Alexandra Kourakli

Genotypic heterogeneity and correlation to intergenic haplotype within high HbF β-thalassemia intermedia

Abstract:  Objectives: A molecular study was carried out of β‐thalassemia intermedia patients, compound heterozygotes for mutations usually found in β‐thalassemia major, with high levels of HbF in the absence of hereditary persistence of fetal hemoglobin (HPFH) syndrome. Our objective was to locate cis‐DNA structures, DNA haplotypes, motifs, or polymorphisms that may correlate with the presence of high HbF. Methods: Allele‐specific oligonucleotide (ASO) hybridization was used for the detection of mutations and restriction fragment length polymorphism (RFLP) analysis and automated sequencing for motifs, haplotypes, and polymorphisms. Southern blot was used for investigating α‐thalassemia and/or α‐ or γ‐globin genes triplications. RNA extracted from burst forming unit‐erythroid (BFU‐e) colonies of peripheral blood mononuclear cell cultures was used in reverse transcriptase‐polymerase chain reaction (RT‐PCR) to investigate intergenic transcription. Results: We established that (i) the combination: T haplotype of the Aγ‐δ‐globin intergenic region, the motif (TA)9N10(TA)10 in the HS2 site of locus control region (LCR), and TAG pre‐Gγ haplotype is sufficient but not necessary for high HbF, (ii) the genetic determinant(s) for high HbF involves an element associated with this combination and must be present in the specific R haplotype occurring in β‐thalassemia intermedia and (iii) the genetic determinant(s) for high HbF does not involve the abolition of intergenic transcription in the Aγ‐δ‐globin intergenic region. Conclusions: The genetic determinant(s) of high HbF in the absence of HPFH is linked to intergenic haplotype T and does not disrupt intergenic transcription.

Combined flash- and glow-type chemiluminescent reactions for high-throughput genotyping of biallelic polymorphisms

The difference in light-emission kinetics between the Ca(2+)-triggered bioluminescent reaction of the photoprotein aequorin (AEQ) and the alkaline phosphatase (ALP)-catalyzed chemiluminescent hydrolysis of dioxetane aryl phosphate substrates was exploited for the analysis of both alleles of biallelic polymorphisms in a single microtiter well. The genotyping of the IVS-1-110 locus of the human beta-globin gene was chosen as a model. Genomic DNA, isolated from whole blood, was first subjected to polymerase chain reaction using primers flanking the polymorphic site. A single oligonucleotide-ligation reaction employing two allele-specific probes, labeled with biotin and digoxigenin, and a common probe carrying a characteristic tail was then performed. The ligation products were captured in a microtiter well through hybridization of the tail with an immobilized complementary oligonucleotide. The products were detected by adding a mixture of streptavidin-aequorin complex and antidigoxigenin-alkaline phosphatase conjugate. AEQ was measured first by adding Ca(2+) and integrating the signal for 3s followed by the addition of the substrate for ALP. The ratio of the luminescence signals obtained from ALP and AEQ gives the genotype of each sample. The coefficient of variation of the dual assay ranged from 7 to 11% for each allele. The reproducibility of the ALP/AEQ signal ratio was about 14%. The proposed assay allows for many samples to be screened in parallel in a single microtiter plate, for single-nucleotide polymorphisms.

Genomic variation in the MAP3K5 gene is associated with β-thalassemia disease severity and hydroxyurea treatment efficacy

AIM In this study we explored the association between genetic variations in MAP3K5 and PDE7B genes, residing on chromosome 6q23, and disease severity in β-hemoglobinopathy patients, as well as the association between these variants with response to hydroxyurea (HU) treatment. Furthermore, we examined MAP3K5 expression in the context of high fetal hemoglobin (HbF) and upon HU treatment in erythroid progenitor cells from healthy and KLF1 haploinsufficient individuals. MATERIALS & METHODS For this purpose, we genotyped β-thalassemia intermedia and major patients and healthy controls, as well as a cohort of compound heterozygous sickle cell disease/β-thalassemia patients receiving HU as HbF augmentation treatment. Furthermore, we examined MAP3K5 expression in the context of high HbF and upon HU treatment in erythroid progenitor cells from healthy and KLF1 haploinsufficient individuals. RESULTS A short tandem repeat in the MAP3K5 promoter and two intronic MAP3K5 gene variants, as well as a PDE7B variant, are associated with low HbF levels and a severe disease phenotype. Moreover, MAP3K5 mRNA expression levels are altered in the context of high HbF and are affected by the presence of HU. Lastly, the abovementioned MAP3K5 variants are associated with HU treatment efficacy. CONCLUSION Our data suggest that these MAP3K5 variants are indicative of β-thalassemia disease severity and response to HU treatment.

New film-coated tablet formulation of deferasirox is well tolerated in patients with thalassemia or lower-risk MDS: Results of the randomized, phase II ECLIPSE study

Once‐daily deferasirox dispersible tablets (DT) have a well‐defined safety and efficacy profile and, compared with parenteral deferoxamine, provide greater patient adherence, satisfaction, and quality of life. However, barriers still exist to optimal adherence, including gastrointestinal tolerability and palatability, leading to development of a new film‐coated tablet (FCT) formulation that can be swallowed with a light meal, without the need to disperse into a suspension prior to consumption. The randomized, open‐label, phase II ECLIPSE study evaluated the safety of deferasirox DT and FCT formulations over 24 weeks in chelation‐naïve or pre‐treated patients aged ≥10 years, with transfusion‐dependent thalassemia or IPSS‐R very‐low‐, low‐, or intermediate‐risk myelodysplastic syndromes. One hundred seventy‐three patients were randomized 1:1 to DT (n = 86) or FCT (n = 87). Adverse events (overall), consistent with the known deferasirox safety profile, were reported in similar proportions of patients for each formulation (DT 89.5%; FCT 89.7%), with a lower frequency of severe events observed in patients receiving FCT (19.5% vs. 25.6% DT). Laboratory parameters (serum creatinine, creatinine clearance, alanine aminotransferase, aspartate aminotransferase and urine protein/creatinine ratio) generally remained stable throughout the study. Patient‐reported outcomes showed greater adherence and satisfaction, better palatability and fewer concerns with FCT than DT. Treatment compliance by pill count was higher with FCT (92.9%) than with DT (85.3%). This analysis suggests deferasirox FCT offers an improved formulation with enhanced patient satisfaction, which may improve adherence, thereby reducing frequency and severity of iron overload‐related complications.

A Single Nucleotide Polymorphism in the HBBP1 Gene in the Human β-Globin Locus is Associated with a Mild β-Thalassemia Disease Phenotype

The rs2071348 (g.5264146A>C) polymorphism on the HBB pseudogene, namely HBBP1, previously emerged as a variant significantly associated with a milder disease phenotype in Asian β0-thalassemia/hemoglobin (Hb) E (β0-thal/Hb E [β26(B8)Glu→Lys, GAG>AAG]) patients. In this study, we aimed to explore the possible association of rs2071348 with β-thalassemia (β-thal) disease severity in a group of β-thal major (β-TM) patients (severe phenotype) and β-thal intermedia (β-TI) patients (mild phenotype) of Hellenic origin and compare the results with normal (non thalassemic) individuals of the same origin. In addition, we explored whether this single nucleotide polymorphism (SNP) can be exploited as a pharmacogenomic marker to predict the outcome of Hb F-augmenting therapy in β-thal patients receiving hydroxyurea (HU). Our data suggest that the rs2071348 polymorphism is associated with higher Hb F levels and a milder β-thal disease phenotype. However, the rs2071348 polymorphism in the HBBP1 gene does not correlate with response to HU treatment.

Region-Specific Genetic Heterogeneity of HBB Mutation Distribution in South-Western Greece

β-Thalassemia (β-thal), is caused by reduced or absent synthesis of β-globin chains resulting in impaired erythropoiesis. It is the most common single gene defect disease in Greece, with heterozygous rates reaching, on average, 8% in the general population. Here, we performed molecular analyses on 199 unrelated β-thal and compound β-thal/sickle cell disease patients, of whom 157 originated from three prefectures of South-Western Greece, namely Achaia, Ilia and Etoloakarnania. Our results indicate that the frequency of specific HBB gene mutations, namely the HBB:c.118C>T (codon 39, C>T), HBB:c.92+6T>C (IVS-I-6, T>C), and HBB:c.20A>T [Hb S, β6(A3)Glu→Val, GAG>GTG], present distinct distribution patterns in the Achaia and Ilia prefectures (p < 0.001, p < 0.003 and p < 0.002, respectively). This detailed analysis of the distribution of the HBB gene mutations is useful for genetic counseling in the region, and illustrates that the identification of the HBB gene mutation spectrum in this region is necessary for population carrier screening and for efficient provision of prenatal diagnosis.

Plasma levels of lipoprotein-associated phospholipase A2 are increased in patients with β-thalassemia

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an independent cardiovascular risk factor. We investigated the plasma levels of Lp-PLA2 activity and mass as a function of plasma lipid levels, LDL subclass profile, and oxidative stress in patients with β-thalassemia. Thirty-five patients with β-thalassemia major (β-TM) and 25 patients with β-thalassemia intermedia (β-TI) participated in the study. Lp-PLA2 activity and mass were measured in total plasma, in apolipoprotein (apo)B-depleted plasma (HDL-Lp-PLA2), and in LDL subclasses. Lp-PLA2 activity produced and secreted from peripheral blood monocytes in culture was also determined. Patients with β-thalassemia are characterized by a predominance of small-dense LDL particles, increased oxidative stress, and very high plasma levels of Lp-PLA2 mass and activity, despite low LDL-cholesterol levels. A significant positive correlation between plasma Lp-PLA2 activity or mass and 8-isoprostane (8-epiPGF2a) and ferritin levels as well as intima-media thickness (IMT) values was observed. An increase in secreted and cell-associated Lp-PLA2 activity from monocytes in culture was observed in both patient groups. The HDL-Lp-PLA2 activity and mass as well as the ratio of HDL-Lp-PLA2/plasma Lp-PLA2 were significantly higher in both patient groups compared with the control group. In conclusion, patients with β-thalassemia exhibit high plasma Lp-PLA2 levels, attributed to increased enzyme secretion from monocytes/macrophages and to the predominance of sdLDL particles in plasma. Plasma Lp-PLA2 is correlated with carotid IMT, suggesting that this enzyme may be implicated in premature carotid atherosclerosis observed in β-thalassemia.

Premature atherosclerosis in non-transfusion-dependent β-thalassemia intermedia.

Objectives: β-Thalassemias are inherited hemolytic anemias with a broad phenotype and an increased rate of vascular complications despite a near absence of coronary artery disease. Methods: We investigated the presence of endothelial dysfunction and subclinical atherosclerosis in non-transfusion-dependent patients with β-thalassemia intermedia (β-TI) by means of flow-mediated (FMD) and flow-independent (FID) brachial artery dilatation and carotid artery intima-media thickness. Results: In 20 nondiabetic young adults with β-TI, FMD (6.6 ± 3.7 vs. 10.3 ± 3.1%; p = 0.002) and FID (14.0 ± 4.7 vs. 18.0 ± 5.6%; p = 0.02) were both lower relative to the values in 20 matched control subjects, whereas the intima-media thickness was increased (0.51 ± 0.09 vs. 0.46 ± 0.07 mm; p = 0.049). Fibrin generation, soluble endothelial activation markers, and proinflammatory proteins were higher in the patient group, while the plasma cholesterol level was lower. Conclusions: These findings indicate premature atherosclerosis among patients with β-TI; this is in accord with the high incidence of noncoronary vascular episodes in β-TI.

Cardiac mortality in β‐thalassemia major: resting but not dobutamine stress echocardiography predicts mortality among initially cardiac disease‐free patients in a prospective 12‐year study

Cardiac death remains the principal cause of mortality in β‐thalassemia major (β‐TM). Echocardiography may provide additional information, incremental to haematological profile, both for guiding chelation therapy and to assess prognosis.

Correlation of SIN3A genomic variants with β-hemoglobinopathies disease severity and hydroxyurea treatment efficacy

AIMS Hemoglobinopathies, particularly β-thalassemia and sickle cell disease, are characterized by great phenotypic variability in terms of disease severity, while notable differences have been observed in hydroxyurea treatment efficacy. In both cases, the observed phenotypic diversity is mostly dependent on the elevated fetal hemoglobin levels, resulting from the persistent fetal globin gene expression in the adult erythroid stage orchestrated by intricate mechanisms that still remain only partly understood. We have previously shown that several protein factors act as modifiers of fetal hemoglobin production, exerting their effect via different pathways. MATERIALS & METHODS Here, we explored whether SIN3A could act as a modifier of fetal globin gene expression, as it interacts with KLF10, a known modifier of fetal hemoglobin production. RESULTS We show that SIN3A genomic variants are associated both with β-thalassemia disease severity (rs11072544) as well as hydroxyurea treatment response (rs7166737) in β-hemoglobinopathies patients. CONCLUSION Our findings further underline that fetal hemoglobin production is the result of a complex interplay in which several human globin gene cluster variants interact with protein factors encoded by modifier genes to produce the observed clinical outcome.