Induction of therapeutic levels of HbF in genome-edited primary ß0 39-thalassaemia haematopoietic stem and progenitor cells.

Hereditary persistence of fetal haemoglobin (HPFH) is the major modifier of the clinical severity of ß-thalassaemia. The homozygous mutation c.-196 C>T in the Aγ-globin (HBG1) promoter, which causes Sardinian δß0 -thalassaemia, is able to completely rescue the ß-major thalassaemia phenotype caused by the ß0 39-thalassaemia mutation, ensuring high levels of fetal haemoglobin synthesis during adulthood. Here, we describe a CRISPR/Cas9 genome-editing approach, combined with the non-homologous end joining (NHEJ) pathway repair, aimed at reproducing the effects of this naturally occurring HPFH mutation in both HBG promoters. After selecting the most efficient guide RNA in K562 cells, we edited the HBG promoters in human umbilical cord blood-derived erythroid progenitor 2 cells (HUDEP-2) and in haematopoietic stem and progenitor cells (HSPCs) from ß0 -thalassaemia patients to assess the therapeutic potential of HbF induction. Our results indicate that small deletions targeting the -196-promoter region restore high levels of fetal haemoglobin (HbF) synthesis in all cell types tested. In pools of HSPCs derived from homozygous ß0 39-thalassaemia patients, a 20% editing determined a parallel 20% increase of HbF compared to unedited pools. These results suggest that editing the region of HBG promoters around the -196 position has the potential to induce therapeutic levels of HbF in patients with most types of ß-thalassaemia irrespective of the ß-globin gene (HBB) mutations.

The Coup-TFII orphan nuclear receptor is an activator of the γ-globin gene.

The human fetal γ-globin gene is repressed in the adult stage through complex regulatory mechanisms involving transcription factors and epigenetic modifiers. Reversing γ-globin repression, or maintaining its expression by manipulating regulatory mechanisms, has become a major clinical goal in the treatment of ß-hemoglobinopathies. Here, we identify the orphan nuclear receptor Coup-TFII (NR2F2/ARP-1) as an embryonic/fetal stage activator of γ-globin expression. We show that Coup-TFII is expressed in early erythropoiesis of yolk sac origin, together with embryonic/fetal globins. When overexpressed in adult cells (including peripheral blood cells from human healthy donors and ß039 thalassemic patients) Coup-TFII activates the embryonic/fetal globins genes, overcoming the repression imposed by the adult erythroid environment. Conversely, the knock-out of Coup-TFII increases the ß/γ+ß globin ratio. Molecular analysis indicates that Coup-TFII binds in vivo to the ß-locus and contributes to its conformation. Overall, our data identify Coup-TFII as a specific activator of the γ-globin gene.

Changes in HbA2 and HbF in alpha thalassemia carriers with KLF1 mutation.

α-thalassemia carriers are common in Mediterranean regions, particularly in the Sardinian population. Their haematological phenotype is characterized by reduced MCV and/or MCH with normal or slightly reduced HbA2 levels and normal HbF. Krüppel-like factor 1 (KLF1) is a pleiotropic erythroid transcription factor that is essential for haematopoiesis. Mutations in the KLF1 gene trigger a series of benign human red blood phenotypes, such as an increase in HbA2 and HBF. Recently, it has been found that KLF1 mutations were a frequent cause of borderline HbA2 levels in a group of Sardinian subjects. Here, we found that KLF1 mutations modulate the phenotype in a cohort of α-thalassemia carriers.

A validated cellular biobank for ß-thalassemia.

BACKGROUND: Cellular biobanking is a key resource for collaborative networks planning to use same cells in studies aimed at solving a variety of biological and biomedical issues. This approach is of great importance in studies on ß-thalassemia, since the recruitment of patients and collection of specimens can represent a crucial and often limiting factor in the experimental planning. METHODS: Erythroid precursor cells were obtained from 72 patients, mostly ß-thalassemic, expanded and cryopreserved. Expression of globin genes was analyzed by real time RT-qPCR. Hemoglobin production was studied by HPLC. RESULTS: In this paper we describe the production and validation of a Thal-Biobank constituted by expanded erythroid precursor cells from ß-thalassemia patients. The biobanked samples were validated for maintenance of their phenotype after (a) cell isolation from same patients during independent phlebotomies, (b) freezing step in different biobanked cryovials, (c) thawing step and analysis at different time points. Reproducibility was confirmed by shipping the frozen biobanked cells to different laboratories, where the cells were thawed, cultured and analyzed using the same standardized procedures. The biobanked cells were stratified on the basis of their baseline level of fetal hemoglobin production and exposed to fetal hemoglobin inducers. CONCLUSION: The use of biobanked cells allows stratification of the patients with respect to fetal hemoglobin production and can be used for determining the response to the fetal hemoglobin inducer hydroxyurea and to gene therapy protocols with reproducible results.

The Problem of Borderline Hemoglobin A2 Levels in the Screening for ß-Thalassemia Carriers in Sardinia.

BACKGROUND: The increase in HbA2 is the most important parameter for the identification of thalassemia carriers. However, in routine screening for hemoglobinopathies, some cases are difficult to classify because the level of HbA2 is not typically elevated. In this work, we report the results of a molecular investigation on a cohort of subjects with borderline HbA2. METHODS: All subjects with a ß-thalassemia carrier partner and a borderline percentage level of HbA2 were investigated for the presence of a pathological mutation in the ß-globin gene. All negative subjects were screened for both the KLF1 mutation and the presence of ααα/ or αααα/ alleles. The subjects with reduced MCV and/or MCH were also screened for deletional and nondeletional α-globin gene defects. RESULTS: Various ß-globin mutations and KLF1 gene defects are the most common genetic determinants responsible for this phenotype in our population. CONCLUSION: KLF1 mutations are important in a screening program for hemoglobinopathies. An increase in HbF in association with borderline HbA2 levels is a useful but not exclusive marker that suggests the investigation of this gene. On the basis of our findings, we are able to suggest the molecular procedure to use in a population characterized by a high prevalence of thalassemia carriers.

A genetic score for the prediction of beta-thalassemia severity.

Clinical and hematologic characteristics of beta(ß)-thalassemia are determined by several factors resulting in a wide spectrum of severity. Phenotype modulators are: HBB mutations, HBA defects and fetal hemoglobin production modulators (HBG2:g.-158C>T polymorphism, HBS1L-MYB intergenic region and the BCL11A). We characterized 54 genetic variants at these five loci robustly associated with the amelioration of beta-thalassemia phenotype, to build a predictive score of severity using a representative cohort of 890 ß-thalassemic patients. Using Cox proportional hazard analysis on a training set, we assessed the effect of these loci on the age at which patient started regular transfusions, built a Thalassemia Severity Score, and validated it on a testing set. Discriminatory power of the model was high (C-index=0.705; R(2)=0.343) and the validation conducted on the testing set confirmed its predictive accuracy with transfusion-free survival probability (P<0.001) and with transfusion dependency status (Area Under the Receiver Operating Characteristic Curve=0.774; P<0.001). Finally, an automatized on-line calculation of the score was made available at http://tss.unica.it. Besides the accurate assessment of genetic predictors effect, the present results could be helpful in the management of patients, both as a predictive score for screening and a standardized scale of severity to overcome the major-intermedia dichotomy and support clinical decisions.

KLF1 gene mutations cause borderline HbA(2).

Increased hemoglobin A(2) (HbA(2); ie, levels > 3.9%) is the most important feature of ß-thalassemia carriers. However, it is not uncommon to find persons with borderline HbA(2) (levels, 3.3%-3.8%), who pose a relevant screening problem. Several genotypes have been associated with borderline HbA(2), but sometimes the reasons for this unusual phenotype are unknown. In this paper, we report, for the first time, that mutations of KLF1 result in HbA(2) levels in the borderline range. Six different KLF1 mutations were identified in 52 of 145 subjects with borderline HbA(2) and normal mean corpuscular volume and mean corpuscular hemoglobin. Two mutations (T327S and T280_H283del) are here reported for the first time. The prevalent mutation in Sardinians is S270X, which accounts for 80.8% of the total. The frequent discovery of KLF1 mutations in these atypical carriers may contribute significantly to the thalassemia screening programs aimed at identification of at risk couples.

Compound heterozygosity for KLF1 mutations associated with remarkable increase of fetal hemoglobin and red cell protoporphyrin.

The persistence of high fetal hemoglobin level in adults may ameliorate the clinical phenotype of beta-thalassemia and sickle cell anemia. Several genetic variants responsible for hereditary persistence of fetal hemoglobin, linked and not linked to the beta globin gene cluster, have been identified in patients and in normal individuals. Monoallelic loss of KLF1, a gene with a key role in erythropoiesis, has been recently reported to be responsible for persistence of high levels of fetal hemoglobin. In a Sardinian family, high levels of HbF (22.1-30.9%) were present only in compound heterozygotes for the S270X nonsense and K332Q missense mutations, while the isolated S270X nonsense (haploinsufficiency) or K332Q missense mutation were associated with normal HbF levels (<1.5%). Functionally, the K332Q Klf1 mutation impairs binding to the BCl11A gene and activation of the γ- and ß-globin promoters. Moreover, we report for the first time the association of KLF1 mutations with very high levels of zinc protoporphyrin.

Delta-Globin Gene Expression Is Enhanced in vivo by Interferon Type I.

Beta hemoglobinopathies are widely spread monogenic lethal diseases. Delta-globin gene activation has been proposed as a possible approach for curing these pathologies. The therapeutic potential of delta-globin, the non-alpha component of Hemoglobin A2 (α2δ2; HbA2), has been demonstrated in a mouse model of beta thalassemia, while its anti-sickling effect, comparable to that of gamma globin, was established some time ago. Here we show that the delta-globin mRNA level is considerably increased in a Deoxyribonuclease II-alpha knockout mouse model in which type 1 interferon (interferon beta, IFNb) is activated. IFNb activation in the fetal liver improves the delta-globin mRNA level, while the beta-globin mRNA level is significantly reduced. In addition, we show that HbA2 is significantly increased in patients with multiple sclerosis under type 1 interferon treatment. Our results represent a proof of principle that delta-globin expression can be enhanced through the use of molecules. This observation is potentially interesting in view of a pharmacological approach able to increase the HbA2 level.