Antonio Amato

Correction of β-thalassemia major by gene transfer in haematopoietic progenitors of pediatric patients

β‐Thalassemia is a common monogenic disorder due to mutations in the β‐globin gene and gene therapy, based on autologous transplantation of genetically corrected haematopoietic stem cells (HSCs), holds the promise to treat patients lacking a compatible bone marrow (BM) donor. We recently showed correction of murine β‐thalassemia by gene transfer in HSCs with the GLOBE lentiviral vector (LV), expressing a transcriptionally regulated human β‐globin gene. Here, we report successful correction of thalassemia major in human cells, by studying a large cohort of pediatric patients of diverse ethnic origin, carriers of different mutations and all candidates to BM transplantation. Extensive characterization of BM‐derived CD34+ cells before and following gene transfer shows the achievement of high frequency of transduction, restoration of haemoglobin A synthesis, rescue from apoptosis and correction of ineffective erythropoiesis. The procedure does not significantly affect the differentiating potential and the relative proportion of haematopoietic progenitors. Analysis of vector integrations shows preferential targeting of transcriptionally active regions, without bias for cancer‐related genes. Overall, these results provide a solid rationale for a future clinical translation.

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”.

Screening and genetic diagnosis of hemoglobinopathies in southern and northern europe: two examples.

Prevention of Hemoglobinopathies has developed around the world based upon the experience done in pioneering endemic countries and is now facing a new phase in non-endemic areas with a recent immigration history. We describe two situations, taking Latium (central Italy) and The Netherlands as two models for endemic and non-endemic countries both confronted with a large multi-ethnic immigrant society. We present prevention results and discuss aspects such as local knowledge and organization. We illustrate the importance of issues like information, carrier diagnostics, screening, counseling and prenatal diagnosis in particular situation of contrasting interest an different ethical opinions. We conclude by underlining the importance of implementing primary prevention at the European level, based upon better information, diagnostics and counseling.

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.

Providing Appropriate Genetic Information to Healthy Carriers of Hemoglobinopathy Can Be a Welcome and Safe Initiative: The Latium Example

AIMS To register the opinions and feelings of (presumed) unaware healthy hemoglobinopathy carriers, receiving information on their carrier status. METHODS We collected 259 interviews from the parents of secondary school students, after their children had been provisionally diagnosed as hemoglobinopathy carriers during the routine school screening campaign imbedded in the public health care program of the Latium region (Central Italy). After screening of the children, all parents received a standard reassuring letter informing them about the presumed healthy carrier status of their children and were invited for a confirmation of the trait and for an additional explanation if needed. RESULTS We have analyzed 219 interviews (84.5%) from indigenous subjects and 40 from allochthonous people (15.5%) being either recent immigrants or mixed couples. The average age of the parents was 45.5 years. Only 51 (19.7%) had previous knowledge of their carrier status, while the rest were unaware. When reading the letter with the provisional diagnostic result of their child, emotions that could be considered undesirable were present in about 60% of the cases. Nevertheless, the information was experienced as welcome, clear, and useful by 100% of the participants. When asked about the option of prenatal diagnosis (PD) in case of genetic risk, 85.7% and 87.5% of the autochthonous and allochthonous interviewed declared either to be in favor or to eventually consider PD, while only 14.3% and 12.5% would not consider it for various reasons. DISCUSSION During our study, we registered undesirable feelings as well as welcome reactions: the first being experienced during the very first reading of the letter and the second after reflection on and understanding of the content during the visit to the center later on. Significantly, satisfaction and understanding of the advantage of knowledge was registered in 100% of the cases during our enquiry.

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).