Search

Patients with beta-thalassemia show 5-fold increase in agestandardized lethality due to SARS-CoV-2 infection, representing a high-risk population compared with age- and sex-matched healthy subjects.(1) Vaccination against SARS-CoV-2 is crucial to reduce mortality and morbidity of frail patients.(2) Up to now, limited data have been available on the responses of patients with beta-thalassemia immunized with anti-SARS-CoV-2 mRNA vaccines.(3)

The purpose of this best practice paper is to review the current recommendations for the identification and prenatal diagnosis of hemoglobinopathies. Methods: The management committee of SITE selected and gathered a multidisciplinary team in order to formulate recommendations based on the available scientific evidence integrated with the opinions of experts, with the purpose of supporting clinicians. Results: We provide recommendations for first level tests (complete blood count, hemoglobin separation and iron balance), second level tests (molecular diagnosis) and prenatal diagnosis. Five Italian experts in hemoglobinopathies were consulted regarding the orientation of prenatal diagnosis, and for each indication, the degree of agreement among the experts has been specified. Conclusions: Best practice recommendations are the final outcome of this translational research and allow transfer to daily clinical practice.

Sickle cell disease (SCD) is a widespread genetic disease associated with severe disability and multi-organ damage, resulting in a reduced life expectancy. None of the existing clinical treatments provide a solution for all patients. Gene therapy and fetal haemoglobin (HbF) reactivation through genetic approaches have obtained promising, but early, results in patients. Furthermore, the search for active molecules to increase HbF is still ongoing. The delta-globin gene produces the delta-globin of haemoglobin A2 (HbA2). Although expressed at a low level, HbA2 is fully functional and could be a valid anti-sickling agent in SCD. To evaluate the therapeutic potential of a strategy aimed to over-express the delta-globin gene in vivo, we crossed transgenic mice carrying a single copy of the delta-globin gene, genetically modified to be expressed at a higher level (activated), with a humanised mouse model of SCD. The activated delta-globin gene gives rise to a consistent production of HbA2, effectively improving the SCD phenotype. For the first time in vivo, these results demonstrate the therapeutic potential of delta-globin, which could lead to novel approaches to the cure of SCD.

The aim of this study is the evaluation of the safety and the efficacy of long-term combination therapy deferasirox plus desferrioxamine and deferasirox plus deferiprone in a large group of transfusion-dependent thalassemia patients with high values of serum ferritin and/or magnetic resonance, indicative of severe liver and cardiac iron accumulation. Sixteen adults with transfusion-dependent thalassemia were treated simultaneously with deferasirox plus desferrioxamine, while another 42 patients (seven children) were treated with deferasirox plus deferiprone. The hepatic and cardiac iron overload was assessed prior to treatment and then annually with magnetic resonance imaging, and the serum ferritin was measured monthly. Adverse events were checked at each transfusion visit. The safety of both the combinations was consistent with established monotherapies. Both treatments were able to decrease the serum ferritin and liver iron concentration over time, depending on the level of compliance with therapy. Cardiac iron measured as R2* did not significantly change in patients treated with deferasirox plus desferrioxamine. Most patients with MRI indicative of myocardial siderosis at the beginning of treatment reached normal values of cardiac iron at the last determination if treated with deferasirox plus desferrioxamine. The greatest limitation of these therapies was low patient adherence to the two drugs, which is not surprising considering that the need for an intensive chelation is generally linked to previous issues of compliance.

Few data are available on the efficacy and safety of splenectomy in patients with transfusion-dependent Beta-Thalassemia Major (β-TM) and on its impact on a patient’s health-related quality of life (HRQoL). We examined the long-term HRQoL of adult patients with β-TM in comparison with those treated with medical therapy by using the Medical Outcomes Study 36-Item Short-Form Health Survey (SF-36). We also evaluated the safety and efficacy of splenectomy. Overall, 114 patients with a median age of 41 years (range 18–62) were enrolled in this cross-sectional study. Twenty-nine patients underwent splenectomy (25.4%) at a median age of 12 years (range 1–32). The median follow-up after splenectomy was 42 years (range 6–55). No statistically significant differences were observed in any of the scales of the SF-36 between splenectomized and not-splenectomized patients. The majority of surgical procedures (96.6%) were approached with open splenectomy. Post-splenectomy complications were reported in eight patients (27.5%): four overwhelming infections, three with pulmonary hypertension, and one with thrombosis. A significantly higher prevalence of cardiovascular comorbidities (58.6 vs. 21.2%, p < 0.001) and diabetes (17.2 vs. 3.5%, p = 0.013) was observed in splenectomized patients. These patients, however, required fewer red blood cell units per month, with only 27.6% of them transfusing more than 1 unit per month, compared with 72.9% of the not-splenectomized group. Overall, our data suggest that physicians should carefully consider splenectomy as a possible treatment option in patients with β-TM.

Pulmonary arterial hypertension (PAH) is a life-threatening complication of β-thalassemia, especially in untransfused patients with thalassemia intermedia. Pinto and colleagues analyzed the outcome of 24 patients with PAH documented by right heart catheterization, and they report that with a median follow-up of 4 years, 54% died, most of which deaths were attributable to PAH. Patients who receive treatment that reduce their pulmonary pressures have improved survival, suggesting that improvement in monitoring and treatment are critical imperatives for these patients.

Background: Although numerous patient specific co-factors have been shown to be associated with worse outcomes in COVID-19, the prognostic value of thalasemic syndromes in COVID-19 patients remains poorly understood. Aims: We studied the outcomes of 137 COVID-19 patients with a history of Transfusion Dependent Thalassemia (TDT) and non-Transfusion Dependent Thalassemia (NTDT) extracted from a large international cohort and compared them with the outcomes from a matched cohort of COVID-19 patients with no history of thalasemia. Results: The mean age of thalassemia 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. 25% of thalassemia 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 the an intensive care unit for CPAP therapy or intubation) and all-cause mortality was significantly lower in the thalassemia group compared to the matched cohort with no history of thalassemia. Amongst thalassemia patients in general, the NTDT 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 NTDT 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 thalassemia (either TDT or NTDT) was found to be independently associated with reduced all-cause mortality. Conclusions: The presence of thalassemia in COVID-19 patients was independently associated with lower in-hospital, all-cause mortality and few in-hospital complications in in our study. The pathophysiology of this is unclear and needs to be studied in vitro and in animal models. Trial Registration for sources of data extraction: NCT: 04334291, 04746066 Funding Statement: This study was funded by a non-conditioned grant from FUNDACION INTERHOSPITALARIA PARA LA INVESTIGACION CARDIOVASCULAR, FIC. (Madrid, Spain). This nonprofit institution had no role in the study design, the collection, analysis and interpretation of data, the writing of the report, or the decision to submit the paper for publication. Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The study was approved by the Ethics Committees in all of the centers involved (Banjarmasin, Bari, Cagliari, Catania, Ferrara, Gela, Genoa, Getafe, Guadalajara, Legan, Madrid, Mannheim, Milan, Monza, Naples, Olbia, Padua, Pavia, Ragusa, Rome, Salerno, Turin, Valladolid and Verona).

To investigate the genetic regulation of platelet (PLT) levels we carried out a whole-genome association analysis in 6,528 Sardinians from the general population of the Lanusei valley. We found 6 variants significantly influencing PLT levels, including a novel rare missense mutation (p.Pro27Ser) in the GP1BB protein that is associated with PLT reduction (P=1.17×10−16). This mutation is rare in the SardiNIA population cohort (frequency of 0.45%), even rarer in the rest of the Sardinian island (frequency of 0.16%), and not reported elsewhere. Notably, GP1BB is involved in Bernard-Soulier syndrome (BSS), a rare autosomal recessive bleeding disorder caused by a defect in the platelet GPIb-IX-V protein complex. Consistently, the 57 identified individuals heterozygous for the p.P27S mutation showed mild thrombocytopenia, morphologically enlarged platelets (P=2.13×10−10), and reduced expression of two GPIb-IX-V-complex components: GPIbα (−26.51%, P=3.66×10−8) and GPIX (−24.69%, P=2.66×10−6). Molecular modeling infers a corresponding reduction in the stability of GP1BB. These observations predict that in homozygosity as well as in individuals carrying specific compound heterozygous configurations, this variant likely causes BSS.

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.

Limited information is available on the hematological characterization of the α-thalassemia carrier in pediatric age. The objective of this report was to evaluate the red cell indices according to the α-globin genotype in a cohort of children evaluated in Sardinia. Moreover, we verified the frequency of different α-globin genotypes in this cohort. A total of 453 subjects were investigated for hematological indices and for the most common α-globin defects present in Sardinia. Of them, 352 with HbA2 ≤ 3.2%, and no iron deficiency anemia were taken into consideration to evaluate the red cell indices according to the α-globin genotype in pediatric age. A total of 11 different α-genotypes were detected, confirming the wide heterogeneity of α-thalassemia in Sardinia. Moreover, our results showed that the hematological parameters in normal children may be conditioned by the clinically occult coinheritance of mild α-thalassemia alleles as already described in the adult population while microcytosis and hypocromia in children without iron deficiency should suggest the coexistence of two α-globin defects. We concluded that recognizing the α-globin gene mutations for a particular population with their particular red cell indices may help pediatricians to perform a correct diagnosis distinguishing among physiological and pathological types of microcytosis and hypocromia.

Iron overload and chronic viral hepatitis underlie the increased incidence of hepatocellular carcinoma in patients with Beta thalassemia, as recently emerged due to the prolonged survival. Conversely, there are very few studies examining the relationship between thalassemia and other malignancies. Although some Authors have indicated an increase in incidence of hematologic and solid tumours such as thyroid cancer and renal cell carcinoma, there are not enough data to make a conclusion. Moreover, there is even less data on a possible relationship between other Hemoglobinopathies and tumors risk. Seven Italian Specialized Centres for the treatment of Hemoglobinopathies, with the patronage of the "Società Italiana Talassemie ed Emoglobinopatie" (SITE), evaluated the incidence of malignant neoplasms in Hemoglobinopathies, their site and features. Each Center considered all consecutive patients of its own historical series; the cohort included 4104 patients (48% males), followed between 1970 and 2021 (Figure 1A), for a total of 143255 person-years of observation. The most common diagnosis was Transfusion-Dependent Beta Thalassemia (TDT) with 2122 subjects (51.7%), followed by Non-Transfusion-Dependent Beta Thalassemia (NTDT, 789 subjects, 19.2%), Sickle Cell Disease (SCD, 787, 19.2%), Hemoglobin H Disease (HbH, 375, 9.1%) and Other (34, 0.8%). Two-hundred-sixty-two patients had undergone bone marrow transplantation (BMT). A total of 157 diagnoses of cancer (153 patients, 52% males) was reported during the period of observation, corresponding to a prevalence of 3.8%, with the mean age at the diagnosis of 47±13 years. Hepatocellular carcinoma (HCC) was the most frequent site (62 cases) in both sexes (males 50%, females 32%) contributing to 41% of total number of cases, excluding non-melanoma skin cancer, and in all type of hemoglobinopathies (except HbH). No significant differences were found in the age of diagnosis of HCC stratifying for gender (males 48±14 years, females 51±9 years) and types of Hemoglobinopathies (TD 48±9 years, NTDT 51±10 years, SCD 50±14 years). After HCC, hematologic tumours (11), kidney (6), and lung (6) were the most frequent sites in men, accounting for 29% of all male cancers. For women, the most common incident cancers were breast (9), thyroid (8) and kidney (7), these representing 33% of all female cancer cases (Figure 1B). The first diagnoses of cancer dated back to the 1980s and their number sharply increased after the 2000s with a peak in the five-year period 2008-2012 and a (not significant) reduction in recent years. A similar trend may be noted considering HCC alone but in this specific case, the reduction reaches the level of significance (Figure 1C). The overall age-adjusted incidence rate of cancer in Hemoglobinopathies was estimated to be 391 cases/100000 person-years (95% C.I. 290-650) which was not statistically different from that of the Italian general population (632 cases/100000 person-years), both considering Hemoglobinopathies in total and each subgroup of patients. However, age-adjusted incidence rate of HCC was more than 7 times lower in general population (22 cases/100000 person-years) than in patients with Hemoglobinopathies (153 cases/100000 person-years, 95% C.I. 96-222, p Although preliminary, these findings provide novel insights into the relationship between cancer and Hemoglobinopathies, suggesting that the overall cancer risk is not increased in these patients. HCC is confirmed as the most frequent tumor, especially in patients with Beta Thalassemia (TD and NTDT), but advances in chelation, with renewed attention to hepatic iron, and the new drugs that have led to the eradication of hepatitis C even in these patients, may explain the recent reduction in the number of diagnoses that we have reported in this study. Acknowledgment. We would like to thank ALT (Associazione per la Lotta alla Talassemia R.Vullo - Ferrara). Figure 1 Figure 1. Disclosures Origa: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; BlueBird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria. Longo: Bristol Myers Squibb: Honoraria; BlueBird Bio: Honoraria. Pinto: BlueBird Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Quarta: Sanofi - Genzyme: Membership on an entity's Board of Directors or advisory committees, Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Speaker at conferences; Blue Bird Bio: Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Celgene: Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; Speaker at conferences; Takeda: Other: collaboration relationships for Advisory boards, Webinar events, editorial projects; speaker at conferences; Bristol Meyer Squibb: Membership on an entity's Board of Directors or advisory committees, Other: Speaker at conferences. Casale: Novartis Farma SpA: Honoraria, Membership on an entity's Board of Directors or advisory committees. Maggio: Novartis: Membership on an entity's Board of Directors or advisory committees; Celgene Corp: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees. Perrotta: Novartis Farma SpA: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Blue Bird Bio: Honoraria, Membership on an entity's Board of Directors or advisory committees. Forni: Celgene: Membership on an entity's Board of Directors or advisory committees; Bluebirdbio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

Introduction Asplenic patients are at high risk of potentially fatal invasive infections, such as sepsis, meningitis, and pneumonia. It has been shown that infection from influenza viruses can precede or increase the risk of bacterial infection and of serious complications of the underlying disease. International and national guidelines recommend annual influenza vaccination in asplenic subjects. Following the Covid-19 pandemic, the major government and medical-scientific institutions in the US and in Europe have been planning how to contain infection during the 2020-2021 influenza season. Extending influenza vaccination is the safest and most effective way to reduce the circulation of influenza virus and to promote the correct diagnosis and management of suspected cases of SARS-CoV-2. Influenza vaccination also reduces complications associated with the underlying disease and visits to Emergency Units. Our study aims to evaluate influenza vaccination in a large population of asplenic patients and explore the main causes for non-vaccination to identify critical areas for improvement in the vaccination programme in these at-risk patients for the 2020-2021 influenza season. Methods The Italian Network of Asplenia (INA) is made up of 88 doctors working in 50 clinical centers in 27 cities and 16 of the 20 regions of Italy. It aims to build a large, prospective cohort of asplenic patients throughout Italy through which to study the interaction between asplenia and its associated underlying conditions, collecting precise, accurate data also in cases of rarer diseases. The study also aims to improve the quality of healthcare for this at-risk population. The number of patients enrolled in the Network who had had at least one dose of influenza vaccine at the time of diagnosis of asplenia was retrieved from the INA database. All participating centers were asked to answer a questionnaire to report the main obstacles for influenza vaccination. Results At 1st August 2020, 1,670 patients had been enrolled in the INA (783 females; 887 males). All underlying causes of asplenia are shown in Table 1. Only 466 (28%) patients had had at least one influenza vaccination, while 1,204 (72%) had never been vaccinated since diagnosis of asplenia. Thirty-five (70%) of the 50 centers answered the questionnaire. Main causes of non-vaccination were physicians' ambivalence concerning vaccination and patients' inadequate awareness or logistical problems. Conclusions These data show very low seasonal influenza vaccination cover even though asplenic patients are considered at-risk of complications associated with infection from influenza viruses. Since the 2020-2021 influenza season could see influenza viruses in circulation with SARS-CoV-2, influenza vaccination must be expanded as widely as possible, in particular to subjects of all ages at high risk. These results reveal important areas of concern in the management of asplenic patients and the need to improve the quality of information to physicians and patients alike. The INA co-ordinating center will launch a campaign to provide information and organize ad hoc meetings to widen influenza vaccination coverage in asplenic patients and reduce the pressure on the national health service during the next influenza season. Disclosures Forni: Novartis: Membership on an entity's Board of Directors or advisory committees. Colombatti:Addmedica: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees. Giona:Sanofi Genzyme: Research Funding, Speakers Bureau; Takeda: Speakers Bureau; Novartis: Research Funding. Ferrero:Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; EUSA Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Research Funding, Speakers Bureau; Servier: Speakers Bureau. Perrotta:Novartis: Consultancy, Research Funding, Speakers Bureau. Casale:Novartis: Membership on an entity's Board of Directors or advisory committees.

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 β-thalassemia carrier partner and a borderline percentage level of HbA2 were investigated for the presence of a pathological mutation in the β-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 β-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.

We report genome-wide association study results for the levels of A1, A2 and fetal hemoglobins, analyzed for the first time concurrently. Integrating high-density array genotyping and whole-genome sequencing in a large general population cohort from Sardinia, we detected 23 associations at 10 loci. Five signals are due to variants at previously undetected loci: MPHOSPH9, PLTP-PCIF1, ZFPM1 (FOG1), NFIX and CCND3. Among the signals at known loci, ten are new lead variants and four are new independent signals. Half of all variants also showed pleiotropic associations with different hemoglobins, which further corroborated some of the detected associations and identified features of coordinated hemoglobin species production.

α-Thalassemia commonly results from deletions or point mutations in one or both α-globin genes located on chromosome 16p13.3 giving rise to complex and variable genotypes and phenotypes. Rarely, unusual non-deletion defects or atypical deletions down-regulate the expression of the α-globin gene. In the last decade of the program for β-thalassemia carrier screening and genetic counseling in Sardinia, the association of new techniques of molecular biology such as gene sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA) to conventional methods has allowed to better define several thalassemic genotypes and the complex variability of the α-cluster with its flanking regions, with a high frequency of different genotypes and compound heterozygosity for two α mutations even in the same family. The exact molecular definition of the genotypes resulting from the interactions among the large number of α-thalassemia determinants and with β-thalassemia, is important for a correct correlation of genotype–phenotype and to prevent underdiagnosis of carrier status which could hamper the effectiveness of a screening program particularly in those regions where a high frequency of hemoglobinopathies is present.

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. Kruppel-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.

Increased hemoglobin A2 (HbA2; ie, levels > 3.9%) is the most important feature of β-thalassemia carriers. However, it is not uncommon to find persons with borderline HbA2 (levels, 3.3%-3.8%), who pose a relevant screening problem. Several genotypes have been associated with borderline HbA2, 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 HbA2 levels in the borderline range. Six different KLF1 mutations were identified in 52 of 145 subjects with borderline HbA2 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.

Since the implementation of deferrioxamine for the treatment of thalassemia major, it has been customary to initiate chelation after the first 10–20 transfusions or when serum ferritin reached over 1000 mcg/L.[1][1] However, in non-chronically-transfused thalassemia patients, ferritin

Background Hepcidin plays a key role in body iron metabolism by preventing the release of iron from macrophages and intestinal cells. Defective hepcidin synthesis causes iron loading, while overproduction results in defective reticuloendothelial iron release and iron absorption. Design and Methods We studied a Sardinian family in which microcytic anemia due to defective iron absorption and utilization is inherited as a recessive character. Five members showed iron deficiency anemia that was not responsive to oral iron and only partially responsive to parenteral iron administration. To investigate the involvement of known genes implicated in iron metabolism we carried out linkage analysis with microsatellite markers mapping close to these genes. Afterwards, a genome-wide search was performed. Results No linkage was found between the phenotype of the patients and several known human genes involved in iron metabolism ( DMT1, TF, TFRC, ZIRTL, HAMP, HJV ). Genome-wide scanning by microsatellites and single nucleotide polymorphisms showed a multipoint LOD score of 5.6 on chromosome 22q12.3–13.1, where the matriptase-2 (also known as transmembrane protease, serine 6 or TMPRSS6 ) gene is located. Its murine counterpart ( Tmprss6 ) has recently been found to be an essential component of a pathway that detects iron deficiency and suppresses hepcidin production. Sequencing analysis of TMPRSS6 revealed a homozygous causal mutation, predicting a splicing error and a truncated TMPRSS6 protein in affected members. Homozygous subjects had inappropriately elevated levels of serum and urinary hepcidin. Conclusions The findings of this study suggest that the observed TMPRSS6 mutation leads to overproduction of hepcidin and, in turn, to defective iron absorption and utilization. More generally, they confirm in humans the inhibitory effect of matriptase-2 on hepcidin synthesis already demonstrated in mice.

In this study, 251 Sardinian patients (187 adults and 64 children) with haemoglobin (Hb) H disease were evaluated. Two-hundred and sixteen patients (86%) had the deletional type (- -/-alpha) and 36 (14%) patients had the non-deletional type (- -/alpha(ND)alpha). A clear genotype-phenotype correlation was found, with the non-deletional type more severe than the deletional type. Diagnosis of Hb H disease was incidental in about 60% of cases. Aplastic crises due to B19 parvovirus infection were found in five patients (2.1%), while 23 patients (9.6%) experienced one or more haemolytic crises. Nineteen patients with Hb H received sporadic red blood cell transfusions and three patients were repeatedly transfused. Forty-seven of 61 married women (77%) had 82 pregnancies. In children, mean serum ferritin was 87 +/-92 mug/l and in adults, was 192 +/- 180 mug/l in females and 363 +/- 303 mug/l in males. For the 98 male patients, a significant correlation was found between ferritin values and age (r2 = 0.33, P < 0.0001). In the Sardinian population, Hb H disease needs regular monitoring for early detection and treatment of possible complications, such as worsening of anaemia that may require red cell transfusion, cholelithiasis and iron overload.

We report the sixth occurrence of Hb Belfast [beta15(A12)Trp--Arg], a mild, unstable beta chain variant, in a large family wherein nine subjects were affected. DNA analysis showed a TUG--AGG mutation at codon 15 of the beta-globin gene, confirming a Trp--Arg amino acid substitution. The oxygen affinity of the isolated variant was increased. The clinical phenotype is silent or very mild, the only clinical finding being an intermittent moderate jaundice.

Historically 15-20% of homozygous ß-thalassemia in Sardinia developed the phenotype of thalassemia intermedia not requiring transfusion for survival. However, this category of patients has broad and heterogeneous spectrum severity, embracing truly mild thalassemia patients and patients with severity similar to thalassemia major. Known genetic modifiers that ameliorate the severity of b-thalassemia are the mild genotypes of ß+-thalassemia, the coinheritance of α-thalassemia, of hereditary persistence of fetal hemoglobin (HPFH) and of polymorphic variants in the BCL11a, MYB and HBG1 genes. All together this modifiers allow the calculation of a genetic score of severity that predicts the possibility of remaining transfusion free at a given age, likely aiding in the clinical decision to start or delay the initiation of a stable transfusion program. In this study we planned to evaluate the ameliorating effects of these genetic modifiers on the thalassemia phenotype by prospectively evaluating 51 homozygous ß-thalassemia newborns followed at our Institution in the last ten years. All newborns were genotyped soon after birth for all known genetic modifiers and monitored clinically for the appearance of anemia or other complications with monthly check-ups from birth until they required transfusions, according to the TIF guidelines. In this decennial cohort, all but 2 patients younger than 20 months started a regular transfusion program before 36 months of age. Hence, the percentage of thalassemia intermedia in Sardinia has dropped in the last decade from 18 to 4% and might even reach 0% if the 2 patients non yet transfused will transfuse within the next year. This shift from thalassemia intermedia to major is mainly accounted for by the great improvement in mortality, morbility and quality of life for thalassemia major compared to thalassemia intermedia patients who frequently develop difficult to manage complications with advancing age. In the cohort, the reason for start of blood transfusions was anemia lower than 7gr/dl in 14 % of cases and the appearance of skeletal modifications or growth failure in the remaining 84% of patients. Thus, differently from the past, most patients were transfused despite an acceptable grade of anemia. As expected, the presence of a greater number of genetic modifiers significantly correlated with a delay in the time lag to the first transfusion. Even better positive correlation (p Disclosures No relevant conflicts of interest to declare.

Summary The risk of developing hepatocellular carcinoma (HCC) in patients with thalassaemia is increased by transfusion-transmitted infections and haemosiderosis. All Italian Thalassaemia Centres use an ad hoc form to report all diagnoses of HCC to the Italian Registry. Since our last report, in 2002, up to December 2012, 62 new cases were identified, 52% of whom were affected by thalassaemia major (TM) and 45% by thalassaemia intermedia (TI). Two had sickle-thalassaemia (ST). The incidence of the tumour is increasing, possibly because of the longer survival of patients and consequent longer exposure to the noxious effects of the hepatotropic viruses and iron. Three patients were hepatitis B surface antigen-positive, 36 patients showed evidence of past infection with hepatitis B virus (HBV). Fifty-four patients had antibodies against hepatitis C virus (HCV), 43 of whom were HCV RNA positive. Only 4 had no evidence of exposure either to HCV or HBV. The mean liver iron concentration was 8 mg/g dry weight. Therapy included chemoembolization, thermoablation with radiofrequency and surgical excision. Three patients underwent liver transplant, 21 received palliative therapy. As of December 2012, 41 patients had died. The average survival time from HCC detection to death was 11·5 months (1·4–107·2 months). Ultrasonography is recommended every 6 months to enable early diagnosis of HCC, which is crucial to decrease mortality.

Objective To evaluate the long-term effect of Deferiprone (DFP) in reducing brain iron overload and improving neurological manifestations in patients with NBIA. Methods 6 NBIA patients (5 with genetically confirmed PKAN), received DFP solution at 15 mg/kg po bid. They were assessed by UPDRS/III and UDRS scales and blinded video rating, performed at baseline and every six months. All patients underwent brain MRI at baseline and during follow up. Quantitative assessment of brain iron was performed with T2* relaxometry, using a gradient multi-echo T2* sequence. Results After 48 months of treatment clinical rating scales and blinded video rating indicated a stabilization in motor symptoms in 5/6 Pts. In the same subjects MRI evaluation showed reduced hypointensity in the globus pallidus (GP); quantitative assessment confirmed a significant increment in the T2* value, and hence reduction of the iron content of the GP. Conclusion The data from our 4-years follow-up study confirm the safety of DFP as a chelator agent for iron accumulation. The clinical stabilization observed in 5/6 of our patients suggests that DFP may be a reasonable therapeutic option for the treatment of the neurological manifestations linked with iron accumulation and neurodegeneration, especially in adult patients at early stage of the disease. (Clinicaltrials.gov identifier: NTC00907283).

Sardinian beta-thalassemia patients all are homozygotes for the same null allele in the beta-globin gene, but the clinical manifestations are extremely variable in severity. Previous studies have shown that the coinheritance of alpha-thalassemia or the presence of genetic variants that sustain fetal hemoglobin production has a strong impact on ameliorating the clinical phenotype. Here we evaluate the contribution of variants in the BCL11A, and HBS1L-MYB genes, implicated in the regulation of fetal hemoglobin, and of alpha-thalassemia coinheritance in 50 thalassemia intermedia and 75 thalassemia major patients. We confirm that alpha-thalassemia and allele C of single nucleotide polymorphism rs-11886868 in BCL11A were selectively represented in thalassemia intermedia patients. Moreover, allele G at single nucleotide polymorphism rs9389268 in the HBS1L-MYB locus was significantly more frequent in the thalassemia intermedia patients. This trio of genetic factors can account for 75% of the variation differences in phenotype severity.

Congenital anaemias due to ineffective erythropoiesis may be associated with excessive iron absorption and progressive iron loading. We investigated whether the soluble transferrin receptor (TfR) level was related to the degree of iron overload in 20 patients with thalassaemia intermedia, six patients with congenital dyserythropoietic anaemia type II (CDA II) and four patients with X-linked congenital sideroblastic anaemia (XLSA). All but two patients had increased serum ferritin levels (median 601 microgram/l, range 105-2855 microgram/l). Multiple regression analysis showed that 62% (P0.0001) of the variation in serum ferritin was explained by age and by changes in soluble TfR.

Differentiation between heterozygous alpha-thalassemia and several phenotypically resembling alleles at the beta-globin gene cluster such as coinherited delta- and beta-thalassemia or gammadelta beta-thalassemia is a critical step in genetic counseling. In this paper we report our experience in the identification of the alpha-thalassemia carrier state using polymerase chain reaction (PCR)-based methods, and the feasibility and simplification of screening for thalassemia using this approach. Alpha-globin genotype was determined by PCR-based method in 526 adult subjects with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal hemoglobin A2 and F, and normal serum iron. To verify the reliability of the protocol used, in 68 of these subjects we performed globin chain synthesis analysis and in 101 we determined alpha-globin genotype by Southern blot analysis. Five hundred twenty-one (99%) of 526 subjects examined were identified as carriers of one or two alpha-thalassemia alleles. The identification of the alpha-thalassemia carrier state may be fast and accurate by PCR-based method, avoiding other cumbersome and expensive methods such as globin chain synthesis and Southern blot analysis.

Hb Puttelange [beta 140(H18)Ala-->Val] was found in a 51-year-old Italian man who had mild polycythemia. The variant eluted from ion exchange high performance liquid chromatography at a position between Hb A and Hb A2. It comprised approximately 34% of the total hemoglobin, was weakly unstable and exhibited an increased oxygen affinity. Amplification of the beta-globin exons and nucleotide sequencing revealed a heterozygosity for a GCC-->GTC mutation in codon 140 corresponding to an Ala-->Val replacement. This substitution accounts for the altered functional properties, probably by producing indirect perturbation of the 2 3-diphosphoglycerate pocket through the nearby lysine residue at beta 82(EF6).