ALBERT

Description: Introduction: Von Willebrand disease (VWD) type III is the second most common inherited bleeding disorder in Pakistan. It is caused by severe quantitative deficiency of plasma Von Willebrand factor (VWF). Absence of VWF increases the severity of disease and is caused by homozygous/ compound heterozygous mutations in Von Willebrand factor gene. Objective: The aim of study is to characterize molecular genetics of Von Willebrand type III in Pakistani population. Setting: NIBD & BMT Karachi, Chughtais lab, Children's Hospital Lahore, PAEC Islamabad and HMC Peshawar. Material And Method: In this cohort study of Von Willebrand disease (VWD) type III, Blood samples of 48 unrelated patients of type III VWD were collected in National Institute Of Blood Disease & Bone Marrow Transplant (NIBD) Karachi, Chughtais lab, Children's Hospital Lahore, PAEC Islamabad and HMC Peshawar. Genomic DNA was extracted from peripheral blood by QIAamp DNA Blood mini kit (Qiagen) and Exon specific PCR was done for VWF gene and Direct gene sequenced on automated ABI-3130 Genetic Analyzer (Applied Biosystems). Sequence Variations in VWF were checked on ISTH-SSC VWD homepage (http://www.vwf.group.shef.ac.uk/), Ensemble genome browser (http://www.ensembl.org/), VWFdb hemobase and biobase biological database (http://www.hgmd.cf.ac.uk/ac/index.php). We have adopted the nomenclature for numbering amino acids of Human genome variation society (HGVS). Statistics analysis was done SPSSv17. Results: Sequence analysis detected mutations in 46 (95.83%) out of 48 samples. VWD type III has led to identification of 28 cases (60.8%) homozygous and 18 (39.1%) were compound heterozygous. We have indentified total 31 Mutations distributed as 17 missense mutations (54%), 7 nonsense mutations (22%) 2 small deletions (6%) , 2 insertion mutations (6%) and 3 splice site mutations (9%).19 of these were newly mutations in this cohort study.. Further method multiplex ligation dependent probe amplification (MLPA) is needed for detection of large deletions in two patients. Nonsense c.3931C〉T, p.Q1311*was founder mutation in Pakistani patients. Conclusion: In cohort study, missense mutations are detected as common in among patients and most of the mutations identified in this cohort were homozygous due to Consanguinity in the family of the patients. Disclosures Oldenburg: SOBI: Consultancy.

Description: INTRODUCTION: Incidence of autosomal recessive disorders is rare, includes deficiencies of clotting factors I, II, V, VII, X, XI, XIII, vitamin K dependent clotting factors, combined factor V & VIII, Von Willebrand disease type 3, Glanzmann Thrombasthenia and Bernard Soulier syndrome. OBJECTIVE: Spectrum of autosomal recessive bleeding disorders (ARBDs] among Pakistani patients. PATIENTS AND METHODS: This cross-sectional study was carried out at Karachi, Lahore, Islamabad and Peshawar. PT,aPTT, BT, and fibrinogen levels done. Patients with prolonged APTT were tested for factors VIII and IX. If FVIII was low, von Willebrand factor: antigen (vWF: Ag) and von Willebrand factor: ristocetin cofactor (vWF: RCo) were performed. When PT and aPTT both were prolonged, FII, FV, and FX were tested. Peripheral film and platelet aggregation studies were done for platelet disorders. Urea clot solubility test was done at the end. RESULTS: Out of429 patients, 148 were diagnosed with Hemophilia A, remaining 281 patients had ARBDs. 95 (33.8%) had VWD type 3. Fibrinogen deficiency was found in 34 (12%), Glanzmann Thrombasthenia in 27 (9.6%), factor XIII in 13 (4.6%), factor VII in 12 (4.3%), factor V in 9 (3.2%), 8 (2.8%) in vitamin K dependent clotting factors, , Bernard Soulier in 7 patient (2.5%),factor X in 2 (0.7%), factor II in 2 (0.7%), factor XI and combined factor V and VIII in 1 (0.4%)patients each. 70 patients (16.3%) remained undiagnosed. CONCLUSION: VWD type 3 is the most common deficiency followed by fibrinogen deficiency. Glanzmann thrombasthenia was the third most common ARBD. Disclosures No relevant conflicts of interest to declare.

Description: Background: Congenital afibrinogenemia (OMIM #202400) is a rare coagulation disorder which was first described in 1920. It is transmitted as an autosomal recessive trait characterized by absent levels of fibrinogen (factor I) in plasma. Consanguinity in Pakistan and its neighboring countries has resulted in higher cases of congenital fibrinogen deficiency in their respective populations. Aims: This study focuses on the detection of mutations in the fibrinogen genes by 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 (tested by Fibrinogen functional assay from Laboratoire Stago, Asnieres, France) were screened for mutations in the Fibrinogen gene 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 FGA mutations, four have FGB mutations and one mutation was detected in FGG. Thirteen of these mutations were novel. The missense mutations are predicted to result in loss of stability since they (a) break ordered regions (b) cause clashes in the hydrophobic core of the protein. Conclusion: Congenital afibrinogenemia is a rapid growing problem in countries such as Pakistan where consanguinity is frequently practiced. This study illustrates the fact that mutations in FGA are relatively more common in our population than those in FGB where as FGG mutations appear rarer. Disclosures Oldenburg: SOBI: Consultancy.

Description: Introduction: Vitamin K dependent factors i.e II, VII, IX and X plays an important role in the clotting mechanisms of the human body. Deficiency or defect in any of these factors, Vitamin K or Gamma glutamyl Carboxylase (GC) and Vitamin K Epoxide Reductase (VKOR) involved in the metabolism of these factors will compromise the clotting mechanisms and can lead to the bleeding disorder. 28 rare single mutations in VKORC1 coding sequence have been reported so far. Objective: To investigate the biochemical, haemostatic and molecular basis of the Vitamin K dependent factor deficiency in Pakistani subjects. Setting: NIBD &BMT, University of Bonn and Children hospital. Method: For this purpose 10 subjects, 9 males and 1 female with age ranging from 7 months to 13years were included from selected region of Pakistan who had a history of bleeding disorder and low levels of all the Vitamin K dependent clotting factors. BT, PT, APTT, Factor II, VII, IX, X,Platelet count and fasted serum bile acids were analysed for exclusion of thromobocytopenia which cause of bleeding and bile acid for cholestatic liver disease respectively. DNA sequencing was done for the GCCX and VKOR encoding gene i.e. GCCX-1 and VKORC-1 respectively to understand the etiology at molecular level. Results: Platelet counts of all the subjects were normal only in one case it was mild low 100x103/ul. Mean± S.E.M value of platelet was 440x103 ± 67. Factor II, VII, IX and X mean levels were 12.7%, 6.9%, 11.2%, 7.1% respectively. Out of ten subjects mean bile acid levels were increased in 3 patients (20 µmole/l) while 3 had low levels (1.2 µmole/l). 3 novel mutations identified in GCCX-1 gene in those patients who had normal levels of bile acids. Conclusion: Out of ten only in three patients novel mutations were identified in GCCX-1 while other 6 patients had abnormal levels of bile acid may be a cause of vitamin K clotting factors deficiency.Better strategies for the diagnosis of vitamin K deficiency are needed. Disclosures Oldenburg: SOBI: Consultancy.

Description: Introduction: Von Willebrand disease is a common genetically inherited bleeding disorder. Types of VWD classified on the basis of coagulation factor assays but it cannot characterize further subtypes or verdict the quality and quantity of VWF. Multimer analysis facilitates the classification and prerequisite parameter to accurate diagnosis of VWD. Objective: To characterize the type and subtypes of VWD type I and II on the basis of laboratory data and analysis of VWF multimer structure. Setting: NIBD Karachi, University of Bonn Germany, Children Hospital & Chughtai Lab Lahore, PAEC Islamabad and HMC Peshawar. Material And Method: VWF Ag, Ricof, FVIII, Glycoprotein Ib and multimer analysis of VWD plasma on 1.6% medium resolution SDS-agarose gel electrophoresis was carried out, and then VWF multimer were transferred on nitrocellulose membrane. Incubated nitrocellulose membrane with using polyclonal rabbit anti-human VWF antibody and Goat anti-rabbit IgG HRP conjugate then protein detected by immune reaction after evaluation of the membrane under luminescence. Results: We studied 57 index patients, 2(3.5%) type I, 4(7.01%) type II, 3(5.26%) unclassified and 48(15.78%) were type III vWD. In type I,II, unclassified and III mean vWF Ag was 52.28, 35.15, 54.5 , 5.7 IU/dl, vWF RCo: 19.5, 18, 38.8, 7.22 IU/dl, vWF Ag/ RCof: 0.655, 0.393, 0.74, 0.55, FVIII 69, 25.7, 61.7, 7.22 IU/dl and GpIb: 27.25, 19.8, 39.8, 4.1. Significant loss of large multimers in type II while multimers were subtle in 2 cases, normal pattern in type I and entirely absence of multimers pattern in case of type III observed. Conclusion: Types and subtypes of VWD classified on the basis of concentration and distribution of vWF multimer by functional and immunological assays but single test is not enough for appropriate diagnosis. We need molecular genetics for von Willebrand disease for further diagnosis. Disclosures Oldenburg: SOBI: Consultancy.