5309 Introduction: β -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 β -thalassemia. To achieve this objective, Identification of the spectrum of genetic mutations, especially for various ethnic backgrounds in Pakistan, since the country is a High Burdon Country. Therefore, we designed a cross sectional study to identify the frequency of various gene mutations in different ethnic groups of Pakistan. Methodology: Over a period of five years, venous blood samples were collected from 466 individuals belonging to different ethnic groups residing in Karachi, having at least one affected family member known to have β-thalassemia major/ HbE- β-thalassemia/ HbE homozygotes/ β-thalassemia trait. Chorionic villus sampling at 11 to 15 weeks gestational age for 143 couples referred by thalassemia clinics as also used to obtain allele information. In all, 648 mutated alleles were identified. The diagnosis of β-thalassemia trait, β-thalassemia major and Hb E thalassemia were established from clinical data, hematological indices and hemoglobin electrophoresis by cellulose acetate method. DNA was extracted from whole blood for detection of mutations. Primers were designed for simultaneous detection of the following previously described mutations in a single reaction: IVS 1–5 (G-C), Fr 8–9, IVS 1-1 (G-T), Cd-30 (G-C), Cd-5 (−CT), Del 619bp, Cd-15 (G-A), Fr 41–42, Fr 16 (−C) and Cap +1 (A-C) along with two Hb variants: HbS and HbE. Results: The genetic heterogeneity in Karachi is reflected by the identification of all the common β-thalassemia alleles and two Hb variants but following eight mutations were more common: IVS 1–5 (G-C), Fr 8–9, Del 619 bp, IVS 1-1 (G-T), Fr 41–42, Cd-30 (G-C), Cd-5 (−CT) and Cd-15 (G-A), accounting for 93.9% of the β-thalassemia alleles. However, the distribution was uneven. Although IVS 1–5 (G-C) was the most common mutation (40.89% of the sample), its frequency varied from 20% in the immigrant (from India) population to 76.9% in the Balochis. The second most frequent mutation was Fr 8–9, constituting 15.7% of the allele pool. Fr 8–9 was the most common mutation in the Pathans (31.3%) as it was in people of Saraikee origin (47%). Discussion: IVSI-5 (G-C),(40.89%), Fr8-9(15.7%), & IVSI-I(G-T),(8.17%), were the most common genetic mutations identified 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 for genetic counseling and pregnancy termination. Disclosures: No relevant conflicts of interest to declare.

HU was found to be safe in patients with β-thalassemia major, and resulted in the reduction of transfusion requirement and in an increase in the interval between transfusions.

BACKGROUND:β -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 β-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 β-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 β-thalassemia mutations were identified in 8 different ethnic groups accounting for 93.9% of the β-thalasemia alleles.CONCLUSIONS:Based on the outcome of this study a cost effective proposal is formulated for detection of β-thalassemia mutations.

3199 Introduction: Packed red blood cell (PRC) transfusion with iron chelation, despite their undesirable effects, has been the mainstay of treatment for patients with beta-thalassaemia major. In the recent past introduction of oral medications that augment Hemoglobin F (HbF) has opened new horizons in the management & prognosis of these patients sparing many of them from the hardtimes of blood transfusions & related adversities. Methods: On the basis of our previous studies evaluating the safety & efficacy of Hydroxyurea (HU) in beta thalassemia patients which is an oral HbF augmentation agent, at 10–15 mg/kg/day was used on 238 patients for 24 months under the guidelines of Helsinki's declaration. Response was measured by using transfusion requirement prior to 6 months period of enrollment in the study as control. Patients were finally divided into 3 groups on the basis of response, Group 1 consisted of Complete Responders, group 2 were partial responders & group 3 were non-responders. Group1 patients needed regular blood transfusions prior to HU therapy while after 24 months they never required transfusion as they maintained their mean Hb at ≥7gm/dL. Partial responders substantially decreased their need for transfusion (less than 50 %), while Non-responders had no decrease in their need for transfusions. All patients' genetic mutation profiles were investigated upon. Results: Most common genetic mutations observed were IVS1-5 (48%) either homozygous or heterozygous, Fr 8–9 (11.8%), IVS1-1 (10.5%), Cd 30 (7.6 %), Fr 41–42 (6.3%), Cap+1 (3.3%) & Del-619 (8%). Homozygous Xmn-polymorphism was found in 20% & heterozygous Xmn polymorphism was observed in 26% of the patients, while rest 54% had no Xmn polymorphism. Astounding results were observed when these mutations were correlated with response of HU. Response rate was found to be most closely related with gene mutations IVS1-1 (68% Responders), Cd-30 (56% Responders), Cap+1 (38% Responders)IVS1-5 (37% Responders). Xmn polymorphism was found to be significantly associated with Response rate 73% (p-value 0.00). Discussion: It was observed from our study that certain genetic mutations in beta-thalassemia & presence of Xmn polymorohism responds exceptionally well to oral HbF augmentation agents, sparing the patients from the adversities of blood transfusion. Most notably presence of IVS1-1 spared 68% of the patients from blood transfusions while Xmn polymorphism spared 73% of them. It is recommended, therefore, that a new classification be made on the basis of genetic profile of beta thalassemia patients for better treatment & prognosis preventing unnecessary blood transfusions. Disclosures: Ansari: National Institute of Blood Diseases & Bone Marrow Transplantation: Consultancy. Off Label Use: We used Hydroxyurea or hydroxy carbamide. Hydroxycarbamide increases the concentration of fetal hemoglobin. The precise mechanism of action is not yet clear, but it appears that hydroxycarbamide increases nitric oxide levels, causing soluble guanylyl cyclase activation with a resultant rise in cyclic GMP, and the activation of gammaglobin synthesis necessary for fetal hemoglobin. Shamsi:National Institute of Blood Diseases & Bone Marrow Transplantation: Consultancy. Bohray:National Institute of Blood Diseases & Bone Marrow Transplantation: Consultancy. Farzana:National Institute of Blood Diseases: Employment. Erum:National Institute of Blood Diseases: Employment.