A Pragmatic Scoring Tool to Predict Hydroxyurea Response Among ß-Thalassemia Major Patients in Pakistan.

Despite high prevalence and incidence of ß-thalassemia in Pakistan, there is very limited work on the use of hydroxyurea (HU) in thalassemia patients in the country. This is the first insight regarding genetic profiling of BCL11A and HU responses in Pakistani ß-thalassemia. It correlates single-nucleotide polymorphisms on BCL11A (rs4671393, rs766432) and HBG2 (XmnI), age at first transfusion, and ß-globin mutations with HU response in ß-thalassemia major (BTM). Of 272 patients treated with HU, 98 were complete responders, 55 partial responders, and 119 nonresponders. Our analysis shows that HU response was significantly associated with patients having IVSI-1 or CD 30 mutation (P<0.001), age at first transfusion >1 year (P<0.001), and with the presence of XmnI polymorphism (P<0.001). The single-nucleotide polymorphisms of BCL11A were more prevalent among responders, but could not show significant association with HU response (P>0.05). Cumulative effect of all 5 predicting factors through simple binary scoring indicates that the likelihood of HU response increases with the number of primary and secondary genetic modifiers (P<0.001). Predictors scoring is a pragmatic tool to foresee HU response in patients with BTM. The authors recommend a score of ≥2 for starting HU therapy in Pakistani patients with BTM.

Prenatal diagnosis of maternal serum from mothers carrying ß-thalassemic fetus.

BACKGROUND: This study focuses on the discovery of protein biomarkers from the maternal serum of ß-thalassemic trait mothers carrying the normal fetus and ß-thalassemic major fetus. METHODS: Serum samples from ß-thalassemic trait mothers carrying major (N = 5) and normal fetuses (N = 5) were studied. The IVS1-5 thalassemia mutation was common among ß-thalassemic trait mothers who were carrying a homozygous ß-thalassemic fetus (IVS1-5/ IVS1-5 mutation) or a normal fetus (no mutation). We employed two-dimensional gel electrophoresis and mass spectrometry analysis to explore differentially expressed maternal serum proteins from thalassemia carrier couples with the same ß-thalassemia mutation. Western blotting was performed for one of the identified proteins to validate our data. RESULTS: Ten proteins were identified in the maternal serum of ß-thalassemic trait mothers carrying the ß-thalassemic major fetus and normal fetus. Among these, serotransferrin, haptoglobin, α-1 anti-trypsin, apo-lipoprotein A1, and the fibrinogen-ß chain were found to be upregulated in mothers carrying major fetuses and are known to be associated with pregnancy-related disorders. The expression of α-1 anti-trypsin was validated through western blotting. CONCLUSIONS: Proteins identified in the current study from maternal serum are reported to contribute to hereditary disorders. We suggest that these can serve as putative screening markers for non-invasive prenatal diagnosis in ß-thalassemic pregnancies.

Antioxidant Activity and Oxidative Stress Status in Maternal Serum of ß-thalassemic Mothers Carrying ß-thalassemic Major and Normal Fetuses.

Objectiveß-thalassemia is a genetic disorder characterized by reduction or absence of ß-globin chain with mutations in both copies (ß-thalassemia major) or in one copy (ß-thalassemia minor). Pregnancies in ß- thalassemic carrier women are considered symptom free but have risk of inheriting ß-thalassemic fetuses. Current study was designed to compare oxidative stress and antioxidants status in maternal serum from ß-thalassemic minor mothers having ß-thalassemic major and normal fetuses. Method: We investigated paraoxonase (PON1) and arylesterase (ARE) activities along with malondialdehyde (MDA) and reactive oxygen species (ROS) in maternal serum of ß-thalassemic carrier women. Results: PON1 and ARE activities were found to be significantly decreased, whereas the concentration of MDA and ROS were significantly increased in ß-thalassemic minor mothers with ß-thalassemic major fetuses. Conclusion: The study concludes that redox imbalance in ß-thalassemic trait mothers carrying thalassemic fetuses is higher than in mothers carrying normal fetuses.

Hydroxyurea (hydroxycarbamide) for transfusion-dependent ß-thalassaemia.

BACKGROUND: Hydroxyurea (hydroxycarbamide) promotes the production of foetal haemoglobin (HbF) by reactivating gamma-genes. Evidence has shown clinical benefits of hydroxyurea in people with sickle cell anemia; however, only a few studies have assessed this treatment in people with beta (ß)-thalassaemia. OBJECTIVES: The primary objective is to review the efficacy of hydroxyurea in reducing or ameliorating the requirement of blood transfusions in people with transfusion-dependent ß-thalassaemia. The second objective is to review the safety of hydroxyurea with regards to severe adverse effects in this population. SEARCH METHODS: We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group's Haemoglobinopathies Trials Register, compiled from electronic database searches and hand searching of journals and conference abstract books. We also searched electronic databases and trial registries, including ClinicalTrials.gov, the WHO ICTRP and PubMed (09 October 2018).Date of last search of the Group's haemoglobinopathies trials register: 04 March 2019. SELECTION CRITERIA: Randomised controlled trials of hydroxyurea in people with transfusion-dependent ß-thalassaemia, compared with placebo or standard treatment or comparing different doses of hydroxyurea. DATA COLLECTION AND ANALYSIS: Two authors independently assessed trials for inclusion in the review, which was verified by a third author. MAIN RESULTS: No trials were eligible for inclusion in this review. AUTHORS' CONCLUSIONS: Currently, there is no high-quality evidence to support or challenge the continued use of hydroxyurea for managing people with transfusion-dependent ß-thalassaemia. Multicentre, randomised controlled trials (compared to placebo or other available treatment, i.e. blood transfusion and iron chelation) are needed in order to assess the efficacy and safety of hydroxyurea for reducing the need for blood transfusion, for maintaining or improving mean haemoglobin levels, as well as for determining its cost-effectiveness.

Sensitivity and specificity of single-tube osmotic fragility test and its different methods as screening test for thalassemia trait: an alternative to expensive laboratory tests for resource-limited countries.

OBJECTIVE: To determine the sensitivity and specificity of single-tube osmotic fragility (SOFT) and its different methods as screening test for thalassemia trait. METHODS: A cross-sectional study was conducted at Omair Sana Foundation. A total of 400 participants were included in the study. Three hundred were known thalassemia carriers (parents with at least one child with thalassemia major), while 100 were healthy blood donors. SOFT was performed on all 400 participants. Serum iron, ferritin, and DNA tests were performed on 100 participants (donors). ARMS technique was used for detecting thalassemia mutations. RESULTS: Sensitivity and specificity of SOFT (venous method) were found to be 99.6% and 86%, respectively, while with EDTA method, sensitivity was 95% and specificity was 96%. For venous and EDTA methods, positive predictive values were 95.5% and 98.6%, respectively, while negative predictive values were 98.8% and 86.6%, respectively. Use of EDTA and storage had an effect on the results. Sensitivity of SOFT was 95% at 5 min, while it decreased to 87% with EDTA method at 240 min. Sensitivity of SOFT for iron deficiency anemia was found to be 14%. CONCLUSION: SOFT can be used as screening test for thalassemia trait in a cost-effective way. Moreover, we also found that SOFT should be performed on venous blood without adding preservatives (EDTA) that can interfere with the results.

Gγ-Xmn I polymorphism: a significant determinant of ß-thalassemia treatment without blood transfusion.

ß-thalassemia is characterized by impaired ß-chain synthesis leading to ineffective erythropoiesis, severe anemia, and a need for blood transfusion. Presence of Xmn I polymorphism (-158 C-T nucleotide change) in γ-globin gene is associated with a higher fetal hemoglobin and a lesser clinical severity. This prospective study attempted to find out the effect of hydroxyurea (HU) on ß-thalassemia patients in the presence or absence of Xmn I polymorphism. A total of 143 consecutive ß-thalassemia patients received HU (16 mg/kg/d). Sixty-four (44.7%) had Xmn I polymorphism (either homozygous or heterozygous). Patients were evaluated at a median duration of 3 years (range, 6 mo to 9 y). Responders became transfusion independent after 6 months, partial responders had a least 50% reduction in transfusion requirement and nonresponders had no significant reduction. Of the 64 patients with Xmn I polymorphism, 44 (69%) showed response (P<0.01), whereas in those who lacked Xmn I polymorphism (n=79), only 17 (21%) were responders. This study showed that the presence of Xmn I polymorphism in ß-thalassemia is a predictor of response to HU and highlights the possibility of managing this subset of patients without blood transfusion.

Molecular epidemiology of ß-thalassemia in Pakistan: Far reaching implications.

BACKGROUND: ß -Thalassaemia, an autosomal recessive hemoglobinopathy, is one of the commonest genetically transmitted disorders throughout the world. Collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing ß-thalassemia. AIM: To achieve this objective, Identification of the spectrum of genetic mutations, especially for various ethnic backgrounds in Pakistan. Therefore, we designed a cross sectional prospective study to identify the frequency of various gene mutations in different ethnic groups of Pakistan. MATERIALS AND METHODS: Over a 5-year period, DNA from 648 blood samples {including specimens of chorionic villus sampling (CVS)} were analyzed for the twelve most common ß-thalassemia mutations found in the Pakistani population by a Multiplex amplification refractory mutation system (ARMS). Each sample was analyzed for the mutation as well as the normal gene, appropriate with negative and positive controls, and reagent blanks. RESULTS: Out of 648 samples mutations were identified in 640 (98.75%) samples by multiplex ARMS. 8 common ß-thalassemia mutations were identified in 8 different ethnic groups accounting for 93.9% of the ß-thalasemia alleles. CONCLUSIONS: Based on the outcome of this study a cost effective proposal is formulated for detection of ß-thalassemia mutations.

Evaluation of the combination therapy of hydroxyurea and thalidomide in ß-thalassemia.

Transfusion-related complications and lack of resources in low-to-middle-income countries have led to a search for novel therapies to reduce the need for blood transfusions in patients with ß-thalassemia. Hydroxyurea (HU) has demonstrated promising outcomes; additionally, thalidomide has also shown improvement in hemoglobin (Hb) levels for patients with ß-thalassemia in some studies. This study presents the findings of a single-arm nonrandomized trial to evaluate the efficacy of combination therapy of HU and thalidomide in children with ß-thalassemia. A total of 135 patients (median age, 6 [interquartile range, 3-10] years), 77 (57%) males and 58 (43%) females, were followed first using HU alone, for 6 months, and then using the combination of HU and thalidomide for another 6 months. The primary outcome was a response to therapy, as measured by the number of transfusions required and Hb levels, for patients while receiving HU alone and then while using the combination therapy. Study findings showed a significant decline in blood transfusion volume (P < .001) and a significant increase in median Hb levels within 3 and 6 months of the combination therapy (P < .001). Eighty-nine (65.93%) participants were good responders, 16 (11.85%) were responders, and 30 (22.22%) were nonresponders, whereas the responders had variable genetic mutations. A total of 38 adverse events were reported that resolved on supportive treatment or temporary hold of the intervention. The combination therapy demonstrated promising results and could be considered for a diverse patient population with ß-thalassemia. This trial was registered at www.clinicaltrials.gov as #NCT05132270.

Efficacy of hydroxyurea in providing transfusion independence in ß-thalassemia.

BACKGROUND: Packed red blood cell (PRC) transfusion with iron chelation is the mainstay of treatment for ß-thalassemia major. This prospective interventional trial serves as a follow up to our similar earlier study that evaluated the efficacy and safety of hydroxyurea (HU) in minimizing PRC transfusions in patients with ß-thalassemia major. METHODS: One hundred fifty-two patients with ß-thalassemia major received HU at a mean dose of 16 mg/kg/d. The results were analyzed at the end of 24 months. Transfusion requirement during the 6 months preceding the study was considered as the control. RESULTS: One hundred forty-six of 152 patients were evaluated after 24 months of follow up; 6 patients were either lost to follow-up or withdrew consent. Grade 1 myelosuppression was observed in 4 patients and diarrhea in 2 patients. Sixty children (41%) did not require any transfusion after using HU; 57 patients (39%) showed partial response with greater than 50% reduction in PRC transfusion; and 29 patients (20%) were nonresponders with less than 50% reduction in PRC transfusion. The mean volume of PRC transfused was reduced for all patients. CONCLUSIONS: HU was found to be safe in patients with ß-thalassemia major, and resulted in the reduction of transfusion requirement and in an increase in the interval between transfusions.

Pharmacoproteomics Profiling of Plasma From ß-Thalassemia Patients in Response to Hydroxyurea Treatment.

ß-Thalassemia is a genetic disorder caused by defects in the ß-globin gene resulting in the absence or reduced synthesis of adult hemoglobin (HbA). Hydroxyurea is an effective drug to increase fetal γ-globin (HbF) expression, replacing the missing adult ß-globin. The mechanism of HbF induction by hydroxyurea and improvement in clinical symptoms are still poorly understood. In the present study we performed comparative analysis of plasma proteome in pre- and post-hydroxyurea-treated ß-thalassemia major transfusion-dependent children (n = 10, mean age = 3.2 years) as well as responders versus nonresponders to hydroxyurea treatment. Plasma was collected before and after 6 months of hydroxyurea treatment, with patients subcategorized on the basis of their response to hydroxyurea. Among 400 identified proteins using a label-free quantitative proteomics approach, 28 proteins were found to be significantly different in pre- versus post-hydroxyurea-treated groups, with transferrin receptor protein-1 being most downregulated and hemopexin and haptoglobin the most upregulated proteins after treatment. In responder versus nonresponder comparison, 26 proteins were found to be differentially expressed, with carbonic anhydrase 1, hemoglobin subunit γ-1, and peroxiredoxin-2 showing the significant changes. The mechanism of hydroxyurea treatment in ß-thalassemia patients appears to be complex, requiring a large sample size and a longer period of treatment to reveal its details.

Serum Paraoxonase Activity and Malondialdehyde Serum Concentrations Remain Unaffected in Response to Hydroxyurea Therapy in ß-Thalassemia Patients.

ß-Thalassemia is the most common hereditary disorder characterized by reduced production of ß-globin chains of hemoglobin A (HbA). In recent years, hydroxyurea (HU) has shown promising therapeutic benefits in patients with ß-thalassemia by fetal hemoglobin augmentation. We have analyzed effects of hydroxyurea treatment on oxidative stress in ß-thalassemia patients by assessing activities of paraoxonase (PON) and arylesterase along with malondialdehyde (MDA) and total reactive oxygen species (ROS) concentrations. Blood samples from 159 individuals including 56 HU-treated and 58 untreated ß-thalassemia patients and 45 healthy controls were analyzed. PON activity was found to be highest in healthy individuals (177.76 ± 4.44 U/mL) as compared to treated (52.67 ± 3.65 U/mL) and untreated (55.11 ± 3.26 U/mL) patients. A similar trend was observed in the case of arylesterase activity in normal, ß-thalassemia-treated, and untreated (210.0 ± 11.25 U/mL, 163.03 ± 9.04 U/mL, 139.77 ± 10.10 U/mL) subjects. Serum MDA concentrations (2.59 ± 0.09 nmol/mL, 2.45 ± 0.08 nmol/mL, and 1.15 ± 0.05 nmol/mL) and total ROS concentrations (3.73 ± 0.20 nmol/mL, 3.54 ± 0.23 nmol/mL, and 2.45 ± 0.14 nmol/mL) were significantly elevated in both groups (untreated and treated) as compared to healthy individuals (P < .01). Oxidative stress was found to be markedly elevated in ß-thalassemia patients as compared to healthy controls. Insignificant differences were, however, observed in mean concentrations of PON1 paraoxonase and arylesterase activities, serum MDA concentration and total ROS concentrations between HU-treated and untreated patients. We propose that HU therapy alone seems to be ineffective in managing oxidative stress and is likely to offer a better clinical outcome when supplemented with efficient iron chelation therapy and antioxidants.

Seropositivity of hepatitis C, hepatitis B and HIV in chronically transfused ß-thalassaemia major patients.

The purpose of this study was to determine the frequency and trend of transfusion transmitted infections (TTI) in chronically transfused ß-thalassaemia major (TM) patients with reference to the duration of transfusions. A cross-sectional study was done on 160 ß-TM patients and 5517 healthy blood donors to find out the prevalence of HCV, HBV and HIV infections. Out of 160 patients, 21 cases (13.1%) were anti-HCV positive, 2 (1.25%) were HBsAg positive. HIV antibodies were not detected in any sample. However, 109 (1.9%) and 104 (1.8%) of 5517 blood donors were positive for HCV and HBV respectively. No donor showed HIV antibodies. Anti-HCV was positive in 9/111(8.4%) thalassaemics (< 10 years of age) while 11/49 (22%) [> 10 years of age] showing significant difference (p = 0.005) among the two groups. For the past 10 - 12 years the screening of blood has reduced the magnitude of the disease significantly as shown by the trend in two age groups. Further improvements need to be done to implement uniform screening throughout the country.

JAK2V617F mutation in chronic myeloid leukemia predicts early disease progression.

OBJECTIVE: To determine the association of JAK2V617F mutation along with BCR-ABL translocation or Philadelphia chromosome in chronic myeloid leukemia with early disease progression to advanced stages (accelerated phase or blast crisis) and poor outcome. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi, from February 2008 to August 2009. METHODOLOGY: All the newly diagnosed cases of BCR-ABL or Philadelphia positive CML were tested for JAK2V617F mutation by Nested PCR. Demographic data, spleen size, hemoglobin levels, white blood cell and platelet counts were recorded. Independent sample t-test was used for age, haemoglobin level and spleen size. Fisher's exact test was applied to compare disease progression in JAK2V617F mutation positive and negative cases. RESULTS: Out of 45 newly diagnosed cases of CML, 40 were in chronic phase, 01 in accelerated phase and 04 in blast crisis. JAK2V617F mutation was detected in 12 (26.7%) patients; 09 (22.5%) in chronic phase, none in accelerated phase and 03 (75%) in blast crisis. During a mean follow-up of 8 months, 03 patients in chronic phase transformed in blast crisis and 02 into accelerated phase. Overall 08 out 0f 11 (73%) JAK2V617F positive patients either had advanced disease or showed disease progression. Only 2 of 20 (10%) available patients, negative for the mutation, showed disease progression by transforming into blast crisis (p < 0.001). No statistically significant difference was seen in the age, spleen size, haemoglobin levels, white blood cells and platelets counts in JAK2V617F positive patients. CONCLUSION: JAK2V617F mutation was detected in 26.7% cases of chronic myeloid leukemia. A significant proportion of them showed early disease progression.

Molecular epidemiology of ß-thalassemia in Pakistan: far reaching implications.

ß-thalassaemia, an autosomal recessive hemoglobinopathy, is one of the commonest genetically transmitted disorders throughout the world. Collective measures including carrier identification, genetic counseling and prenatal diagnosis are required for preventing ß-thalassemia. To achieve this objective, Identification of the spectrum of genetic mutations, especially for various ethnic backgrounds in Pakistan is necessary. Therefore, we designed a cross sectional prospective study to identify the frequency of various gene mutations in different ethnic groups of Pakistan. Over a 5-year period, DNA from 648 blood samples [including specimens of chorionic villus sampling (CVS)] were analyzed for the twelve most common ß-thalassemia mutations found in the Pakistani population by a Multiplex amplification refractory mutation system (ARMS). The most common mutation identified was Intervening Sequence 1-5 (IVS 1-5 (G-C)); accounting for 40.89% mutated alleles, and was represented in all ethnic groups. 15.7 % of the ß-thalassemia alleles were found to have Frameshift 8-9 (Fr 8-9) as the second most common mutation Other common genetic defects responsible for ß-thalassemia: IVS 1-1 (G-T) was found in 8.17%, Codon-30 (Cd-30 (G-C)) 8.02%, Codon-5(Cd-5 (-CT)) contributed 2.16% and Deletion 619 base pair (Del 619bp) affected 11.11% were found in Pakistan. This large study adds to the pre-existing data in Pakistan. Knowledge of the predominant mutation in a given ethnic group will not only help in developing a short panel of (population-specific) primers of mutations thereby providing a cost-effective method for prenatal diagnosis and also help the clinicians to counsel regarding blood transfusion regimen/ pregnancy termination.

Efficacy of hydroxyurea (HU) in reduction of pack red cell (PRC) transfusion requirement among children having beta-thalassemia major: Karachi HU trial (KHUT).

BACKGROUND: Packed red blood cell (PRC) transfusion with iron chelation is the mainstay of treatment for patients with beta-thalassemia major. Hemoglobin F augmentation is another approach to treat this hemoglobinopathy. This study evaluates the efficacy and safety of hydroxyurea (HU) in minimizing PRC transfusions in patients with beta-thalassemia major. METHOD: Twenty-three patients with beta-thalassemia major received HU at a mean dose of 16 mg/kg/d. The results were analyzed at the end of 24 months. Transfusion requirement during the 6 months preceding the study was considered as the control. RESULT: Twenty patients were evaluable after 24 months. The mean volume of PRC transfused was reduced from 2126.45 mL to 1489.59 mL (P<0.001). The interval between transfusions was increased by 68.7%. Grade I myelosuppression was observed in 4 patients and diarrhea in 2 patients. CONCLUSIONS: HU was found to be safe in patients with beta-thalassemia major, and resulted in reduction in the transfusion requirements and in increase of the intervals between transfusions.