Iron chelation therapy for children with transfusion-dependent ß-thalassemia: How young is too young?

In this review, we provide a summary of evidence on iron overload in young children with transfusion-dependent ß-thalassemia (TDT) and explore the ideal timing for intervention. Key data from clinical trials and observational studies of the three available iron chelators deferoxamine, deferiprone, and deferasirox are also evaluated for inclusion of subsets of young children, especially those less than 6 years of age. Evidence on the efficacy and safety of iron chelation therapy for children ≥2 years of age with transfusional iron overload is widely available. New data exploring the risks and benefits of early-start iron chelation in younger patients with minimal iron overload are also emerging.

RP1 Dominant p.Ser740* Pathogenic Variant in 20 Knowingly Unrelated Families Affected by Rod-Cone Dystrophy: Potential Founder Effect in Western Sicily.

Background and Objectives. Retinitis pigmentosa (RP) is the most common inherited rod-cone dystrophy (RCD), resulting in nyctalopia, progressive visual field, and visual acuity decay in the late stages. The autosomal dominant form (ADRP) accounts for about 20% of RPs. Among the over 30 genes found to date related to ADRP, RP1 pathogenic variants have been identified in 5-10% of cases. In a cohort of RCD patients from the Palermo province on the island of Sicily, we identified a prevalent nonsense variant in RP1, which was associated with ADRP. The objective of our study was to analyse the clinical and molecular data of this patient cohort and to evaluate the potential presence of a founder effect. Materials and Methods. From 2005 to January 2023, 84 probands originating from Western Sicily (Italy) with a diagnosis of RCD or RP and their relatives underwent deep phenotyping, which was performed in various Italian clinical institutions. Molecular characterisation of patients and familial segregation of pathogenic variants were carried out in different laboratories using Sanger and/or next-generation sequencing (NGS). Results. Among 84 probands with RCD/RP, we found 28 heterozygotes for the RP1 variant c.2219C>G, p.Ser740* ((NM_006269.2)*, which was therefore significantly prevalent in this patient cohort. After a careful interview process, we ascertained that some of these patients shared the same pedigree. Therefore, we were ultimately able to define 20 independent family groups with no traceable consanguinity. Lastly, analysis of clinical data showed, in our patients, that the p.Ser740* nonsense variant was often associated with a late-onset and relatively mild phenotype. Conclusions. The high prevalence of the p.Ser740* variant in ADRP patients from Western Sicily suggests the presence of a founder effect, which has useful implications for the molecular diagnosis of RCD in patients coming from this Italian region. This variant can be primarily searched for in RP-affected subjects displaying compatible modes of transmission and phenotypes, with an advantage in terms of the required costs and time for analysis. Moreover, given its high prevalence, the RP1 p.Ser740* variant could represent a potential candidate for the development of therapeutic strategies based on gene editing or translational read-through therapy for suppression of nonsense variants.

Incidence of cancer and related deaths in hemoglobinopathies: A follow-up of 4631 patients between 1970 and 2021.

BACKGROUND: The correlation between thalassemia and malignancies other than hepatocellular carcinoma (HCC) and the possible relationship between other hemoglobinopathies and tumor risk have been poorly evaluated. METHODS: Eight Italian specialized centers evaluated the incidence of malignant neoplasms in hemoglobinopathies as well as their sites and features. The study cohort included 4631 patients followed between 1970 and 2021 (transfusion-dependent ß-thalassemia, 55.6%; non-transfusion-dependent thalassemia, 17.7%; sickle cell disease, 17.6%; hemoglobin H disease, 8.3%). RESULTS: A total of 197 diagnoses of cancer were reported (incidence rate, 442 cases per 100,000 person-years). The liver was the most frequent site of tumors in both sexes, with a higher incidence (190 cases per 100,000 person-years) in comparison with the general population found in all types of hemoglobinopathies (except hemoglobin H disease). In recent years, tumors have become the second cause of death in patients with transfusion-dependent thalassemia. A lower risk of breast and prostate cancer was observed in the whole group of patients with hemoglobinopathies. The first cancer diagnoses dated back to the 1980s, and the incidence rate sharply increased after the 2000s. However, although the incidence rate of cancers of all sites but the liver continued to show an increasing trend, the incidence of HCC showed stability. CONCLUSIONS: These findings provide novel insights into the relationship between cancer and hemoglobinopathies and suggest that the overall risk is not increased in these patients. HCC has been confirmed as the most frequent tumor, but advances in chelation and the drugs that have led to the eradication of hepatitis C may explain the recent steadiness in the number of diagnoses that is reported here.

Development of a Thalassemia International Prognostic Scoring System (TIPSS).

A prognostic scoring system that can differentiate ß-thalassemia patients based on mortality risk is lacking. We analysed data from 3145 ß-thalassemia patients followed through a retrospective cohort design for the outcome of death. An a priori list of prognostic variables was collected. ß Coefficients from a multivariate cox regression model were used from a development dataset (n = 2516) to construct a formula for a Thalassemia International Prognostic Scoring System (TIPSS) which was subsequently applied to a validation dataset (n = 629). The median duration of observation was 10.0 years. The TIPSS score formula was constructed as exp (1.4 × heart disease + 0.9 × liver disease + 0.9 × diabetes + 0.9 × sepsis + 0.6 × alanine aminotransferase ≥42 IU/L + 0.6 × hemoglobin ≤9 g/dL + 0.4 × serum ferritin ≥1850 ng/mL). TIPSS score thresholds of greatest differentiation were assigned as <2.0 (low-risk), 2.0 to <5.0 (intermediate-risk), and ≥5.0 (high-risk). The TIPSS score was a good predictor for the outcome of death in the validation dataset (AUC: 0.722, 95%CI: 0.641-0.804) and survival was significantly different between patients in the three risk categories (P < 0.001). Compared to low-risk patients, the hazard ratio for death was 2.778 (95%CI: 1.335-5.780) in patients with intermediate-risk and 6.431 (95%CI: 3.151-13.128) in patients with high-risk. This study provides a novel tool to support mortality risk categorization for patients with ß-thalassemia that could help management and research decisions.

National networking in rare diseases and reduction of cardiac burden in thalassemia major.

AIMS: A tailored chelation therapy guided by magnetic resonance imaging (MRI) is a strategy to improve the prognosis in iron-loaded patients, in many cases still hampered by limited MRI availability. In order to address this issue, the Myocardial Iron Overload in Thalassemia (MIOT) network was established in Italy and we aimed to describe the impact of 10-year activity of this network on cardiac burden in thalassemia major (TM). METHODS AND RESULTS: Within the MIOT network, 1746 TM patients (911 females; mean age 31.2 ± 9.1 years) were consecutively enrolled and prospectively followed by 70 thalassemia and 10 MRI centres. Patients were scanned using a multiparametric approach for assessing myocardial iron overload (MIO), biventricular function, and myocardial fibrosis. At the last MRI scan, a significant increase in global heart T2* values and a significantly higher frequency of patients with no MIO (all segmental T2* ≥20 ms) were detected, with a concordant improvement in biventricular function, particularly in patients with baseline global heart T2* <20 ms. Forty-seven percentage of patients changed the chelation regimen based on MRI. The frequency of heart failure (HF) significantly decreased after baseline MRI from 3.5 to 0.8% (P < 0.0001). Forty-six patients died during the study, and HF accounted for 34.8% of deaths. CONCLUSION: Over 10 years, continuous monitoring of cardiac iron and a tailored chelation therapy allowed MIO reduction, with consequent improvement of cardiac function and reduction of cardiac complications and mortality from MIO-related HF. A national networking for rare diseases therefore proved effective in improving the care and reducing cardiac outcomes of TM patients.

Antioxidant, Pro-Survival and Pro-Regenerative Effects of Conditioned Medium from Wharton's Jelly Mesenchymal Stem Cells on Developing Zebrafish Embryos.

Conditioned media harvested from stem cell culturing have the potential to be innovative therapeutic tools against various diseases, due to their high content of growth, trophic and protective factors. The evaluation in vivo of the effects and biosafety of these products is essential, and zebrafish provides an ideal platform for high-throughput toxicological analysis, concurrently allowing the minimization of the use of mammalian models without losing reliability. In this study, we assessed the biological effects elicited by the exposure of zebrafish embryos to a conditioned medium derived from Wharton's jelly mesenchymal stem cells. By a multiparametric investigation combining molecular, embryological, behavioural and in vivo imaging techniques, we found that exposure to a conditioned medium at a non-toxic/non-lethal dosage triggers antioxidant, anti-apoptotic and pro-regenerative effects, by upregulation of a set of genes involved in antioxidant defence (nrf2, brg1, sirt1, sirt6, foxO3a, sod2 and cat), glycolysis (ldha) and cell survival (bcl2l1, mcl1a and bim), coupled to downregulation of pro-apoptotic markers (baxa, caspase-3a and caspase-8). To our knowledge, this is the first study comprehensively addressing the effects of a conditioned medium on a whole organism from a developmental, molecular and behavioural perspective, and we are fairly confident that it will pave the way for future therapeutic application.

Primary HBB gene mutation severity and long-term outcomes in a global cohort of ß-thalassaemia.

In ß-thalassaemia, the severity of inherited ß-globin gene mutations determines the severity of the clinical phenotype at presentation and subsequent transfusion requirements. However, data on associated long-term outcomes remain limited. We analysed data from 2109 ß-thalassaemia patients with available genotypes in a global database. Genotype severity was grouped as ß0 /ß0 , ß0 /ß+ , ß+ /ß+ , ß0 /ß++ , ß+ /ß++ , and ß++ /ß++ . Patients were followed from birth until death or loss to follow-up. The median follow-up time was 34·1 years. Mortality and multiple morbidity outcomes were analyzed through five different stratification models of genotype severity groups. Interestingly, ß0 and ß+ mutations showed similar risk profiles. Upon adjustment for demographics and receipt of conventional therapy, patients with ß0 /ß0 , ß0 /ß+ , or ß+ /ß+ had a 2·104-increased risk of death [95% confidence interval (CI): 1·176-3·763, P = 0·011] and 2·956-increased odds of multiple morbidity (95% CI: 2·310-3·784, P < 0·001) compared to patients in lower genotype severity groups. Cumulative survival estimates by age 65 years were 36·8% for this subgroup compared with 90·2% for patients in lower genotype severity groups (P < 0·001). Our study identified mortality and morbidity risk estimates across various genotype severity groups in patients with ß-thalassaemia and suggests inclusion of both ß+ and ß0 mutations in strata of greatest severity.

Celocentesis for Early Prenatal Diagnosis in Couples at-Risk for ß-Thalassemia and Sicilian (δß)0-Thalassemia.

The procedures commonly used for prenatal diagnosis (PND) of thalassemia are villocentesis or amniocentesis, respectively, at the 11th and 16th weeks of gestation. Their main limitation is essentially due to the late gestation week in which diagnosis is performed. The celomic cavity is accessible between the 7th and 9th weeks of gestation and it has been demonstrated that it contains embryonic erythroid precursor cells as a source of fetal DNA for earlier invasive PND of thalassemia and other monogenic diseases. In this study, we report the use of celomatic fluids obtained from nine women with high-risk pregnancies for Sicilian (δß)0-thalassemia [(δß)0-thal] deletion (NG_000007.3: g.64336_77738del13403) and ß-thalassemia (ß-thal). Fetal cells were isolated by a micromanipulator, and nested polymerase chain reaction (PCR) and short tandem repeats (STRs) analysis were performed. Prenatal diagnosis was successfully performed in all examined cases. One fetus was a compound heterozygote for (δß)0- and ß-thal, three fetuses were found to be carriers of ß-thal, four fetuses carriers of a Sicilian 뫧 deletion, and one fetus without parental mutations. Accidentally, a rare case of paternal triploidy was observed. The genotypic analysis, carried out both by amniocentesis and on abortive tissue or after birth, showed concordance with results obtained on fetal celomic DNA. Our results unequivocally show that fetal DNA can be obtained by nucleated fetal cells present in the celomatic fluid and demonstrate, for the first time, that PND of Sicilian (δß)0-thal and ß-thal is feasible at an earlier time in pregnancy than other procedures.

Very early prenatal diagnosis of Cockayne's syndrome by coelocentesis.

Cockayne's syndrome (CS) is a rare autosomal recessive multisystem disease characterised by early severe progression of symptoms. This study reports the feasibility of earlier prenatal diagnosis of CS by coelocentesis at 8 weeks of gestation respect to amniocentesis or villocentesis. Three couples at risk for CS asked to perform prenatal diagnosis by coelocentesis. Coelomic fluid was aspired from coelomic cavity in four singleton pregnancy at 8 weeks of gestation and 40 foetal cells were recovered by micromanipulator. Maternal DNA contamination was evaluated by quantitative fluorescent PCR (QF-PCR) and target regions of foetal DNA containing parental mutations of ERCC6 gene were amplified and sequenced. In all these cases, molecular analysis was possible. One foetus resulted affected of CS and the diagnosis was confirmed on placental tissue after voluntary abortion. In three cases, foetuses resulted carrier of a parental mutation and the results were confirmed after the birth. This study suggests that reliable prenatal diagnosis of CS could be performed using foetal cells present in coelomatic fluid in earlier pregnancy. Coelocentesis could be applied in prenatal diagnosis of CSs as well as for other monogenic diseases, at very early stage of pregnancy, if parental mutations are already known.Impact StatementWhat is already know on this subject? Previous studies utilising coelocentesis for prenatal determination of foetal sex reported variable success ranging from 58% to 95%, because of low total DNA content and presence of maternal cell contamination. This procedure has never been reported for early prenatal diagnosis at 8 weeks of gestation for rare genetically transmitted diseases such as Cockayne's syndrome.What do the results of this study add? This study demonstrates that coelomic fluid sampling combined with well-standardised laboratory procedures can be applied for prenatal diagnosis at eight weeks of gestation for any rare monogenic disease if molecular defects are known.What are the implications of these findings for clinical practice and/or further research? The findings of this study in at risk couples for monogenic diseases investigated by coelocentesis demonstrate that embryo-foetal cell selection from CF allows reliable and early prenatal diagnosis of diseases. This technique is attractive to parents because it provides prenatal diagnosis of genetic disease at least 4 weeks earlier than what can be achieved by the traditional procedures reducing anxiety of parents and provides the option for medical termination of affected cases at 8-10 weeks' gestation, which is less traumatic and safer than second-trimester surgical termination. Further research concerns the possibility to obtain foetal karyotype at eight weeks of gestation and the possibility of intrauterine corrective therapy.

A complication risk score to evaluate clinical severity of thalassaemia syndromes.

The thalassaemia syndromes (TS) show different phenotype severity. Developing a reliable, practical and global tool to determine disease severity and tailor treatment would be of great value. Overall, 7910 patients were analysed with the aim of constructing a complication risk score (CoRS) to evaluate the probability of developing one or more complications. Nine independent variables were included in the investigation as predictors. Logistic regression models were used for Group A [transfusion-dependent thalassaemia (TDT)], Group B [transfused non-TDT (NTDT)] and Group C (non-transfused NTDT). Statistically significant predictors included age (years), haemoglobin levels, hepatic transaminases [alanine aminotransferase (ALT) and aspartate aminotransferase] and left-ventricular ejection fraction (LVEF) for Group A; age (years), age at first chelation (months), ALT and LVEF for Group B; and age (years), mean serum ferritin (SF) levels and LVEF for Group C. The area under the receiver operating characteristic curve was 84·5%, 82·1% and 80·0% for Groups A, Group B and Group C respectively, suggesting the models had good discrimination. Finally, the CoRS for each group was categorised into four risk classes (low, intermediate, high, and very high) using the centiles of its distribution. In conclusion, we have developed a CoRS for TS that can assist physicians in prospectively tailoring patients' treatment.

Antimicrobial prophylaxis in patients with immune thrombocytopenia treated with rituximab: a retrospective multicenter analysis.

The primary aim of this study was to describe the use of primary anti-infective prophylaxis (AP) in common clinical practice in patients affected by immune thrombocytopenia (ITP) and treated with RTX. Population studied consisted of patients affected by ITP (age ≥ 18 years) who had received at least one dose of RTX from January 2008 to June 2018. Five Italian haematology centres participated in the current study. Data were retrospectively collected: demographic data (age, gender), concomitant comorbidities and previous therapies for ITP, characteristics of AP, the occurrence of infections and their management. The ITP cohort consisted of 67 patients sub-grouped into two categories according to the administration of AP: (1) treated with AP (N= 34; 51%) and (2) not treated with AP (N=33, 49%). AP consisted of combined trimethoprim/sulfamethoxazole (TMP/SMX) and acyclovir (AC) in half of patients. TPM/SMX as a single agent was adopted in 32% patients and one patient received only AC. Overall, infections were experienced in 15% of patients during follow-up with a similar proportion in the 2 groups (treated and not treated) of patients (14.7% vs 15%). Clinical course of infections was however, less severe in patients treated with AP, where all infections were grade 2 and did not require hospitalization. In neither group of patients was reported Pneumocystis pneumonia. In conclusion, despite the absence of clear evidence, our analysis shows that AP in patients with ITP receiving RTX is frequently adopted, even if in the absence of well-defined criteria. Prophylaxis administration is quite consistent within the same haematological Center; thus, it seems related to clinicians' experience.

Longitudinal follow-up of patients with thalassaemia intermedia who started transfusion therapy in adulthood: a cohort study.

We longitudinally evaluated the effects of regular blood transfusions (BTs), in the real-life context of the Myocardial Iron Overload in Thalassaemia network, in patients with thalassaemia intermedia (TI). We considered 88 patients with TI (52 females) who started regular BTs after the age of 18 years. Magnetic resonance imaging was used to quantify iron overload and biventricular function. For 56·8% of the patients there were more than two indications for the transition to regular BTs, with anaemia present in 94·0% of the cases. A significant decrease in nucleated red blood cells, platelets, lactate dehydrogenase, bilirubin, and uric acid levels was detected 6 months after starting regular BTs. After the transition to the regular BT regimen there was a significant increase only in the frequency of hypothyroidism and osteopenia, and a significant decrease in liver iron and cardiac index. The percentage of chelated patients increased significantly after starting regular BTs. The decision to regularly transfuse patients with TI may represent a way to prevent or slow down the natural progression of the disease, despite the more complex initial management.

CMR for myocardial iron overload quantification: calibration curve from the MIOT Network.

OBJECTIVES: R2* cardiac magnetic resonance (CMR) allows the non-invasive measurement of myocardial iron. We calibrated cardiac R2* values against myocardial tissue-measured iron concentration by using a segmental approach and we assessed the iron distribution. METHODS: Five hearts of thalassemia patients were donated after death/transplantation to the CoreLab of the Myocardial Iron Overload in Thalassemia Network. A multislice multiecho R2* approach was adopted. After CMR, used as guidance, the heart was cut in three short-axis slices and each slice was cut into different equiangular segments according to AHA segmentation and differentiated into endocardial and epicardial layers. Tissue iron concentration was measured by atomic absorption spectrometer technique. RESULTS: Fifty-five samples were used since only for two hearts all the 16 samples were analyzed. Mean iron concentration was 4.71 ± 4.67 mg/g dw. Segmental iron levels ranged from 0.24 to 13.78 mg/g dw. The coefficient of variability of iron for myocardial segments ranged from 8.08 to 24.54% (mean 13.49 ± 6.93%). Iron concentration was significantly higher in the epicardial than in the endocardial layer (5.99 ± 6.01 vs 4.84 ± 4.87 mg/g dw; p = 0.042). Four different circumferential regions (anterior, septal, inferior, and lateral) were defined. A circumferential heterogeneity was noted, with more iron in the anterior region, followed by the inferior region. The direct nonlinear fitting of R2* and [Fe] data led to the calibration curve: [Fe] = 0.0022 ∙ (R2*-ROI)1.462 (R-square = 0.956). CONCLUSIONS: Our data further validate R2* CMR using a segmental approach as a sensitive and early technique for quantifying iron distribution in the current clinical practice. KEY POINTS: • Calibration in humans for cardiovascular magnetic resonance R2* against myocardial iron concentration was provided. • A circumferential heterogeneity in cardiac iron distribution was detected: more iron was observed in the anterior region, followed by the inferior region. This finding corroborates the use of a segmental T2* CMR approach in the clinical practice to detect a heterogeneous iron distribution. • The comparison between the cardiac T2* values obtained with the region-based and the pixel-wise approaches showed a significant correlation and no significant difference but, in presence of significant iron load, the region-based approach resulted in significantly higher T2* values.

Cardiac involvement by CMR in different genotypic groups of thalassemia major patients.

Beta thalassemia major (ß-TM) displays a great deal of phenotypic heterogeneity, not fully investigated in terms of cause-effect. We aimed to detect if different genotypic groups could be related to different levels of cardiac impairment, evaluated by cardiovascular magnetic resonance (CMR). We considered 671 ß-TM patients (age 30.1 years, 52.9% females) consecutively enrolled in the Myocardial Iron Overload (MIO) in Thalassemia network. MIO was assessed by T2* technique. Biventricular function was quantified by cine images. Myocardial fibrosis was evaluated by late gadolinium enhancement (LGE) technique. Three groups of patients were identified: heterozygotes ß+/ß° (N = 279), homozygotes ß + (N = 154), homozygotes ß° (N = 238). Transfusional needs resulted significantly lower in homozygous ß + TM patients when compared to the other groups. The homozygous ß + group versus the heterozygous and homozygous ß° groups showed higher global heart T2* values (P < 0.0001) and a lower number of patients with a global heart T2* value<20 ms (P < 0.001). The homozygotes ß + showed a lower number of patients with a pathological left ventricular ejection fraction (LVEF) than the other two groups (P < 0.05). The ß+/ß + TM patients showed less MIO and a concordant better systolic heart function. These data support the knowledge of different genotypic groups in the management of ß-TM patients.

Double Heterozygosity for Hb Durham-N.C. (HBB: c.344T>C) [ß114(G16)Leu→Pro] and the IVS-I-110 (HBB: c.93-21G>A) Causing a Severe ß-Thalassemia Phenotype.

The evaluation of a 10-month-old girl of Sicilian origin with a clinical phenotype of severe thalassemia led to the identification of two ß-globin gene defects, a ß-thalassemia (ß-thal), mutation at IVS-I-110 (HBB: c.93-21G>A) and a variant hemoglobin (Hb) mutation at codon 114 (HBB: c.344T>C) on the other allele, reported as Hb Durham-N.C. (also known as Hb Brescia) [ß114(G16)Leu→Pro] in the HbVar database. A very low Hb level (Hb 3.5 g/dL), microcytosis [mean corpuscular volume (MCV) 63.2 fL] and hypocromia [mean corpuscular Hb (MCH) 19.6 pg], increased red blood cell (RBC) distribution width (RDW) (36.0%), higher reticulocytes (6.2%), anisocytosis, poikilocytosis, hypocromia, basophilic stippling and inclusion body formation, were present in the affected subject. Analysis of other family components showed the presence of HBB: c.93-21G>A defect in the mother and in her brother, while Hb Durham-N.C. was absent in all other relatives, thus, this mutation has arisen as a de novo defect. This is the first case described as a severe thalassemic phenotype in a compound heterozygote carrier of this unstable Hb and a common ß-thalassemic allele. The important information gained from this case is that a rare dominant or recessive mutation may arise in every individual, even if this is a very rare event.

Longitudinal changes in LIC and other parameters in patients receiving different chelation regimens: Data from LICNET.

OBJECTIVES: The liver remains the primary site of iron storage, with liver iron concentration (LIC) being a strong surrogate of total body iron. MRI-R2 can accurately measure LIC. The LICNET (Liver Iron Cutino Network) was established to diagnostics of liver iron overload by MRI-R2 subjects with hemochromatosis in hematological disorders. The aims of the study were to look at variation in LIC measurements during time across different chelation regimens. METHODS: This was a cross-sectional study of 130 patients attending 9 Italian centers participating in the LICNET. LIC comparisons over time (T0 and T1 ) were made using t test and/or Wilcoxon test. RESULTS: LIC significantly decreased from MRI1 to MRI2 although at high variance (median change -0.8 mg Fe/g dw, range: -29.0 to 33.0; P = .011) and 7.7% of patients shifted from LIC values of high risk (>15 mg Fe/g dw) to an intermediate-risk category (7-15 mg Fe/g dw). Median change in LIC and correlation with serum ferritin levels (SF), during different chelation regimens, is reported. CONCLUSIONS: These findings suggest as longitudinal variation in the LIC is possible, across all chelation regimens. It confirms as SF levels not always can be used for estimating changes in LIC.