Reactivation of developmentally silenced globin genes by forced chromatin looping.

Distal enhancers commonly contact target promoters via chromatin looping. In erythroid cells, the locus control region (LCR) contacts ß-type globin genes in a developmental stage-specific manner to stimulate transcription. Previously, we induced LCR-promoter looping by tethering the self-association domain (SA) of Ldb1 to the ß-globin promoter via artificial zinc fingers. Here, we show that targeting the SA to a developmentally silenced embryonic globin gene in adult murine erythroblasts triggers its transcriptional reactivation. This activity depends on the LCR, consistent with an LCR-promoter looping mechanism. Strikingly, targeting the SA to the fetal γ-globin promoter in primary adult human erythroblasts increases γ-globin promoter-LCR contacts, stimulating transcription to approximately 85% of total ß-globin synthesis, with a reciprocal reduction in adult ß-globin expression. Our findings demonstrate that forced chromatin looping can override a stringent developmental gene expression program and suggest a novel approach to control the balance of globin gene transcription for therapeutic applications.

Effect of Migalastat on cArdiac InvOlvement in FabRry DiseAse: MAIORA study.

BACKGROUND: A small but significant reduction in left ventricular (LV) mass after 18 months of migalastat treatment has been reported in Fabry disease (FD). This study aimed to assess the effect of migalastat on FD cardiac involvement, combining LV morphology and tissue characterisation by cardiac magnetic resonance (CMR) with cardiopulmonary exercise testing (CPET). METHODS: Sixteen treatment-naïve patients with FD (4 women, 46.4±16.2 years) with cardiac involvement (reduced T1 values on CMR and/or LV hypertrophy) underwent ECG, echocardiogram, troponin T and NT-proBNP (N-Terminal prohormone of Brain Natriuretic Peptide) assay, CMR with T1 mapping, and CPET before and after 18 months of migalastat. RESULTS: No change in LV mass was detected at 18 months compared to baseline (95.2 g/m2 (66.0-184.0) vs 99.0 g/m2 (69.0-121.0), p=0.55). Overall, there was an increase in septal T1 of borderline significance (870.0 ms (848-882) vs 860.0 ms (833.0-875.0), p=0.056). Functional capacity showed an increase in oxygen consumption (VO2) at anaerobic threshold (15.50 mL/kg/min (13.70-21.50) vs 14.50 mL/kg/min (11.70-18.95), p=0.02), and a trend towards an increase in percent predicted peak VO2 (72.0 (63.0-80.0) vs 69.0 (53.0-77.0), p=0.056) was observed. The subset of patients who showed an increase in T1 value and a reduction in LV mass (n=7, 1 female, age 40.5 (28.6-76.0)) was younger and at an earlier disease stage compared to the others, and also exhibited greater improvement in exercise tolerance. CONCLUSION: In treatment-naïve FD patients with cardiac involvement, 18-month treatment with migalastat stabilised LV mass and was associated with a trend towards an improvement in exercise tolerance. A tendency to T1 increase was detected by CMR. The subset of patients who had significant benefits from the treatment showed an earlier cardiac disease compared to the others. TRIAL REGISTRATION NUMBER: NCT03838237.

Therapeutic perspective for children and young adults living with thalassemia and sickle cell disease.

Hemoglobinopathies, including thalassemias and sickle cell disease, are the most common monogenic diseases worldwide, with estimated annual births of more than 330,000 affected infants. Hemoglobin disorders account for about 3.4% of deaths in children under 5 years of age. The distribution of these diseases is historically linked to current or previously malaria-endemic regions; however, immigration has led to a worldwide distribution of these diseases, making them a global health problem. During the last decade, new treatment approaches and novel therapies have been proposed, some of which have the potential to change the natural history of these disorders. Indeed, the first erythroid maturation agent, luspatercept, and gene therapy have been approved for beta-thalassemia adult patients. For sickle cell disease, molecules targeting vaso-occlusion and hemoglobin S polymerization include crizanlizumab, which has been approved for patients ≥ 16 years, voxelotor approved for patients ≥ 12 years, and L-glutamine for patients older than 5 years.    Conclusion: We herein present the most recent advances and future perspectives in thalassemia and sickle cell disease treatment, including new drugs, gene therapy, and gene editing, and the current clinical trial status in the pediatric populations. What is Known: • Red blood cell transfusions, iron chelation therapy and hematopoietic stem cell transplantation have been the mainstay of treatment of thalassemia patients for decades. • For sickle cell disease, until 2005, treatment strategies were mostly the same as those for thalassemia, with the option of simple transfusion or exchange transfusion. In 2007, hydroxyurea was approved for patients ≥ 2 years old. What is New: • In 2019, gene therapy with betibeglogene autotemcel (LentiGlobin BB305) was approved for TDT patients ≥ 12 years old non ß0/ß0 without matched sibling donor. • Starting from 2017 several new drugs, such as L-glutamine (approved only by FDA), crizanlizumab (approved by FDA and EMA for patients ≥ 16 years), and lastly voxelotor (approved by FDA and EMA for patients ≥ 12 years old).

Thalassaemia is paradoxically associated with a reduced risk of in-hospital complications and mortality in COVID-19: Data from an international registry.

Although numerous patient-specific co-factors have been shown to be associated with worse outcomes in COVID-19, the prognostic value of thalassaemic syndromes in COVID-19 patients remains poorly understood. We studied the outcomes of 137 COVID-19 patients with a history of transfusion-dependent thalassaemia (TDT) and transfusion independent thalassaemia (TIT) extracted from a large international cohort and compared them with the outcomes from a matched cohort of COVID-19 patients with no history of thalassaemia. The mean age of thalassaemia patients included in our study was 41 ± 16 years (48.9% male). Almost 81% of these patients suffered from TDT requiring blood transfusions on a regular basis. 38.7% of patients were blood group O. Cardiac iron overload was documented in 6.8% of study patients, whereas liver iron overload was documented in 35% of study patients. 40% of thalassaemia patients had a history of splenectomy. 27.7% of study patients required hospitalization due to COVID-19 infection. Amongst the hospitalized patients, one patient died (0.7%) and one patient required intubation. Continuous positive airway pressure (CPAP) was required in almost 5% of study patients. After adjustment for age-, sex- and other known risk factors (cardiac disease, kidney disease and pulmonary disease), the rate of in-hospital complications (supplemental oxygen use, admission to an intensive care unit for CPAP therapy or intubation) and all-cause mortality was significantly lower in the thalassaemia group compared to the matched cohort with no history of thalassaemia. Amongst thalassaemia patients in general, the TIT group exhibited a higher rate of hospitalization compared to the TDT group (p = 0.001). In addition, the rate of complications such as acute kidney injury and need for supplemental oxygen was significantly higher in the TIT group compared to the TDT group. In the multivariable logistic regression analysis, age and history of heart or kidney disease were all found to be independent risk factors for increased in-hospital, all-cause mortality, whereas the presence of thalassaemia (either TDT or TIT) was found to be independently associated with reduced all-cause mortality. The presence of thalassaemia in COVID-19 patients was independently associated with lower in-hospital, all-cause mortality and few in-hospital complications in our study. The pathophysiology of this is unclear and needs to be studied in vitro and in animal models.

The role of hypoxia and inflammation in the regulation of iron metabolism and erythropoiesis in COVID-19: The IRONCOVID study.

Coronavirus Disease (COVID-19) can be considered as a human pathological model of inflammation combined with hypoxia. In this setting, both erythropoiesis and iron metabolism appear to be profoundly affected by inflammatory and hypoxic stimuli, which act in the opposite direction on hepcidin regulation. The impact of low blood oxygen levels on erythropoiesis and iron metabolism in the context of human hypoxic disease (e.g., pneumonia) has not been fully elucidated. This multicentric observational study was aimed at investigating the prevalence of anemia, the alterations of iron homeostasis, and the relationship between inflammation, hypoxia, and erythropoietic parameters in a cohort of 481 COVID-19 patients admitted both to medical wards and intensive care units (ICU). Data were collected on admission and after 7 days of hospitalization. On admission, nearly half of the patients were anemic, displaying mild-to-moderate anemia. We found that hepcidin levels were increased during the whole period of observation. The patients with a higher burden of disease (i.e., those who needed intensive care treatment or had a more severe degree of hypoxia) showed lower hepcidin levels, despite having a more marked inflammatory pattern. Erythropoietin (EPO) levels were also lower in the ICU group on admission. After 7 days, EPO levels rose in the ICU group while they remained stable in the non-ICU group, reflecting that the initial hypoxic stimulus was stronger in the first group. These findings strengthen the hypothesis that, at least in the early phases, hypoxia-driven stimuli prevail over inflammation in the regulation of hepcidin and, finally, of erythropoiesis.

Epidemiological shift of glucose-6-phosphate dehydrogenase mutations in northern Italy in the last 15 years.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked recessive hemolytic anemia caused by mutations in G6PD gene. The distribution and frequency of genetic variants differ depending on ethnicity and geographical areas. Because of new migrations different variants are now present in Europe. This retrospective study aims to identify variants among the G6PD deficient subjects referred since 2004 to IRCCS Ca' Granda Foundation Hospital in Milan. The subjects were divided into 3 groups: group 1 (2004-2008), group 2 (2009-2013), and group 3 (2014-2018). During 15 years a significant decrease of the Mediterranean and an important increase of the African, Asian, and uncommon variants (classified as Others) have been observed. Three new mutations were found: in group 2 heterozygosity for c.[1454G > A] (Gly485Asp) in an adult female with severe anemia, high bilirubin levels and G6PD activity of 0,69 (IU/gHb) and heterozygosity for c.[584A > G] (Gln195Arg) in an elderly woman of Italian origin showing only anemia and enzymatic activity of 1,54 (IU/gHb) were detected. In group 3 hemizygosity for c.[670A > T] (Ile224Phe) in an adult Chinese man without anemia but with total absence of G6PD activity was found. These data reflect the appearance of uncommon G6PD mutations in northern Italy, probably due to new migrations, as consequence G6PD characterization becomes a diagnostic issue.

Liver steatosis is highly prevalent and is associated with metabolic risk factors and liver fibrosis in adult patients with type 1 Gaucher disease.

BACKGROUND AND AIMS: Gaucher disease (GD) is associated with peculiar metabolic abnormalities (ie hypermetabolic state, peripheral insulin resistance, dyslipidaemia), partially reverted by enzyme replacement therapy (ERT) at the expense of weight gain. Such metabolic alterations together with an unhealthy lifestyle acquired by an ageing GD population may favour the development of liver steatosis. We aimed at evaluating the prevalence of significant liver steatosis and at identifying the factors associated with liver steatosis in a cohort of patients with type 1 GD. METHODS: Twenty adult type 1 GD patients from an Italian academic referral centre were prospectively submitted to vibration-controlled transient elastography (Fibroscan®) with controlled attenuation parameter (CAP); significant steatosis was defined as CAP values ≥250 dB/min. RESULTS: Median CAP values were 234 [165-358] dB/min and 8 patients (40%) had significant steatosis. Significant steatosis was associated with indices of adiposity (weight, BMI and waist circumference), high blood pressure, insulin resistance and metabolic syndrome. GD-related variables and dose and duration of ERT were not associated with significant steatosis. In the subgroup of 16 patients on stable ERT for at least 24 months, CAP resulted significantly and positively associated with liver stiffness (rho 0.559, P = .024). CONCLUSIONS: Significant steatosis is highly prevalent in adult type 1 GD patients and is strongly associated with a worse metabolic profile, featuring metabolic dysfunction-associated fatty liver disease (MAFLD). MAFLD may determine liver fibrosis progression in GD patients on stable ERT and may be a risk factor for long-term liver-related complications.

2'-O-methoxyethyl splice-switching oligos correct splicing from IVS2-745 ß-thalassemia patient cells restoring HbA production and chain rebalance.

ß-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for ß-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects ß-globin synthesis in ß-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the ß-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits ß-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between ß-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of ß-thalassemia caused by mutations leading to aberrant splicing.

Iron deficiency from the standpoint of cardiac rehabilitation: novel therapeutic opportunities.

Anemia is one of the most frequent comorbidities found in patients with coronary artery disease and chronic heart failure (CHF) who are being followed in cardiac rehabilitation facilities. The more frequent type of anemia is caused by iron deficiency (IDA, iron-deficiency anemia): this review summarizes the state of the art of this topic. First of all, the mechanisms of IDA will be analyzed. Subsequently, a description of the main conditions where IDA can unfavorably affect the clinical course, and of its more frequent complications, will be presented (percutaneous interventions, heart surgery, CHF). Special attention will be paid in the description of anemia in the setting of CHF. To this regard, in recent years a relevant amount of research has been carried out, to determine whether treating anemia (either by directly stimulating erythropoiesis or by correcting iron deficiency by oral or intravenous route) is of any clinical and prognostic relevance in patients with CHF. The results of this research will, therefore, be summarized and critically discussed. Finally, we will outline the promising role of cardiac rehabilitation facilities and of its network of experts in the diagnosis, prognostic stratification, and treatment of anemia and iron deficiency.

Prevalence and predictors of liver fibrosis evaluated by vibration controlled transient elastography in type 1 Gaucher disease.

BACKGROUND & AIMS: Long-term liver-related complications of Gaucher disease (GD) include cirrhosis, portal hypertension and hepatocellular carcinoma. Although liver fibrosis is the main determinant of adverse liver-related clinical outcomes, it has rarely been evaluated in previously published cohorts of GD patients. We aimed at: assessing the prevalence of significant liver fibrosis in a cohort of patients with type 1 GD; identifying its predictors among GD-related variables, enzyme replacement therapy (ERT) and metabolic features. METHODS: 37 adult type 1 GD patients from two Italian academic referral centers were prospectively submitted to vibration controlled transient elastography (Fibroscan®); significant fibrosis was defined as liver stiffness ≥7 kPa. RESULTS: Median liver stiffness was 4.6 [3-15.1] kPa and 7 patients (19%) had significant fibrosis. Significant fibrosis was associated with splenectomy (p = .046) and with scores (DS3: p = .002; SSI: p = .026) and biomarkers (ACE: p = .016; HDL cholesterol: p = .004) of GD severity. Length of ERT was significantly lower in GD patients with significant fibrosis. In the subgroup of 29 patients who were on stable ERT for at least 24 months, further to splenectomy, GD severity and non-N370S GBA1 genotypes, also diastolic blood pressure, BMI and the number of metabolic syndrome (MetS) components emerged as factors significantly associated with significant fibrosis. CONCLUSIONS: Significant fibrosis is present in a remarkable proportion of adult type 1 GD patients. Splenectomy, GD severity and GBA1 genotypes are major GD-related predictors of liver fibrosis. Length of ERT is inversely correlated with liver disease in GD patients, suggesting a beneficial effect of ERT on liver fibrosis. However, GD patients on stable ERT should be monitored for metabolic complications, since MetS features may enhance liver disease progression despite optimal GD control.

Forced chromatin looping raises fetal hemoglobin in adult sickle cells to higher levels than pharmacologic inducers.

Overcoming the silencing of the fetal γ-globin gene has been a long-standing goal in the treatment of sickle cell disease (SCD). The major transcriptional enhancer of the ß-globin locus, called the locus control region (LCR), dynamically interacts with the developmental stage-appropriate ß-type globin genes via chromatin looping, a process requiring the protein Ldb1. In adult erythroid cells, the LCR can be redirected from the adult ß- to the fetal γ-globin promoter by tethering Ldb1 to the human γ-globin promoter with custom-designed zinc finger (ZF) proteins (ZF-Ldb1), leading to reactivation of γ-globin gene expression. To compare this approach to pharmacologic reactivation of fetal hemoglobin (HbF), hematopoietic cells from patients with SCD were treated with a lentivirus expressing the ZF-Ldb1 or with chemical HbF inducers. The HbF increase in cells treated with ZF-Ldb1 was more than double that observed with decitabine and pomalidomide; butyrate had an intermediate effect whereas tranylcypromine and hydroxyurea showed relatively low HbF reactivation. ZF-Ldb1 showed comparatively little toxicity, and reduced sickle hemoglobin (HbS) synthesis as well as sickling of SCD erythroid cells under hypoxic conditions. The efficacy and low cytotoxicity of lentiviral-mediated ZF-Ldb1 gene transfer compared with the drug regimens support its therapeutic potential for the treatment of SCD.

A multicentre observational study for early diagnosis of Gaucher disease in patients with Splenomegaly and/or Thrombocytopenia.

Gaucher disease (GD) is the most common lysosomal disorder resulting from deficient activity of the ß-glucosidase enzyme that causes accumulation of glucosylceramide in the macrophage-monocyte system. Notably, because of non-specific symptoms and a lack of awareness, patients with GD experience long diagnostic delays. The aim of this study was to apply a diagnostic algorithm to identify GD type 1 among adults subjects referred to Italian haematology outpatient units because of splenomegaly and/or thrombocytopenia and, eventually, to estimate the prevalence of GD in this selected population. One hundred and ninety-six subjects (61 females, 135 males; mean age 47.8 ± 18.2 years) have been enrolled in the study and tested for ß-glucosidase enzyme activity on dried blood spot (DBS). Seven of 196 patients have been diagnosed with GD, (5 females and 2 males) with mean age 31.8 ± 8.2 years, with a prevalence of 3.6% (with a prevalence of 3.6% (I95% CI 1.4-7.2; 1/28 patients) in this population. These results show that the use of an appropriate diagnostic algorithm and a simple diagnostic method, such as DBS, are important tools to facilitate the diagnosis of a rare disease even for not disease-expert physicians.