Introduction

The ABO blood group has been described as one of the most important systems in transfusion and transplantation practice. Antibodies in the ABO system are produced as naturally occurring antibodies to non-self ABO antigens due to a result of individual exposure to substances present in nature [1], individuals greater than six months old have clinically significant anti-A /B antibodies or both. They may result in an immediate haemolytic transfusion reaction if they receive an ABO-incompatible blood. The frequency distribution for ABO groups depend on the population’s ethnic background; however, O and A groups are the most frequent in human, whereas the lowest reported group was AB [2].

ABH antigens are generated by the combination of three different genes (ABO, Hh, and Se) that create glycosyltransferases to add certain monosaccharides at precise linkages to oligosaccharide precursor chains, with the terminal sugar determining antigen specificity [3]. The antigens A, B, and H are all formed from the same basic precursor material (paragloboside or glycan), to which sugars are linked in response to a transferase enzyme elicited by an inherited gene [4]. 

The H antigen is a product of inheritance from the H gene and is characterized as an O blood group in which the precursor for A and B antigens is made. The H(FUT1) and Se (FUT2) genes are both found on chromosome 19q13.3, while the ABO gene is located on chromosome 9q34.2 [5,6]. The fucosyltransferase gene H(FUT1) encodes a fucosyltransferase expressed on red blood cells (RBCs) and adds fucose to type 2 glycoproteins in a (1,2) link to create the H antigen. Se (FUT2) is a fucosyltransferase that is expressed on epithelial cells and adds fucose to type 1 glycoprotein chains in the ratio (1,2) [7]. The ABH antigens are synthesized in low quantities on the surfaces of RBCs during early fetal life and subsequently reach the maximum development capacity by 2 to 4 years of age. Race, genetic interaction, and illness conditions may influence the expression of ABH. 

The ABO gene is divided into seven exons ranging between 28 and 688 bp, exons 6 and 7 are the longest and contain the catalytic domain for ABO glycosyltransferases and predominantly affect the amino acid changes [8-11]

ABO blood group antigens are expressed on other body cells such as platelets, vascular endothelial cells, mucus secretions, and epithelial tissues contributing to elevating the risk of diseases [12]. ABO blood group studies have shown great interest in health and disease, significant association between SNPs present within the glycosyltransferase gene and the risk of pancreatic cancer was confirmed [13,14]. Also, Blood group antigens modify the innate immune response by enhancing the intracellular uptake, adhesion molecules, or signal transduction that affects the infection course in the host body [15]

A Saudi study by Mohamed et al. used a pre-designed multiplex polymerase chain reaction and agarose gel electrophoresis for to detect ABO blood groups. A cohort study by Alzahrani, et al. used in-house molecular techniques to genotype the ABO and Rhesus blood for potential Saudi stem cell donors.

In this study, we aimed to characterize the ABO blood groups in a Saudi sample utilizing the sequence-based typing (SBT) method. The purpose was to identify the single nucleotide polymorphisms (SNPs) within exons 6 and 7 of the ABO gene and constitute reference control data for future genetic disease association studies.

Material and methods

We recruited 96 healthy Saudi individuals for this genetics experimental study with known ABO group phenotypes, as shown in Table 1. IRB clearance is approved by the IRB committee at King Fahad Medical City (KFMC). Whole blood samples were collected in EDTA and the informed consent was obtained. We used the MagNa Pure Compact instrument (Roche Diagnostics) for DNA extraction. DNA quality and quantity were measured using Nanodrop^® 2000c spectrophotometer (Thermo Fisher Scientific).

Primer pairs mol-46/mol-57 and mol-71/mol-101, as described by Yu et al. [18], were utilized to amplify the sequences of interest in exons 6 (251 bp) and 7 (843 bp) and the PRC products were visualized in 3% gel electrophoresis. For the PCR reaction, we used 5 μL of AmpliTaq Gold^™ 360 Master Mix (Thermofisher), 1 μL RNase-Free Distilled Water, 3 μL pooled PCR primers (forward and reverse) and 1 μL DNA template (10ng). Amplification is carried out in Veriti™ 96-Well Fast Thermal Cycler using the following program: 1 cycle of 95°C for 10 min; then 10 cycles of 94°C for 60 s, 63°C for 90 s, and 72°C for 60 s and more 25 cycles composed of 94°C for 60 s, 61°C for 90 s and 72°C for 60 s followed by a final cycle of 72°C for 10 min. We treated amplicons with ExoSAP-IT and proceeded into the second stage for the genotyping reaction (PCR sequencing reaction) using 2 μl of BigTerminatorator v3.1, 1 μl of 5x sequencing buffer, 3 μl of deionized water, and 1 μl forward primer (3.2 μM) or reverse primer (3.2 μM) and 3 μl of clean purified PCR product (PCR product+ExoSAP-IT). The following program: 96°C for 7 min, 25 cycles composed of 96°C for 10 s, 58°C for 5 s and 72°C for 4 min, then hold at 4°C was used. SAM/BigDyeX terminator to purify sequencing reactions was used. The Genetic Analyser 3130xl was used for data collection.

We implemented SNPstat software ((https://www.snpstats. net/start.htm) to detect Hardy-Weinberg equilibrium (HWE) for the expected and observed distribution of polymorphic sites and the Linkage disequilibrium (LD) tests, including D statistic, D’ statistic, r statistic was also calculated.

Results

Based on the serological phenotypes, we have chosen A, B, AB and O blood groups considering male and female representation, as shown in Table 1. Thirteen different locations, including 261 (A>AG) and 267 (C>CT) in exon 6 and 526 (C>CG), 646 (T>TA), 657 (C>CT), 681(G>GA), 703 (G>GA), (771 (C>CT), 796 (C>CA), 803 (G>GC), 829 (G>GA), 1023 (C>AC) and 1061 (del/C) in exon 7 were demonstrated as shown in Table 2.

For the A blood group has shown conformation to HWE (p-value <0.05) except for 261 and 526 locations. The number of polymorphisms not conforming to HWE was increased in the B blood group individuals including 297, 526, 703, 796 and 803 sites. Additionally, three polymorphisms among the AB blood group individuals 261, 657 and 703 also were not observed conforming to HWE and only 803 (G>GC) site in the O blood group not conforming to HWE.

Among the A blood group, we observed more than 90% of individuals were homozygous within the sites of the proteincoding region at exon 6 and exon 7 including 297A, 657C, 703G, 796C, 803G, 1023C, and 1061C. The 261(A>AG) position at exon 6, demonstrated 100% heterozygous (wt/G) in the female group, while only 75% were heterozygous in males and 25% were homozygous (G/G). At positions 526CTA, 681 G>GA, 771 C>CT and 829 G>GA, we observed multiple polymorphisms in both groups but no significant differences between the male and female in the genotype frequencies. 

For the B blood group people, we observed monomorphic genotypes among the male group at 297 (A/A), 526 (C/G), 703 (G/A), 796 (C/A), 803 (G/C) and 1023 (C/C) locations and only 1061 (C/C) in the female group. Variable genotypes (homozygous/ heterozygous) in other locations for exon 6 and exon 7 were detected, but there were no significant differences between males and females when frequency distributions were compared. For the AB blood group, 10/13 locations showed monomorphic genotypes in the female group, including 297 (G/A), 526 (G/G), 646 (T/T), 657 (C/C), 681(G/G), 703 (G/A), 771(C/C), 829 (G/G), 1023(C/C) and 1061(C/C), whereas, only 3/13 location have shown monomorphic genotypes in male. Other polymorphic sites were identified in both male and female groups, but there were no significant differences between the two groups in terms of genotype frequency distributions.   

O blood group genotypes

For the O blood group, similar genotypes in males and females were demonstrated in three genetic sites, including 261(del/del), 1023(C/C) and 1061(C/C), except in the 261 position, one male was observed with del/G genotype. Four more monomorphic sites were detected among the male group but not in females including 526 (C/C), 657 (C/C), 703 (G/G) and 796 (C/C). No statistical significance was observed between males and females in regional sites with polymorphisms. 

Table 1: shows the number and phenotypes on the gender base.