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Allele and Genotype Frequencies of the ABO Blood Group System in a Palestinian Population

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Allele and Genotype Frequencies of the ABO Blood Group System in a Palestinian Population Allele and Genotype Frequencies of the ABO Blood Group System in a Palestinian Population DECLARATION The work provided in this thesis, unless otherwise referenced, is the researcher's own work, and has not been submitted elsewhere for any other degree or qualification اسم الطالب: لمياء صبحي صقر :Student's name التوقيع: Signature: Lamia'a التاريخ: Date: 2013 The Islamic University – Gaza Deanery of Higher Education Faculty of Science Master of Biology Sciences Medical Technology Allele and Genotype Frequencies of the ABO Blood Group System in a Palestinian Population Prepared by Lamia'a Sobhi. Saqer Supervisor Prof. Fadel A. Sharif A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of Master in Biological Sciences/ Medical Technology 1434هـ -2013م ii iii Abstract The ABO blood group antigens are of clinical importance in blood transfusion, organ transplantation, autoimmune hemolytic anemia and fetomaternal blood group incompatibility. The ABO locus are located on chromosome 9. Till now, more than 200 ABO alleles have been identified by molecular investigations. The objective of this study was to determine the major ABO alleles' and genotypes' frequencies in a Palestinian population residing in Gaza Strip. A four separate–reaction multiplex allele specific polymerase chain reaction (AS-PCR) was used to determine the ABO genotypes. Our study population consisted of 201 unrelated subjects (50 males and 151 females) whose DNA extracted from peripheral blood was subjected to genotyping. The genotypes of 201 samples were found to be A1A1 (n=3), A1O 1(n=24), A1O2 (n=25), A1A2 (n=4), A2A2 (n=2), A2O1 (n=13), A2O2 (n=2), B1B1 (n=5), B1O1 (n=26), B1O2 (n=14), A1B (n=11), A2B (n=4) , O1O1 (n=31) , O1O2 (n=26) and O2O2 (n=11), from which the deduced phenotypes were A (n=73), B (n=45), AB (n=15) and O (n=68).Moreover, there was no significant difference between observed and expected genotypes and the genotyping results were consistent with Hardy-Weinberg law. The frequencies of A1 , A2 , B1, O1 and O2 alleles were: 0. 174, 0.067, 0.162, 0.376 and 0.221 respectively. The rare cis-ABO1 allele was not encountered in the study population. The genotype results were compared with serologically determined phenotypes and there were no deviation. To our knowledge, this is the first study in Gaza strip investigating the ABO genotypes. ABO genotyping has practical applications in blood transfusion, tissue/organ transplantation, blood typing discrepancies and forensic/paternity testing investigations. Key words ABO alleles ; ABO genotypes ; AS-PCR; allele frequencies ; ABO phenotype . i ABO ABO ABO Antisera . AS-PCR DNA A1O1 A1A1 B1O2 B1O1 B1B1 A2O2 A2O1 A2A2 A1A2 A1O2 A O2O2 O1O2 O1O1 A2B A1B AB B A2 A1 O cis-ABO1 O2 O1 B ii TABLE OF CONTENTS CONTENTS Page ABSTRACT (English)------------------------------------------------------------------------- i ABSTRACT (Arabic)-------------------------------------------------------------------------- ii TABLE OF CONTENTS--------------------------------------------------------------------- iii LIST OF TABLES----------------------------------------------------------------------------- v LIST OF FIGURES---------------------------------------------------------------------------- vi ABBREVIATIONS----------------------------------------------------------------------------- vii DEDICATION---------------------------------------------------------------------------------- x ACKNOWLEDGEMENTS------------------------------------------------------------------ xi CHAPTER 1 INTRODUCTION------------------------------------------------------------------------------ 1 1.1. Background-------------------------------------------------------------------------------- 2 1.2. Objectives of the Study------------------------------------------------------------------ 4 1.2.1. General Objective-------------------------------------------------------------------- 4 1.2.2. Specific objectives------------------------------------------------------------------- 4 CHAPTER 2 LITIRATURE REVIEW--------------------------------------------------------------------- 6 2.1. Background-------------------------------------------------------------------------------- 6 2.2. Biosynthesis of ABH antigens---------------------------------------------------------- 6 2.2.1. H antigen------------------------------------------------------------------------------ 8 2.2.2. A and B antigens------------------------------------------------------------------- 9 2.3. ABO Glycosyltransferases------------------------------------------------------------- 11 2.4. ABO Subgroups-------------------------------------------------------------------------- 11 2.4.1. A and B Subgroups ----------------------------------------------------------------- 11 2.4.1.1. A Subgroups --------------------------------------------------------------------- 12 2.4.1.2. B Subgroups---------------------------------------------------------------------- 13 2.4.2. O Subgroups-------------------------------------------------------------------------- 13 2.4.3. Weak subgroups-------------------------------------------------------------------- 14 2.4.3.1. Weak A alleles-------------------------------------------------------------------- 15 2.4.3.2. Weak B alleles-------------------------------------------------------------------- 15 2.5. ABO antibodies-------------------------------------------------------------------------- 17 2.6. Studies on ABO Genotypes------------------------------------------------------------ 18 2.7. ABO Genotyping and susceptibility to diseases------------------------------------- 22 CHAPTER 3 MATERIALS and METHODS-------------------------------------------------------------- 24 3.1. Materials---------------------------------------------------------------------------------- 24 3.1.1. PCR primers-------------------------------------------------------------------------- 24 3.1.2. Kits------------------------------------------------------------------------------------- 25 3.1.3. Reagents and Chemicals------------------------------------------------------------ 25 3.1.4. Apparatus and Equipments--------------------------------------------------------- 25 3.2. Methods------------------------------------------------------------------------------------ 26 3.2.1. Study population--------------------------------------------------------------------- 26 iii TABLE OF CONTENTS CONTENTS Page 3.2.2. Sample collection------------------------------------------------------------------- 26 3.2.3. Ethical Considerations------------------------------------------------------------- 26 3.2.4. Data Analysis-------------------------------------------------------------------------- 26 3.3.Blood ABO-typing---------------------------------------------------------------------- 26 3.3.1. Forward blood group----------------------------------------------------------------- 26 3.4. ABO Genotyping------------------------------------------------------------------------ 27 3.4.1. DNA Extraction---------------------------------------------------------------------- 27 3.4.2. Detection of extracted DNA-------------------------------------------------------- 28 3.5. PCR reactions---------------------------------------------------------------------------- 28 3.5.1. Temperature cycling program------------------------------------------------------ 29 3.5.2. Expected PCR results---------------------------------------------------------------- 29 CHAPTER 4 RESULTS---------------------------------------------------------------------------------------- 31 4.1. Study Population------------------------------------------------------------------------- 31 4.2. Phenotypic Frequency of ABO Blood Groups--------------------------------------- 31 4.3. The Allele Frequencies of ABO Antigens----------------------------------------- 32 4.4. PCR Results------------------------------------------------------------------------------- 33 4.4.1 Quality of the isolated DNA-------------------------------------------------------- 33 4.4.2. Blood group genotyping by allele specific PCR-------------------------------- 33 4.4.3. Genotype Frequencies--------------------------------------------------------------- 36 CHAPTER 5 DISCUSSION----------------------------------------------------------------------------------- 39 CHAPTER 6 CONCLUSION and RECOMMENDATIONS------------------------------------------- 43 CHAPTER 7 REFERENCES---------------------------------------------------------------------------------- 45 iv List of Tables Table Page Table 2.1. Some of Human blood group systems recognized by the ISBT----- 6 Table 2.2. Serological reaction patterns------------------------------------------------- 15 Table 2.3. Characteristics of some more frequent, B weak phenotypes----------------- 16 Table 3.1. PCR primers sequences used for ABO genotype---------------------- 24 Table 3.2. Sample / Anti A, Anti B reaction for forward blood grouping------- 27 Table 3.2. Composition of PCR master mix----------------------------------------- 29 Table 4.1. Phenotypic frequencies of various blood groups in the studied 31 population------------------------------------------------------------------- Table 4.2. Observed and expected genotypes for the 201 samples -------------- 32 Table 4.3. PCR products according to the genotype-------------------------------- 34 Table 4.4. The frequency of recognized genotypes using multiplex AS-PCR 37 method for population residing in Gaza Strip-------------------------- Table 5.1. Frequency of ABO alleles
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