Ilham Youssry

Enzyme replacement therapy and bony changes in Egyptian paediatric Gaucher disease patients

SummaryBackground. In Gaucher disease, the infiltration of the bone marrow by glucocerebroside-laden macrophages (Gaucher cells) triggers a diverse pattern of skeletal disease that results in crippling complications. Reliable ascertainment of the severity and pattern of skeletal disease is essential to determine disease status and the response to enzyme replacement therapy (ERT). Although there is ample documentation of reversal of haematological and visceral disease by ERT, there is a paucity of data on skeletal response to ERT in children.Aim. To delineate the pattern of bone disease in children with Gaucher disease in Egypt and to evaluate its response to ERT.Method. Twenty-two children with Gaucher disease were treated with ERT. Phenotyping by clinical, laboratory and radiological criteria was performed at baseline and following 11.2 ± 4 months of ERT. Genotyping for glucocerebrosidase (GBA) mutations was performed by gene sequencing, and genotype–phenotype correlations were performed.Results. Two-thirds of the patients were from consanguineous pedigrees and 14/22 patients were homozygous or compound heterozygous for L444P and D409H mutations. Bone involvement was detected by plain radiology in 11 children (50%) and in 16 (73%) by magnetic resonance imaging (MRI). There was no correlation of severity of bone involvement and GBA genotype. ERT ameliorated bone disease: 10 of the 11 children with abnormal radiographic findings at baseline showed improvement in skeletal lesions; while 9/16 showed improvement of marrow disease by MRI. Radiographic sensitivity and specificity were 62% and 82% compared to MRI for detection of bone involvement in this patient population. At baseline, bone pain was present in 5 patients and ERT resulted in complete symptomatic remission in all of them. ERT was associated with significant improvement in growth parameters and amelioration of haematological and visceral involvement.Conclusion. Symptomatic and radiological skeletal disease is common in children with Gaucher disease in Egypt. MRI is the most accurate technique for detecting early skeletal involvement. There was no correlation between severity of skeletal involvement and GBA genotype. ERT was effective in ameliorating radiological manifestations of skeletal disease and achieving complete remission of bone pain.

PULMONARY HYPERTENSION IN β-THALASSEMIA MAJOR AND THE ROLE OF L-CARNITINE THERAPY

Cardiac complications, such as pulmonary hypertension (PHT), are the leading cause of death in β-thalassemia patients. L-Carnitine, due to its role in fatty acid oxidation, might help control the elevation in pulmonary artery systolic pressure (PASP). The objectives of this study were to assess the prevalence of PHT in β-thalassemia major patients, identify clinical predictors for its development, and determine the potential effects of L-carnitine. In total, 32 patients with β-thalassemia major were recruited; 16 age- and sex-matched children constituted the control group. Cardiac evaluation was performed by using echocardiography. The patients with PHT received 50 mg/kg/day L-carnitine orally for 3 months and were then reevaluated. Based on PASP, the patients were divided into group A without PHT and group B with PHT. The prevalence of PHT was 37.5%. The other echocardiographic measurements were not significantly different between groups A and B. PASP did not have any significant correlation with the following variables: age, total number of blood units received, splenic status, serum ferritin level, and ejection fraction. Following the administration of L-carnitine, there was a significant decrease in the mean PASP from 33.96 ± 7.85 to 24.11 ± 7.61. All cardiac dimensions decreased following L-carnitine, but the changes were not statistically significant. Even though β-thalassemia major resulted in an elevation in the PASP in only a fraction of the patients, it seems to have an impact on the heart dimensions and function of all patients. No clinical predictors were identified. Oral administration of L-carnitine appears to significantly improve PASP.

Abnormal Glucose Tolerance in β-Thalassemia: Assessment of Risk Factors

In β-thalassemia (β-thal) major, the pathogenetic mechanisms leading from siderosis to diabetes are poorly understood. We assessed the glycometabolic status in transfusion-dependent Egyptian β-thal patients and evaluated their possible risk factors for abnormal glucose tolerance (AGT). An oral glucose tolerance test (OGTT) was done on 54 multi-transfused patients and 28 age-matched normal controls, measuring their serum insulin levels at 0 and 120 min. Insulin sensitivity and insulin release indices were calculated. Indicators of iron overload and liver status were recorded. Thirteen patients (24.1%) had AGT. Cases with AGT had significantly higher mean postprandial insulin, fasting insulin resistance index (FIRI) and homeostasis model assessment (HOMA) insulin resistance (IR), p = 0.0001 for all, and significantly lower mean HOMA β cell, p = 0.007, when compared with normal glucose tolerance (NGT) cases. Abnormal glucose tolerance is common in multi-transfused β-thal major patients and could be attributed to early impaired β-cell function with increasing IR.

Apoptosis in Thalassemia major Reduced by a Butyrate Derivative

Background and Objectives: In cases of β-thalassemia major, apoptosis appears to be greatly enhanced in the early-stage erythroid precursors in the bone marrow leading to ineffective erythropoiesis. L-Carnitine is found to strongly reduce apoptosis in different diseases. We investigated the effect of oral L-carnitine therapy on apoptosis in thalassemia major patients. Methods: Eighteen thalassemia major patients with a mean age of 12.2 ± 6.6 years were included. Detection of apoptosis was done by photometric enzyme immunoassay (ELISA) and agarose gel electrophoresis before and after 6 months of oral therapy with L-carnitine (50 mg/kg/day). Results: A significant decrease of apoptosis frequency in the erythroid precursors in the bone marrow of studied cases was noted after therapy. The quantity of nucleosomes measured by ELISA dropped from 3.65 ± 1.338 to 1.60 ± 0.65 after therapy (p = 0.005). A positive ladder pattern reflecting apoptosis on agarose gel electrophoresis was detected in 88.9% of cases prior to treatment versus 16.7% after therapy (p = 0.006). Patients also had a significant decrease in the frequency of transfusions and increase in the pre-transfusion hemoglobin levels after therapy. Conclusion:L-Carnitine seems to be a good modulator of apoptotic processes in thalassemic patients leading to a decreased frequency of programmed erythroblast death and general improvement of the disease condition.

Correction of Aberrant Pre-mRNA Splicing by Antisense Oligonucleotides in ??-Thalassemia Egyptian Patients With IVSI-110 Mutation

The splicing mutation in intron 1 of β-globin gene (IVS1-110) is the most common mutation in Egyptian thalassemics that causes aberrant splicing of pre-mRNA and deficient β-globin chain synthesis. Antisense oligonucleotides (ASONs) are compounds that redirect pre-mRNA splicing and modify gene expression. Our aim was ex vivo correction of the aberrant splicing of β-globin110 pre-mRNA by ASON against the 3′ aberrant splice site. Peripheral blood mononuclear cells of 10 thalassemic patients with IVS1-110 mutation were duplicated and 1 was treated with 20 μmoL/mL morpholino ASON targeted against the 3′ aberrant splice site. The level of total hemoglobin (Hb), fetal Hb, and mRNA were estimated in the duplicate samples. Five cases (50%) showed correction with ASON treatment, of which 2 cases showed the appearance of corrected mRNA band with absence of the aberrant band and 3 cases showed an increased ratio of the corrected to the aberrant mRNA band from 2:1 to 3:1, and 4:1. The total Hb showed significant increase in the 5 corrected cases. In conclusion, ASON can restore correct splicing of β-globin pre-mRNA leading to correct gene product in cultured erythropoietic cells. These results suggest the applicability of ASON for the treatment of thalassemia.

Genetic polymorphisms of glutathione-S-transferase and microsomal epoxide hydrolase in egyptian acquired aplastic anemia patients.

Exposure to various environmental toxins with a reduced ability to metabolize them may lead to acquired aplastic anemia (AA). Genetic polymorphism of the detoxifying enzymes, the glutathione-S-transferase (GST) and microsomal epoxide hydrolase (mEh), with alteration in their activities could explain the genetic interindividual risks for AA. We aimed to characterize the genetic polymorphisms of the GST and mEh and to test their impact on the susceptibility, disease severity, and prognosis in Egyptian patients with AA. The GST and mEh genotypes were determined by multiplex-polymerase chain reaction and polymerase chain reaction-restriction fragment length polymorphism analysis, respectively, in 21 patients with AA and 20 healthy control subjects. The mEh functional phenotypes were assessed. The frequency of GST &thgr;1-null genotype was found significantly higher in AA patients compared with the controls (odds ratio=2.8, 95% confidence interval=1.1-7.8; P=0.001). The frequency of heterozygous 139A---G of the mEh gene was significantly higher in AA patients compared with the controls (odds ratio=3.07, 95% confidence interval=1.23-7.7; P=0.018). Moreover, the patients with normal functional phenotype of the mEh had significantly favorable prognosis than those with abnormal enzyme activity (P=0.027). Thus, the GST &thgr;1-null genotype and the139A---G mEh gene polymorphism may enhance the susceptibility to AA and provide an evidence of gene-environmental interaction.

Skin Iron Concentration: a Simple, Highly Sensitive Method for Iron Stores Evaluation in Thalassemia Patients

Iron overload is a potentially fatal complication in thalassemia patients. Accurate assessment of body iron is of utmost importance for these patients. The available methods for iron stores evaluation have limitations. We assessed biochemically the skin iron concentration (SIC) and determined the relation between the hepatic and skin iron level in thalassemia major patients to develop a simple, sensitive, quantitative measure of the body iron stores. Thirty-one cases with thalassemia major were assessed for iron overload. Liver and skin biopsies were performed for the patients and skin biopsies were taken from the 31 controls. The biopsies were subjected to biochemical assay of iron and histologic sections were examined. The SIC of the studied cases was significantly higher than that of the control group with a mean of 2.705 ± 1.14 and 0.275 ± 0.13 mg/g dry skin weight, respectively, p < 0.001. There was significant correlation between the SIC and the liver iron concentration (LIC) (r = 0.43, p = 0.01). The amount of liver iron is equivalent to [(3.5 × SIC) + 12.9]. With the use of this equation, we could reliably estimate an LIC value as high as 21.2 mg/g dry liver weight with a standard error of 4.07. Biochemical assay of the skin iron concentration is a reliable quantitative indicator of the body iron stores in patients with thalassemia major.

βS globin gene haplotype and the stroke risk among Egyptian children with sickle cell disease

ABSTRACT Background and aim of work: Sickle cell disease (SCD) is an inherited disease of the beta globin gene. The βS globin gene haplotypes are Senegal, Benin, Bantu, Cameroon, Arab-Indian and atypical haplotypes. In SCD, stroke is a life-threatening event in both adults and children. In light of paucity of studies on βS globin gene haplotypes in Egypt, we aimed to determine βS globin gene haplotypes in children with SCD and study their impact on stroke risk. Methods: Fifty-two SCD patients were included in the study, they were 26 males and 26 females with age range from 3 to 18 years old. The PCR-RFLP technique was used for the determination of βS globin gene haplotypes. Transcranial Doppler (TCD) was done to identify patients at risk of stroke. Results: Benin/Benin was the most prevalent haplotype detected in 50% followed by Benin/Bantu in 30.8% of studied patients. TCD study showed that 14/52 (26.9%) patients had abnormally high TCD flow velocities (TCD velocities ≥170 cm/s) and thus considered high stroke risk group, whereas 38/52 (73.1%) patients had TCD flow velocities <170 cm/s and are considered low stroke risk group. Stroke risk was not found to be associated with βS globin gene haplotype (p = .532). Conclusion: This study provides a relevant contribution to our understanding of the anthropological and historical background of the population in Egypt where Benin haplotype is the commonest βS globin gene haplotype and homozygous Benin/Benin is associated with higher stroke risk than other haplotypes.

Chimerism in pediatric hematopoietic stem cell transplantation and its correlation with the clinical outcome

Hematopoietic stem cell transplantation (HSCT) is the only hope to cure many inherited and acquired hematological disorders in children. Monitoring of chimerism helps to predict the post-transplantation events, with the intention to enhance the long-term disease free survival (DFS). The study aimed to investigate the importance of early chimerism detection to predict the clinical outcome following HSCT. The study included nine recipients (six β-thalassemia and three severe aplastic anemia patients) and their 10/10 HLA identical sibling donors. Chimerism detection was performed by analysis of short tandem repeat (STR) polymerase chain reaction (PCR) for detection and quantification of the relative amounts of donor and recipient cells present on day +28. Peripheral blood (PB) was the main stem cell source for HSC transplantation. Disease free survival (DFS) was 71.4% while overall survival was 85.7% for PBSC transplants at the median follow up period of 4years. The early detection of chimerism by PCR-STR analysis for children with β-thalassemia and aplastic anemia correlated with the outcome of HSCT in 8 (88.8%) patients. Complete chimerism was associated with disease-free survival while mixed chimerism and autologous patterns were associated with poor prognosis. In conclusion, early chimerism testing is clinically important in prediction of outcome after allogeneic HSC transplantation.

Enhancing Effect of Hydroxyurea on Hb F in Sickle Cell Disease: Ten-Year Egyptian Experience

Abstract Patients with sickle cell disease experience hemolytic anemia and vaso-occlusions that result in pain, organ injury, and premature mortality. Several prospective studies have verified the efficacy and tolerability of hydroxyurea (HU), and demonstrated its efficacy in reducing painful vaso-occlusive crises (VOCs) in addition to its ability to increase Hb F levels. We aimed to evaluate the long-term effects of HU therapy on Hb F and assess its long term efficacy and safety in sickle cell disease patients. A retrospective study on 60 sickle cell disease patients was conducted. We studied the laboratory changes, frequency of VOCs per year, frequency of hospital admisions per year and number of transfusions per year, both before and after HU therapy. The follow-up period was 4 to 120 months. Hb F levels after HU therapy positively correlated with the duration of HU therapy, baseline Hb F levels and baseline total hemoglobin (Hb) (r = 0.4, p = 0.04; r = 0.45, p = 0.001; r = 0.5, p = 0.019, respectively) and inversely correlated with baseline total leucocyte count (r = –0.33, p = 0.034). Hydroxyurea therapy was associated with an increase in the total Hb and mean corpuscular volume (MCV) (p = 0.009, p = 0.000; respectively) and with a decrease in total leucocyte count, platelet count and reticulocyte count (p = 0.00, p = 0.03, p = 0.02, respectively). Moreover, a significant reduction in the frequency of VOCs, transfusion frequency and hospital admissions per year after HU therapy was shown in the studied subjects. Hydroxyurea induced an increase in Hb F level, which was maintained over time and was associated with clinical efficacy and acceptable safety.