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Sequence variations in rare ABO alleles identified in this study (base changes shown with reference to the A 1 allele)
Source publication
The ABO blood group is clinically the most important blood group system. Elucidation of the molecular basis of the ABO polymorphism allows genotype determination without family studies. Described here is a new method based on the simultaneous amplification by polymerase chain reaction (PCR) of 3 fragments from exon 6, and 5' and 3' ends of exon 7 o...
Contexts in source publication
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... 15 samples were sequenced for exons 6 and 7. (The nucleotide sequences for new alleles identified in this study have been submitted to GenBank [accession numbers: AF182745 to AF182756].) The SSCP banding patterns for each distinct new allele are shown in Figure 1C, and the corresponding base changes underlying these alleles in Table 5. ...
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... SSCP pattern of 2b/1c/3c identified 1 new allele as an A allele (A v1 ) because of its 1c pattern in F1. Apparently, this A v1 allele seemed to be a hybrid A 1 -A 2 allele and resembled the classical A 2 allele in carrying the single C deletion at nt 1059 to 1061, but not the base change at nt 467 ( Table 5). It has been shown that the decreased A transferase activity of the A 2 allele is due to the single C deletion. ...
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... other new alleles had patterns 1a/3b or 1b/3b for F1 and F3 ( Figure 1C and Table 5). F1 producing patterns 1a or 1b had the single-base deletion at nt 261 ( Figure 1B and Table 1), which would result in frameshift and thus produce a truncated and enzymatically inactive protein product. ...
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... summary, 1 A v1 and 7 distinct O v alleles (O v1 -O v7 ) were identified in a total of 18 chromosomes (Table 5). A v1 and O v1 appeared to be hybrid alleles. ...
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... is expected that this method can also detect some known rare alleles with the base changes within the regions bounded by the 3 primer pairs (Table 2), eg, A x and B 3 . 20 This technique is the simplest, quickest, most cost-effective method reported to date to be able to discriminate 7 common ABO alleles in a single-tube single-lane format, and at the same time readily identifies new alleles (Table 5). ...
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... rare alleles identified in this study were characterized by additional base changes on an O 1 or O 1v background. On the other hand, alleles A v1 and O v1 each had one of the standard SSCP patterns in their respective fragments F1 to F3, but in unexpected combinations: 2b/1c/3c and 2e/1a/3b ( Figure 1C and Table 5). These unexpected combinations and the underlying nucleotide sequence data suggest that these 2 alleles might be hybrid alleles (A 1 -A 2 and O 1 -O 1v , respectively) probably generated by intragenic recombination between common alleles. ...
Citations
... Thus, genetic approaches to analyze single nucleotide polymorphism (SNP) present in ABO genes are favored. The conventional methods to identify genotypes of the ABO blood group include PCR restriction length polymorphism (PCR-RFLP) [2], PCR employment sequence-specific primers (PCR-SSP) [3], PCR strand-conformation polymorphism (PCR-SSCP) [4,5], and multiplex single-base primer extension reaction (SNaPshot) [6,7]. Other studies were conducted to accurately identify ABO blood types in one tube at a time through melting curve analysis using a PNA (peptide nucleic acid) probe [8] and four tubes using loop-mediated isothermal amplification (LAMP) [9]. ...
Analyzing all biological evidence at a crime scene presents serious time, budget, and labor constraints. Therefore, selecting valid evidence is crucial for efficient screening. The ABO blood group is a marker that can serve as valid evidence for identifying investigative leads in criminal case. Conventional identification of ABO blood groups using serological methods has only been for blood and is difficult to apply to other body fluids. ABO genotyping was conducted by analyzing single nucleotide polymorphisms (SNP) representative of each blood group. However, this method is time-consuming, expensive, and requires sophisticated instruments. In this study, we developed rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and multiplex real-time polymerase chain reaction (PCR). Three SNP sites in the ABO gene (nt 261, 526, and 803) were selected to classify the ABO genotypes. For the specificity test, we performed sequencing of 60 saliva samples to confirm that the genotyping. We conducted experiments to apply ABO genotyping using two amplification methods to mock forensic sample using cotton swab and filter paper. As a result, using LAMP, we successfully identified six ABO genotypes within 30 min at a constant temperature (65 ℃). Moreover, by using multiple real-time PCR, it was possible to detect not only the major group but also the subgroup of the ABO genotype (ex. cis-AB). The amplification results using the new methods were in concordance with the sequencing results. Therefore, these ABO genotyping methods are expected to select valid evidence successfully and efficiently at the crime scene.
... A Rapid Method for ABO Genotyping in a Closed Tube Transfus Med Hemother DOI: 10.1159/000530013 reaction (PCR) have been developed, including PCR sequence-specific primer (PCR-SSP) assay [5,6], PCR restriction fragment length polymorphism [7], PCR single-strand conformation polymorphism [8], PCR sequencing-based typing [9], and real-time PCR [10][11][12]. Various methodologies have both advantages and disadvantages. ...
... The detection process comprised PCR amplification, SSCP analysis, and direct PCR product sequencing. Simply, SNPs are detected based on electrophoretic mobility differences of single-stranded DNA fragments with different sequences, and the addition of sequencing technology makes new allele identification easy [8]. However, the whole process is relatively time-consuming. ...
Introduction
The molecular biology detection technology of the human ABO blood group system makes up for the limitations in many aspects compared with conventional serological typing technology. This study aimed to establish a new method to identify seven common ABO alleles (ABO*A1.01, ABO*A1.02, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02, and ABO*O.02.01) by two-dimensional polymerase chain reaction (2D PCR). 2D PCR can identify multiple target genes in a closed test tube by labeling specific primers with tags homologous to the sequence of fluorescently labeled probes, and melting curve analysis is performed after the fluorescent probes are hybridized with tag complementary sequences in PCR-specific products. In this study, 2D PCR and PCR sequence-specific primer (PCR-SSP) were combined to discriminate different alleles in a single reaction, which has the characteristics of high throughput, and compared with other typing techniques; the typing results can be obtained without additional operations.
Methods
The ABO*A1.01 allele genetic sequence was used as the reference sequence. The specific sense and antisense primers for seven common ABO alleles were designed on exons 6 and 7 according to the principle of 2D PCR and PCR-SSP. Single nucleotide polymorphism sites for identifying seven alleles were detected in FAM and HEX channels, respectively. Two hundred sixty DNA samples were enrolled for rapid ABO genotyping by 2D PCR, and 95 of them were selected for Sanger sequencing. The Kappa test was used to analyze the agreement of the methodologies.
Results
These 7 alleles each had four characteristic melting valleys at different single nucleotide polymorphism loci. A total of 15 genotypes were detected, including ABO*A1.01/A1.02, ABO*A1.01/O.01.01, ABO*A1.01/O.01.02, ABO*A1.02/A1.02, ABO*A1.02/O.01.01, ABO*A1.02/O.01.02, ABO*B.01/B.01, ABO*B.01/O.01.01, ABO*B.01/O.01.02, ABO*O.01.01/O.01.01, ABO*O.01.01/O.01.02, ABO*O.01.02/O.01.02, ABO*A1.01/B.01, ABO*A1.02/B.01, and ABO*B.01/O.01. v (containing a rare ABO*O allele, based on the sequencing results). The Kappa test showed completely consistent results for 2D PCR and Sanger sequencing (Kappa = 1).
Conclusion
The 2D PCR technique could be used for molecular typing of the ABO blood group, which was efficient, rapid, accurate, and economical.
... In particular, we wondered if universal bases could be incorporated into crRNAs so as to enable Cas9 recognition of polymorphic target sequences. To test this possibility, we selected a highly polymorphic sequence from the ABO gene that determines the most clinically important blood group system in mammals 43 . We generated a series of 16 DNA target sequences (ABO-T1-16), derived from prevalent alleles in the human population, containing naturally occurring single nucleotide polymorphisms (SNPs) within that region (Fig. 1b, Supplementary Fig. 2a). ...
... HLA-C crRNAs were designed as described in the manuscript. Two of the four SNPs chosen for the ABO target site are found in the most common ABO alleles and are linked to changes in blood type 43 . The polymorphisms seen in the HIV gene set are linked to the formation of drugresistant mutations in a domain of the viral protease 52 . ...
CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases.
... в работе [3]). Для определения группы крови использованы четыре позиции в гене AB0: 261 (C>del), 297 (A>G), 657 (G>A) и 681 (C>T) [20], -для определения пола -различающиеся фрагменты гена AMEL на X-и Y-хромосомах [13]. При выборе SNP, ассоциированных с гаплогруппами Y-хромосомы, мы руководствовались данными ISOGG (Y-DNA Haplogroup Tree 2019-2020, ver. ...
В работе представлен метод генотипирования панели из 60 однонуклеотидных полиморфизмов (SNP) с помощью одностадийной ПЦР с последующей гибридизацией на гидрогелевом биочипе. Пул анализируемых полиморфизмов состоит из 41 SNP, входящих в панель HIrisPlex-S, 4 SNP гена AВ0 (261G>Del, 297A>G, 657C>T, 681G>A), маркеров генов AMELX и AMELY и 14 SNP-маркеров гаплогрупп Y-хромосомы: B (M60), C (M130), D (CTS3946), E (M5388), G (P257), H (M2920), I (U179), J (M304), L (M185), N (M231), O (M175), Q (M1105), R (P224) и T (M272). Получаемые генетические данные позволяют прогнозировать фенотип искомого лица по признакам цвета глаз, волос, кожи, группе крови АВ0, половой принадлежности, геногеографического происхождения по мужской линии. Протокол постановки максимально упрощен для облегчения внедрения метода в практику. Установлено распределение частот аллелей исследуемых полиморфизмов, а также групп крови АВ0 среди славян (N = 482), происхождением преимущественно из центральной России.
... 1-41 are combined into the general group "Pigmentation of the iris, hair and skin", since some of them has cross effects on several traits (for more details, see [3]). To determine the blood group, four positions in the AB0 gene were used: 261 (C>del), 297 (A>G), 657 (G>A), and 681 (C>T) [20], for sex determination, different fragments of the AMEL gene on the X and Y chromosomes [13]. When choosing SNPs associated with Y chromosome haplogroups, we were guided by the ISOGG data (Y-DNA Haplogroup Tree 2019-2020, ver. ...
This paper presents a method for genotyping a panel of 60 single nucleotide polymorphisms
(SNPs) using single-stage PCR followed by hybridization on a hydrogel biochip. The pool of analyzed polymorphisms
consists of 41 SNPs included in the HIrisPlex-S panel, 4 SNPs of the AB0 gene (261G>Del,
297A>G, 657C>T, 681G>A), markers of the AMELX and AMELY genes, and 14 SNP markers of the Y chromosome
haplogroups: B (M60), C (M130), D (CTS3946), E (M5388), G (P257), H (M2920), I (U179),
J (M304), L (M185), N (M231), O (M175), Q (M1105), R (P224) and T (M272). These genetic data allow
one to predict the phenotype of the desired person according to the characteristics of eye, hair, skin color,
AB0 blood group, sex, and genogeographic origin in the male line. The setting protocol is simplified as much
as possible to facilitate the introduction of the method into practice. The distribution of allele frequencies of
the studied polymorphisms, as well as AB0 blood groups among the Slavs (N = 482), originating mainly from
central Russia, was established.
... Major alleles Sub-alleles Essawy, et al. Gene Reports 21 (2020) 100760 including; Kuwaitis (EL-zawahri and Luqmani, 2008), Bahraini (Al-Arrayed et al., 2001) and Saudi Arabian populations (Mohamed et al., 2016) and from White European (Yip, 2000) and Chinese populations (Zhang et al., 2015). The frequencies ABO sub-alleles A 1 , A 2 , B, O 1 and O 2 were 0.2500, 0.0417, 0.2083, 0.2917 and 0.2083 respectively. ...
Background
Genetic profiles of ABO blood group system was reported to have clinical importance in predisposition to certain malignancies. This study was attempted to investigate any possible association between ABO genotypes and the risk of developing hepatocellular carcinoma (HCC) and to determine the major ABO allele's frequencies in a cohort of Egyptian population.
Subjects and methods
One hundred patients (HCC group) and 100 healthy subjects (control group) were enrolled in this study. ABO Genotyping was done using Real-time PCR-melting curve analysis.
Results
Egyptians with BO genotype were 3.38 times more likely to develop HCC. This association was gender related and noted in hepatitis-free HCC cases. In elderly subjects HCC risk increased not only with BO genotype but also with AO and AB genotypes. In the healthy Egyptian sample, frequencies of the three major alleles (A, B and O) were 0.2917, 0.2083 and 0.5000 respectively. Frequencies of sub-alleles (A¹, A², B, O¹ and O²) were 0.2500, 0.0417, 0.2083, 0.2917 and 0.2083 respectively. Serological and genotyping results showed a concordance rate of 97.9%.
Conclusion
BO genotype is associated with a higher risk for HCC in Egyptians independent of the established HCC risk factors. Egyptian ABO allele frequencies are closely linked to Iranian and Palestinian populations.
... Multiplex PCR-single-strand conformation polymorphism (SSCP) analysis [67] Denaturation of PCR product followed by fragment analysis in polyacrylamide gel 9 polymorphisms associated with 1 blood group PCR-RFLP [68][69][70][71][72][73][74] Amplification of target sequence followed by enzyme digestion (PCR-RFLP) ...
Blood transfusion is an effective therapeutic approach for several hematological conditions including sickle cell disease (SCD), thalassaemia, myelodysplastic syndrome (MDS), and autoimmune hemolytic anemia. It is also often indicated for transplantation and for patients receiving medical treatments for cancer. However, transfusion treatment can lead to the red blood cell (RBC) alloimmunization when an incompatible antigen is inadvertently present in the transfused blood. Alloantibodies can cause RBC destruction and many other complications defeating the purpose of the treatment. The risk of development of multiple alloantibodies increases with the frequency of transfusions in transfusion-dependent patients and can be mitigated by transfusing blood type negative for multiple antigens to prevent hemolysis. This chapter discusses the transfusion's risk of RBC alloimmunization as an adverse event; consequences of alloimmunization in patients' care; approaches to prevent and/or mitigate alloimmunization and enhance transfusion efficacy; application of RBC genotyping to supplement serology for preventing alloimmunization. The currently available techniques for RBC genotyping and the importance of reference reagents for determining the genotyping accuracy will also be discussed.
... Moreover, one of the novel O alleles without a 261G deletion but with a 467C＞T SNP was found among one hundred of the Chinese Han population [11]. Different ethnic groups have their own O allele genetic characteristics [12][13][14][15]. Won et al. [16], who examined ABO discrepancies, reported that various kinds of O alleles, such as O02var, O04, O04var, and O07 existed in the Korean population. ...
... This allele has been reported at low frequencies in several populations, and interestingly, its frequency increases from north to south for Basques (from France and Spain, 0% ±5% of all O alleles), Berbers (from Morocco, 9%) and Akans (from Ivory Coast, 19%). [9,10] ...
Weaker subgroups of ABO blood group system give rise to discrepancies between forward and reverse grouping and cause diagnostic difficulties in routine blood banking. Weaker subgroups of A blood group that have been reported so far include A3, Aend, Ax, Am, Ay, and Ael. We report a case of a 54-year-old patient whose red cells showed a discrepancy between cell and serum grouping on initial testing. Serological investigation included absorption elution tests and saliva testing after performing initial blood grouping. Molecular genotyping of the ABO gene was performed by DNA sequencing of exons 6 and 7 of the ABO gene. The serological characteristics of the patient's red cells were similar to Ax subtype. The patient was a secretor and only H substance was present in the saliva. Serum did not show the presence of anti-A1. Molecular genotyping confirmed the ABO status as Aw06/O13. The weak A phenotype identified in the propositus had serological characteristics similar to Ax and showed the ABO genotype Aw06/O13. Although Aw06 allele has been previously reported in the Indian population, this is the first study to report O13 allele in the Indian population.
... Our study has identified one SNP at nucleotide position 1061 with single deletion of C nucleotide of A201 background allele from sample 2 without substitution of 467 C>T. In regards to our findings, sample 2 would probably be best identified-as A205 allele as described by Olsson & Chester in 1996and Yip in 2000(Olsson & Chester, 1996, Yip, 2000b. However, sample 1 without both of these mutations would probably belong to the other A2 alleles or another subgroup of A such as A3, Aw, Ae, Am. ...
Introduction: ABO blood grouping is an important antigenic blood typing tools in blood transfusion and organ transplants. Mismatching of blood during transfusion would lead to undesired transfusion reactions. Due to rare occurrence of rare blood group such as A2 subtype, regular blood grouping technique would have missed the identification of blood group. Objectives: In this study, the identification of A2 subgroup using routine serological technique was validated via DNA sequencing technique. Materials and Methods: A total of 656 students participated in this study consist of Malay (87.0 %), Chinese (0.4 %), Indian (11.4 %) and others ethnic group (0.9%) respectively. Monoclonal antisera A, B, AB, D, A1 lectin and H lectin were used to identify the antigen on red blood cells. DNA sequence analysis was applied to examine single nucleotide polymorphisms (SNPs) at position 467 (substitution of C>T) and 1061 (deletion of C) on coding region of ABO gene. Results: Our findings showed of 656 blood samples, 256 (39.0%) were blood group O, 190 (29.0%) were blood group B, 179 (27.3%) were blood group A and 31(4.7%) were blood group AB. The frequency of A1 subgroup is 177 (99.0%) and A2 subgroup is 2 (1.0%). From 179 A blood group, only 2 samples showed negative reaction towards anti-A1 lectin. DNA sequence analysis revealed the SNPs at nucleotide 1061 position in sample 2, however this mutation was absence in sample 1, suggesting presence of another mutation that may result in the A2 phenotype. Conclusion: The current study reported the absence of 1061C deletion in A2 blood group sample among Malaysian population.
