Efficacy and Safety of a Dispersible Tablet of GPO-Deferasirox Monotherapy among Children with Transfusion-Dependent Thalassemia and Iron Overload.

The study aimed to determine efficacy and safety of generic deferasirox monotherapy. Deferasirox was administered in transfusion-induced iron overloaded thalassemia. Efficacy was defined as responders and nonresponders by ≤ 15 reduced serum ferritin from baseline. Adverse events were also monitored. Fifty-two patients with mainly Hb E/ß-thalassemia at the mean (SD) age of 8.7 (4.1) years, were enrolled. The mean (SD) daily transfusion iron load was 0.47 (0.1) mg/kg and maximum daily deferasirox was 35.0 (6.2) mg/kg. Altogether, 52, 40 and 18 patients completed the first, second and third years of study, respectively. The median baseline serum ferritin 2,383 ng/mL decreased to 1,478, 1,038 and 1,268 ng/mL at the end of first, second and third years, respectively, with overall response rate at 73.1% (38/52). Patients with baseline serum ferritin >2,500 ng/mL showed a change in serum ferritin higher than those ≤2,500 ng/mL starting from the 9th month of chelation. Adverse events were found in 5 of 52 patients (9.6%) including transaminitis (n = 2), one each of proteinuria, rash and proximal tubular dysfunction which resolved after transient stopping or decreasing the chelation dose. Generic deferasirox was effective and safe among pediatric patients with transfusion-induced iron overloaded thalassemia.

Response to Prolonged Duration of Therapeutic Dose Oral Iron Therapy in a Girl With Novel TMPRSS6 Gene Variants: A Case Report and Review Literature.

Iron-refractory iron deficiency anemia (IRIDA) is an autosomal recessive disorder caused by mutations in the TMPRSS6 gene, which impair iron homeostasis. We reported a 4-year-old girl who presented with a 1-year history of iron deficiency anemia. Her hemoglobin level increased from 6.5 g/dL to 12.6 g/dL with a prolonged duration of therapeutic dose oral iron therapy (5 mg/kg/d), and the level remained quite stable during the therapy. Genetic analysis of the TMPRSS6 gene revealed compound heterozygotes of 2 novel pathogenic variants: c.811C> T (NM_153609.3) in exon 7 (NP_705837: p.R271Ter) and c.1254C> G in exon 11 (p.Y418Ter). The results highlight the significance of genetic investigation and long-term iron therapy in iron-refractory iron deficiency anemia patients.

Severity scoring system to guide transfusion management in pediatric non-deletional HbH.

BACKGROUND: Hemoglobin (Hb) H is generally recognized as mild thalassemia, despite its actual phenotypic diversity. A disease severity scoring system to guide initiation of regular transfusion among severely affected pediatric patients has not previously been reported. METHODS: Patients with HbH were classified into transfusion-dependent thalassemia (TDT) and non-transfusion-dependent thalassemia (NTDT) as a surrogate for disease severity. Alpha-globin genotypes and relevant clinical parameters associated with TDT were identified. Univariate and multiple logistic regression analyses were performed to yield the most suitable severity scoring system. RESULTS: From 246 patients with a median age of 14.3 (interquartile range 9.9-18.4) years initially enrolled into the study, the chance of having severe disease and developing TDT was remarkable only among patients with non-deletional HbH, for whom the scoring system was developed. Univariate and multiple logistic regression analyses resulted in three retained parameters associated with TDT, ß-coefficients of which were used to develop the score. The final scoring system comprised age at diagnosis <2 years (score = 1), spleen size ≥3 cm (score = 1) and Hb at steady-state <7 (score = 4) or 7-8 g/dL (score = 3). A cutoff score ≥4 was associated with severe disease likely requiring regular transfusion (sensitivity 89.3%, specificity 81.4%), given regular transfusion resulted in maintained growth. The scoring system was validated in the second cohort of 77 non-deletional HbH, from which comparable sensitivity and specificity were obtained. CONCLUSION: The newly developed scoring system was practical and helpful to highlight severely affected pediatric non-deletional HbH patients with potential needs of regular transfusion. This can be used as a guide for optimal treatment and disease monitoring in the future.

Whole-exome sequencing uncovered genetic diagnosis of severe inherited haemolytic anaemia: Correlation with clinical phenotypes.

Next-generation sequencing has shed light on the diagnosis of previously unsolved cases of inherited haemolytic anaemia (IHA). We employed whole-exome sequencing to explore the molecular diagnostic spectrum of 21 unrelated Thai paediatric patients with non-thalassemic IHA, presenting hydrops fetalis and/or becoming transfusion-dependent for 1 year or more or throughout their lifespan. Anaemia was detected prenatally, within the first month and the fifth year of life in three, 12 and six patients respectively. Molecular diagnosis obtained from all patients revealed SPTB as the most frequently mutated gene (four reported, three novel), found in 31 of 42 studied alleles. The other two mutated genes identified were ANK1 (three novel) and KLF1 (two reported). Four recurring mutations within exon 29/30 (NM_001024858.2) accounted for the vast majority (90%) of mutated SPTB alleles, biallelic inheritance of which resulted in the most severe phenotypes: hydrops fetalis and life-long transfusion dependency. Dominant ANK1 (n = 3) and SPTB (n = 2) mutations and biallelic class 2 KLF1 mutations (n = 1) led to a shorter period of transfusion dependency. Our study demonstrated that mutated SPTB causing red-cell membranopathy is likely the most common cause of severe non-thalassemic IHA among Thai patients. This urges carrier screening in the population to prevent subsequent, severely affected births.

Haploidentical Hematopoietic Stem Cell Transplantation in Thalassemia.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only established treatment that is potentially curative, but it is limited by the availability of donors and the medical condition of the patient. To expand the donor pool to include haploidentical related donors, we introduced a program consisting of a pharmacologic pre transplant immune suppression phase (PTIS) and two courses of dexamethasone (DXM) and fludarabine (FLU) followed by pre transplant conditioning with intravenous FLU busulfan (BU) and post transplant graft-vs.-host disease (GvHD) prophylaxis with cyclophosphamide (CPM), tacrolimus, and mycophenolate mofetil. We transplanted 83 consecutive transfusion-dependent patients with thalassemia; the 3-year projected overall and event-free survival is over 96.0%, and there have been no secondary graft failures. Of the first 31 patients, we had two graft failures, both of them occurring in patients with extremely high titers of anti-donor-specific human leukocyte antigen (HLA) antibodies [anti-donor specific antibodies (DSAs)], but after adjusting the PTIS to include bortezomib (BORT) and rituximab (RIX) for patients with high titers of anti-DSAs and using pharmacologic dose guidance for BU, we had no graft failures in the last 52 patients. Six (7.0%) of 83 patients developed severe GvHD. We conclude that this is a safe and efficacious approach to allogeneic HSCT in thalassemia.

Real-world evidence on health resource use among patients with haemophilia and inhibitor exhibiting severe bleeding episodes.

OBJECTIVE: This study aimed to explore real-world evidence on health resource use (HRU) spending on patients with haemophilia and inhibitor. MATERIALS AND METHODS: Medical records from 1990 to 2019 of patients with haemophilia and inhibitor from three comprehensive haemophilia treatment centres were retrospectively retrieved. RESULTS: In all, 31 patients with haemophilia (A = 30, B = 1) and inhibitor ≥5 BU were included. The mean initial inhibitor of 95.4 BU was detected at the mean age of 6.7 years. The mean number of annual hospitalisations was 3.9. A total of 795 bleeding episodes (major =125, minor =670) were evaluated. The treatment included bypassing agents or plasma exchange before administering high-dose factor VIII concentrate and intervention or surgery. Six patients succumbed to bleeding at the mean age of 17.2 years. Nineteen surviving patients experienced multiple morbidity except six patients with successful and partially successful immune tolerance induction (ITI). The mean (SD) annual total medical consumption for episodic treatment and successful ITI per patient with haemophilia A were 30,804 (81,332) USD and 55,531 (100,566) USD, respectively. Only episodic treatment was paid by the government budget for limited amounts of bypassing agents. CONCLUSION: Management for patients with haemophilia and inhibitor exhibiting severe bleeding is challenging for medical personnel in countries having limited resources over decades. The real-world data will be used to negotiate with the government to increase budget for adequate bypassing agents or nonreplacement therapy and to include ITI in the national haemophilia treatment.

Hematopoietic Stem Cell Transplantation for Severe Thalassemia Patients from Haploidentical Donors Using a Novel Conditioning Regimen.

Patients with severe thalassemia commonly have a survival that is significantly shorter than that of the general population. Allogeneic hematopoietic stem cell transplantation (allo-SCT) is the only established treatment that is potentially curative, but it is limited by the availability of donors and the medical condition of the patient. To expand the donor pool to include haploidentical related donors, we introduced a program consisting of a pharmacologic pretransplant immune suppression phase (PTIS) and 2 courses of dexamethasone and fludarabine, followed by pretransplant conditioning with fludarabine-i.v. busulfan and post-transplant graft-versus-host disease (GVHD) prophylaxis with cyclophosphamide, tacrolimus, and mycophenolate mofetil. We transplanted 83 consecutive transfusion-dependent patients with thalassemia (median age, 12 years; range, 1 to 28 years) with a minimum follow-up of 6 months (median, 15 months; range, 7 to 53 months); the 3-year projected overall and event-free survival is over 96%, and there have been no secondary graft failures. Of the first 31 patients, we had 2 graft failures, both of them occurring in patients with extremely high titers of anti-donor-specific HLA antibodies (anti-DSAs), but after adjusting the PTIS to include bortezomib and rituximab for patients with high titers of anti-DSAs and using pharmacologic dose guidance for busulfan, we had no graft failures in the last 52 patients. Six (7%) of 83 patients developed severe GVHD. We conclude that this is a safe and efficacious approach to allogeneic SCT in thalassemia, yielding results comparable to those available for patients with fully matched donors.

No differences in hemostatic and endothelial activations between haploidentical and matched-donor hematopoietic stem cell transplantation in thalassemia disease.

Hemostatic changes and endothelial activations have been recognized in ß-thalassemic patients after matched-donor hematopoietic stem cell transplantation (HSCT) but there are limited studies for haploidentical HSCT. This report demonstrates that the levels of hemostatic and endothelial markers, including thrombin antithrombin complex, prothrombin fragment, D-dimer, von Willebrand factor antigen and thrombomodulin levels, were not significantly different between haploidentical and matched-donor HSCT patients.

Increased endothelial activation in α-thalassemia disease.

One complication of thalassemia is thromboembolism (TE), which is caused by an abnormal red blood cell surface, as well as endothelial and platelet activation. These findings are commonly observed in severe ß-thalassemia. However, limited information on α-thalassemia exists. This study enrolled subjects with deletional and non-deletional α-thalassemia and normal controls (NC). Plasma and serum of subjects were tested for endothelial activation markers including thrombomodulin (TM), vascular cell adhesion molecule-1 (VCAM-1), and von Willebrand factor antigen as well as platelet activation markers including thromboxane B2 and platelet factor 4. A total of 179 subjects were enrolled: 29 in the deletional group (mean age 13.3 ± 4.4 years), 31 in the non-deletional group (mean age 12.9 ± 4.8 years), and 119 in the NC group (mean age 13.6 ± 3.0 years). Twenty nine percent of subjects in the non-deletional group received regular red blood cell transfusion and iron chelator administration. Serum ferritin level was higher in the non-deletional group than that in the deletional group. Multivariate analysis demonstrated that VCAM-1 and TM levels were increased significantly in α-thalassemia compared with NC group (816.8 ± 131.0 vs 593.9 ± 49.0 ng/ml, and 4.9 ± 0.7 vs 4.0 ± 0.4 ng/ml, P < 0.001 respectively). VCAM-1 and TM levels in the non-deletional group were significantly higher than that in the deletional group. The present study demonstrated endothelial activation in children with α-thalassemia disease, especially those in the non-deletional group, which might be one risk factor for TE in α-thalassemia disease.

Reticulocyte hemoglobin equivalent in a thalassemia-prevalent area.

BACKGROUND: Reticulocyte hemoglobin equivalent (Ret-He), a direct measure of the hemoglobin (Hb) in the young red blood cells, has been reported to be useful in the diagnosis of iron deficiency anemia (IDA) but may have some limitations in thalassemia trait. This study evaluated the differences in Ret-He in school-aged children, and assessed the diagnostic value of Ret-He in identifying IDA in a thalassemia-prevalent area. METHODS: Blood samples underwent complete blood count analysis, including Ret-He, ferritin, serum iron and total iron binding capacity. Blood samples also underwent Hb typing and a molecular study for α-thalassemia. Receiver operating characteristic analysis was performed to determine the predictive capacity of Ret-He in the diagnosis of IDA. ID was defined as serum ferritin <30 ng/mL and/or transferrin saturation (TSAT) <16%; IDA was defined as serum ferritin <12 ng/mL and/or TSAT <16% with low Hb for age. Normal healthy children (normal controls: NC) had normal iron study, without the thalassemia trait. RESULTS: Ninety-eight children with a mean age of 12.9 ± 0.6 years were included. Ret-He in the thalassemia trait group (26.7 ± 2.4 pg), ID group (29.0 ± 2.9 pg), IDA group (25.4 ± 2.7 pg), ID + thalassemia trait group (26.6 ± 2.8 pg), and the IDA + thalassemia trait group (24.6 ± 2.3 pg) was significantly lower than in the NC group (30.8 ± 1.7 pg; P < 0.001, 0.01, 0.006, 0.002 and <0.001, respectively). Ret-He had an area under the curve of 0.904 in diagnostic ability for IDA, while a cut-off ≤27 pg had a sensitivity of 91.7% and a specificity of 81%. CONCLUSION: Ret-He was lowest in subjects with IDA + thalassemia trait. A Ret-He cut-off ≤27 pg was suggestive of IDA in the present study.

The Effect of Blood Transfusion on Growth of Patients with Hb E/ß-Thalassemia.

A retrospective evaluation of growth in 112 patients (68 males, 44 females) with Hb E (HBB: c.79G>A)/ß-thalassemia (ß-thal), classified as 88 transfusion-dependent thalassemia (TDT) and 24 non transfusion-dependent thalassemia (NTDT), is reported. Patients with TDT have received regular transfusions of red blood cells (RBCs) 15 mL/kg every 4 weeks to maintain pre transfusion hemoglobin (Hb) levels of at least 9.0 g/dL and were categorized according to age at initiation of regular RBC transfusion as subgroup 1, <4 years; subgroup 2, 4-10 years, and subgroup 3, >10 years. Iron chelation was initiated at the mean age of 7 years. The results revealed that patients in subgroups 1 and 2, receiving RBC transfusions at a young age (2.9 and 6.9 years, respectively), had normal prepubertal growth at enrollment and last follow-up. Patients in subgroup 3, with the lowest initial height Z-score of -2.10, were able to achieve comparable final adult height as those in subgroups 1 and 2. The mean final height of 21 males and 13 females with TDT at the ages of 18.9 and 18.7 years was 168.1 and 157.7 cm, respectively, which did not significantly differ from their midparental height and those with NTDT. Early initiation of optimal transfusion and iron chelation promoted normal prepubertal growth. However, delayed initiation of transfusion at age 12 years impaired prepubertal growth but they could achieve normal final adult height.

Differences in activation and tissue homing markers of natural killer cell subsets during acute dengue infection.

Dengue virus (DENV) infection is considered one of the most important mosquito-borne diseases. It causes a spectrum of illness that could be due to qualitative and/or quantitative difference(s) of the natural killer (NK) cell responses during acute DENV infection. This view prompted us to perform a detailed phenotypic comparative characterization of NK cell subsets from DENV-infected patients with dengue fever (DF), patients with dengue haemorrhagic fever (DHF) and healthy controls. The activation/differentiation molecules, CD69 and CD57 and a variety of tissue homing molecules were analysed on the CD56hi CD16- and CD56lo CD16+ NK cells. Although there was no increase in the frequency of the total NK cells during DENV infection compared with the healthy individuals, there was a significant increase in the frequency of the CD56hi CD16- subset and the frequency of CD69 expression by both NK cell subsets during the febrile phase of infection. We also found an increase in the frequencies of cells expressing CD69 and CD57 in the CD56lo CD16+ subset compared with those in the CD56hi CD16- subset. Moreover, although the CD56lo CD16+ subset contained a high frequency of cells expressing skin-homing markers, the CD56hi CD16- subset contained a high frequency of cells expressing bone marrow and lymph node trafficking markers. Interestingly, no differences of these NK cell subsets were noted in samples from patients with DF versus those with DHF. These findings suggest that activation and differentiation and the patterns of tissue homing molecules of the two major NK cell subsets are different and that these might play a critical role in the immune response against acute DENV infection.

Factor VII deficiency: Unveiling the cellular and molecular mechanisms underlying three model alterations of the enzyme catalytic domain.

Activated factor (F) VII is a vitamin K-dependent glycoprotein that initiates blood coagulation upon interaction with tissue factor. FVII deficiency is the most common of the rare congenital bleeding disorders. While the mutational pattern has been extensively characterized, the pathogenic molecular mechanisms of mutations, particularly at the intracellular level, have been poorly defined. Here, we aimed at elucidating the mechanisms underlying altered FVII biosynthesis in the presence of three mutation types in the catalytic domain: a missense change, a microdeletion and a frameshift/elongation, associated with severe or moderate to severe phenotypes. Using CHO-K1 cells transiently transfected with expression vectors containing the wild-type FVII cDNA (FVIIwt) or harboring the p.I289del, p.G420V or p.A354V-p.P464Hfs mutations, we found that the secretion of the FVII mutants was severely decreased compared to FVIIwt. The synthesis rate of the mutants was slower than the FVIIwt and delayed, and no degradation of the FVII mutants by proteasomes, lysosomes or cysteine proteases was observed. Confocal immunofluorescence microscopy studies showed that FVII variants were localized into the endoplasmic reticulum (ER) but were not detectable within the Golgi apparatus. These findings suggested that a common pathogenic mechanism, possibly a defective folding of the mutant proteins, was triggered by the FVII mutations. The misfolded state led to impaired trafficking of these proteins causing ER retention, which would explain the low to very low FVII plasma levels observed in patients carrying these mutations.

B cell subset alteration and the expression of tissue homing molecules in dengue infected patients.

BACKGROUND: B cells play an essential role during dengue viral infection. While a major expansion of antibody secreting cells (ASCs) was observed, the importance of these increased frequencies of ASCs remains unclear. The alteration of B cell subsets may result from the expression of tissue specific homing molecules leading to their mobilization and distribution to different target organs during acute dengue viral infection. METHODS: In this study, whole blood samples were obtained from thirty pediatric dengue-infected patients and ten healthy children and then stained with fluorochrome-conjugated monoclonal antibodies against CD3, CD14, CD19, CD20, CD21, CD27, CD38, CD45, CD138 and homing molecules of interest before analyzed by polychromatic flow cytometry. B cell subsets were characterized throughout acute infection period. RESULTS: Data shows that there were no detectable differences in frequencies of resting, activated and tissue memory cells, whereas the frequency of ASCs was significantly increased and associated with the lower frequency of naïve cells. These results were found from patients with both dengue fever and dengue hemorrhagic fever, suggesting that such change or alteration of B cells was not associated with disease severity. Moreover, several homing molecules (e.g., CXCR3 and CCR2) were found in ASCs, indicating that ASCs may distribute to inflamed tissues and various organs. CONCLUSIONS: Findings from this study provide insight into B cell subset distribution. Furthermore, organ mobilization according to homing molecule expression on different B cell subsets during the course of dengue viral infection also suggests they are distributed to inflamed tissues and various organs.

Preventable Severe Thalassemia among Children.

This retrospective study analyzed 27 children with preventable severe thalassemia born to 24 at-risk couples between 1997 and 2017. The couples were categorized into two groups: the prenatal diagnosis (PND) group (n = 8) and the non PND group (n = 16). In the PND group, following comprehensive counseling on having a fetus with thalassemia, six couples decided to continue the pregnancy (n = 6). Termination of the two remaining fetuses was excluded as the thalassemia status was reported at a gestational age of 24 weeks. In the non PND group, medical errors were found in the misdiagnosis of couples as non thalassemia carriers (n = 4) and not offering PND to couples with known thalassemia carrier status when attending the antenatal clinic (ANC) (n = 2). Additionally, parental ignorance was found in parents experiencing their own thalassemia, or that of their spouse or child (n = 6). The remaining couples (n = 4) with known carrier status either directly refused PND or were ineligible for it. A total of five divorces (5/24 = 20.8%) occurred in the PND (n = 2) and the non PND (n = 3) groups. Knowledge, beliefs, religion, experience of thalassemia, as well as the sex of the at-risk fetus all influenced parental decisions. Therefore, both medical personnel and parents are key in preventing new cases of thalassemia. Parents should be aware of the consequences of having children with severe thalassemia, while medical personnel should provide accurate carrier detection and PND.

A molecular study on the role of alpha-hemoglobin-stabilizing protein in hemoglobin H disease.

The clinical course of hemoglobin H (HbH) disease is remarkably variable. It is not completely clear how genetic and environmental factors interplay to modify clinical severity in affected individuals. Previous studies suggested that altered structure or function of alpha-hemoglobin-stabilizing protein (AHSP) could modify the clinical phenotypes of thalassemias. The present study attempted to explore the potential role of AHSP in the pathophysiology of HbH disease in 95 Chinese and Thai/Sino-Thai patients with deletional and non-deletional form of this disease. We identified six polymorphic sites in AHSP which were subgrouped into major haplotype clades. No association between AHSP genotypes or haplotypes and clinical phenotypes was observed. Instead, multiple linear regression analysis indicated that expression of AHSP correlated negatively with age (P < 0.001) and hemoglobin (P = 0.007), but positively with reticulocyte count (P = 0.003) and severity score (P = 0.003). Subgroup analysis showed that AHSP expression was higher in the non-deletional form than in the deletional form (P < 0.001). Moreover, specific types of non-deletional HbH disease with production of mutant alpha-globin chains that do not bind to AHSP (Hb Constant Spring and Hb Pakse) showed the highest AHSP expression. The present findings demonstrate that AHSP expression is a biomarker of HbH disease severity and infer an important role of AHSP in modulating the pathophysiology of this disease. Pharmacological or genetic means to alter AHSP expression may be a novel approach for amelioration of disease severity in HbH disease.