Christina Vrettou

Thalassaemia was the first human disease for which a genetic basis was elucidated. The marked clinical heterogeneity of this monogenic disorder has attracted research interest, particularly towards the identification of genetic and other modifiers of disease phenotype. The aim of the present study was to investigate genotype–phenotype correlations in pediatric patients with thalassaemia followed at a Pediatric Thalassaemia Unit. Medical records of patients with thalassaemia followed at the Pediatric Thalassaemia Unit of Northern Greece over a 30-year period were retrospectively reviewed for demographic characteristics, genetic findings, transfusion dependency, and disease-related complications. Among 82 pediatric patients with β-thalassaemia, 54 were transfusion-dependent. Thirty different β-globin variants were identified, with HBB:c.93-21G>A (IVSI-110 G>A) being the most frequent and associated with a significantly younger age of transfusion initiation. Among the small number of patients carrying the HBB:c.-151C>T (−101 C>T) promoter variant in combination with either a β⁰ or β⁺ variant, an association with an NTDT phenotype was observed. All 36 patients carrying homozygous β⁰/β⁰ or β⁺/β⁺ variants, or compound heterozygous β⁰/β⁺ mutations were transfusion-dependent, whereas the remaining TDT patients carried β⁰ or β⁺ and silent β-globin variants or α-globin gene triplication. Four genotypes found in 16 patients were associated with both transfusion-dependent and non-transfusion-dependent phenotypes. Notably, two pairs of related patients carrying the same genotype exhibited discordant clinical phenotypes. The clinical heterogeneity of β-thalassaemia is further highlighted by the discordant phenotypes observed among patients carrying the same genotype, underscoring the need to identify additional factors that influence clinical expression. Characterization of these factors is important, not only for more accurate genetic counseling and prognostic assessment, but also for individualization of treatment strategies and appropriate selection of candidates for emerging therapeutic approaches.

Haemoglobinopathies are among the most prevalent genetic disorders globally. In the context of these conditions, preimplantation genetic testing (PGT) plays a pivotal role in preventing genetic diseases in the offspring of carrier parents, reducing the need for pregnancy termination and enabling the selection of compatible sibling donors for potential stem cell transplantation in cases of thalassemia or sickle cell disease. This review explores the evolving role of PGT as a reproductive option for haemoglobinopathy carriers, tracing the development of PGT protocols from patient-specific to comprehensive testing enabled by advanced technologies like next-generation sequencing (NGS). We discuss key technical, biological, and practical limitations of PGT, as well as the ethical considerations specific to haemoglobinopathies, such as the complexity of interpreting genotypes. Emerging technologies, such as whole-genome sequencing, non-invasive PGT, and gene editing, hold significant promise for expanding applications but also raise new challenges that must be addressed. It will be interesting to explore how advancements in technology, along with the changing management of haemoglobinopathies, will impact reproductive choices. It is anticipated that continued research will improve genetic counseling for PGT for haemoglobinopathies, while a careful evaluation of ethical and societal implications is also required. Responsible and equitable implementation of PGT is essential for ensuring that all families at risk can make informed reproductive choices.

It is well known that modifiers play a role in ameliorating or exacerbating disease phenotypes in patients and carriers of recessively inherited disorders such as sickle cell disease and thalassemia. Here, we give an overview of the literature concerning a recently described association in carriers of SUPT5H Loss-of-Function variants with a beta-thalassemia-like phenotype including the characteristic elevated levels of HbA₂. That SUPT5H acts as modifier in beta-thalassemia carriers became evident from three reported cases in whom combined heterozygosity of SUPT5H and HBB gene variants was observed to resemble a mild beta-thalassemia intermedia phenotype. The different SUPT5H variants and hematologic parameters reported are collected and reviewed to provide insight into the possible effects on hematologic expression, as well as potential disease mechanisms in carriers and patients.

In this short review, we presented and discussed studies on the expression of globin genes in β-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of β-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of β-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by β-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with β-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.

The Corfu δ⁰β⁺ thalassemic allele is a unique thalassemic allele consisting of the simultaneous presence in cis of a deletion of the δ-globin (Hemoglobin Subunit Delta, HBD) and a single nucleotide variant in the β-globin gene (Hemoglobin Subunit Beta, HBB). The allele has, so far, been described in individuals of Greek origin. The objectives of the study are to ascertain the prevalence of the Corfu δ⁰β⁺ allele in comparison to other β-thalassemia variants encountered in Greece using our in-house data repository of 2558 β-thalassemia heterozygotes, and to evaluate the hematological phenotype of Corfu δ⁰β⁺ heterozygotes in comparison to heterozygotes with the most common β⁺- and deletion α⁰- thalassemia variants in Greece. The results of the study showed a relative incidence of heterozygotes with Corfu δ⁰β⁺ at 1.56% of all β-thalassemic alleles, and a distinct hematological phenotype of the heterozygotes characterized by microcytic, hypochromic anemia with normal levels of HbA₂ (Hemoglobin A2) and elevated HbF (Hemoglobin F) levels. The application of a specific methodology for the identification of the Corfu δ⁰β⁺ allele is important for precise prenatal and antenatal diagnosis programs in Greece.

The inherited disorders of hemoglobin synthesis constitute the most common monogenic diseases worldwide. The clinical severity of β-thalassemia major and the sickle cell syndromes targets them as priority genetic diseases for prevention programs, which incorporates population screening to identify heterozygotes, with the option of prenatal diagnosis for carrier couples. Rapid genotype characterization is fundamental in the diagnostic laboratory, especially when offering prenatal diagnosis. The application of real-time PCR provides a means for rapid and potentially high throughput assays, without compromising accuracy. It has several advantages over end-point PCR analysis, including the elimination of post-PCR processing steps and a wide dynamic range of detection with a high degree of sensitivity. Although there are over 200 mutations associated with the β-thalassemia and sickle cell syndromes, the relatively small size of the β-, HBB gene (less than 2000 base-pairs) and the close proximity of most mutations facilitates the design of a minimal number of real-time PCR assays using the LightCycler™ system, which are capable of detecting the majority of most common β-gene mutations world-wide. These assays are highly appropriate for rapid genotyping of parental and fetal DNA samples with respect to β-thalassemia and sickle cell syndromes.

Human leukocyte antigen (HLA) typing of in vitro fertilization (IVF) embryos, aims to establish a pregnancy that is HLA compatible with an affected sibling who requires hematopoietic stem cell transplantation (HSCT). It can be performed with or without preimplantation genetic diagnosis (PGD) for exclusion of a single-gene disorder (SGD) and it is a multistep, technically challenging procedure at every stage. Our purpose was to address the difficulties of genetic analysis by developing a fast, reliable and accurate PGD-HLA protocol, to simplify patient work-up and PGD application, while providing high flexibility for combination with any SGD. Requests included PGD-HLA for β-thalassemia (β-thal)/sickle cell disease (most common request), Diamond-Blackfan anemia (DBA), chronic granulomatous disease (CGD) and preimplantation-HLA typing only. For HLA haplotyping, we selected a panel of 26 short tandem repeats (STRs) distributed across the entire HLA locus, following PGD guidelines. When required, mutation detection was performed by both a direct and indirect approach. To support concurrent SGD exclusion and HLA typing, a one-step, single-tube, multiplex fluorescent touchdown-polymerase chain reaction (PCR) was optimized. The described touchdown-PCR was successfully applied for all PGD-HLA protocols. Eight clinical cycles were performed with a diagnosis achieved for 94.7% of amplified biopsied blastomeres. Embryo transfer took place in six cycles, with two pregnancies achieved and two healthy female infants (from a twin pregnancy) born so far. Our protocol enables HLA typing in a single PCR, reducing the risk of contamination and the cost, and providing faster results. It requires minimum optimization before clinical application, irrespective of the SGD involved, decreasing the waiting time from referral to treatment for all PGD-HLA cases.

The most frequent defects resulting in alpha-thalassemia (thal) include large deletions that remove one or both of the duplicated alpha-globin genes on chromosome 16. Less commonly, alpha-thal mutations involve single nucleotide substitutions or micro-deletions, leading either directly to decreased alpha-globin chain synthesis by the affected allele, or indirectly through production of hyperunstable variant alpha-globin chains. Here we describe the characterization of a 33 bp in-frame deletion within the alpha1-globin gene, in a woman with hematological findings consistent with an alpha-thal trait. The amino acids predicted to be missing as a result of the 33 bp deletion are at the end of the E helix and the EF corner of the alpha-globin protein chain, and are not normally involved in the heme contact, although it is presumed that alpha-globin chain folding and hemoglobin (Hb) formation will be disrupted. The observation of inclusion and Heinz bodies indicates the synthesis of some abnormal Hb (or globin chains). An identical mutation has been previously observed in a single case, a Canadian individual of Greek descent, indicating that it is a rare mutation, and probably of the same origin. Possible mechanisms underlying the mutation at the DNA level are discussed.