Clinico-haematological characteristics in Pakistani patients of primary myelodysplastic syndrome according to World Health Organization classification.

OBJECTIVE: To assess the applicability of WHO classification on a cohort of Pakistani myelodysplastic syndrome (MDS) patients, and determine their epidemiological and clinico-pathological features. STUDY DESIGN: Case series. PLACE AND DURATION OF STUDY: Haematology Department, Shaikh Zayed Hospital, Lahore, from April 2004 to March 2006. METHODOLOGY: Forty six patients of primary MDS diagnosed by World Health Organization (WHO) criteria were included in the study by nonprobability purposive sampling. The cohort was classified accordingly and the epidemiological, clinical and haematological parametres were assessed. Descriptive statistics were used to describe the data. RESULTS: Forty six patients (28 males and 18 females) of primary MDS were included in the study. The mean age was 46.21 years. According to the WHO classification, 12 cases of refractory anaemia, 24 cases of refractory cytopenia with multi lineage dysplasia, 1 case of refractory cytopenia with multi lineage dysplasia and ring sideroblasts, 3 cases of MDS unclassified and 3 cases each of refractory anaemia with excess of blasts I and II were diagnosed. Symptomatic anaemia was seen in 37 cases and pancytopenia was documented in 33 cases. Dyserythropoiesis affected 41 cases. Grade III reticulosis was seen in 7 cases. ALIP was present in 13 cases. CONCLUSION: MDS presented at a young age. Refractory cytopenia with multi lineage dysplasia was the dominant disease category. Further studies are suggested for identifying the cytogenetic abnormalities and del 5q- category.

Clinicopathological analysis of myelodysplastic syndrome according to French-American-British classification.

OBJECTIVE: To evaluate the age of onset, gender ratio, clinical presentation of Myelodysplastic syndrome patients, and to classify these patients according to French-American-British classification on the basis of morphological features in blood and bone marrow. STUDY DESIGN: A case series. PLACE AND DURATION OF STUDY: The Department of Haematology, Shaikh Zayed Hospital, Lahore, from April 2004 to March, 2007. METHODOLOGY: Fifty patients of primary Myelodysplastic syndrome (MDS) were studied. The patients were classified according to French-American-British (FAB) criteria and the epidemiological, clinical and haematological features of MDS patients were evaluated. Descriptive statistics were used to describe data. RESULTS: There were 31 males and 19 females. The mean age was 41 years. According to FAB classification, 39 cases of refractory anaemia, 1 case of refractory anaemia with ring sideroblast, 6 cases of refractory anaemia with excess of blasts and 4 cases of refractory anaemia with excess of blasts in transformation were identified. The commonest complaint was easy fatiguability affecting 41 cases (82%). Anaemia was the most common finding seen in 47 patients (94%). Pancytopenia was seen in 33 cases (66%). Dyserythropoeisis was present in 42 (84%); dysmyelopoeisis was seen in 21 (42%) and morphologically abnormal megakaryocytes were identified in 29 (58%) of the bone marrow aspirates. Grade- III reticulosis was seen in 9 bone marrow trephine biopsies. Abnormal localization of immature precursors (ALIP) were present in 18 cases. CONCLUSION: MDS was more frequent in young males. Refractory anaemia constituted a major chunk of the disease entity.

Cystic fibrosis detection in high-risk Egyptian children and CFTR mutation analysis.

BACKGROUND: Knowledge about Cystic Fibrosis (CF) in Egypt is very limited. The objective of this study was to screen for CF in Egyptian children with suggestive clinical features and to identify causative genetic mutations. METHODS: Sixty-one patients from the Chest Unit, Cairo University Children's Hospital, Egypt, were included. Subjects presented with persistent or recurrent respiratory symptoms, failure to thrive, diarrhea and/or steatorrhea and unexplained persistent jaundice. Patients were screened using the CF Indicatortrade mark sweat test system (PolyChrome Medical, Inc., Brooklyn Center, MN). A quantitative sweat testing was conducted on 10 of the 12 positive patients. Seven probands and one sibling underwent molecular analysis by direct DNA sequencing of the coding region and of the intronic sequences adjacent to the 27 exons of the CFTR gene. RESULTS: Of 61 patients, 12 (20%) had positive sweat chloride screening. Ten of the 12 patients underwent quantitative sweat testing and were positive. Eight CFTR sequence changes were identified in seven affected probands and two were confirmed in one sibling by direct DNA sequencing. CONCLUSION: The study results suggest that CF is more common in Egypt than previously anticipated. Larger studies are warranted to identify the incidence, molecular basis and clinical pattern of CF in the Egyptian population.

Upshaw-Schulman Syndrome.

A13 years girl presented with history of sudden onset fits, altered sensorium and anuria for 2 days. There was also history of right sided weakness of the whole body. Past history revealed repeated episodes of similar complaints since early childhood. On the basis of history, physical examination and extensive investigations, patient was diagnosed as Upshaw-Schulman syndrome, a rare case of congenital Thrombotic Thrombocytopenic Purpura (TTP). She is now in remission and being maintained on twice weekly transfusions of Fresh Frozen Plasma (FFP).

Relationship between codon 249 mutation in exon 7 of p53 gene and diagnosis of hepatocellular carcinoma.

INTRODUCTION: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. Multiple genetic and epigenetic changes are involved in the molecular pathogenesis of HCC. Heat shock proteins have essential roles in protecting cells from the potentially lethal effects of stress. Among them, HSP70 are often overexpressed in cells of various cancers and have been suggested to contribute to tumourigenesis. p53 mutations in codon 249 have also been identified in HCC. MATERIAL AND METHODS: Fifty patients with liver disease were enrolled in this study compared to 10 healthy volunteers. The studied patients were divided into 2 groups: group I includes those suffering from HCC, group II includes those suffering from post-hepatitis B and C liver cirrhosis. The presence of p53 gene mutation was detected by DNA extraction from whole blood of patients and controls followed by polymerase chain reaction then restriction fragment length polymorphism (RFLP) analysis of codon 249 of exon 7. We also studied the genotypes of the HSP70 gene by PCR followed by RFLP analysis. RESULTS: Our results revealed no statistical difference between group I, group II, and the control group as regards exon 7 mutation of the p53 gene. Also the frequency of polymorphic genotypes of HSP70 showed no significant difference between the 3 studied groups. CONCLUSIONS: The present study supports the view that the incidence of point mutation of p53 codon 249 mutations in exon 7 of the p53 gene may not play a role in carcinogenesis of HCC in Egyptian patients. Also, genetic polymorphism in HSP70 was not associated with high risk of future development of HCC.

Effect of infrared laser irradiation on amino acid neurotransmitters in an epileptic animal model induced by pilocarpine.

OBJECTIVE: The aim of the present study was to investigate the effect of daily laser irradiation on the levels of amino acid neurotransmitters in the cortex and hippocampus in an epileptic animal model induced by pilocarpine. BACKGROUND DATA: It has been claimed that at specific wavelengths and energy densities, laser irradiation is a novel and useful tool for the treatment of peripheral and central nervous system injuries and disorders. MATERIALS AND METHODS: Adult male albino rats were divided into three groups: control rats, pilocarpinized rats (epileptic animal model), and pilocarpinized rats treated daily with laser irradiation (90 mW at 830 nm) for 7 d. The following parameters were assayed in cortex and hippocampus: amino acid neurotransmitters (excitatory: glutamic acid and aspartate; and inhibitory: gamma-aminobutyric acid [GABA], glycine, and taurine) by high-performance liquid chromatography (HPLC), glucose content, and the activity of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), using a spectrophotometer. RESULTS: Significant increases in the concentrations of glutamic acid, glutamine, glycine, and taurine were recorded in the cortices of pilocarpinized rats, and they returned to initial levels after laser treatment. In the hippocampus, a moderate increase in aspartate accompanied by a significant increase in glycine were observed in the epileptic animal model, and these dropped to near-control values after laser treatment. In addition, a significant increase in cortical AST activity and a significant decrease in ALT activity and glucose content were obtained in the pilocarpinized animals and pilocarpinized rats treated with laser irradiation. In the hippocampus, significant decreases in the activity of AST and ALT and glucose content were recorded in the epileptic animals and in the epileptic animals treated with laser irradiation. CONCLUSION: Based on the results obtained in this study, it may be suggested that nearinfrared laser irradiation may reverse the neurochemical changes in amino acid neurotransmitters induced by pilocarpine.

Correction of aberrant pre-mRNA splicing by antisense oligonucleotides in beta-thalassemia Egyptian patients with IVSI-110 mutation.

The splicing mutation in intron 1 of beta-globin gene (IVS1-110) is the most common mutation in Egyptian thalassemics that causes aberrant splicing of pre-mRNA and deficient beta-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 beta-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 micromoL/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 beta-globin pre-mRNA leading to correct gene product in cultured erythropoietic cells. These results suggest the applicability of ASON for the treatment of thalassemia.

Effect of three different intensities of infrared laser energy on the levels of amino acid neurotransmitters in the cortex and hippocampus of rat brain.

OBJECTIVE: The aim of this study is to investigate the effects of three different intensities of infrared diode laser radiation on amino acid neurotransmitters in the cortex and hippocampus of rat brain. BACKGROUND DATA: Lasers are known to induce different neurological effects such as pain relief, anesthesia, and neurosuppressive effects; however, the precise mechanisms of these effects are not clearly elucidated. Amino acid neurotransmitters (glutamate, aspartate, glutamine, gamma-aminobutyric acid [GABA], glycine, and taurine) play vital roles in the central nervous system (CNS). MATERIALS AND METHODS: The shaved scalp of each rat was exposed to different intensities of infrared laser energy (500, 190, and 90 mW) and then the rats were sacrificed after 1 h, 7 d, and 14 d of daily laser irradiation. The control groups were exposed to the same conditions but without exposure to laser. The concentrations of amino acid neurotransmitters were measured by high-performance liquid chromatography (HPLC). RESULTS: The rats subjected to 500 mW of laser irradiation had a significant decrease in glutamate, aspartate, and taurine in the cortex, and a significant decrease in hippocampal GABA. In the cortices of rats exposed to 190 mW of laser irradiation, an increase in aspartate accompanied by a decrease in glutamine were observed. In the hippocampus, other changes were seen. The rats irradiated with 90 mW showed a decrease in cortical glutamate, aspartate, and glutamine, and an increase in glycine, while in the hippocampus an increase in glutamate, aspartate, and GABA were recorded. CONCLUSION: We conclude that daily laser irradiation at 90 mW produced the most pronounced inhibitory effect in the cortex after 7 d. This finding may explain the reported neurosuppressive effect of infrared laser energy on axonal conduction of hippocampal and cortical tissues of rat brain.

Study of beta-Thalassemia mutations using the polymerase chain reaction-amplification refractory mutation system and direct DNA sequencing techniques in a group of Egyptian Thalassemia patients.

The aim of this study was the molecular characterization of beta-thalassemia (thal) mutations in a group of 95 Egyptian thalassemic patients from Fayoum in Upper Egypt, Cairo, Alexandria and Tanta in Lower Egypt and the Nile Delta. To identify these anomalies, the polymerase chain reaction-amplification refractory mutation system (PCR-ARMS) technique was used, complemented by direct DNA sequencing for uncharacterized cases. In 80 of the 95 patients, the beta-thal mutation was detected by PCR-ARMS. The most common allele encountered in our study was IVS-I-6 (T-->C) (36.3%); the second most common mutation was IVS-I-110 (G-->A) (25.8%). In addition, we report three homozygous cases for the promoter region -87 (C-->G) allele with a frequency of 3.2%. DNA sequencing of uncharacterized cases (14 cases, 15 alleles) revealed six cases (six alleles) of codon 27 (G-->T), and three cases (three alleles) of the IVS-II-848 (C-->A) mutation. Codon 37 (G-->A) in the homozygous state was found in one patient with positive consanguinity. The frameshift codon 5 (-CT) mutation was detected in two of our uncharacterized cases. The codon 15 (TGG-->TGA) mutations was detected in one patient (one allele, 0.5%). All studied cases were fully characterized by this strategy. Screening for beta-thalassemic mutations using ARMS-PCR for the seven most frequent alleles in Egypt succeeded in determining the beta-globin genotype in 84.2% of our patients (91.6% of the expected alleles). To improve the efficiency of routine screening, the PCR-ARMS mutation panel should be updated to include the reported rare alleles. Direct DNA sequencing is an additional way to allow a full characterization of beta-thal patients in the Egyptian population.

Genetic polymorphism of folate and methionine metabolizing enzymes and their susceptibility to malignant lymphoma.

BACKGROUND: Folate and methionine metabolism is involved in DNA synthesis and methylation. Polymorphisms in the genes of folate metabolism enzymes have been associated with some forms of cancer. In the present study, 2 polymorphisms were evaluated for a folate metabolic enzyme, methylene-tetrahydrofolate reductase (MTHFR), and one was evaluated for methionine synthase (MS). The 2 polymorphisms MTHFR 677 C-->T and MTHFR 1298 A-->C, are reported to reduce the enzyme activity, which causes intracellular accumulation of 5, 10- methylene-tetrahydrofolate and results in a reduced incidence of DNA double strand breakage. The MS 2756 A-->G polymorphism also reduces the enzyme activity and results in the hypomethylation of DNA. PATIENTS AND METHODS: To test this hypothesis, genetic polymorphisms in the folate metabolic pathway were investigated using the DNA from a case-control study on 31 patients having malignant lymphoma from the Oncology Outpatient Clinic of the New Children's Hospital, Cairo University and 30 controls who were actually normal children attending for vaccination to the same hospital. RESULTS: We found that there is a higher susceptibility with the MTHFR 677CC and MTHFR 1298 AA genotypes (OR=4.3, 95% CI 1.12-16). When those harbor at least one variant allele in either polymorphism of MTHFR they were defined as reference. For the MS 2756 AG genotype polymorphism there was also a higher susceptibility to developing malignant lymphoma (OR=2.6; 95% CI 1.1- 6.4). CONCLUSION: Results suggest that folate and methionine metabolism may play an important role in the pathogenesis of malignant lymphoma. Further studies to confirm this association and detailed biologic mechanisms are now required.

Immune-mediated liver injury: prognostic value of CD4+, CD8+, and CD68+ in infants with extrahepatic biliary atresia.

BACKGROUND: Hepatic fibrosis and cirrhosis develop progressively in extrahepatic biliary atresia (EHBA) despite timely surgical intervention. PURPOSE: The aim of the study was to define CD4+ helper T lymphocytes, cytotoxic CD8+ T lymphocytes, and CD68+ (macrophages) infiltration of portal tracts and lobules and hepatic fibrosis as possible predictive measures of outcome of infants having EHBA. METHODS: The outcome of 32 infants with EHBA was correlated to their percutaneous biopsy and postportoenterostomy core liver tissue infiltration by CD4+, CD68+, and CD8+ cells and to the degree of detected fibrosis. RESULTS: Portoenterostomy cores were heavily infiltrated by CD4+, CD8+, and CD68+, compared with the preoperative liver biopsy (P = .008, .004, and .017, respectively). Infants having favorable outcome had more macrophage infiltration in portoenterostomy core compared with those having an unfavorable outcome (25.66 +/- 29.77 per HPF compared with 11.62 +/- 4.58, P = .000). Mean CD4+/CD8+ ratio was 1.54 +/- 1.37 in those who died within 18 months postoperatively and 0.733 +/- 0.48 in others (P = .021). CONCLUSION: Immune-mediated destruction of portal tracts is an integral part of pathogenesis of EHBA.

Prevalence of factor V Leiden mutation and other hereditary thrombophilic factors in Egyptian children with portal vein thrombosis: results of a single-center case-control study.

No identifiable cause can be found in more than half of the cases of portal vein thrombosis (PVT). Our aim was to assess the prevalence of factor V Leiden mutation and other thrombophilic factors as risk factors in the development of PVT in the pediatric age group. From March 2001 to January 2002, 40 children with PVT were enrolled in the study, in addition to 20 age-matched and sex-matched controls. Protein C, protein S, antithrombin III, and activated protein C resistance (APCR) were assayed. Molecular study of factor II and factor V mutations was carried out. Of the patients, 25 had detectable hereditary thrombophilia (62.5%), 12 had factor V Leiden mutation (30%), 11 had protein C deficiency (27.5%), 6 had factor II mutation (15%), 1 had antithrombin III deficiency (2.5%), and none had protein S deficiency. Five children had concurrence of more than one defect. Factor V Leiden mutation is the most common hereditary thrombophilia associated with PVT and the relative risk of factor V Leiden mutation, as a cause of PVT, was six times more than in controls (odds ratio=6). Concurrence of more than one hereditary thrombophilic factor was seen in 12.5% of our patients. Circumstantial risk factors (neonatal sepsis, umbilical sepsis, umbilical catheterization) were not more significantly prevalent among patients with hereditary thrombophilia than among those with no detectable abnormalities in anticoagulation.