535.0 ± 2.252.8 ± 0.360.4 ± 0.51
3HBB:c.364G>C
(n = 1)
HbD Punjab
HbD
4.7012.237.479.926.337.72.360.37
4HBB:c.79G>AHbE5.2013.138.273.525.234.3 1.6
5HBB:c.364G>AHb O-Arab5.2113.439.474.025.434.43.70.8
Table 5. Comparison between pathogenic variants and biochemical data in compound heterozygotes for both α- and β-thalassaemia traits. Of the total subjects studied, these represent approximately 1.7%. Furthermore, 25 subjects were found to be carriers of both a mutation in the α gene and a mutation in the β gene at the same time.
Table 5. Comparison between pathogenic variants and biochemical data in compound heterozygotes for both α- and β-thalassaemia traits. Of the total subjects studied, these represent approximately 1.7%. Furthermore, 25 subjects were found to be carriers of both a mutation in the α gene and a mutation in the β gene at the same time.
HGVS Nomenclature
Presence of both β- and α-thalassaemia traits
Nomenclature IthanetRBC 106/μLHB g/LHCT %MCV fLMCH pgMCHC/dLHBA2%HBF %
1NG_000006.1:g.34164_37967del3804/NM_000518.5(HBB):c.118C>T
(n = 8)
α−3.7/CODON 395.33 ± 0.6610.9 ± 1.3733.5 ± 4.2062.8 ± 3.0320.5 ± 1.2632.6 ± 1.145.3 ± 0.591.95 ± 1.16
2NG_000006.1:g.34164_37967del3804/HBB:c.20A>T
(n = 3)
α−3.7/CODON 6 [A>T]4.41 ± 0.4510.9 ± 0.4633.4 ± 0.3574.3 ± 5.0126.9 ± 1.8532.663 ± 0.850.7 ± 0.62
3HbA2: c.95+2_95+6delTGAGG/
NM_000518.5(HBB):c.118C>T
(n = 3)
α2 IVS1 -5nt/CODON 395.77 ± 0.2311.5 ± 0.6135.4 ± 1.7061.4 ± 0.4519.4 ± 0.9131.5 ± 1.235.7 ± 0.610.9 ± 0.83
4NG_000006.1:g.34164_37967del3804/NM_000518.5(HBB):c.93-21G>A
(n = 2)
α−3.7/IVS 1.1104.84 ± 0.7013.1 ± 3.440.3 ± 8.285.5 ± 2.9828.3 ± 4.3232.6 ± 1.204.9 ± 1.701.6 ± 0.63
5NG_000001:g.34247_38050dup/NM_000518.5(HBB):c.93-21G>A
(n = 2)
ααα−3.7/IVS 1.1105.6 ± 0.4611.0 ± 1.1932.5 ± 3.1757.3 ± 1.6319.4 ± 0.5033.9 ± 0.694.7 ± 0.682.1 ± 0.17
6NG_000006.1:g.34164_37967del3804/NM_000518.5(HBB):c.92+6T>C/
(n = 1)
α−3.7/IVS 1.65.0611.436.171.322.531.63.90.5
7HbA2: c.95+2_95+6delTGAGG/NM_000518.5(HBB):c.93-21G>A
(n = 1)
α2 IVS1 -5nt/IVS 1.1105.2411.836.569.722.532.35.55.4
8HbA2: c.95+2_95+6delTGAGG/NM_000518.5(HBB):c.92+1G>A/
(n = 1)
α2 IVS1 -5nt/IVS 1.16.3913.740.96421.433.56.30.8
9α−4.2/NG_000007.3:g63632_7104 del
(n = 1)
α−4.2/HB LEPORE-BW5.8114.94577.425.633.13.52.5
10NG_000006.1: g.15164_37864del22701
/NM_000518.5(HBB):c.118C>T
(n = 1)
-20.5 KB/CODON 395.0311.83671.623.532.84.70.3
11NG_000006.1:g.34164_37967del3804/HBB:c.20A>T
(n = 1)
α−3.7/CODON 6 [A>T]4.813.138.580.528.633.52.90.1
12NG_000001:g.34247_38050dup/NM_000518.5(HBB):c.118C>T
(n = 1)
ααα−3.7/CODON 394.7910.930.160.222.134.55.41.1
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Dell’Edera, D.; Persia, B.; La Rocca, F.; Centoducati, C. Haemoglobinopathies: Integrated Biochemical and Molecular Diagnosis in 5243 Patients. Hemato 2025, 6, 36. https://doi.org/10.3390/hemato6040036

AMA Style

Dell’Edera D, Persia B, La Rocca F, Centoducati C. Haemoglobinopathies: Integrated Biochemical and Molecular Diagnosis in 5243 Patients. Hemato. 2025; 6(4):36. https://doi.org/10.3390/hemato6040036

Chicago/Turabian Style

Dell’Edera, Domenico, Brunilde Persia, Francesco La Rocca, and Carmela Centoducati. 2025. "Haemoglobinopathies: Integrated Biochemical and Molecular Diagnosis in 5243 Patients" Hemato 6, no. 4: 36. https://doi.org/10.3390/hemato6040036

APA Style

Dell’Edera, D., Persia, B., La Rocca, F., & Centoducati, C. (2025). Haemoglobinopathies: Integrated Biochemical and Molecular Diagnosis in 5243 Patients. Hemato, 6(4), 36. https://doi.org/10.3390/hemato6040036

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