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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 Student's name: Signature: Date: لمياء صبحي صقر:اسم الطالب Lamia'a :التوقيع 2013 :التاريخ 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 م2013- هـ1434 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 A1 O 1 B1 O 2 DNA A1 A1 B1 O 1 B1 B1 A A2 O 2 A2 O 1 O2 O2 O1 O2 A2 A2 A 1 A2 O1O1 A2 B AB A2 A1 O 2 A1 A1 B B O cis-ABO1 O2 ii O1 B TABLE OF CONTENTS CONTENTS Page ABSTRACT (English)------------------------------------------------------------------------ABSTRACT (Arabic)-------------------------------------------------------------------------TABLE OF CONTENTS--------------------------------------------------------------------LIST OF TABLES----------------------------------------------------------------------------LIST OF FIGURES---------------------------------------------------------------------------ABBREVIATIONS----------------------------------------------------------------------------DEDICATION---------------------------------------------------------------------------------ACKNOWLEDGEMENTS-----------------------------------------------------------------CHAPTER 1 INTRODUCTION-----------------------------------------------------------------------------1.1. Background-------------------------------------------------------------------------------1.2. Objectives of the Study-----------------------------------------------------------------1.2.1. General Objective-------------------------------------------------------------------1.2.2. Specific objectives------------------------------------------------------------------CHAPTER 2 LITIRATURE REVIEW--------------------------------------------------------------------2.1. Background-------------------------------------------------------------------------------2.2. Biosynthesis of ABH antigens---------------------------------------------------------2.2.1. H antigen-----------------------------------------------------------------------------2.2.2. A and B antigens------------------------------------------------------------------2.3. ABO Glycosyltransferases------------------------------------------------------------2.4. ABO Subgroups-------------------------------------------------------------------------2.4.1. A and B Subgroups ----------------------------------------------------------------2.4.1.1. A Subgroups --------------------------------------------------------------------2.4.1.2. B Subgroups---------------------------------------------------------------------2.4.2. O Subgroups-------------------------------------------------------------------------2.4.3. Weak subgroups-------------------------------------------------------------------2.4.3.1. Weak A alleles-------------------------------------------------------------------2.4.3.2. Weak B alleles-------------------------------------------------------------------2.5. ABO antibodies-------------------------------------------------------------------------2.6. Studies on ABO Genotypes-----------------------------------------------------------2.7. ABO Genotyping and susceptibility to diseases------------------------------------CHAPTER 3 MATERIALS and METHODS-------------------------------------------------------------3.1. Materials---------------------------------------------------------------------------------3.1.1. PCR primers-------------------------------------------------------------------------3.1.2. Kits------------------------------------------------------------------------------------3.1.3. Reagents and Chemicals-----------------------------------------------------------3.1.4. Apparatus and Equipments--------------------------------------------------------3.2. Methods-----------------------------------------------------------------------------------3.2.1. Study population--------------------------------------------------------------------- iii i ii iii v vi vii x xi 1 2 4 4 4 6 6 6 8 9 11 11 11 12 13 13 14 15 15 17 18 22 24 24 24 25 25 25 26 26 TABLE OF CONTENTS CONTENTS Page 3.2.2. Sample collection------------------------------------------------------------------3.2.3. Ethical Considerations------------------------------------------------------------3.2.4. Data Analysis-------------------------------------------------------------------------3.3.Blood ABO-typing---------------------------------------------------------------------3.3.1. Forward blood group----------------------------------------------------------------3.4. ABO Genotyping-----------------------------------------------------------------------3.4.1. DNA Extraction---------------------------------------------------------------------3.4.2. Detection of extracted DNA-------------------------------------------------------3.5. PCR reactions---------------------------------------------------------------------------3.5.1. Temperature cycling program-----------------------------------------------------3.5.2. Expected PCR results---------------------------------------------------------------CHAPTER 4 RESULTS---------------------------------------------------------------------------------------4.1. Study Population------------------------------------------------------------------------4.2. Phenotypic Frequency of ABO Blood Groups--------------------------------------4.3. The Allele Frequencies of ABO Antigens----------------------------------------4.4. PCR Results------------------------------------------------------------------------------4.4.1 Quality of the isolated DNA-------------------------------------------------------4.4.2. Blood group genotyping by allele specific PCR-------------------------------4.4.3. Genotype Frequencies--------------------------------------------------------------CHAPTER 5 DISCUSSION----------------------------------------------------------------------------------CHAPTER 6 CONCLUSION and RECOMMENDATIONS------------------------------------------CHAPTER 7 REFERENCES---------------------------------------------------------------------------------- iv 26 26 26 26 26 27 27 28 28 29 29 31 31 31 32 33 33 33 36 39 43 45 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. 31 Table 4.2. Phenotypic frequencies of various blood groups in the studied population------------------------------------------------------------------Observed and expected genotypes for the 201 samples -------------- Table 4.3. PCR products according to the genotype-------------------------------- 34 Table 4.4. The frequency of recognized genotypes using multiplex AS-PCR method for population residing in Gaza Strip-------------------------Frequency of ABO alleles in Gaza Strip in comparison to some other countries-------------------------------------------------------------- Table 5.1. v 32 37 41 List of Figures Figure Page 2 Figure 2.1 Schematic representation of the genomic organization of the ABO locus-------------------------------------------------------------------------Biosynthesis of ABO Antigens------------------------------------------- 8 Figure 2.2 H antigen structure--------------------------------------------------------- 9 Figure 2.3 A, B, and O(H) blood group structures and their synthesis----------- 10 Figure 2.4 A and B antigen structure------------------------------------------------- 10 Figure 4.1 Distribution of the subjects according to gender----------------------- 13 Figure 4.2 Gel electrophoresis for DNA extracted from human blood 33 samples---------------------------------------------------------------------The electrophoresis pattern of recognized genotypes using the 36 multiplex AS-PCR method------------------------------------------------ Figure 1.1 Figure 4.3 vi ABBREVIATIONS A Adenine a.a Amino acid Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid AS-PCR Allele Specific – Polymerase Chain Reaction BGMUT Blood Group antigen gene Mutation database bp Base pair °C Celsius C Cytosine C-2 position Carbon -2 position CAZy Carbohydrate Active enZyme cDNA Complementary DNA C-terminus Carbon terminus dATP deoxyadenosine Triphosphate dCTP deoxyacytidine Triphosphate dGTP deoxyguanosine Triphosphate dNTPs Deoxynucleotide Triphosphates dTTP deoxytymidine triphosphate Del deletion E. coli Escherichia coli EDTA Ethylenediaminetetraacetic acid Fuc Fucose FUT fucosyltransferases G Guanine Gal Galactose GalNAc N-acetylgalactosamine GalNAcα3 N-acetylgalactosylamine Galα3 galactosyl GDP guanosine diphosphate vii Glc glucose GlcNAc N-acetylglucosamine Glu Glutamic acid Gly Glycine GTA Glycosyltransferases A GTB Glycosyltransferases B GTs Glycosyltransferases Ile Isoleucine ISBT International Society of Blood Transfusion kb Kilo base Leu Leucine Met Methionine mf Mixed field ml Milliliter Mn++ Manganese µl Micro liter nt nucleotide MTHFR Methylenetetrahydrofolate reductase PCR Polymerase chain reaction PCR-SSCP PCR- Single-Strand Conformation Polymorphism Phe Phenylalanine pmol picomole Pro Proline RBCs Red Blood Cells RFLP Restriction Fragment Length Polymorphism rpm Round per minute Ser Serine SNP Single – Nucleotide polymorphism SSP Sequence-Specific Primers SPSS Statistical Package for Social Sciences T Thymine Trp Tryptophan U.V Ultra Violet viii UDP uridine diphosphate UDP-Gal uridine diphosphate galactose UDP-GalNAc uridine diphosphate N-acetylgalactosamine Val Valine VTE Venous thromboembolism vWF von Willebrand factor w weak ix To my family , especially my parents , for their continuous encouragement and unlimited support , to all those who enliven my days and brighten my ways. To whom who change my life . x Acknowledgments This work has been carried out in the Genetic Diagnosis Laboratory at the Islamic university of Gaza, Palestine. First and for most, I am thankful to his almighty God for this work. To those people who directly or indirectly helped a lot to make this work perfect. I wish to express my gratitude and deepest thanks to his Excellency Professor Fadel A. Sharif to his initiating, planning, supervision and scientific guidance of this work. He stand with me step by step and he was very keen to teach me every thing right. My thanks and appreciation to V. Dean for Academic Affairs–College of science and technology Mr. Niddal S. Abu hujayer for his great help and support. I would like to extend my thanks to the staff of the college of science and technology especially the Medical Sciences department , Khan Younis , where I am working , for their support . I am grateful too for the support and advise from Mr. Ahmad S. Silmi for his assistance, advice, and support . I am deeply grateful to Mr. Mohammad J. Ashour for his helpful and friendly support during the laboratory work . Finally , I want to thank my family ,parents , brothers and sisters, who they always stood beside me and gave me encouragement all the time . xi CHAPTER 1 INTRODUCTION 3 CHAPTER 1 INTRODUCTION 1.1. Background In clinical practice, the ABO blood group system is one of the most important since the A and B epitopes may provoke a strong immune reaction. With the introduction of blood typing and cross-matching techniques, blood transfusion became not only a simple but also a much safer procedure. Furthermore, although ABO typing reduced the occurrence of transfusion reactions, they still occurred, indicating the presence of other genetic differences in blood groups of importance in transfusion medicine, as well as in the later emerging field of organ transplantation(1). Human ABO locus is located in chromosome 9q34.1-q34.2 (2-5) and consists of 7 exons distributed over 18 kb of genomic DNA, ranging in size from 28 to 688 base pairs (bp) and 6 introns with 554 to 12982 bp (6-8) (Figure 1.1). Exon 7 contains most of the largest coding sequence whereas, exon 6 contains the deletion found in most O alleles (9). Figure 1.1: Schematic representation of the genomic organization of the ABO gene (10) 2 Molecular genetic studies of human ABO genes have demonstrated that ABO genes have two critical single base substitutions in the last coding exon that result in amino acid substitutions responsible for the different donor nucleotide sugar substrate specificity between A- and B-transferases. A single base deletion in exon 6 was ascribed to shift the reading frame of codons and to abolish the transferase activity of Atransferase in most O alleles (4,5,9,11) ABO genotyping is important not only for blood transfusion, but also for tissue/cell and organ transplantations. Also, ABO genotypes are important evidence at crime scenes, and for personal identification in forensic investigations and paternity testing. To our knowledge, this is the first study in Gaza Strip investigating the ABO gene polymorphism. In this study, multiplex allele-specific PCR is used for determining the ABO genotypes and the corresponding allele frequency in a group of 201 unrelated Palestinians residing in Gaza Strip . 1 1.2 Objectives of the Study 1.2.1 General Objective To determine the major ABO alleles and genotypes frequencies in a Palestinian population residing in Gaza Strip. 1.2.2 Specific objectives 1- To employ PCR and Allele-specific (AS)-PCR for molecular genotyping of ABO blood system. 2- To correlate ABO genotypes with phenotypes in blood samples of Gaza Strip population. 3- To compare the frequency of ABO genotypes with other populations. 4 CHAPTER 2 LITIRATURE REVIEW 5 CHAPTER 2 LITIRATURE REVIEW 2.1. Background The International Society of Blood Transfusion (ISBT) Working Committee on Terminology for Red Cell Surface Antigens was set up in 1980 to establish and define a meaningful nomenclature for different blood groups (12). Every valid blood group antigen is given a six digit identification number. There are 29 different systems to date, and the first three digits represent the systems (001-029). The symbol for a gene or cluster of genes controlling a blood group system is often the italicized symbol for the system. The ABO genotypes should consequently be written in italicized capital letters (Table 2.1). Table 2.1 Some of Human blood group systems recognized by the ISBT (12) No. Name Symbol No. of antigens Gene name(s) Chromosome 001 ABO ABO 4 ABO 9 002 MNS MNS 43 GYPA, GYPB, GYPE 4 003 P P1 1 P1 22 004 Rh RH 49 RHD, RHCE 1 005 Lutheran LU 19 LU 19 006 Kell KEL 25 KEL 7 007 Lewis LE 6 FUT3 19 008 Duffy FY 6 DARC 1 009 Kidd JK 3 SLC14A1 18 2.2. Biosynthesis of ABH antigens The biochemical basis of the ABO and H antigens is well understood due to intensive studies during the 1950s and 1960s by the pioneering work on ovarian cyst fluids (which contain large amounts of water-soluble blood-group-active glycoproteins) by Morgan & Watkins and Kabat. The ABO antigens are not limited to erythroid tissues, but are also found in different tissues and on some epithelial cells (13).The antigens are also present in the secretory fluids in the majority of humans therefore, they 6 can sometimes be noted as histo-blood group antigens (14). ABH antigens are carbohydrate structures. These oligosaccharide chains are generally conjugated with polypeptides to form glycoproteins. Oligosaccharides are synthesized in a stepwise fashion, the addition of each monosaccharide being catalyzed by a specific glycosyltransferase enzyme (15). The glycoproteins contain a peptide backbone to which multiple oligosaccharide chains are attached through an alkali-labile glycosidic bond (16,17) to the hydroxyl group of serine or threonine (18). Most of the oligosaccharide chains are linked to the backbone through an N-acetylgalactosamine residue. The carbohydrate moiety of the ABH glycoproteins consists primarily of four sugars, D-galactose, L-fucose, N-acetylD-galactosamine and N-acetyl-D-glucosamine (19). The amino acid compositions of the different blood group glycoproteins are similar to each other, and unrelated to bloodgroup specificity. Expression of H, A, and B antigens is dependent on the presence of specific monosaccharides attached to various precursor disaccharides at the non-reducing end of a carbohydrate chain (14). A transferase, product of A allele, transfers the monosaccharide N acetylgalactosamine from the donor substrate uridine diphosphate (UDP)-N-acetylgalactosamine to the fucosylated galactosyl residue of the H antigen, to produce an active structure. The B transferase, product of B allele, transfers galactose from UDP-galactose to the fucosylated galactose of H, to produce a B-active structure (20).Individuals with the gene for Glycosyltransferases A have blood group A; those with the gene for Glycosyltransferases B have blood group B; those with genes for both enzymes or a cis-acting form of GTA or GTB have blood type AB; and those with a mutated inactive form of enzyme have blood group O (Figure 2.1 ). 7 Figure 2.1 : Biosynthesis of ABO Antigens (2) The major alleles at the ABO locus are A, B and O and to-date a number of ABO blood groups variants have been reported, with approximately 250 different alleles registered in the Blood Group antigen gene Mutation database "BGMUT" (21). 2.2.1. H antigen In nature, there exists at least four H antigens on glycolipids and glycoproteins that are recognized by GTA and GTB (22).The most common are the type I and the type II H antigens (23).H antigen is produced when an α1,2-Lfucosyltransferase catalyses the transfer of L-fucose from a guanosine diphosphate (GDP)-L-fucose donor to the C-2 position of the terminal galactose of one of the precursor structures (Figure 2.1). Two α1,2-L-fucosyltransferases, produced by two genes, FUT1 (H) and FUT2 (Se), catalyze 8 the biosynthesis of H-active structures in different tissues (24) mainly in epithelial cells and body fluids such as saliva. The H antigen (Figure 2.2) is the natural precursor of A and B antigens and its fucose residue is required for A and B glycosyltransferases to recognize it as the acceptor and transfer GalNAc or Gal to its terminal Gal. Depending on the disaccharide precursor core chain on which ABH determinants are synthesized, they can be further divided into different types. Figure 2.2 : H antigen structure Everybody expresses H antigen on his red cells, but only about 80% of Europeans have H antigen in their body secretions. These people are called ABH secretors because, if they have an A and/or B gene, they also secrete A and/or B antigens. The remaining 20% are called ABH non-secretors as they do not secrete H, A, or B regardless of ABO genotype (25,26). 2.2.2. A and B antigens A and B antigens can be produced by the presence of the appropriate A- or B transferase (Figure 2.1). The A gene product is an α1,3-N-acetyl galactosaminyltransferase, which transfers N-acetyl-galactosamine from a uridine diphosphate (UDP)N acetylgalactosamine donor to the fucosylated galactosyl residue of H antigen. The B gene product, an α 1,3-D-galactosyltransferase, transfers D-galactose from UDPgalactose to the fucosylated galactose of H. A and B are alleles at the ABO locus on chromosome 9. A third allele, O, does not produce an active enzyme and in persons homozygous for O the H antigen remains unmodified (Figure 2.3). 9 Figure 2.3: Summary of A, B, and O(H) blood group structures and their synthesis(28) The A and B antigens are carbohydrate molecules built stepwise from saccharides such as galactosamine (GalNAc), glucosamine (GlcNAc), fucose (Fuc), galactose (Gal), and glucose (Glc). H-antigen is the requisite precursor and galactosylamine (GalNAcα3) or galactosyl (Galα3) with α1-3 linkage onto H antigen become the A and B antigens respectively (5,14) (Figure 2.4). Figure 2.4: A and B antigen structure The majority of ABO antigens on red cells are linked to glycoproteins (approximately 70%), thus they very much influence the blood group activity on the red cells. At each step in the biosynthesis of ABO antigens and carbohydrate chains, synthesis is facilitated by glycosyltransferases, which are competing for available precursors and substrates. This represents approximately 80% of the total complement of red cell ABH determinants. Another 5 × 105 ABH determinants localize to the red 31 cell glucose transport protein (Band 4.5). Small numbers of ABH antigens are also expressed by other red cell glycoproteins (28). 2.3. ABO Glycosyltransferases Glycosyltransferases (GTs) constitute a large family of enzymes that are involved in the biosynthesis of oligosaccharides, polysaccharides, and glycoconjugates (29).Particularly abundant are the GTs that transfer a sugar residue from an activated nucleotide sugar donor, to specific acceptor molecules, forming glycosidic bonds. Glycosyltransferases are classified into 87 different families based on substrate/product stereochemistry according to the CAZy database (30). Knowledge of the sequences of the ABO genes (9) have established that mammalian Glycosyltransferases A and Glycosyltransferases B are type II integral membrane proteins containing 354 amino acids and are localized in the lumen of the Golgi apparatus. These enzymes typically have a short amino-terminal cytoplasmic tail, a hydrophobic membrane domain, a short protease-sensitive stem region and a large catalytic domain that includes the carboxy terminus(31). GTA and GTB are very similar in the coding regions and the soluble enzyme can be found in serum (32),urine (33)and milk (34).The enzyme contains an acceptor recognition domain that binds H antigen and a donor recognition domain that binds UDP-GalNAc or UDP-Gal. GTA and GTB require the metal ion Mn++ (manganese) for activity (35). Glycosyltransferases are antigenic structures. Human antibodies to blood group transferases are often produced following organ transplantation (36-39). 2.4. ABO Subgroups 2.4.1. A and B Subgroups An ABO blood group subtype is called a subgroup and/or a variant. Subgroups of ABO are distinguished by decreased amounts of A, B or O (H) antigens on red blood cells. The most common are subgroups of A and B. Blood type A appears to have the most variation in subgroups. The two most common subgroups of blood group A are A1 and A2 expressing on average, 1 million and 250 000 A determinants, respectively (40). 33 2.4.1.1. A Subgroups The A1 (A101) allele is the reference allele often denoted as the "consensus sequence" in an ABO genotype context (9),but has additionally eight variant alleles. There are variants of A1 alleles, one of which is very common in Asian populations with a 467C→T (A102) polymorphism resulting in the substitution Pro156Leu (41).Two minor A1 alleles (A103 and A104) have been described and differ as follows. The first one also has the 467C→T point mutation but an additional silent mutation 567C→T, the second allele contains a silent polymorphism in nucleotide 297A→G (42). A105 is like A102 with the same mutation in exon 7, 467C→T but analysis of intron 6 showed additional single-nucleotide polymorphism (SNP) compared to A102. Another A1 allele (A106) contains both 297A→G and 467C→T (43). The A2 subgroup is the most common A phenotype after the A1 subgroup. The main genetic difference between A1 and A2 alleles is one point mutation in exon 7, 467C→T (Pro156Leu), and a deletion of one of the three cytosines at nt. 1059-1061 (CCC to CC). The latter mutation results in an extension of the reading frame by 64 nucleotides. This deletion occurs in the codon before the translation stop codon (TGA), resulting in a gene product with an extra 21 amino acid at its C-terminus. The glycosyltransferases encoded by the A2 allele have lower efficiency, leading to a weaker A phenotype. The enzyme activity is decreased by 30-50 times compared to A1 (22).Other variants of A2 alleles have subsequently been elucidated (A202-206). Three A2-like alleles were shown to have three different single mutations near the 3´ end of exon 7 by Ogasawara, A2-2 (A202) contains 1054C→T (Arg352Trp), A2-3 (A203) with 1054C→G (Arg352Gly) (42) and A2-4 (A205) with both 467C→T and an additional new mutation 1009A→G (Arg337Gly) (44). A204 with four common B-related base substitutions (297A→G, 526C→G, 657C→T and 703G→A) seems to be a hybrid with two extra substitutions 771C→T (silent mutation) and 829G→A (Val277Met). One other rare A2 subgroup, A2-5 (A206) carried only the single deletion (1061delC) (45,46). 32 2.4.1.2. B Subgroups Subtypes of blood type B are classified by the quantity of B antigen, and the amount of B antigen decreases in the order B, B3, Bx, Bm, Bel . B1 allele (B101) showed seven single nucleotide substitutions, 297, 526, 657 , 703, 796, 803 and 930 throughout exon 6 and 7 and later one extra mutation outside the coding region at the 3´ end at nt. 1096 (33). The four amino acids substitutions governed by nt. 526C→G (Arg176Gly), 703G→A (Gly235Ser), 796C→A (Leu266Met) and 803G→C (Gly268Ala) discriminate GTA from GTB (41,47) while the substitutions 297, 657 and 930 are silent. ABO glycosyltransferases can accordingly be described by using the letters A and B to illustrate the derivation of the amino acid at these four residues. GTA would be represented by AAAA indicating the presence of Arg/Gly/Leu/Gly and similarly BBBB would describe the GTB with Gly/Ser/Met/Ala at residues 176/235/266 and 268, respectively. The substitutions at positions 266 and 268 were shown to be responsible for the nucleotide/donor specificity of the transferases (47) and the other residues may have a role in acceptor binding and turnover (48).Some other variants have been reported afterwards that differ from B1 (B101) by lacking the 930 substitution for B2 (B102), the 657 substitution for B3 (B103) (47), the 526 substitution for B4 (B107) (44) and 297 for B108 (43). 2.4.2. O Subgroups The blood group O demonstrates the absence of A and B antigens on the RBC surface in forward blood typing. Reverse blood typing indicates the presence of both anti-A and -B in the plasma. The first O allele (O1-1, [O01]) was shown to be identical to the consensus A allele (A101) except for a nucleotide deletion, 261delG, in exon 6. This results in a shift in the reading frame, giving rise to a truncated protein that alters the protein sequence after amino acid 88. A stop codon halts translation after amino acid 117 and the resulting protein is enzymatically inactive (41). Some other O1-1-like alleles that are characterized by the presence of the 261delG and at least one additional point mutation. A second kind of O allele has the same inactivating deletion (261delG) as the original O allele (O1-1, [O01]), but in addition has nine point mutations spread 31 throughout exons 3 to 7 (41,45) and a further 13 mutations have been found amongst the intron 6 (50). Additional polymorphisms associated with blood group O have been found up to approximately 4300 bp from ABO exon 7 (51), some of which correlated with O1 and O1v alleles (52). Other O alleles not due to 261delG also exist that are caused by other inactivating mutations along the reading frame. The ( O2-1 [O03]) was the first allele described of this type (53,54) which has a critical mutation (802G → A) causing an amino acid change (Gly268Arg) that prevents the enzyme from utilizing the nucleotide sugar donor (55). This O allele comprises approximately 2 to 5 percent of O alleles in Caucasian persons but seems to be absent, or at least very rare, in other populations (54,56). Other rare O alleles, such as O3 [O08] that does not have 261delG but instead contains both the common A2 allele polymorphisms 467C→T and C-deletion at nt. 1059-1061 and an insertion of an extra guanosine in the 7-guanosine sequence at nt. 798-804 (45). Two other rare O alleles lacking 261delG were reported in the Japanese population by Ogasawara O301 [O14] has the missense mutation 893C→T (Ala268Val) on an A102 background whereas O302 [O15] has the nonsense mutation 927C→A (Tyr309Stop) on an A101 background (43). 2.4.3. Weak subgroups In addition to the major phenotypes characterized by either strong or absent haemagglutination with anti-A/-A1 and -B reagents, the ABO blood group system also includes phenotypes in which erythrocytes react weakly with the anti-A and –B reagents, for example A3, Ax, Afinn, Ael, B3, Bx, Bv, Bel, cis-AB (57). It is difficult to determine clearly their specific ABO subgroup by conventional serological methods. The weak subgroups are important in more than one way. First, they risk complicating patient and donor blood group determination. At worst, the wrong group can be assigned if e.g., a weak antigen is missed. Second, they allow us to characterize and understand glycosyltransferase mechanisms by studying the results of mutations in the underlying alleles. 34 2.4.3.1. Weak A alleles The RBCs from individuals with the A3 phenotype agglutinate strongly with anti-A and anti-A,B in vitro but show a large number of free cells. One A3B individual had a novel point mutation, 871G→A (Asp291Asn), on the A1 [A101] (58) and this allele was named A301. Ax alleles are responsible for the rare subgroup Ax and the RBCs typically show a weak positive reaction with anti-A,B and anti-H. There are four base substitutions involved in these alleles; 646T→A (Phe216Ile), 681G→A (silent), 771C→T (silent), 829G→A (Val277Met). The encoded transferase is expected to be 37 a.a. longer than the normal consensus allele, and 16 a.a. longer than A2-encoded transferase. The serological characteristics of some other minor phenotypes included among A subgroups e.g,. Aend, Am, Ay, Afinn, Abantu and the collective description Aw are more unclear. Table 2.2 shows the serological reaction patterns of weak A alleles. Table 2.2 :Serological reaction patterns. RBC reactions with Subgroup of A Anti-A1 in serum Anti-A Anti-A,B Anti-A1 Anti-H A1 4+ 4+ 4+ 0 No A2 4+ 4+ 0 4+ Sometimes Aint 4+ 4+ 2+/3+ 2+/3+ No A3 2+/+mf 2+/+ mf 0 4+ Ax 0/+ 2+/+ 0 4+ No Often Ael 0 0 0 4+ Sometimes Aend + + 0 4+ Afinn + + 0 4+ Sometimes Yes Abantu +(+) +(+) 0 4+ Yes Am 0/+ 0/+ 0 4+ No Ay 0 0 0 4+ No A negative reaction is noted by 0 and positive reactions are denoted from + (very weak agglutination) to 4+ (maximal agglutination).mf mixed field agglutination. 2.4.3.2. Weak B alleles B variants are much more uncommon than A variants, this may reflect the relatively low frequency of the B blood group in many populations. These phenotypes 35 often appear to result from missense mutations at the ABO locus causing single a.a. changes in the GTB. Characteristics of some B variants are summarized in Table 2.3 (57). Table 2.3 : Characteristics of some more frequent, B weak phenotypes RBC reactions with Subgroup of B Anti-B in serum Anti-A Anti-A,B Anti-B Anti-H B 0 ++++ ++++ ++ None B3 1 mf mf +++ None Bx 1 w w +++ Yes Bel 0 w 0/w +++ None Bm 0 0 0 +++ Sometimes mf, mixed field; w, very weak agglutination. The B3 phenotype have missense mutation 1054C→T (Arg352Trp) on a B1 (B101) background and named B301 (58). A Bx allele responsible for the Bx phenotype had a point mutation at nt. 871G→A (Asp291Asn) (59) which was also found in an A3 sample (58). The Bel phenotype was divided into two suballeles, Bel-1 and Bel-2 (Bel01 and Bel02), which had substitutions at 641T→G (Met214Arg) and 669G→T (Glu223Asp), respectively (59), these missense mutations reduce the enzymatic activities of the GTB. Ogasawara et al. (1996) also found another B3 allele, B302, which differs from B consensus, by two nucleotide substitutions, 646T→A (Phe216Ile) and 657T→C. CisAB alleles rare phenotype was described by Seyfried in 1964. It represents a very interesting phenomenon that proved that it is possible for a child with blood group O to have a parent with blood group AB. Seyfried hypothesized that both A and B determinants of the AB blood group could be located on the same chromosome. Later on, this hypothesis confirmed in that the existence of an exceptional ABO allele encoding a glycosyltransferase is indeed able to produce both A and B enzymes at the same time (57). Cis-ABO1 was sequenced and showed the substitution 803G→C (Gly268Ala) on the A1-2 (A102) background and thus can be described as AAAB (60) . Another allele named cis-ABO2 was discovered when a Vietnamese man who was to undergo organ transplantation showed irregular blood grouping results. The sequencing 36 showed that his ABO genes were nearly identical to the normal B allele except for a 796A→C (Met266Leu) substitution (61). 2.5. ABO antibodies Antibodies are immunoglobulin proteins secreted by B-lymphocytes after stimulation by a specific antigen. The antibody formed binds to the specific antigen in order to mark the antigen for destruction. The type of antigenic exposure occurring in the body determines if the antibody is a naturally occurring or immune antibody. The term ‘naturally occurring’ is used for blood group antibodies produced in individuals who have never been transfused with red cells carrying the relevant antigen or been pregnant with a fetus carrying the relevant antigen. Naturally occurring antibodies can be formed after exposure to environmental agents that are similar to red cell antigens, such as bacteria, dust or pollen (62). Most of these antibodies are not clinically significant with the exception of ABO antibodies. It is possible that food and environmental antigens (bacterial, viral, or plant antigens) have epitopes similar enough to A and B glycoprotein antigens. The antibodies created against these environmental antigens in the first years of life can cross-react with ABO-incompatible red blood cells (RBCs) that it comes in contact with during blood transfusion later in life. Anti-A antibodies are hypothesized to originate from immune response towards influenza virus, whose epitopes are similar enough to the α-D-N-galactosamine on the A glycoprotein to be able to elicit a cross-reaction. Anti-B antibodies are hypothesized to originate from antibodies produced against Gram-negative bacteria, such as E. coli, cross-reacting with the α-D-galactose on the B glycoprotein (49). Anti-A and anti-B antibodies (called isohaemagglutinins), which are not present in the newborn, appear in the first years of life. They are isoantibodies, that are produced by an individual against antigens produced by members of the same species (isoantigens). Anti-A and anti-B antibodies are usually IgM type, which are not able to pass through the placenta to the fetal blood circulation and react best at room temperature or lower. The ABO antibodies may also be found in various body fluids including saliva, milk, cervical secretions, tears, and cysts. These antibodies are detected at about 3 months and increase their titer until the 5th to 10th year of life . 37 Sera from A individuals contain anti-B antibody while B individual's sera contain two types of antibody against A antigens. The first is anti-A and the second one is specific towards A1 RBCs. Anti-A reacts with both A1 and A2 cells whereas the second only does with A1 RBCs. Anti-A1 is also present in some A2 and A2B individuals (63). Group O people produce an antibody, anti-A,B able to cross-react with both A and B RBCs. 2.6. Studies on ABO Genotype Olsson et al (1995) studied the ABO genotype in 300 Danish blood donors with a method using six restriction enzyme digestions following three different PCRs detecting polymorphism at nucleotide positions (nt) 261, 526 and 703. The results of the study showed that about 3% of the O alleles were of the new type of O gene, O1. The common O allele was assigned O2. They found a restriction enzyme site for HpaIl that is only present in non-B/ O2 alleles (64). Fukumori et al (1995) performed the genotyping of ABO blood groups on a Japanese population using the polymerase chain reaction (PCR) method. The 4 DNA fragments containing the nucleotide position 261, 526, 703 and 796 of cDNA from Atransferase were amplified by PCR, and the amplified DNA was subjected to restriction fragment length polymorphism (RFLP) analysis. The different nucleotide at position 803 was distinguished by electrophoresis of the PCR products amplified with allelespecific primers. By analyzing the electrophoresis patterns. The frequencies of ABO genotypes found in Japanese blood donors with A and B phenotypes were as follows: in the phenotype A group, AA =19.8 % and AO = 80.2%; and in the phenotype B group, BB =12.8% and BO=87.2% (65). Ogasawara et al (1996) investigated the polymorphism of the ABO blood group gene in 262 healthy Japanese donors by a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) method, and 13 different alleles were identified. The number of alleles identified in each group was 4 for A1 (called ABO* A101, A102, A103 and A104), 3 for B (ABO* B101, B102 and B103), and 6 for O (ABO* O101, O102, O103, O201, O202 and O203). Nucleotide sequences of the amplified fragments with different SSCP patterns were determined by direct sequencing. These alleles were classified into three major lineages, A/O1, B and O2. In 38 Japanese, A102 and B101 were the predominant alleles with frequencies of 83% and 97% in each group, respectively, whereas in group O, two common alleles, O101 (43%) and O201 (53%), were observed (44). Akane et al (1996) isolated DNA from peripheral blood leukocytes of 24 unrelated Japanese individuals, ABO phenotypes of these samples were identified by serological methods. Using primers, 200 base-pair (bp) fragment of ABO locus was amplified by PCR, which spans the site of the single nucleotide deletion associated with O allele. O allele identified by Kpn I digestion of the PCR product. A and B alleles were distinguished by Mae II digestion of the product. The nucleotide substitution in the 200bp product between A and B alleles was also found in O allele, resulting in 2 different suballeles OA and OG (66). Al-Bustan et al (2002) genotyped the ABO blood group system in a Kuwaiti population sample using polymerase chain reaction—restriction fragment length polymorphism (PCR-RFLP) analysis. The positions of nucleotides 258 and 700 of cDNA from A transferase were amplified by PCR. The amplified DNA was subjected to RFLP analysis to distinguish A, B, and O alleles. Blood samples of known ABO phenotype from 101 healthy unrelated Kuwaiti individuals (A, 29; B, 23;AB, 14; O, 35) were used. Two DNA fragments of the ABO locus were designed to be amplified by 2 pairs of primers. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with KpnI. For the 700th nucleotide, a 128-bp DNA fragment was amplified by PCR and digested with AluI. By analyzing the electrophoresis patterns the DNA fragments were examined. The result of the study showed that ABO genotypes of the known 101 samples were as follows : AA, 4.30% ; AO, 24.41% ;BB , 4.16% ; BO, 24.2% ; AB, 8.46% ; and OO, 34.65% (67). Seltsam et al (2003) analyzed the complete genomic sequences, except intron 1, and 2 regulatory regions of 6 common (ABO*A101, ABO*A201, ABO*B101, ABO*O01, ABO*O02, and ABO*O03) and 18 rare ABO alleles, by phylogenetic analysis and correlating sequence data with the ABO phenotypes. They revealed multiple polymorphisms in noncoding regions. The analysis revealed 5 main lineages: ABO*A, ABO*B, ABO*O01, ABO*O02, and ABO*O03. Phenotype-genotype correlation showed that sequence variations within the complete coding sequence can 39 affect A and B antigen expression. All variant ABO*A/B alleles and one new ABO*O03- like allele were associated with weak ABO phenotypes (10). Natsuko et al (2004) studied ABO genotypes from samples obtained from 1134 randomly selected Japanese peripheral blood samples. A simple ABO genotyping method using multiplex sequence-specific PCR and capillary electrophoresis was developed as a supplement to serological ABO typing. They found a concordance rate of 99.82% (1132/1134 samples) between genotypes and phenotypes defined as groups A, B, AB, and O. Sequencing analysis revealed that one discrepant sample contained an O allele having a point mutation at the primer binding site in exon 6, and another discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the O301 allele) (68). K. Honda et al (2004) determined the ABO genotypes of 958 DNA samples extracted from individuals living in Japan, Mongolia, and Colombia by using SingleStrand Conformation Polymorphism (SSCP), which detects only one-base difference between different genotypes. The denatured single-stranded amplicons were electrophoresed in sequencing gel, analyzed by laser detector, and visualized the peak patterns of chromatogram. As a result, they were able to classify ABO genotypes into 15 groups and additional subtypes. Ten kinds of fundamental genotypes (AA, AOA, AOG, BB, BOA, BOG, OAOA, OAOG, OGOG, and AB) by the combination of a base substitution of np261 (G/del) and np297 (A/ G) were detected. In addition, examination of 400 DNA samples from Japan, Mongolia, and Columbia revealed a remarkable regional deviation in allele frequency of A101 versus A102, and OA vs. OG (69). Hanania et al (2007) determined the phenotypic, allelic frequencies and the genotypes of ABO blood groups in a Jordanian population. Samples of 12215 randomly healthy Jordanian voluntary blood donors during the period 1998-2003 were taken from the National Blood Bank donor registry, Amman, Jordan. The results of the phenotypic distribution indicated that 4686 (38.36%) of the donors were type A, 4473 (36.62%) O, 2203 (18.04%) B and 853 (6.98%) AB. The gene frequencies were 0.6052 for Io allele, 0.2607 for Ia allele and 0.1341 for Ib allele. Using PCR-RFLP technique, two separate segments of the transferase gene containing nucleotide 261 in exon 6 and nucleotide 703 in exon 7 of the ABO gene locus were amplified and their products were analyzed 21 with two restriction enzymes (KpnI and AluI). The electrophoresis patterns of 105 samples showed that ABO genotypes were AA: 6 (5.714%), AO: 35 (33.333%), BB: 1 (0.953%), BO: 14 (13.333%), AB: 10 (9.524%) and OO: 39 (37.143%) (70). EL-Zawahri and Luqmani (2008) examined the genotype of a 355 unrelated blood donors of phenotype A1 (46), A2 (31), A1B (6), A2B (4), B (97) and O (171) by using a multiplex PCR-RFLP technique in a Kuwaiti Arab cohort. DNA fragments of 252 (251 for O1) and 843 (842 for A2) bp spanning the two major exons, 6 and 7, of the ABO gene were amplified and digested with HpaII and KpnI. They identified 13 different genotypes combining the A1, A2, B, O1 and O2 alleles from the digestion patterns: 1 A1A1 (0.28%), 6 A1A2 (1.69%), 38 A1O1 (10.71%), 1 A1O2 (0.28%), 1 A2A2 (0.28%), 30 A2O1 (8.45%), 6 A1B (1.69%), 4 A2B (1.13%), 12 BB (3.38%), 79 BO1 (22.25%), 6 BO2 (1.69%), 167 O1O1 (47.04%) and 4 O1O2 (1.13%). Two of the combinations (A2O2, O2O2) were not found (71). Sung et al (2009) evaluated ABO genotypes via multiplex allele-specific PCR (ASPCR) amplification using whole blood samples without DNA purification of 127 randomly chosen samples. The genotypes of the 127 samples were found to be A1A2 (n=1), A2A2 (n=9), A1O1 (n=3), A2O1 (n=12), A2O2 (n=14), B1B1 (n=5), B1O1 (n=18), B1O2 (n=15), O1O1 (n=14), O2O2 (n=8), O1O2 (n=14) and A2B1 (n=14), from which phenotypes were calculated to be A (n=39), B (n=38), O (n=36) and AB (n=14). They found no discrepancies when the multiplex AS-PCR assay results were compared with the serologically determined blood group phenotypes and genotypes determined by DNA sequencing (72). Nojavan et al (2012) examined the genotype of 744 randomly selected samples from Azari donors of East Azerbaijan province (Iran) using multiplex allele-specific PCR ABO genotyping technique. As a result, the ABO blood group genotypes were: 1.2% A1A1,0.4% A1A2, 4%A1B1, 2.4% A1O1, 14.1% A1O2, 3.2% A2A2, 6%A2B1,5.2% A2O1, 6.9% A2O2 , 1.6% B1B1, 11.3% B1O1, 10.5 % B1O2, 9.3% O1O1, 15.3% O1O2, 8.5% O2O2 (73). 23 Bugert et al (2012) determined the major ABO alleles by PCR amplification with sequence-specific primers (PCR-SSP) in a representative sample of 1,335 blood donors in Germany. The genotypes were compared to the ABO blood groups registered in the blood donor files. Then the ABO phenotypes and genotypes were determined in 95 paternity trio cases that have been investigated. They found that the prevalence of the major ABO alleles and genotypes corresponded to the expected occurrence of ABO blood groups in a Caucasian population. In 12 of 35 exclusion cases (34.3%) the ABO genotype also excluded the alleged father, whereas the ABO phenotype excluded the alleged father only in 7 cases (20%) (74). 2.7. ABO Genotyping and Susceptibility to Diseases The presence of the A and B blood group antigens, expressed on red blood cells and other cells and molecules within the body, has been associated with susceptibility to diseases like cancer, leukemia, cardiovascular disease and risk of both arterial and venous thrombosis. Most studies indicated an increased risk of thrombosis associated with the non-O blood group (75,76). Non–group O patients have a greater risk of venous thromboembolism (VTE) than patients of group O and have greater levels of von Willebrand factor (vWF) and factor VIII (75,77). The risk of VTE is probably related to the level of vWF and factor VIII because patients of group A2 have lower levels of these proteins than A1, B, and AB and have a lower risk of VTE (76). A, B, and H blood group antigens are expressed on N-glycans of vWF and influence the halflife of the protein, providing an explanation for the greater levels in non-O patients, which increase clot formation in non-O patients (78). 22 CHAPTER 3 MATERIALS & METHODS 21 CHAPTER 3 MATERIALS & METHODS 3.1. Materials 3.1.1. PCR primers The nucleotide sequence of the PCR primers used in the current study was as described by Yamamoto et al (79). The nucleotide substitutions in the primers are focused on the nucleotide positions 261, 297, 467, and 803, so as to discriminate between the A101, A102, B101, O01, O02, and cis-ABO1 alleles. Table 3.1 shows the oligonucleotide primer sequences, their combinations, amplification product lengths, and allele specificities. The 3′ base of each primer (except int6) was designed to correspond to the nucleotides at positions 261, 297, 467, and 803, which define the polymorphisms. Table 3.1: PCR primers sequence used for ABO genotype PCR reaction 1 2 3 4 Primer pair Fragment size (bp) Allele specificity 261G: 5′-GCAGTAGGAAGGATGTCCTCGTGTTG-3′ int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′ 467C: 5′-CCACTACTATGTCTTCACCGACCATCC-3′ 803G: 5′-CACCGACCCCCCGAAGATCC-3′ 205 A101, A102, B101, cis-ABO1 381 A101, O01, O02 297A: 5′-CCATTGTCTGGGAGGGCCCA-3′ int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′ 467C: 5′-CCACTACTATGTCTTCACCGACCATCC-3′ 803C: 5′-CACCGACCCCCCGAAGATCG-3′ 164 A101, A102, O01, cis-ABO1 381 B101 261A: 5′-GCAGTAGGAAGGATGTCCTCGTGTTA-3′ int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′ 467T: 5′-CCACTACTATGTCTTCACCGACCATCT-3′ 803G: 5′-CACCGACCCCCCGAAGATCC-3′ 205 O01, O02 381 A102 297G: 5′-CCATTGTCTGGGAGGGCCCG-3′ 164 B101, O02 int6: 5′-AGACCTCAATGTCCACAGTCACTCG-3′ 467T: 5′-CCACTACTATGTCTTCACCGACCATCT-3′ 381 cis-ABO1 803C: 5′-CACCGACCCCCCGAAGATCG-3′ The primers are named based on the position of their 3 ′ end relative to the cDNA sequence of A101 allele. 24 3.1.2. Kits DNA extraction kit (Promega , USA) 3.1.3. Reagents and Chemicals Anti A (Plasmatec, Monoclonal, UK) Anti B (Plasmatec, Monoclonal, UK) Agarose Molecular biology grade (Promega, USA) DNA molecular weight marker 50 bp-ladder (Promega, USA) EDTA disodium salt (Promega, USA) Absolute Ethanol (Sigma, USA) Ethidium bromide (Promega, USA) Absolute Isopropanol (Sigma, USA) Tris base " hydroxyl methyl amino methane" (Promega, USA) DNAse , RNAse free water (Promega, USA) Acetic acid (Sigma, USA) PCR Master mix (Promega, USA) 3.1.4. Apparatus and Equipments Thermal Cycler (Biometra, Germany) L.G Microwave Oven Electrophoresis Apparatus Vortex Mixer Digital Camera Power Supply (Biorad) Freezer, Refrigerator Micro-Centrifuge Hoefer shortwave UV light table (Transilluminator) Computer Electrical Balance Automatic Micropipettes 25 3.2. Methods 3.2.1. Study population 3.2.2. Sample collection Blood samples were collected from 201 subjects recruited from the Islamic University –Genetics Laboratory. Each sample was collected into EDTA tube .The EDTA samples were kept at 4°C and were used within 24 hours for forward blood grouping and DNA extraction and subsequent PCR analysis. 3.2.3. Ethical Considerations An authorization to carry out the study was obtained from a local ethics committee using an agreement letter prepared by the Islamic University of Gaza. All the information that were obtained about the subjects were kept confidential. 3.2.4. Data Analysis The data were entered, stored and analyzed by personal computer using the Statistical Package for Social Sciences (SPSS) version 16.0. Allele frequencies were calculated under the assumption of Hardy–Weinberg equilibrium and expressed as percentages. Chi-square test was used to compare observed allelic and genotypic frequency distributions of the blood group antigens to that expected under the Hardy– Weinberg equation . P values >0.05 were considered statistically significant. The frequency of ABO in the studied population were compared with the frequency of ABO in some neighbor countries. 3.3. Blood ABO-typing 3.3.1. Forward blood group Whole blood sample (50 µl) was mixed with Anti A and Anti B reagents by using slide method in the proportions shown in Table 3.2. below. 26 Table 3.2. Sample / Anti A, Anti B reaction for forward blood grouping Sample Anti -A Anti -B 50 µl 50 µl ــــــ 50 µl ــــــ 50 µl The contents of each slide were rotated, then the agglutination (if present) was read by naked eye after 30 seconds. 3.4. ABO Genotyping 3.4.1. DNA Extraction DNA was isolated from fresh EDTA whole blood cells by using Promega kit for human DNA isolation. The kit contains the following components that are enough for purifying genomic DNA from 200 samples of human blood: Cell lysis solution Nuclei lysis solution Protein precipitation solution DNA rehydration solution RNase solution The human genomic DNA was isolated from human blood sample according to the kit instructions and was as follows: Three hundred µl EDTA blood were transferred into a sterile 1.5 ml microcentrifuge tube containing 900 µl of cell lysis solution. The tube was inverted 5-6 times to mix the components. The mixture was incubated for 10 minutes at room temperature (with gentle mixing once during the incubation) to lyse the red blood cells . The tube was then centrifuged at 13,000 rpm for 20 seconds at room temperature. Supernatant was removed and discarded as much as possible without disturbing the visible white pellet. Approximately 10-20 µl of residual liquid should be left in tube. The tube was vortexed vigorously until the white blood cells were completely resuspended. Three hundred µl of nuclei lysis solution was then added to the tube 27 containing the resuspended cells and the suspension was mixed by pipetting the solution 5-6 times to lyse the white blood cells; the solution should become very viscous. RNase solution (1.5 µl) was added to nuclear lysate and the sample was mixed by inverting the tube 2-5 times. The mixture was incubated at 37°C for 15 minutes, and then cooled to room temperature. One hundred µl of protein precipitation solution was added to the nuclear lysate, and the mixture was vortexed vigorously for 10–20 seconds. Small protein clumps may be visible after vortexing, and were removed by centrifuge precipitation at 13,000 rpm for 3 minutes at room temperature. The supernatant was transferred to a clean 1.5 ml micro-centrifuge tube containing 300µl isopropanol at room temperature. The mixture was gently mixed by inversion until the white thread-like strands of DNA form a visible mass. The DNA was then precipitated by centrifugation at 13,000 rpm for 1 minute at room temperature. The DNA would be visible as a small white pellet. The supernatant decanted and one volume of 70% ethanol was added to the DNA and kept at room temperature and gently inverted several times to wash the DNA pellet and the sides of the micro-centrifuge tube . After centrifugation at 13,000 rpm for 1 minute , the ethanol was aspirated using a suitable pipette . The DNA pellet is very loose at this point and care must be used to avoid aspirating the pellet into the pipette. The tube was inverted on a clean absorbent paper for 10–15 minutes in order to air-dry the pellet. DNA rehydration solution was added to the dry pellet and the DNA was rehydrated by incubation at 65°C for 1 hour. Periodically the solution was mixed by gently tapping the tube. The DNA was then stored at 2-8°C. 3.4.2. Detection of extracted DNA The quality of the isolated DNA was determined by running 5 µl of each sample on ethidium bromide stained 3.0% agarose gels and the DNA was visualized on a short wave U.V. transilluminator, the results were documented by photography. 3.5. PCR reactions PCR was performed using the primers listed in Table 3.1 in 4 micro-tubes as described by Sung Ho Lee et al (70). For each PCR, 5 μl master mix (Promega), 2 μl deionized water, 1 μl DNA template and 0.5 μl of each allele specific primer (5 pmol) in 28 one micro-tube (0.2 ml) were mixed. The volume and concentration of a typical PCR reaction are shown in Table 3.2. PCR was performed in a thermal cycler. The cycling conditions were as described below. In each PCR, MTHFR gene specific primers with the following sequences: (Forward: 5′-ACGATGGGGCAAGTGATGCCC -3' and reverse: 5'GAGAAGGTGTCTGCGGGATC-3') were used as a positive control that produces a 95 bp fragment. Upon completion of PCR, the products were analyzed by electrophoresis on 2% ethidium bromide stained agarose gel, or stored at 4°C until analysis. Table 3.3. Composition of PCR master mix. Reagent dNTPs Taq DNA Polymerase MgCl2 Composition 400µM each :dATP, dGTP , dCTP , dTTP 50 units/ ml 2mM 3.5.1. Temperature cycling program The thermal cycler program was set as follows: Step 1 . Denaturation for 3 minutes at 95°C Step 2 . 35 cycles of: 2.1. Melting for 40 seconds at 95°C 2.2. Annealing for 40 seconds at 58.5°C 2.3. Extension for 40 seconds at 72°C Step3. Final elongation for 5 minutes at72°C 3.5.2. Expected PCR results The PCR product size was estimated by comparing it with DNA molecular size marker (50 bp ladder DNA ) run on the same gel. 29 CHAPTER 4 RESULTS 11 CHAPTER 4 RESULTS 4.1. Study Population The study population consisted of 201 subjects (50 males , 151 females ). The percentage of males was 24.9% while that of females was 75.1% ( figure 4.1). 75.3 % 80 70 60 50 24.9 40 30 20 10 0 Female male Figure 4.1: Distribution of the study subjects according to gender. 4.2. Phenotypic Frequency of ABO Blood Groups Blood grouping was done by antigen antibody agglutination test by using commercial monoclonal antisera . The distribution of phenotypes in the total sample were 36.3% (73), 22.4% (45) , 7.5% (15) , 33.8% (68) for groups A, B, AB and O, respectively. Group A was the dominant one in both genders, and AB was the rarest in both males and females ( table 4.1). Table 4.1: Phenotypic frequencies of various blood groups in the study population Phenotype Subject Sex Total A B AB O Male 16 10 4 20 50 Female 57 35 11 48 151 Total 73 45 15 68 201 13 4.3. The Allele Frequencies of ABO Antigens Allele frequency for the antigens was computed by the Hardy-Weinberg law, on the basis of the number of subjects with blood groups, ABO . The distribution of the alleles in the total samples was 0.25, 0.17, 0.58 for IA, IB and IO, respectively. All genotyping results were compatible with the determined phenotypes by serological method. The observed genotypes as compared with the expected genotypes are shown in Table 4.2. The frequencies of the five alleles in the our sample population were : O1 and O2 alleles : 0.376 and 0.221, respectively , while A1 : 0.174 , A2: 0.067 , B : 0.162. Table 4.2 : Observed and expected genotypes for the 201 samples . Observed Expected Phenotype Genotype P-Value Number Percent Number Percent AA 9 4.5 12.6 6.3 0.230 AO 64 31.8 58.3 29 0.4762 BB 5 2.5 5.8 2.9 0.7205 BO 40 19.9 39.6 19.7 0.9496 AB AB 15 7.5 17.08 8.5 0.5915 O OO 68 33.8 67.6 33.6 0.9613 201 100 201 100 A B Total The statistical analysis ( using chi square test ) indicated that molecular data were in good agreement with the ratio calculated from the estimated gene frequencies of the ABO blood group system in the Gaza Strip population. 12 4.4. PCR Results 4.4.1 Quality of the isolated DNA Regarding the method of DNA isolation that was described in chapter 3, the quality and quantity of DNA were suitable for PCR processing . The quality of the isolated DNA from human subjects is represented in the following figure (4.2). Figure 4.2: A representative photograph of DNA isolated from human blood. The samples were run on 1% agarose gel stained with ethidium bromide. 4.4.2. Blood group genotyping by Allele Specific PCR Multiplex Allele Specific PCR (ASPCR) assay contains four independent PCR reactions (Table 3.1). In the first reaction, 261G-int6 primer pair was used to amplify the 205 bp fragment to detect the A101, A102, B101, cis-ABO1 alleles, and the 467C803G primer pair was selected to amplify the 381 bp fragment to detect the A101, O01 and O02 alleles. The 297A-int6 primer pair and the 467C-803C primer pair in the second reaction were selected to amplify the 381 bp fragment to detect the B101 allele. In the third reaction, the 261A-int6 primer pair was selected to amplify the 205 bp product to detect the O01 and O02 alleles, and the 467T-803G primer pair was selected to amplify the 381 bp product to detect the A102 allele. Finally in the fourth reaction, the 297G-int6 primer pair were applied to produce the 164 bp fragment to detect the B101 and O02 alleles, and the 467T-803C primer pair to produce the 381 bp fragment to recognize the cis-ABO1 allele. Table 4.3 show the products of PCR reactions according to the genotype . 11 Table 4.3: PCR products according to the genotype. PCR Reactions Genotype R1 R2 R3 R4 381 164 205 _ O1O1 381 _ 205 164 O2O2 381 164 205 164 O1O2 205 381 _ 164 B1B1 381,205 381,164 205 164 B1O1 381,205 381 205 164 B1O2 381,205 381,164 _ 164 A1B1 205 381,164 381 164 A2B1 381,205 164 _ _ A1A1 381,205 164 205 _ A1O1 381,205 164 205 164 A1O2 381,205 164 381 _ A1A2 205 164 381 _ A2A2 381,205 164 381,205 _ A2O1 381,205 164 381,205 164 A2O2 Random mating, with the six different alleles at the ABO locus, can result in 21 different genotype combinations. Only 15 different genotype combinations were detected from the 201 investigated samples (Figure 4.3) A- 1. O1O1 2. 14 O2O2 B- C- D- E- 1. 1. B1B1 2. O1O2 B1O1 2. A1B 1. 1. A1A2 2. A1O1 2. 15 A2O2 A1A1 F- G- 1. 1. A1O2 2. B1O2 3. 2. A2O1 A2A2 A2B1 Figure 4.3: The electrophoresis pattern of recognized genotypes using the multiplex ASPCR method .This figure shows 15 different genotypes . lane M indicates the 50 bp ladder. The R1R4 show the PCR reaction number. 4.4.3. Genotype Frequencies The frequency of ABO recognized genotypes belonging to the population residing in Gaza Strip are shown in table 4.4. As shown in the table the highest allele frequencies in A , B , AB and O phenotypes were A1O2 (12.4%) , B1O1 (12.9%) , A1B (5.5%) and O1O1 (15.4%), respectively. 16 Table 4.4: The frequency of recognized genotypes using multiplex ASPCR method for a population residing in Gaza Strip Genotype Total A1A1 Frequency 3 Percent 1.5 A1O1 24 11.9 A1O2 25 12.4 A1A2 4 2.0 A2A2 2 1.0 A2O1 13 6.5 A2O2 2 1.0 B1B1 5 2.5 B1O1 26 12.9 B1O2 14 7.0 A1B 11 5.5 A2B 4 2.0 O1O1 31 15.4 O1O2 26 12.9 O2O2 11 5.5 201 100.0 17 CHAPTER 5 DISCUSSION 18 CHAPTER 5 DISCUSSION Since the first delineation of the molecular basis of the ABO blood group by Yamamoto et al. (11, 79), it has become possible to determine the ABO genotypes using molecular methods without the need for family investigations. ABO genotyping is commonly used in cases of an ABO discrepancy between cell typing and serum typing, as well as in forensic practices for personal identification and paternity testing (72). The ABH antigens are ubiquitously expressed in humans and are present primarily as glycoproteins and partly as glycolipids. The ABO locus is found on the long arm of chromosome 9 (9q34.1-q34.2). Multiplex allele specific PCR (the technique we used here) has advantages over PCR-RFLP in terms of cost effectiveness, reaction time and simplicity of handling. It is a rapid method that is based on detecting four single nucleotide polymorphisms at nucleotides 261, 297, 796, and 803 of the ABO locus which in turn discriminate the major ABO alleles. In this study, the ABO genotypes of 201 samples recruited from the Islamic University –Genetics Diagnosis Laboratory were determined using multiplex AS-PCR method. The objectives of this study were to correlate ABO genotypes with phenotypes in blood samples in a Gaza Strip population and to compare the frequency of ABO genotypes with other populations. 5.1. The Allele Frequencies of ABO Antigens in Gaza Strip The frequency of the alleles in the total samples were 0.250 for the IA allele, 0.170 for the IB allele and 0.580 for the IO allele. This distribution is in agreement with the distribution with the samples reported in an Iranian study were they reported: 0.23974 for IA allele, 0.18147 for IB allele and 0.57879 for IO allele (73). The frequencies of IA , IB and IO alleles in our subjects are also comparable to those reported in Iraqi and Jordanian studies where they found 0.212 for IA allele , 0.177 for IB allele and 0.6611 for IO allele and 0.270 for IA allele, 0.130 for IB allele and 0.60 for 19 IO allele, respectively (82,83). The results showed that the frequency of IO allele is higher than either IA or IB and that of IA is higher than IB i.e., the trend is Io ˃ IA ˃ IB . Our results showed that the distribution of ABO alleles in Gaza Strip is similar to that reported for other Arabian and Iranian populations as shown in Table 4.4 which represents the distribution of ABO alleles in different human populations. This result may imply that those populations share common ancestry. 5.2. ABO Genotype Frequencies The method employed here for blood group genotyping discriminates A1, A2, O1, O2, and B alleles. All expected alleles and allelic combinations were observed in this group except cis-ABO1 allele and its pertinent genotypes. The results obtained from AS-PCR for the investigated 201 samples showed that the frequencies of the various ABO genotypes were: AA: 9 (4.48%), AO: 64 (31.84%), BB: 5 (2.49%), BO: 40 (19.9%), AB: 15 (7.46%) and OO:68 (33.83%). When compared to other studies, these percentages are close to those reported by Irshaid et al (2007) in Jordan where they have reported the following frequencies: AA:5.71%; AO:33.333%; BB:0.953%; BO:13.333%; AB: 9.52% and OO: 37.143% (83). According to our results it was found that the highest frequency of A phenotype (64/73,87.67%) had the AO genotype, while (9/73,12.33%) were A phenotype homozygotes (AA). Forty samples with phenotype B were recognized as heterozygous (BO) and 5 samples with phenotype B recognized as homozygous (BB). On the other hand, (68/201, 33.8%) were homozygous (OO) and 15 (7.46%) samples were AB heterozygotes. The high heterozygosity observed in this study is mainly due to the high frequency of the IO allele (0.58) in our population as compared to that of IA (0.250) and IB (0.170) alleles. Additionally, the samples investigated here were collected from unrelated individuals and that allowed for the random assortment of the alleles according to their frequencies in the population. This is further confirmed by finding that the observed genotypes did not deviate significantly from Hardy-Weinberg equilibrium. 41 Regarding the genotypes observed (Table 4.4), the highest frequency belonged to O1O1 genotype ( as a consequence of the high frequency of O1 allele ) with a frequency of 15.4% (31/201) and the lowest frequency belonged to A2A2 and A2O2 genotypes ( as a result of the low frequency of A2 allele ) with a frequency of 1.0% (2/201) each. In addition, 145 (72.14%) out of all the samples were heterozygous and 56 samples (27.86%) were homozygous. In our population the calculated frequencies of the O1 and O2 alleles were 0.3756 and 0.2214, respectively. These values differ from those reported in other populations e.g., in Kuwaiti population the reported frequencies were 0.6831 and 0.0155 for O1 and O2, respectively (80). Our findings are, however, consistent with many other studies where they reported higher prevalence of the O1 allele (73,80) (Table 5.1). Table 5.1: Frequency of ABO gene Alleles in Gaza Strip in comparison to some other countries Population A Allele frequencies qB 0.170 Reference rO 0.580 Current study Gaza Strip p 0.250 Kuwait 0.1338 0.1676 0.6986 Mokhtar and Yunus 2008 (80) Bahrain 0.141 0.157 0.704 Al-Aarrayed et al 2001 (81) Iraq 0.212 0.177 0.6611 Tills et al 1983 (82) Jordan 0.270 0.130 0.60 Irashaid et al 2002 (83) Saudi Arabia 0.1663 0.1197 0.714 Bashwar et al 2001 (84) Egypt 0.188 0.149 0.663 Khalil et al 1989 (85) Sudan 0.192 0.140 0.668 Khalil et al 1989 (85) 0.23974 0.18147 0.57879 Iran Nojavan et al 2012 (73) Meanwhile, the calculated frequencies of A1, A2 and B alleles were : A1 : 0.17413 , A2: 0.06716 , B : 0.16171 where these are also different in frequency from those of the Kuwaiti study. cis-ABO1 allele was not observed in this study. It is a rare allele which was reported in Korea (0.0354%) among blood donors (86). This allele also was not detected in Kuwaiti or Iranian populations. 43 CHAPTER 6 CONCLUSION and RECOMMENDATIONS 42 CHAPTER 6 CONCLUSION and RECOMMENDATIONS The present study focused on detection of the major ABO genotypes in a Palestinian population residing in Gaza Strip. The results of this study can be summarized as follows: In Gaza Strip , the A phenotype was the most common blood group followed by O,AB and B. The frequencies of ABO alleles in the investigated subjects were 0.25 for IA, 0.17 for IB and 0.58 for IO. These frequencies are comparable to those obtained from ABO genotyping. No statistically significant differences were found between the frequency of observed and expected genotypes .This proved that the ABO genotypes of the randomly collected samples were in Hardy- Weinberg equilibrium data. Molecular data indicated that Hardy-Weinberg equation can be used to detect the percentage of the major blood group in our population . The distribution of the ABO genotypes in Gaza Strip population is similar to that of many Asian populations. There is no significant difference between male and female in terms of ABO phenotypes . Our study indicated that the most common genotype is O1O1 and the lowest are A2A2 and A2O2. The cis ABO1 was not encountered. The homozygous genotype of A and B alleles (AA , BB ) were less than the heterozygous(AO, BO) genotype. The frequency of O2 allele in Gaza strip seems to be higher than that reported in many neighboring countries . This study determined the exact phenotypic frequency of ABO blood groups in Gaza Strip and the frequencies of the prevalent ABO alleles namely, A1, A2, B1, O1 and O2. When both serological typing and ABO genotyping are performed and full compatibility between a phenotype and a genotype is observed, examiners can determine the ABO phenotype with an even higher level of confidence. 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