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.

β-thalassemia is characterized by impaired β-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 β-thalassemia patients in the presence or absence of Xmn I polymorphism. A total of 143 consecutive β-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 β-thalassemia is a predictor of response to HU and highlights the possibility of managing this subset of patients without blood transfusion.

Background Long survival with improve quality life in treatment of thalassaemia patients can be procure by blood transfusion and sedulous chelation but, it is cumbrous. Therapeutic manoeuvers designed to stabilize or increase hemoglobin without transfusion and to reduce the blood transfusion. Efforts have been undertaken to find an alternative approach to transfusion in β-thalassaemia. Methods On the basis of our previous studies evaluating the safety & efficacy of Hydroxyurea (HU) in beta thalassemia patients which is an oral Hb-F augmentation agent, at 10-15 mg/kg/day was used on 473 patients for Mean duration of 3 years 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 Complete Responders (CR), partial responders (PR) and non-responders(NR).Patients’ genetic profile was investigated. Chi square test was applied to check association between variables, p-value ≤0.05 considered significant. Results Complete responders were 176, PR were 194 while 103 were NR.  Most common genetic mutations observed were IVS1-5 (46%) either homozygous or heterozygous, Fr 8-9 (14.3%), IVS1-1 (11%), Cd 30 (5.5%), Fr 41-42 (5.5%), Del-619 (5%) &Cap+1 (1.9%). Astounding results were found when association of these mutations with response of HU was observed. Response was found to be closely related with gene mutations Cd-30 (61.5 % Responders), IVS1-1 (58 % Responders), Cap+1 (44.4 % Responders), IVS1-5 (33.6 % Responders). Homozygous Xmn-I polymorphism was observed in 21% & heterozygous in 24% of the patients, while 55 % had no Xmn-I polymorphism. Xmn-I polymorphism was found to be significantly associated with Response 67 % (p-value 0.00). Conclusion Hydroxyurea showed promising results in this long term follow up of large cohort of patients. We suggest screening all newly diagnosed thalassaemic children for Genetic characteristic in order to offer Hb-F Augmenting agent as an alternative to blood transfusion. Disclosures: No relevant conflicts of interest to declare.

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.

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