Maria Pia Cappabianca

Application of MLPA assay to characterize unsolved α-globin gene rearrangements

α-thalassemia belongs to those inherited diseases in which large genomic deletions/duplications represent a significant proportion of causative mutations. Until recently, large α-globin gene cluster rearrangements have been mainly detected by gap-PCR and Southern blotting, methods that have significant drawbacks. We tested the recently developed multiplex ligation-dependent probe amplification (MLPA) assay for deletional screening of the α-globin gene cluster in a cohort of 25 individuals suspected of having α-globin alteration(s), in which no or doubtful mutations had been found using conventional methods. In 13 out of 18 α-thalassemia carriers and in all 5 patients with HbH we found the causative α-globin defects. In 2 thalassemia intermedia patients, carriers of heterozygous β-globin mutations, the co-inheritance of homozygous α-genes triplication was detected. MLPA results were subsequently confirmed by real-time PCR. This study shows that MLPA can effectively identify different and unknown types of α-globin gene rearrangements, to allow characterizing previously unsolved α-thalassemia genotypes.

Prevention strategies for severe hemoglobinopathies in endemic and nonendemic immigration countries: the Latium example

To review prevention data for hemoglobinopathies from Latium, a large Italian region with a considerable immigrant population and with a well‐established regional prevention program.

Factors regulating Hb F synthesis in thalassemic diseases

BackgroundThe thalassemic syndromes originate from mutations of the globin genes that cause, besides the characteristic clinical picture, also an increased Hb F amount. It is not yet clear if there are more factors, besides the beta globin genotype, determining the Hb F production. We have tried to find out if there are relations between total Hb and Hb F, between erythropoietin (Epo) and Hb F, between Hb F and point mutations of the gamma gene promoters.Materials and MethodsHematologic parameters, iron status, alpha/non-alpha globin ratio, Epo level, and thalassemic defects of the alpha-, beta-, and gamma-globin genes were explored using standard methods in patients affected by thalassemic diseases. Ninety-five non thalassemic individuals have been examined as controls.ResultsTwo clinical variants of beta-thalassemia intermedia referred to as beta-thal int sub-silent and evident are associated with distinct sets of mutations of the beta-globin gene. Silent beta thal mutations are invariably associated with sub-silent beta thal int; beta° or severe beta+ thal mutations are associated with evident beta thal int (88%) and almost invariably (98%) with thalassemia major. A positive correlation was observed between the severity of the disease and the Hb F level, but no correlation was found between the Hb F and erythropoietin (Epo) level. The mutation Ggamma -158 C→T was detected in 26.9% of patients affected by beta-thal int sub-silent and evident, respectively, but only in 2% of patients with thalassemia major.ConclusionsThe severity of beta-thal int and the increased Hb F level are strictly dependent from the type of beta-globin gene mutations. No relation is found between Hb F synthesis and Epo secretion. The mutation Ggamma -158 C→T, common among patients affected by beta-thal int and very rare in thal major patients, does not seem, in this study, to influence the Hb F content in beta thal int patients.

Interpreting elevated fetal hemoglobin in pathology and health at the basic laboratory level: new and known γ- gene mutations associated with hereditary persistence of fetal hemoglobin.

Fetal hemoglobin may be slightly or significantly elevated in post‐natal life due to a number of causes. We report two novel mutations found on the promoter of the Aγ gene and summarize all common and rare determinants associated with hereditary persistence of fetal hemoglobin (HPFH) described thus far. Hematological and molecular analysis of the Aγ globin gene in two cases of HPFH. Comparison of the novel cases with all those described in the literature. We have found two novel mutations in three Italian patients with HbF values between 5.9% and 6.5% without an elevated HbA2 and with normal hemoglobin parameters. In two probands (mother and son), a −197 C>T transition was observed, while in a single individual, a −113 A>G transition was present on the distal CCAAT box of the Aγ gene. As no other abnormalities were present in both γ‐gene promoters and the changes are located on regulatory sequences, we may conclude that these mutations are responsible for the HPFH phenotype shown by the carriers. The laboratory should be able to discriminate between elevated HbF due to artifacts or to serious causes including bone marrow malignancies, aplastic anemia, and β‐thalassemia major or recessive traits such as β‐thalassemia minor, δβ‐thalassemia, or nonpathological conditions induced by mutations or polymorphisms of the γ‐gene promoters that may even be beneficial when present in patients with thalassemia major or sickle cell disease and, in particular, when these patients are treated with hydroxyurea.

Two new β-thalassemia deletions compromising prenatal diagnosis in an Italian and a Turkish couple seeking prevention

Two novel deletions in the beta gene cluster were identified by Multiplex Ligation-dependent Probe Amplification in two at-risk couples seeking prevention. This study exemplifies a successful diagnostic approach in case one member of the couple is an atypical thalassemia carrier. When the molecular background of couples requesting prevention is unclear, family analysis and tools to define rare mutations are essential. We report two novel deletion defects observed in an Italian and in a Turkish couple. The first proband presented with microcytic hypochromic parameters without iron deficiency, a normal HbA2 and an elevated HbF (10.6%). His father presented with a similar phenotype and his wife was heterozygous for the common Mediterranean codon 39 (HBB:c.118C>T) mutation. Having excluded point mutations and common deletions, Multiplex Ligation-dependent Probe Amplification was performed revealing an unknown Gγ(Aγδβ)0-thalassemia defect spanning from the Aγ gene to downstream of the β-globin gene provisionally named Leiden 69.5 kb deletion. In the second case, the wife presented with a mild thalassemic picture, normal HbA2, elevated HbF (18.5%) and a β/α globin chain synthesis ratio of 0.62, without iron deficiency or any known β-thalassemia defect, while the husband was a simple carrier of the common Mediterranean IVS-I-110 (HBB:c.93-21 G>A) mutation. A new large deletion involving the β-gene and part of the δ-gene was identified by Multiplex Ligation-dependent Probe Amplification provisionally named “Leiden 7.4 kb”.

A new beta-chain haemoglobin variant with increased oxygen affinity: Hb Roma [beta115(g17)Ala-->Val].

BACKGROUND Haemoglobin Roma [beta115(G17)Ala-->Val] is a new adult haemoglobin variant found in a patient presenting a mild hypochromia and microcytosis. We studied this previously uncharacterised variant in order to evaluate the effect on the structural and funcional properties of the Ala-->Val substitution at the alpha1beta1 interface. METHODS AND RESULTS The variant chain was identified by direct DNA sequencing of the beta-globin gene, which revealed a GCC-->GTC mutation in codon 115. This mutation was confirmed by mass spectrometric analysis of the tetramers and peptides. The oxygen-binding properties of the haemoglobin A/haemoglobin Roma mixture, in which the variant makes up 25% of the haemoglobins, showed a significant increase in oxygen affinity with respect to normal haemoglobin A, both in the absence and presence of 2,3-bisphosphoglycerate. The role of the betaG17 position, situated at the alpha(1)beta(1) interface, has been examined using computational models of haemoglobin Roma and other known betaG17 variants, in comparison with normal haemoglobin A. CONCLUSIONS This study suggests that the beta115(G17)Ala-->Val substitution at the alpha1beta1 interface is responsible for increased oxygen affinity and mild destabilisation of the haemoglobin Roma. GENERAL SIGNIFICANCE An amino acid substitution at the G17 position of the alpha1beta1 interface may result in stabilisation of the high affinity R-state of the haemoglobin molecule.

Detection of a Rare β-Globin Nonsense Mutation [Codon 59 (AAG→TAG)] in an Italian Family

In this study we report on the hematological and molecular findings of a family from Central Italy, whose 33-year-old male proband presented with a β0-thalassemia (thal) trait associated to a relevant Hb F level. The proband and his family (parents and a sister) were investigated by hematological analysis. The two β-thal carriers of the β-globin nonsense mutation [codon 59 (AAG→TAG)] (the proband and his father) showed the hematological picture of a β0-thal trait: the only hematological difference between the two β-thal carriers was in the Hb F level (3.3% in the proband and 1% in his father).

Current Genetic Epidemiology of β-Thalassemias and Structural Hemoglobin Variants in the Lazio Region (Central Italy) Following Recent Migration Movements

The aim of this study was to describe the changing pattern of mutational spectrum of β-thalassemia (β-thal) in the Lazio region (Central Italy), as consequence of recent demographic variations. From 1994 until present, 256 immigrant subjects with hemoglobin disorders (including 191 heterozygotes and 65 homozygotes or compound heterozygotes) coming from 44 different foreign countries, have been molecularly characterized. 14 β-globin gene mutations were identified and their frequencies reflect different ethnic origins: 8 of these mutations account for 76.97% of all molecular defects, while 6 of them are much rare, representing less than 2% of the total. These data differ, both in type and percentage, from the mutational spectrum detected in the native population in 1995. Since a few defects are prevalent in each country, a proper strategy for the identification of mutations in immigrant individuals relies on the prior knowledge of their frequency in native ethnic group.

Hb Filottrano [codon 120 (–A)]: A Novel Frameshift Mutation in Exon 3 of the β-Globin Gene Causing Dominantly Inherited β-Thalassemia Intermedia

We report a novel frameshift mutation in exon 3 of the β-globin gene, that, in the heterozygous state, leads to a β-thalassemia intermedia (β-TI) phenotype (marked anemia, splenomegaly, hyperbilirubinemia, jaundice, unbalanced synthesis of α/non-α chains in a 34-year-old Italian woman. This frameshift mutation, due to the deletion of the first nucleotide (–A) at codon 120, results in a β-globin chain that is elongated to 156 amino acid residues. These highly unstable abnormal chains precipitate in the erythroblasts as inclusion bodies, thus causing inefficient erythropoiesis and ultimately resulting in the observed dominant clinical phenotype.

A Clinical Update of the Hb Siirt [β27(B9)Ala→Gly; HBB: c.83C>G] Hemoglobin Variant

Abstract We report a clinical update of the hemoglobin (Hb) variant [β27(B9)Ala→Gly; HBB: c.83C>G], named Hb Siirt, that was previously described as a silent variant in a 23-year-old Kurdish female. The patient was also a carrier of the codon 5 (–CT) (HBB: c.17_18delCT) frameshift mutation and of the ααα anti 3.7 triplication. Her initial moderate β-thalassemia intermedia (β-TI) phenotype worsened with time, causing the patient to become a transfusion-dependent subject at the age of ∼40 years. Subsequent molecular characterization of both parents revealed that the Hb Siirt variant was inherited by the mother, while the other two globin alterations (HBB: c.17_18delCT and αααanti 3.7 triplication) were genetically transmitted by the father. The latter remained a carrier of a mild β-TI phenotype throughout his life, at least until the age of 65 years. We hypothesize that the worsened clinical conditions in the daughter were due to the additional, maternally inherited Hb Siirt variant. However, protein 3D conformational analysis did not seem to reveal substantial overall structural changes. Among the other three described variants [Hb Volga (HBB: c.83C>A), Hb Knossos (HBB: c.82 G>T), Hb Grange-Blanche (HBB: c.83C>T] that are due to nucleotide substitutions at codon 27 of the β-globin gene; only Hb Knossos causes a β+-thalassemia (β+-thal) phenotype.