Thal Patients Research Articles

A retrospective study of glucose homeostasis, insulin secretion, sensitivity/resistance in non- transfusion-dependent β-thalassemia patients (NTD- β Thal): reduced β-cell secretion rather than insulin resistance seems to be the dominant defect for glucose dysregulation (GD).

Non-transfusion - dependent β-thalassemias (NTD-βThal) can cause iron overload and serious iron-related organ complications as endocrine dysfunction, including glucose dysregulation (GD). We retrieved data of all NTD- β Thal patients referred consecutively to a single Outpatient Italian Clinic from October 2010 to April 2023. All patients underwent a standard 3-h oral glucose tolerance test (OGTT) for analysis of glucose homeostasis, insulin secretion and sensitivity/resistance (IR), using conventional surrogate indices derived from the OGTT. The collected data in NTD- β Thal patients were compared to 20 healthy subjects. Seventeen of 26 (65.3 %) NTD- β Thal patients (aged: 7.8 -35.1 years) had normal glucose tolerance, 1/26 (3.8 %) had impaired fasting glucose (IFG), 5/26 (19.2 %) impaired glucose tolerance (IGT), 1/26 (3.8%) IFG plus IGT and 2/26 (7.6%) plasma glucose (PG) level ≥155 mg/dL 1-h after glucose load. GD was observed exclusively in young adult patients; none of them had diabetes mellitus (DM). These findings were associated with a low insulinogenic index (IGI) and oral disposition index. HOMA-IR and QUICKI were not significantly different compared to controls. Interestingly, in young adult patients, ISI-Matsuda index was statistically higher compared to the control group, suggesting an increased insulin sensitivity. This study reported a high prevalence of GD in young adults with NTD- β Thal. The documented reduction of IGI rather than the presence of IR, indicates reduced insulin secretory capacity as the pathophysiological basis of dysglycemia that may represent a novel investigational path for future studies on the mechanism(s) responsible for GD in NTD- β Thal patients.

Mitapivat Treatment Increases β-Thalassemic Erythroblasts Energy Production and Responsiveness to Oxidative Stress

β thalassemia (β thal) is a worldwide red blood cell (RBC) genetic disorder with limited therapeutic tools. Standard of care includes chronic RBC transfusions and iron chelation. In β thal, pathophysiologic studies have shown that oxidation plays a key role in both ineffective erythropoiesis and reduced survival of mature red cells in the peripheral circulation (Matte et al ARS 28: 1-14, 2018; Matte A et al. ARS 23: 1284, 2015). Recently, in a mouse model of β thal, we showed that the investigational pyruvate kinase (PK) activator mitapivat significantly improved anemia by targeting both ineffective erythropoiesis and hemolysis (Matte et al. JCI 131: e144206, 2021). We also found that mitapivat ameliorated the erythroid cell maturation index for in vitro CD34+ derived erythroblasts from β thal (cod b039) patients (Matte et al. JCI 131: e144206, 2021). The results of phase 2 proof-of-concept study (NCT03692052) in non-transfusion-dependent thalassemic patients support the beneficial effects of mitapivat on β thal (Kuo KHM et al Lancet 400: 493-501, 2022). Here, we study CD34+ derived erythroblasts from either healthy controls or β thal (cod b039) patients (n=10). Mitapivat (2uM) was added to the cell cultures as previously described (Matte et al. JCI 131: e144206, 2021). Compared to healthy controls, we found up-regulation of PKM gene expression in the late phase of β thal erythropoiesis (14 days of culture). Whereas the expression of PKLR gene declined during erythroid maturation in both healthy and β-thal erythroblasts. We found higher total PK activity in β thal erythroblasts than in healthy cells. Using recombinant PKR and PKM2 isoforms (R&D systems), we found that mitapivat activates both PKR and PKM2 (EC50 13.6±3.3 and 30.1±1.5, respectively) (Kung C et al Blood 2017). Studying erythropoiesis in vitro we confirmed that mitapivat significantly increased β thal erythroid maturation as supported by increased late-erythroblasts at 11 days of cell culture (Matte et al. JCI 131: e144206, 2021). The metabolomic analysis of β-thal erythroblasts at 14 days of culture confirmed that mitapivat activated PK, as gleaned by PGLY/DPG levels and increased fluxes through glycolysis, as inferred from steady-state measurements of glycolytic metabolites, DPG, ATP, and GTP. In addition, we observed higher levels of methionine oxidation in both mitapivat-treated healthy and β-thal erythroblasts, suggestive of a regulatory PTM effect secondary to higher ATP availability and, possible increased longevity of the cells. The improvement in the intracellular milieu by mitapivat is also supported by the downregulation of peroxiredoxin-2 (Prx2) and heme-oxygenase-1 (Hmox1) gene expression, which are up-regulated in response to oxidative stress and free heme accumulation in vehicle treated β-thal erythroblasts (Matte et al. ARS 23: 1284, 2015). Noteworthy, we also observed a reduction in heat shock protein-70 (Arlet JB et al Nature 514: 242, 2014) in mitapivat treated β-thal erythroblasts, further supporting the protective effect of mitapivat during β-thal erythropoiesis. In conclusion, our data show that both pklr and pkm isoforms are expressed in the late phase of β-thal erythropoiesis. The ability of mitapivat to activate both PKLR and PKM2 might represent an added value to limit oxidation during b-thal erythropoiesis, ensuring an improved β-thal erythroblasts maturation and survival.

PSUN160 Transfusion Related Endocrinopathies and Thalassemia (TREAT) Study

Abstract Thalassemia (Thal), the most common form of inherited anemia worldwide, may require regular blood transfusions and life-long medical care. The introduction of oral chelators and imaging methods to monitor iron overload transformed Thal from a rapidly fatal disease of childhood to a chronic illness compatible with prolonged life expectancy. Regular blood transfusions resulting in tissue iron overload and oxidative stress may lead to liver disease and polyendocrinopathy. In this observational descriptive study, we described the patient characteristics and prevalence of endocrinopathies (growth failure, hypothyroidism, hypogonadism, low bone mass, and impaired glucose intolerance) in a contemporary group of transfusion dependent Thal patients cared for our tertiary care center since 2000. A total of 253 transfusion dependent Thal patients were identified. Of those patients, 51.8% were female; 22% (n=56) were adopted, mainly from China. The following ethnicities were represented: Chinese 19.9%, Indian 12.6%, Laotian 7.8%, Asian 7.8%, Italian 7.8%, Vietnamese 3.9%, Afgani 3.4%, Pakistani 3.4%, Thai 2.9%, Greek 2.4%, Cambodian 2.4%, Filipino, Caucasian, Iranian 1.9% each. Majority of the diagnoses were Beta Thal Major (69.9%), followed by E Beta 0 Thal (14.6%). The median age at first visit was 10.6 years (IQR1-IQR3: 2.2-25), last visit was 27 years (IQR1-IQR3: 12.1–39.4) with 33.5% of the patients ≤18 years of age. Median follow-up time was 10 years. Median final adult height (height z-score) for females was 159.5 cm (-0.53) (IQR1-IQR3: 152.4,160.3cm; height z-score: -1.68, -0.47) and for males 167.5cm (-1.3) (IQR1-IQR3: 161.5, 171cm; height z-score: -2.13, -0.81). Only, 16.5% (n=14) of children were on growth hormone treatment due to low-normal growth hormone surrogates or peak growth hormone <10ng/ml in growth hormone stimulation test. Hypothyroidism was diagnosed in 9.9% (n=25) of the patients and 5 patients had central hypothyroidism. Hypogonadism was diagnosed in 32.4% of adult female (n=24), and 44.6% of adult male (n=29) patients who were subsequently prescribed sex steroid replacement. Low bone mass in those with z-scores ≤2.0 was noted in 22.9% (n=43) female and 26% (n=49) males. The mean bone mineral density z-score for spine was -2.1±1.1 for females and -2.5±1.3 for males. Either impaired glucose tolerance or insulin-dependent DM was observed in 29% of the patients (n=54). Endocrinopathies are common and continue to be a significant morbidity in transfusion dependent Thal patients despite improved chelation agent availability and tissue iron monitoring. Future long-term longitudinal and intervention studies elucidating and mitigating potential effects of iron overload on developing children's developing endocrine organs are needed. Presentation: Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.

Sickle cell disease patients with COVID-19 in Guadeloupe: Surprisingly favorable outcomes.

We investigate risk factors for hospitalization and difference between sickle cell syndromes in a cohort of COVID‐19 sickle cell disease (SCD) adult patients managed in the Reference Center of Guadeloupe. We retrospectively collected data of symptomatic SCD adult patients infected with SARS‐CoV‐2 between March and December 2020. Thirty‐eight SCD adult patients with symptomatic COVID‐19 infection were included during the first wave, representing 9.6% of the active patient file at our center. The median age (IQR) was 39 years (24–47). Four patients were obese and two had moderate renal failure. The median duration of symptoms (IQR) was 10 days (5–15). Seventeen (44.7%) patients were hospitalized, including two in intensive care unit (ICU) for acute chest syndrome. An 85‐year‐old SC patient with prostate cancer died. No difference was detected between inpatient and outpatient groups in terms of age, gender, BMI, SCD clinical complications, and in history SCD treatment. There was no difference for severity, hospitalization, length of stay, ICU stay, or death between SS or Sβ°‐thal patients and SC or Sβ+‐thal patients. These overall favorable outcomes among symptomatic patients may be related to the low prevalence of comorbidity known to be linked to the more severe forms of COVID‐19, but also to the prompt coordinated management of SCD patients in the Reference Center.

ERN-EuroBloodNet European Registry of Patients Affected by Red Blood Cell Disorders and COVID-19

ERN-EuroBloodNet European Registry of Patients Affected by Red Blood Cell Disorders and COVID-19

Determinants Of Tissue Iron Loading In Transfused and Non-Transfused β-Thalassemia and Sickle Cell Disease Patients – Biomarkers Of Iron Regulation and Inflammation

IntroductionTransfused thalassemia (Thal) and sickle cell disease (SCD) patients develop tissue iron loading and organ injury. Even non-transfused Thal patients develop iron overload and iron-induced organ injury. In general, Thal patients are known to have greater extra-hepatic iron loading than SCD patients. This study investigated biomarkers that could modulate tissue iron loading in Thal and SCD. Patients and MethodsSeventy-eight Thal and SCD patients were enrolled including transfused (17 Tx Thal, and 19 Tx SCD) and non-transfused (12 nTx Thal and 16 nTx SCD). The following were analyzed: markers of erythroid expansion GDF15 and sTfR; markers of iron regulation and loading including hepcidin, NTBI, transferrin saturation, serum ferritin, ferritin/hepcidin and liver iron concentration (LIC); and a marker of inflammation, high sensitivity C-reactive protein (CRP). LIC was measured by SQUID Ferritometry®. Serum hepcidin was determined by competitive ELISA (Intrinsic LifeSciences, La Jolla, CA). ResultsLIC is similar between Tx Thal, nTx Thal and Tx SCD patients. Only nTx SCD patients had lower levels of LIC. However, all thal patients had an 83% increase in GDF15 levels and 87% higher NTBI levels (p<0.001) compared to all SCD patients. nTx thal had lower NTBI compared to TX thal despite similar LIC and not being on chelation therapy. Also sTfR was 80% higher in nTx thal and SCD compared to Tx Thal. Inflammation, on the other hand, as measured by CRP was 62% higher in all SCD patients (p=0.038) relative to all thal. Also Tx SCD patients had a strong negative relationship between NTBI and sTfR (r=-0.598, p=0.018) indicating that both inflammation and erythropoietic drive may be important in extra-hepatic tissue iron loading. Tx Thal and SCD patients had at least 70% higher levels of hepcidin. nTx thal had 93% higher levels of the ratio of ferritin to hepcidin (p=0.023). NTBI and LIC were found to positively correlate in nTx Thal patients (r=0.788, p=0.012), whereas no correlation between NTBI and LIC was found in Tx Thal patients. In nTx Thal, GDF15 is also negatively correlated with hepcidin (r=-0.631, p=0.038). In Tx SCD, GDF15 positively correlates with hsCRP (r=0.784, p=0.001). Tx SCD patients also show positive correlations between GDF15 and LIC (r=0.692, p=0.003) and serum ferritin (r=0.545, p=0.024). Finally, there is a negative relationship between GDF15 and hemoglobin in both Tx Thal and nTx Thal (r=-0.574, p=0.016 ; r=-0.643, p=0.033, respectively). ConclusionsThe data presented here suggests that more effective erythropoiesis in nTx SCD, Tx SCD and nTx Thal seems to protect from excess NTBI and possible extra-hepatic iron loading. In Tx SCD the positive correlation between CRP and GDF15 and the negative correlation between NTBI and sTfR suggests both inflammation and effective erythropoiesis may be important in extra-hepatic iron loading. Furthermore, GDF15 could be related to ineffective erythropoiesis while sTfR is reflecting effective erythropoiesis in this study cohort. Disclosures:Walter:Novartis: Research Funding. Nemeth:Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Ganz:Intrinsic LifeSciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Porter:Shire: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Celgene: Consultancy. Harmatz:Novartis: Research Funding; Shire: Consultancy, Honoraria, Research Funding, travel, travel Other.

Extracellular vesicles from thalassemia patients carry iron-containing ferritin and hemichrome that promote cardiac cell proliferation.

Extracellular vesicles (EVs) are bioactive, submicron-sized membrane vesicles released from all cell types upon activation or apoptosis. EVs including microparticles (MPs) and exosomes have emerged as important mediators of cell-to-cell communication in both normal and pathological states including thalassemia (thal). However, the role of EVs derived from β-thal patients with iron overload (+ IO) and without iron overload (-IO) on cardiac cells is unclear. We hypothesized plasma EVs in thal patients containing ferritin (iron storage protein) and a denaturated hemoglobin-hemichrome that induce cardiac cell proliferation. The origins and numbers of EVs isolated from plasma of normal, thal (+ IO), and (- IO) patients were compared and determined for their iron and iron-containing proteins along with their effects on cardiac and endothelial cells. Data shows that MPs were originated from many cell sources with marked numbers of platelet origin. Only the number of RBC-derived MPs in thal (+ IO) patients was significantly high when compared to normal controls. Although MPs derived from both normal and thal patients promoted cardiac cell proliferation in a dose-dependent manner, only exosomes from thal patients promoted cardiac cell proliferation compared to the untreated. Moreover, the exosomes from thal (+ IO) potentially induce higher cardiac cell proliferation and angiogenesis in terms of tube number than thal (- IO) and normal controls. Interestingly, ferritin content in the exosomes isolated from thal (+ IO) was higher than that found in the MPs isolated from the same patient. The exosomes of thal patients with higher serum ferritin level also contained greater level of ferritin inside the exosomes. Apart from ferritin, there were trends of increasing hemichrome and iron presented in the plasma EVs and EV-treated H9C2 cells. Findings from this study support the hypothesis that EVs from β-thal patients carry iron-load proteins that leads to the induction of cardiac cell proliferation.

Oxidant and antioxidant status of patients with sickle cell -β thalassemia in Thi-Qar Province, Iraq

BackgroundSickle cell/β thalassemia (HbS/ β thal) is a compound heterozygous state for βS and either β+ thalassemia or β0 thalassemia. In sickle cell/ β0 thalassemia, there is no hemoglobin A, whereas, in sickle cell/ β+thalassemia, a variable amount of hemoglobin A is present. Aim of study was to shed a light on the expected association between sickle cell-thalassemia and oxidative stress.MethodsA total of one hundred and fifty (150) subjects were recruited for this study which consists of hundred (100) sickle cell-thalassemia subjects who 53were males and 47 were females and fifty (50) apparently healthy subjects which served as control who matched by sex and age. The present study is designed to determine (MDA) (malondialdehyde) concentration as a marker for lipid peroxidation and measurement some parameters of antioxidants including: transferrin (Tf), ceruloplasmin (Cp), and albumin (Alb), in patients with Sickle cell/β thalassemia.The means MDA and Alb were increase significant while means TF and CP decrease significant in HbS/ β thal patients as compared to control Also it showed same result in both male and female patients with HbS/ β thal as compared with their healthy counterparts. There were no significant difference between male and female patients in means MDA, CP and Alb except mean TF. The result revealed that no significant difference in means MDA while significant difference in TF, CP and Alb in patient among age group. The means MDA, CP and Alb were not significant difference between male and female patients in each age group while there was found significant difference in mean TF between male and female patient in 1-10 age group only.The increase in the oxidative stress level, especially MDA in sickle cell thalassemia patients compared to healthy individuals, confirms that oxidative stress is involved in the pathogenesis of Sickle cell/β thalassemia disease.

Clinical phenotypes and outcomes of precapillary pulmonary hypertension of sickle cell disease.

Precapillary pulmonary hypertension (PH) is a devastating complication of sickle cell disease (SCD). Little is known about the influence of the SCD genotype on PH characteristics. To describe clinical phenotypes and outcomes of precapillary PH due to SCD according to disease genotype. A nationwide multicentre retrospective study including all patients with SCD-related precapillary PH from the French PH Registry was conducted. Clinical characteristics and outcomes according to SCD genotype were analysed. 58 consecutive SCD patients with precapillary PH were identified, of whom 41 had homozygous for haemoglobin S (SS) SCD, three had S-β0 thalassaemia (S-β0 thal) and 14 had haemoglobin SC disease (SC). Compared to SC patients, SS/S-β0 thal patients were characterised by lower 6-min walk distance (p=0.01) and lower pulmonary vascular resistance (p=0.04). Mismatched segmental perfusion defects on lung scintigraphy were detected in 85% of SC patients and 9% of SS/S-β0 thal patients, respectively, and 50% of SS/S-β0 thal patients had heterogeneous lung perfusion without segmental defects. After PH diagnosis, 31 patients (53%) received medical therapies approved for pulmonary arterial hypertension, and chronic red blood cell exchange was initiated in 23 patients (40%). Four patients were managed for chronic thromboembolic PH by pulmonary endarterectomy (n=1) or balloon pulmonary angioplasty (n=3). Overall survival was 91%, 80% and 60% at 1, 3 and 5 years, respectively, without influence of genotype on prognosis. Patients with precapillary PH related to SCD have a poor prognosis. Thrombotic lesions appear as a major component of PH related to SCD, more frequently in SC patients.

TIF 2.0: The Thal e-Course and TIF Expert Patients’ Programme for Disease-Related Education and Self-Management Skills in Thalassaemia

In response to the fundamental shift that has been taking place in the way chronic diseases are perceived and managed and the increasingly established role of patients as equal partners in the management of their condition, the Thalassaemia International Federation (TIF) has undertaken the design and development of a comprehensive online Expert Patients’ Programme (EPP) for patients with thalassaemia. Focusing particularly on β-thalassaemia, the most severe form of thalassaemia, the goal of the programme is to develop patients’ disease-related knowledge and self-care skills and enable them to co-manage their disease in a meaningful partnership with their treating physicians. An important goal of this ecourse is to empower patients to advocate for the improvement of national treatment services in every affected country. The aim of this article is threefold: (1) Relate TIF’s EPP with the goals and outcomes of other EPPs, as they are made available in the literature. (2) Describe the rationale and distinguishing features of TIF’s EPP on the basis of learning theories of knowledge acquisition and attrition, and best practices from the scientific disciplines of Human Computer Interaction (HCI) and Technology-Assisted Learning (TEL). (3) Relay the objectives of TIF’s EPP and the intended international impact in relation to TIF’s mission.

Prospective evaluation of chronic organ damage in adult sickle cell patients: A seven-year follow-up study.

Organ damage in sickle cell disease (SCD) is a crucial determinant for disease severity and prognosis. In a previous study, we analyzed the prevalence of SCD-related organ damage and complications in adult sickle cell patients. We now describe a seven-year follow-up of this cohort.All patients from the primary analysis in 2006 (n = 104), were included for follow-up. Patients were screened for SCD-related organ damage and complications (microalbuminuria, renal failure, elevated tricuspid regurgitation flow velocity (TRV) (≥2.5 m/seconds), retinopathy, iron overload, cholelithiasis, avascular osteonecrosis, leg ulcers, acute chest syndrome (ACS), stroke, priapism and admissions for vaso-occlusive crises (VOC) biannually. Upon 7 years of follow-up, progression in the prevalence of avascular osteonecrosis (from 12.5% to 20.4%), renal failure (from 6.7% to 23.4%), retinopathy (from 39.7% to 53.8%) was observed in the whole group. In HbSS/HbSβ0 -thal patients also progression in microalbuminuria (from 34% to 45%) and elevated TRV (from 40% to 48%) was observed while hardly any progression in the prevalence of cholelithiasis, priapism, stroke or chronic ulcers was seen. The proportion of patients with at least one episode of ACS increased in the group of HbSS/HbSβ0 -thal patients from 32% to 49.1%. In conclusion, 62% of the sickle cell patients in this prospective cohort study developed a new SCD-related complication in a comprehensive care setting within 7 years of follow-up. Although the hospital admission rate for VOC remained stable, multiple forms of organ damage increased substantially. These observations underline the need for continued screening for organ damage in all adult patients with SCD.

Assessing Bone Quality Using Trabecular Bone Score in Patients with Hemoglobinopathies

Introduction: Osteoporosis is characterized by a decrease in bone mass and density with enlarged trabecular space resulting in porosity and bone fragility. It has been described in as many as 70 to 80% of adults with thalassemia (Thal) and sickle cell disease (SCD). Though assessment by DXA scan is now part of routine clinical practice, bone quality has been poorly characterized, particularly in SCD. Trabecular bone score (TBS) is a new textural analysis of lumbar spine DXA scans that reflects bone microarchitecture, shown to be highly predictive of fracture in adults. The objectives of this study were 1) to determine the prevalence of poor bone quality as assessed by TBS in patients with Thal and SCD, compared to healthy individuals and 2) to assess the relationship between bone quality and clinical predictors (age, transfusion status, liver iron concentration, diet, BMI, endocrinopathies).Methods: A retrospective chart review was conducted in patients > 10 years and > 40 Kg with Thal or SCD who had a spine bone mineral density (BMD) scan performed in the previous 5 years. Patients had on average 1.7±0.9 spine scans during the collection period (range 1-5); all scans were reanalyzed using the TBS software (Insight, MediMaps v2.2, France). Optimal bone quality was defined as TBS >1.35; subnormal TBS= 1.34-1.20; abnormal <1.20. Liver iron concentration (LIC) was assessed by SQUID. Data from healthy controls without Thal or SCD were collected from previously completed research studies. Statistical analysis was performed using STATA, v. 9.0 (College Station, TX). This study was approved by the Institutional Review Board at UCSF Benioff Children’s Hospital Oakland.Results: Data from 251 patients were abstracted which included, 162 females, 173 adults; 81 Thal, 102 SCD, and 68 healthy controls. Thal patients were older than SCD or controls (29.7 vs. 23.8, 25.8 years, p<0.05) and had lower LIC (2303 vs. 3014 µg Fe/g wet wt., p=0.004) but higher incidence of hypogonadism (31% vs. 1%, p<0.001). No differences were observed in vitamin D status, fracture history or family history of osteoporosis. On average, Thal patients had greater deficits in spine BMD Z-score (-2.1±1.2, Mean±SD), as compared to SCD (-1.0±1.5) and controls (-0.1±0.8), as well as a higher prevalence of abnormal bone quality by TBS (29.7%) vs. 12.2% in SCD, 4.6% in control, (p<0.001). TBS was positively correlated with BMD (r=0.7, p<0.001) and negatively correlated with age (r=-0.28, p<0.001). After controlling for age, BMI, hypogonadism and diagnosis, LIC was negatively associated with bone quality (r=0.30, p=0.001).Conclusions: These data support the relationship between reduced bone mass and bone quality in adult and adolescent patients with hemoglobinopathies. Older patients with low bone mass appear to be at particular risk for abnormal bone quality. TBS may be a valuable clinical tool in the assessment of true fracture risk in this group of patients with extremely low bone mineral density. However, future research is needed to develop models that include BMD and TBS for prediction of absolute fracture risk and need for treatment of low bone mass in patients with hemoglobinopathies. DisclosuresNo relevant conflicts of interest to declare.

Assessment of Cardiac Iron Overload in Chonically Transfused Patients with Thalassemia, Sickle Cell Anemia, and Myelodysplastic Syndromes

▪Background. Cardiac iron overload is the major cause for death in regularly transfused thalassemic (Thal) patients. Its impact in myelodysplastic syndrome (MDS) patients is debated. Heart seems to be spared in regularly transfused patients with sickle cell anemia (SCA), which is supposed to be related to a later onset on transfusions and to the use of erythrocytapheresis rather than simple transfusion. Our aim was to assess the prevalence of cardiac iron overload, defined as a T2*cardiovascular magnetic resonance (MR) <20ms, and to look for predisposing factors.Patients and methods. Patients were enrolled if they were regularly followed in a center where the exact number of erythrocyte concentrates (EC) could be obtained, had received in the previous year more than 8 EC, were older than 6 years (limit for MRI without sedation), had no known heart disease related to another pathology, and had given informed consent.All patients underwent 1.5T myocardial T2*MR imaging after validation of the procedures in the different sites. Assessment of Liver Iron Content (LIC) used two signal intensity ratio of gradient echo imaging (R2*) MRI protocols. Serum Non-Transferrin Bound Iron (NTBI) was measured by the FeROSTM eLPI kit, and serum hepcidin by LC-MSMS.Results. 20 Thal, 41 SCA and 25 MDS patients were evaluable. We divided SCA in 2 groups, according to the procedure recorded at the time of the study, manual exchange transfusion (G1, N = 30 patients), or erythrocytapheresis (G2, N=11 patients).We found cardiac overload in 0, 3 (15%), and 4 (16%) of SCA, Thal, and MDS patients respectively. Serum ferritins at beginning of chelation were not statistically different in all categories of patients, as well as Ferritin and LIC at the time of the study. Increased LIC and abnormal T2* were associated in Thal and MDS patients (p=0.04), with no correlation between abnormal T2* and parameters of transfusion and chelation.Plasmatic iron level was increased in Thal and MDS patients but remained at normal range in SCA patients. NTBI level was high in Thal and MDS but completely absent in SCA groups.The major discrepancy was in the values of hepcidin, which were collapsed in Thal, at normal range in SCA, and highly elevated in MDS patients.Discussion and conclusion.We confirm that SCA patients are relatively protected from cardiac iron overload. This results probably from massive consummation of iron through effective erythropoiesis, making toxic free iron (NTBI) less available in the circulation. In addition, since iron overload in SCA results from a massive outflow of hemoglobin (Hb) due to intravascular hemolysis and transfusion, the heme/Hb-bound iron must be efficiently handled in liver macrophages, limiting its release in the bloodstream.In Thal patients, underlying defects in erythropoietic processes, together with low hepcidin that stimulates intestinal iron absorption and increases NTBI, must provoke more organ damages. Hepcidin levels were high in MDS patients, suggesting that transfusion-dependent iron overload was a more effective regulator of hepcidin production than dyserythropoiesis. The % of T2*<20 ms we observed in MDS patients (16%) was quite comparable with previous publications.Finally, we observe that, in opposition with previous reports, SCA patients undergoing eythrocytapheresis may experience severe iron overload and need iron chelation.Table 1ThalSCA G1SCA G2MDSpAge at beginning of transfusion (yrs)8.5[0-45]7[0-45]16.5[1-55]66[38-83]<0.001Duration of transfusion (yrs)10[1-39]7[1-22]10.5[0-25]3[1-10]<0.001N EC since diagnosis359[21-1360]139[24-791]201[14-888]77[16-544]0.0005N CE/yr24[8-67]21[4-62]35[17-58]27[7-65]0.09G1vsG2=0.03% patientschelated959072.7720.12Age at beginning of chelation (yrs)11[1-48]9[2-47]18[6-31]68[38-84]<0.0005Ferritin at beginning of chelation (ng/ml)1148[713-2400]2075[448-3670]1500[905-2804]2398[482-5140]0.22N T2*<20 ms3(15%)004(16%)0.01LIC (mg/g d.w.)10.4[0.8-20.2]10.7[0.8-37.1]14[0.8-19.7]15.2[3.0-45.3]0.29Plasmatic iron(μmol/l)36.9[31-57]22.5[6-42.2]21[13-46]38.2[11.9-72]<0.001NTBI (mg/ml)7.1[0-31.1]0[0-18.3]0[0-12.4]4.45[0-25.5]0.0005Ferritin (ng/ml)870[169-4339]2739[393-5596]2404[33-20030]1611[223-6813]0.08Hepcidin (ng/ml)1.35[0-12.3]9.95[0-67.9]2.10[0-52.4]36.35[3-143.2]<0.001Deferasirox dosage<0.5 μg/ml3/8(38%)3/10(29%)3/5(60%)0/11(0%)0.03 DisclosuresDe Montalembert:Addmedica: Membership on an entity's Board of Directors or advisory committees; Novartis: Speakers Bureau. Guerci-Bresler:ARIAD: Speakers Bureau; BMS: Speakers Bureau; Novartis: Speakers Bureau; PFIZER: Speakers Bureau.

Zinc status affects glucose homeostasis and insulin secretion in patients with thalassemia.

Up to 20% of adult patients with Thalassemia major (Thal) live with diabetes, while 30% may be zinc deficient. The objective of this study was to explore the relationship between zinc status, impaired glucose tolerance and insulin sensitivity in Thal patients. Charts from thirty subjects (16 male, 27.8 ± 9.1 years) with Thal were reviewed. Patients with low serum zinc had significantly lower fasting insulin, insulinogenic and oral disposition indexes (all p < 0.05) and elevated glucose response curve, following a standard 75 g oral load of glucose compared to those with normal serum zinc after controlling for baseline (group × time interaction p = 0.048). Longitudinal data in five patients with a decline in serum zinc over a two year follow up period (−19.0 ± 9.6 μg/dL), showed consistent increases in fasting glucose (3.6 ± 3.2 mg/dL) and insulin to glucose ratios at 120 min post glucose dose (p = 0.05). Taken together, these data suggest that the frequently present zinc deficiency in Thal patients is associated with decreased insulin secretion and reduced glucose disposal. Future zinc trials will require modeling of oral glucose tolerance test data and not simply measurement of static indices in order to understand the complexities of pancreatic function in the Thal patient.

Very mild forms of Hb S/beta(+)-thalassemia in Brazilian children.

The clinical phenotype of sickle cell/beta+-thalassemia (Hb S/ +-Thal) is highly variable, and severity is associated with the quantitative degree of decrease in the production of the beta globin chains.1 Evidence shows that differences in the production of hemoglobin A (Hb A) and severity correspond to different molecular beta-thalassemia ( -Thal) mutations.2 A previous report proposed a classification of Hb S/ +-Thal phenotypes based on the relative concentration of Hb A: Type I: 1–7% of Hb A; Type II: 7–14% of Hb A; and Type III: 14–25% of Hb A.2 However, some -Thal mutations lead to low impairment of -globin production and the resulting phenotype does not fit this proposed classification. In a single patient case report, the −92 (C>T) mutation was associated with a high level of Hb A (45%) in an adult Sicilian patient with Hb S/ +-Thal.3 Recently, a combination of two sequence variants, IVS-II839 (T>C) and IVS-II-844 (C>A), was associated with a very mild phenotype of sickle cell disease (SCD).4 Because of the marked clinical variability of Hb S/ +-Thal patients, molecular

Spectral detection of thalassemia: a preliminary study

BackgroundThalassemias (Thal) are forms of inherited autosomal recessive blood disorders arising out of mutations in the chromosomes 11 or 16. These disorders lead to poor oxygen delivery to blood vessels and consequent splenomegaly, bone deformities, and shorter life spans. The most common detection methods for Thal are complete blood count (CBC) followed by electrophoresis and molecular diagnosis methods, such as high-performance liquid chromatography (HPLC) and polymerase chain reaction (PCR) genotyping. These methods involve sophisticated instrumentations and are cumbersome and expensive.ResultsIn this study an innovative spectral detection method, based on the fluorescence spectra of a set of biomolecules (tyrosine, tryptophan, nicotinamide adenine dinucleotide, and flavin adenine dinucleotide and porphyrins) found in blood components is presented. An algorithm based on the spectral features of such biomolecules of blood components of 20 Thal patients (10 female and 10 male) and 18 age adjusted normal controls (4 female and 14 male) demonstrate reasonable level of classification with sensitivity and specificity values exceeding 90%.ConclusionThis new technique could be of significant value for Thal detection, diagnosis, and subsequent genetic counselling and could be adapted for use in small primary health centres.

Zinc supplementation improves bone density in patients with thalassemia: a double-blind, randomized, placebo-controlled trial

Patients with thalassemia major (Thal) frequently have low plasma zinc, which has been associated with low bone mass. The objective was to determine the effect of zinc supplementation on bone mass in patients with Thal. Forty-two subjects (21 females aged 10-30 y) with Thal and low bone mass were randomly assigned to receive 25 mg Zn/d or placebo. Bone mineral content (BMC) and areal bone mineral density (aBMD) were assessed by using dual-energy X-ray absorptiometry, and fasting blood was collected for the measurement of plasma zinc at 0, 12, and 18 mo. Thirty-two subjects, 81% of whom were transfusion dependent, completed the study (mean ± SD: 17.1 ± 5.2 y). Plasma zinc was ≤70 μg/dL in 11 subjects at baseline and increased significantly with zinc supplementation (P = 0.014). Use of intention-to-treat analysis and linear models for longitudinal data, adjusted for baseline and pubertal stage, showed that the zinc group had significantly greater increases in whole-body BMC (adjusted mean ± SE: 63 ± 15 g; P = 0.02), and aBMD (0.023 ± 0.006 g/cm(2); P = 0.04) than did the placebo group after 18 mo. Furthermore, adjusted spine and hip aBMD z scores each decreased by 0.3 SDs (both P = 0.04) in the placebo compared with the zinc group over the 18-mo study. In young patients with Thal, zinc supplementation resulted in greater gains in total-body bone mass than did placebo. Zinc was well tolerated and is worthy of investigation in larger trials in Thal patients across a range of ages and disease severity. This trial was registered at clinicaltrials.gov as NCT00459732.

Beta-Globin Cluster Cis-ACTING Sequence and BCL11A Variants in the Control of Hemoglobin F Expression

Abstract 1091Fetal hemoglobin (Hb F: α2γ2.) is the most important modulating factor of sickle cell disease(SCD) and beta-major thalassemias (β°thal). Due to the expected benefit from an active control of gamma gene expression, studies of the regulation of Hb F expression are very intense with the recent focus on BCl11 A, a transacting factors strongly involved in the control of its expression. Studies have subsequently established that Bcl 11A is a repressor of HbF expression through different mechanisms including a direct action “in trans” on the β-globin locus. Our work aims to look for Bcl 11A coding sequence’s variations in a cohort of homozygous β°thal patients whose clinical status is less severe than that expected from their globin genotype because of high Hb F level (12 patients). Interest has also been given to a cohort of patients (12 patients) displaying a small deletions of the Ψβ-δ intergenic region for which it has been noted significant Hb F rate differences in possible correlation with a Bcl 11A binding site located1.5 Kb 5 ’to the δ gene previously described (Sankaran, 2008). Methods:Sequencing of Bcl 11A (promoter, XL and L isoforms exons and flanking regions) was performed and the determination of the four main SNPs cited in the literature as predictors of the rate of HbF: rs4671393 and rs11886868 (Bcl 11A), rs9399137 (HSB1L-MYB), and rs7482144-XmnI (β-globin cluster), was carried out. The precise study of deletions was performed using a custom CGH array chip and the subsequent determination of breakpoints. In both patients’ cohorts, the absence of the deletion of one BCL11A allele was checked by a tailored semi-quantitative Multiplex PCR, and the hypothesis of a sequence variation inside the “Sankaran region” was screened using a High Resolution Melting procedure (HRM). Results and discussion:Analysis of BCL11A: We have not found mutation inside the coding sequence. However, A “CCG” triplet insertion within a CCG-rich region located in the 5'UTR, immediately 5' to the ATG codon was observed in three patients. That triplet insertion was not found in the other patients of our cohort of 12 patients with high expression of Hb F, and neither in another cohort of high F patients (23 patients). It was also absent in low Hb F patient ( 15) or in controls (50 patients not known to have a red cell disease), highlighting the rarity of this insertion. The effects on transcription or RNA translation could not be predicted by “in silico” analysis.The deletion analysis: We found in this cohort patients two kind of deletions: a first group where the 5’ breakpoint of the deletion is located close to the δ-globin gene and for which the presumed BCL11A binding domain was not removed and a second group for which the 5’ deletion breakpoint extends 5’ and removes that region. Interestingly the second group has a higher Hb F expression calculated as the absolute level of Hb f (Hb value * Hb F %) than the first group with a ratio of 2.54. That result highlight the role of this domain for Hb F control. We, also, cannot found sequence variation in that domain using HRM analysis. In conclusion:The presence of BCL 11A binding sites immediately 5’ of d gene appears to be necessary for Hb F control. However, investigating the role of Bcl 11A in Hb F dysregulation (ie: the absence of down regulation) either by looking at protein variants or at variations in the supposed target of Bcl11A in β globin cluster, we found that this lack of regulation is not linked to mutated Bcl 11a and nor to SNPs into its binding region. These results support the hypothesis that the control of Hb F is due to variation of the level of Bcl 11 A expression and the finding of a variation in the 5’UTR region which may interfere with the Bcl 11A RNA translation is another supporting evidence. Further studies of the consequence of this triplet insertion are ongoing to estimate its possible involvement in BCL11A transcription and translation and thus Bcl11A level.We confirmed that the “Sankaran region” is an important element of Bcl11A control of Hb F expression. A study of chromatin conformation (3C-qPCR) using patient bearing these small deletions are needed to test the hypothesis of a non-dissociation of the beta-globin LCR (locus control region) from its attachment point in the genes Gγ Aγ region confirming the major role of this site in the HbF downregulation. Disclosures:No relevant conflicts of interest to declare.

Exploring Vertebral Height Deficits in Patients with Thalassemia and Sickle Cell Disease,

Abstract 3198Low bone mass has been described in as many as 60% of patients with thalassemia (Thal) and sickle cell disease (SCD). Bone pain is also frequently reported in each disorder, however few have explored the relationship between low bone mass and bone pain. The objectives of this study were to determine the prevalence of vertebral height abnormalities in patients with Thal and SCD at the Children’s Hospital & Research Center, Oakland (CHRCO). The secondary objective was to evaluate the relationship between vertebral height abnormalities and low bone mass and patient assessed bodily pain. Methods:Data for this analysis were collected from two sources: the Thalassemia Clinical Research Network Pain Survey study and the CHRCO Clinical Bone Density database. The TCRN Pain study used a validated pain survey to collect data on 45 patients from CHRCO at multiple time points. Pain data was then analyzed along with bone mineral density (BMD) scans if performed within 6 months. BMD Z-scores were abstracted from the CHRCO database along with patient demographics. Full lateral spine scans conducted at the time of BMD scan were re-analyzed by one observer using the Vertebral Fracture Analysis (VFA) software (Hologic v12.6.1.), and scored according to Genant for vertebral abnormalities (crush, wedge, biconcavity, Grades 1 to 3). Results:A total of 254 VFA scans were re-analyzed from 93 patients with Thal (21.5 ± 10.7 yrs, 55% female), 45 SCD (38.1 ± 12.7 yrs, 61% Female) and 11 healthy control subjects (29.9 ± 10.2 yrs, 64% female). The patients with Thal were primarily Asian (78%), with 68.8% B-thal, 24%E-Bthal and 12.1% Hb H; while 98% of the SCD patients were Black, all HbSS. Patients with Thal were more likely to have low bone mass (any BMD Z-score ≧ −2.0) compared to those with SCD (73% vs. 33%, p<0.01). Of the patients with Thal who had VFA scans, 25 (26.9%) had at least one vertebral abnormality, compared to 16 (34.8 %) of the 45 patients with SCD (p=NS). SCD patients had significantly more vertebral abnormalities compared to healthy controls (p=0.04). However, a similar percentage of Thal patients had substantial vertebral height deficits (Grade 2 or 3; 15.1%) compared to those with SCD (13.3%). The average number of vertebrae with abnormalities within an individal scan was lower in Thal (1.6±0.2) compared to SCD (2.8 ± 0.4; p=0.003). Thal were more likely to have wedge-type deformities in the lumbar region, while SCD had biconcavity-type deformed vertebrae in the thoracic region. Of the patients with multiple VFA scans, 12 progressed toward vertebral abnormality during a 7 year period, all with Thal. The presence of a vertebral abnormality was positively related to age in Thal but not SCD (24.2 ± 1.2 vs. 20.6 ± 0.8 yrs, p=0.029) with a trend towards a relationship to low bone mass (p=0.12). Abnormal VFA was unrelated to gender and vitamin D level. In a sub-set of the Thal subjects who participated in the TCRN pain study, two-thirds reported significant pain within the past month, 16% severe and 27% reported pain localized in the lower back. However in the patients for which both VFA scans and pain studies were performed (n=32), bodily pain was not related to vertebral abnormalities. Discussion:A surprisingly high percentage of both SCD and Thal patients had significant vertebral abnormalities. In many cases the etiology of the observed abnormal morphology unclear but appears to be more developmental in SCD whereas in Thal a consequence of collapse secondary to inherent bone fragility and/or trauma. It is also yet to be determined if this phenomenon results from skeletal effects of the underlying disease or the various treatments (i.e., chelation toxicity). The prevalence of vertebral abnormality observed in this cohort of Thal patients (27%) is twice that previously reported from the TCRN (12%). The prevalence of vertebral abnormality by DXA scan has never before been reported in SCD. It is conceivable that the high rate of lower back pain in Thal is related to the observed abnormal vertebrae, however we were unable to explore this thoroughly given the small sample size. This study was limited by the absence of multiple VFA scans for all subjects and the discordance of a temporal relationship between the pain studies and VFA scans. Further research should focus on the clinical relevance of vertebral abnormalities and its etiology in these at risk patient populations. Disclosures:No relevant conflicts of interest to declare.

Atrial and ventricular function in thalassemic patients with supra-ventricular arrhythmias.

The aims of this study were to evaluate through Color Doppler Myocardial Imaging (CDMI) echocardiography if atrial or ventricular myocardial alterations could be detectable in patients with thalassemia major (THAL) and if these alterations could be considered as predictive elements for supra-ventricular arrhythmic events. Twenty-three patients with THAL underwent clinical and electrocardiographic evaluation; patients were grouped in THAL1 (9 with supra-ventricular arrhythmias) and THAL2 (14 without arrhythmias); 12 healthy subjects were considered as control group (C). We examined through conventional 2D Color Doppler echocardiography some morphological and functional parameters regarding left ventricular (LV) systolic and diastolic function, and through CDMI the velocities at mitral annulus level, the regional LV and left atrial (LA) strain and strain rate. All THAL patients had LV dimension (p<0.05), LA area (p<0.01) and E/Em ratio (p<0.001) to be significantly higher than controls. The mitral annulus longitudinal velocities were significantly lower in THAL1 than in THAL2 (p<0.001); the E/Em ratio was higher in THAL1 than THAL2 (p<0.001). The THAL1 showed a lower systolic strain rate of atrial wall than THAL2 and C (p<0.05). The multiple regression highlighted a significantly inverse correlation among E/Em and atrial strain (p<0.02). CDMI showed both THAL subgroups had subtle systolic and diastolic left ventricular myocardial alterations, which could represent the onset of developing “iron cardiomyopathy” and are related to supra-ventricular arrhythmia. Monitoring these parameters in the THAL patients could contribute to decisions about follow-up and therapy.