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.

Cell Population Data-Driven Acute Promyelocytic Leukemia Flagging Through Artificial Neural Network Predictive Modeling.

A targeted and timely offered treatment can be a benefitting tool for patients with acute promyelocytic leukemia (APML). Current round of study made use of potential morphological and immature fraction-related parameters (cell population data) generated during complete blood cell count (CBC), through artificial neural network (ANN) predictive modeling for early flagging of APML cases. We collected classical CBC items along with cell population data (CPD) from hematology analyzer at diagnosis of 1067 study subjects with hematological neoplasms. For morphological assessment, peripheral blood films were examined. Statistical and machine learning tools including principal component analysis (PCA) helped in the evaluation of predictive capacity of routine and CPD items. Then selected CBC item-driven ANN predictive modeling was developed to smartly use the hidden trend by increasing the auguring accuracy of these parameters in differentiation of APML cases. We found a characteristic triad based on lower (53.73) platelet count (PLT) with decreased/normal (4.72) immature fraction of platelet (IPF) with addition of significantly higher value (65.5) of DNA/RNA content-related neutrophil (NE-SFL) parameter in patients with APML against other hematological neoplasm's groups. On PCA, APML showed exceptionally significant variance for PLT, IPF, and NE-SFL. Through training of ANN predictive modeling, our selected CBC items successfully classify the APML group from non-APML groups at highly significant (0.894) AUC value with lower (2.3 percent) false prediction rate. Practical results of using our ANN model were found acceptable with value of 95.7% and 97.7% for training and testing data sets, respectively. We proposed that PLT, IPF, and NE-SFL could potentially be used for early flagging of APML cases in the hematology-oncology unit. CBC item-driven ANN modeling is a novel approach that substantially strengthen the predictive potential of CBC items, allowing the clinicians to be confident by the typical trend raised by these studied parameters.

Correction to: Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population.

[This corrects the article DOI: 10.1186/s12959-017-0143-3.].

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.

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.

Complex Karyotype in Hematological Diseases: A 6-Year Single Centre Study from Pakistan.

BACKGROUND: Most of the hematological disorders are heterogenous with regard to morphology, immunophenotype, and genetic rearrangements. Multiple recurrent chromosomal aberrations have been identified by conventional cytogenetic analysis, which is now widely recognized as one of the most important diagnostic and prognostic determinants in these patients. Though rarer, complex karyotype has been associated with worst prognosis. MATERIALS AND METHODS: A total of 1185 bone marrow or peripheral blood cytogenetics samples were taken with different hematological diseases. They included both benign and malignant disease entities. In each case, cells were cultured and conventional cytogenetic analysis was performed. RESULTS: Among 1185 subjects, 41 (3.4%) patients possessed complex cytogenetic abnormalities. Out of these 41, 33 (80%) were males. The mean age was 37 years (median age 39 years). Myelodysplastic syndromes had the most numbers of complex karyotypes (8%), followed by acute myeloid leukemia (7%) and acute lymphoblastic leukemia (4%). Also we found few patients with acute promyelocytic leukemia, aplastic anemia , chronic myeloid leukemia, and diffuse large B cell Lymphoma possessing complex karyotype. Frequencies of different cytogenetic abnormalities were assessed with respect to disease as well as independently. Trisomy 21 was the most common chromosomal abnormality found in 28% of patients. CONCLUSION: Complex karyotype was most frequently associated with myelodysplastic syndromes and acute myeloid leukemia. Trisomy 21 and deletion 5q were the commonest cytogenetic abnormalities found. We also assessed complex karyotype in benign diseases and detected one patient of aplastic anemia with complex karyotype. This is the first study highlighting the presence of complex karyotypes in hematological disorders in our region.

Identification of novel mutations in congenital afibrinogenemia patients and molecular modeling of missense mutations in Pakistani population.

BACKGROUND: Congenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder that was first described in 1920. It is transmitted as an autosomal recessive trait that is characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in a higher number of cases of congenital fibrinogen deficiency in their respective populations. This study focused on the detection of mutations in fibrinogen genes using DNA sequencing and molecular modeling of missense mutations in all three genes [Fibrinogen gene alpha (FGA), beta (FGB) and gamma (FGG)] in Pakistani patients. METHODS: This descriptive and cross sectional study was conducted in Karachi and Lahore and fully complied with the Declaration of Helsinki. Patients with fibrinogen deficiency were screened for mutations in the Fibrinogen alpha (FGA), beta (FGB) and gamma (FGG) genes by direct sequencing. Molecular modeling was performed to predict the putative structure functional impact of the missense mutations identified in this study. RESULTS: Ten patients had mutations in FGA followed by three mutations in FGB and three mutations in FGG, respectively. Twelve of these mutations were novel. The missense mutations were predicted to result in a loss of stability because they break ordered regions and cause clashes in the hydrophobic core of the protein. CONCLUSIONS: Congenital afibrinogenemia is a rapidly growing problem in regions where consanguinity is frequently practiced. This study illustrates that mutations in FGA are relatively more common in Pakistani patients and molecular modeling of the missense mutations has shown damaging protein structures which has profounding effect on phenotypic bleeding manifestations in these patients.

Outcome of Inversion 16 in TKD Positive and Negative Acute Myeloid Leukemia Patients.

abstract.

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.

Oxidative stress and total antioxidant status in acute leukemia at diagnosis and post remission induction phase.

This study evaluated the activity of superoxide dismutase (SOD1), glutathione reductase (GR) and total antioxidant status (TAS) in the hemolysate and sera of patients with acute leukemia (AL) at diagnosis, post remission induction phase and in healthy controls. However, total antioxidant status and glutathione reductase activities normalized after remission induction phase in acute myeloid leukemia (AML) only whereas levels of SOD were reduced but not achieved the normal level in acute lymphoblastic leukemia (ALL). TAS activity showed no difference in either sex among any subtype of acute leukemia but glutathione reductase level was significantly higher in female ALL patients. Activity of SOD was elevated in T-cell ALL and acute myelomonocytic leukemia however; no significant difference in the activity of GR and TAS was noted. Levels of antioxidant were reduced insignificantly in patients who achieved complete remission.

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.

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.

Successful Management of Acute Catastrophic Juvenile Vaginal Bleeding in Glanzmann's Thromboasthenia by Uterine Tamponade: A Case Report and Review of The Literature.

Glanzmann's thromboasthenia (GT) is a rare platelet disorder, due to membrane defects involving glycoprotein GP IIb/IIIa complex. Symptoms appear in infancy with episodes of bruising, gingival bleeding, epistaxis, or at the time of menarche acute episode of uterine bleeding. Hormonal therapy and antifibrinolytic agents are first-line treatment. Platelet transfusion is given to control hemorrhage when medical treatment fails. However, repeated transfusions may result in development of platelet refractioness, due to development of antibodies against membrane glycoprotein. Activated recombinant FVII is licensed for use in acute control of bleeding in GT. Here we report a case of acute juvenile uterine bleeding at menarche, which responded successfully to uterine tamponade. To our knowledge, this is the first case report on use of balloon tamponade for control of acute catastrophic juvenile bleeding at menarche.

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.