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Blood Group ABO Genotyping in Paternity Testing
Abstract
BACKGROUND: The ABO blood groups result from DNA sequence variations, predominantly single nucleotide and insertion/deletion polymorphisms (SNPs and indels), in the ABO gene encoding a glycosyltransferase. The ABO blood groups A(1), A(2), B and O predominantly result from the wild type allele A1 and the major gene variants that are characterized by four diallelic markers (261G>del, 802G>A, 803G>C, 1061C>del). Here, we were interested to evaluate the impact of ABO genotyping compared to ABO phenotyping in paternity testing. METHODS: The major ABO alleles were determined by PCR amplification with sequence-specific primers (PCR-SSP) in a representative sample of 1,335 blood donors. 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 by 12 short tandem repeat (STR) markers before. We compared statistical parameters (PL, paternity likelihood; PE, power of exclusion) of both blood grouping approaches. RESULTS: The prevalence of the major ABO alleles and genotypes corresponded to the expected occurrence of ABO blood groups in a Caucasian population. The low resolution genotyping of 4 diallelic markers revealed a correct genotype-phenotype correlation in 1,331 of 1,335 samples (99.7%). In 60 paternity trios with confirmed paternity of the alleged father based on STR analysis both PL and PE of the ABO genotype was significantly higher than of the ABO phenotype. 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%). CONCLUSION: In paternity testing ABO genotyping is superior to ABO phenotyping with regard to PL and PE, however, ABO genotyping is not sufficient for valid paternity testing. Due to the much lower mutation rate compared to STR markers, blood group SNPs in addition to anonymous SNPs could be considered for future kinship analysis and genetic identity testing.
... Data about ABO blood groups were taken from the blood donor files, and all donors had regular ABO blood groups (A, B, AB, or O) without discrimination of the A 1 and A 2 phenotypes. The low-resolution ABO genotypes were determined in a previous study [7] and included the variants c.261delG (for deletional *O.01 alleles), c.802G>A (for the non-deletional O alleles *O.02), c.803G>C (for *B alleles), and c.1061delC (for *A2 alleles). The A 1 , A 2 , B, and O phenotypes were deduced from the low-resolution genotype. ...
... Because of the lack of a specific marker, the A 1 phenotype was diagnosed by exclusion of alleles encoding A 2 , B, or O phenotypes. Based on the genotyping of the 4 major variants c.261delG, c.802G>A, c.803G>C, and c.1061delC, a genotype-phenotype correlation was achieved for 99.7% of the samples [7]. However, the significant diversity of the ABO gene and the increasing number of mutations associated with aberrant blood group phenotypes impeded the molecular diagnosis. ...
Introduction:
The molecular diagnosis of the A1 blood group is based on the exclusion of ABO gene variants causing blood groups A2, B, or O. A specific genetic marker for the A1 blood group is still missing. Recently, long-read ABO sequencing revealed four sequence variations in intron 1 as promising markers for the ABO*A1 allele. Here, we evaluated the diagnostic values of the 4 variants in blood donors with regular and weak A phenotypes and genotypes.
Methods:
ABO phenotype data (A, B, AB, or O) were taken from the blood donor files. The ABO genotypes (low resolution) were known from a previous study and included the variants c.261delG, c.802G>A, c.803G>C, and c.1061delC. ABO variant alleles (ABO*AW.06,*AW.08,*AW.09,*AW.13, *AW.30, and *A3.02) were identified in weak A donors by sequencing the ABO exons before. For genotyping of the ABO intron 1 variants rs532436, rs1554760445, rs507666, and rs2519093, we applied TaqMan assays with endpoint fluorescence detection according to a standard protocol. Genotypes of the variants were compared with the ABO phenotype and genotype. Evaluation of diagnostic performance included sensitivity, specificity, positive (PPV), and negative predictive value (NPV).
Results:
In 1,330 blood donors with regular ABO phenotypes and genotypes, the intron 1 variants were significantly associated with the proposed A1 blood group. In 15 donors, we found discrepancies to the genotype of at least one of the 4 variants. For the diagnosis of the ABO*A1 allele, the variants showed 98.79-99.48% sensitivity, 99.66-99.81% specificity, 98.80-99.31% PPV, and 99.66-99.86% NPV. Regarding the A phenotype, the diagnostic values were 99.02-99.41% sensitivity, 99.63-99.76% specificity, 99.41-99.61% PPV, and 99.39-99.63% NPV. The *A1 marker allele of all intron 1 variants was also associated with the *AW.06, *AW.13, and *AW.30 variants. Samples with *AW.08, *AW.09, and *A3.02 variants lacked this association.
Conclusion:
The ABO intron 1 variants revealed significant association with the ABO*A1 allele and the A phenotype. However, the intron 1 genotype does not exclude variant alleles causing weak A phenotypes. With the introduction of reliable tag, single nucleotide variants for the A1, A2, B, and O blood groups and the genotyping instead of phenotyping of the ABO blood group are getting more feasible on a routine basis.
... Blood group analyses. We classified haplotypes into blood groups on the basis of genotypes at three SNPs (rs8176747, rs41302905 and rs8176719) 13,39 . A deletion at rs8176719 confers a type O haplotype, as does a T allele at rs41302905. ...
... We note that the blood group assignment methodology described above is incomplete and there are other rare variants that can influence blood group 39 . To understand the accuracy of the genetic blood group assignments, we compared them to self-reported blood groups from over 1.47 million research participants. ...
COVID-19 presents with a wide range of severity, from asymptomatic in some individuals to fatal in others. Based on a study of 1,051,032 23andMe research participants, we report genetic and nongenetic associations with testing positive for SARS-CoV-2, respiratory symptoms and hospitalization. Using trans-ancestry genome-wide association studies, we identified a strong association between blood type and COVID-19 diagnosis, as well as a gene-rich locus on chromosome 3p21.31 that is more strongly associated with outcome severity. Hospitalization risk factors include advancing age, male sex, obesity, lower socioeconomic status, non-European ancestry and preexisting cardiometabolic conditions. While non-European ancestry was a significant risk factor for hospitalization after adjusting for sociodemographics and preexisting health conditions, we did not find evidence that these two primary genetic associations explain risk differences between populations for severe COVID-19 outcomes. A study of more than one million 23andMe research participants identifies genetic and nongenetic associations with COVID-19 susceptibility and severity.
... We classified haplotypes into blood groups on the basis of genotypes at three SNPs: rs8176747, rs41302905, and rs8176719 14,15 . A deletion at rs8176719 confers a type O haplotype, as does a T allele at rs41302905. ...
... We note that the blood group assignment methodology described above is incomplete, and there are other rare variants that can influence blood group 14 . In order to understand the accuracy of the genetic blood group assignments, we compared to self-reported blood groups from over 1.47 million research participants. ...
COVID-19 presents with a wide range of severity, from asymptomatic in some individuals to fatal in others. Based on a study of over one million 23andMe research participants, we report genetic and non-genetic associations with testing positive for COVID-19, respiratory symptoms, and hospitalization. Risk factors for hospitalization include advancing age, male sex, elevated body mass index, lower socio-economic status, non-European ancestry, and pre-existing cardio-metabolic and respiratory conditions. Using trans-ethnic genome-wide association studies, we identify a strong association between blood type and COVID-19 diagnosis, as well as a gene-rich locus on chr3p21.31 that is more strongly associated with outcome severity. While non-European ancestry was found to be a significant risk factor for hospitalization after adjusting for socio-demographics and pre-existing health conditions, we did not find evidence that these two primary genetic associations explain differences between populations in terms of risk for severe COVID-19 outcomes.
... 6 The pathogenesis of severe Covid-19 and the associated respiratory failure is poorly understood, but higher mortality is consistently associated with older age and male sex. 7,8 Clinical associations have also been reported for hypertension, diabetes, and other obesity-related and cardiovascular disease traits, but the relative role of clinical risk factors in determining the severity of Covid- 19 has not yet been clarified. [7][8][9][10][11] Observational data on lymphocytic endotheliitis and diffuse microvascular and macrovascular thromboembolic complications suggest that Covid-19 is a systemic disease that involves injury to the vascular endothelium but provide little insight into the underlying pathogenesis. ...
... On the basis of the results from the TOPMed genotype imputation, we selected three ABO SNPs (rs8176747, rs41302905, and rs8176719) 19,20 to infer the ABO blood type and calculated odds ratios according to blood type To assess in detail the HLA complex at locus 6p21, we performed sequencing-based HLA typ- Shown is a Manhattan plot of the association statistics from the main meta-analysis (controlled for potential population stratification). The red dashed line indicates the genomewide significance threshold of a P value less than 5×10 −8 . ...
Background
There is considerable variation in disease behavior among patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (Covid-19). Genomewide association analysis may allow for the identification of potential genetic factors involved in the development of Covid-19.
Methods
We conducted a genomewide association study involving 1980 patients with Covid-19 and severe disease (defined as respiratory failure) at seven hospitals in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe. After quality control and the exclusion of population outliers, 835 patients and 1255 control participants from Italy and 775 patients and 950 control participants from Spain were included in the final analysis. In total, we analyzed 8,582,968 single-nucleotide polymorphisms and conducted a meta-analysis of the two case–control panels.
Results
We detected cross-replicating associations with rs11385942 at locus 3p21.31 and with rs657152 at locus 9q34.2, which were significant at the genomewide level (P<5×10⁻⁸) in the meta-analysis of the two case–control panels (odds ratio, 1.77; 95% confidence interval [CI], 1.48 to 2.11; P=1.15×10⁻¹⁰; and odds ratio, 1.32; 95% CI, 1.20 to 1.47; P=4.95×10⁻⁸, respectively). At locus 3p21.31, the association signal spanned the genes SLC6A20, LZTFL1, CCR9, FYCO1, CXCR6 and XCR1. The association signal at locus 9q34.2 coincided with the ABO blood group locus; in this cohort, a blood-group–specific analysis showed a higher risk in blood group A than in other blood groups (odds ratio, 1.45; 95% CI, 1.20 to 1.75; P=1.48×10⁻⁴) and a protective effect in blood group O as compared with other blood groups (odds ratio, 0.65; 95% CI, 0.53 to 0.79; P=1.06×10⁻⁵).
Conclusions
We identified a 3p21.31 gene cluster as a genetic susceptibility locus in patients with Covid-19 with respiratory failure and confirmed a potential involvement of the ABO blood-group system. (Funded by Stein Erik Hagen and others.)
... Historically, the first biological markers used to genetically distinguish between individuals were the human ABO blood groups. 1 The ABO blood group phenotyping was introduced to paternity or maternity assessment very early, but soon it started playing a complementary role due to the fact that it is not very informative. While it is useful for excluding an individual from the paternity or maternity test, it is not useful when an inclusion is required. ...
The need for confirmation or exclusion of biological father and / or biological mother is a social phenomenon, which is imposed by socioeconomic and, sometimes , by moral-psychological factors. Modern science has significantly contributed to solving this problem, as many medical methods have been applied for this purpose. Biological markers that have been conducted for distinguishing between individuals were the human ABO blood groups, the Rh, MNS, Duffy, Kidd, and Kell systems, as well as the human leukocyte antigens (HLA) system. For a long time the HLA testing represented the standard testing in forensic genetics, but, due to the linkage disequilibrium and the predominance of certain HLA alleles and as the demand for parentage investigations is rapidly increasing during the recent years, this serologi-cal era has been replaced by molecular markers through the introduction of "DNA profiling", which is based on polymorphisms of short tandem repeats (STRs) loci. Nowadays, "DNA profiling" by analysis of STR loci is the method of choice for human identification and parentage investigations. This technique is the most informative , accurate, robust, rapid, cost-effective method of genotyping and has worldwide acceptance in the courts, as the probability of parentage will typically be greater than 99.99999%. I N t r O D U c t I O N Nowadays, most parentage investigations are usually conducted in order to establish legal responsibility and support for a child. In addition, several other cases are investigated in order to prevent disputes in adoption, in in vitro fertilization (IVF) cases, in prenatal diagnosis, in bone marrow transplantation (follow ups), as well as cases in which a record for immigration is required. Historically, the first biological markers used to genetically distinguish between individuals were the human ABO blood groups. 1 The ABO blood group phenotyping was introduced to paternity or maternity assessment very early, but soon it started playing a complementary role due to the fact that it is not very informative. While it is useful for excluding an individual from the paternity or maternity test, it is not useful when an inclusion is required. When for example, conventional ABO blood group typing has a power of exclusion (PE) of only 17%, this value increases to 53% using revIew AbbreviAtions AF(s) = alleged father(s) CPI = combined paternity index HLA = human leukocyte antigens IMGT = international ImMunoGeneTics (project) IVF = in vitro fertilization MHC = major histocompatibility complex PCR = polymerase chain reaction PE = power of exclusion PI = paternity index RFLP = fragment length polymorphism STRs = short tandem repeats VNTRs = variable number of tandem repeats W = probability of paternity Conflict of Interest: none declared
... For COVID-19, we selected nine common (minor allele frequency >0.05 in EUR) GWAS SNPs for COVID susceptibility and severity [11,[20][21][22] (Supplemental Table S1). To complement the ABO susceptibility locus, we additionally inferred blood type (O, A, B, AB) using three SNPs (rs8176747, rs41302905, and rs8176719) as described previously [21][22][23]. For pneumonia susceptibility, we selected common GWAS SNPs near SUCNR1 (rs11708673) [24] and the HLA class I region (rs3131623) [25]. ...
Background: Based on our recently reported associations between specific dietary behaviors and the risk of COVID-19 infection in the UK Biobank (UKB) cohort, we further investigate whether these associations are specific to COVID-19 or extend to other respiratory infections. Methods: Pneumonia and influenza diagnoses were retrieved from hospital and death record data linked to the UKB. Baseline, self-reported (2006–2010) dietary behaviors included being breastfed as a baby and intakes of coffee, tea, oily fish, processed meat, red meat (unprocessed), fruit, and vegetables. Logistic regression estimated the odds of pneumonia/influenza from baseline to 31 December 2019 with each dietary component, adjusting for baseline socio-demographic factors, medical history, and other lifestyle behaviors. We considered effect modification by sex and genetic factors related to pneumonia, COVID-19, and caffeine metabolism. Results: Of 470,853 UKB participants, 4.0% had pneumonia and 0.2% had influenza during follow up. Increased consumption of coffee, tea, oily fish, and fruit at baseline were significantly and independently associated with a lower risk of future pneumonia events. Increased consumption of red meat was associated with a significantly higher risk. After multivariable adjustment, the odds of pneumonia (p ≤ 0.001 for all) were lower by 6–9% when consuming 1–3 cups of coffee/day (vs.
... Europeans and Chinese (AF > 0.39). Notably, this variant is one of the three major variants determining the haplotypes of the ABO blood group 15,24,25 . Carriers of the T/T homozygote belong to blood group O, while carriers of the risk allele TC belong to the non-O group unless they also carry an extremely rare allele T at SNP rs41302905 (absence in our Chinese samples). ...
COVID-19 has caused numerous infections with diverse clinical symptoms. To identify human genetic variants contributing to the clinical development of COVID-19, we genotyped 1457 (598/859 with severe/mild symptoms) and sequenced 1141 (severe/mild: 474/667) patients of Chinese ancestry. We further incorporated 1401 genotyped and 948 sequenced ancestry-matched population controls, and tested genome-wide association on 1072 severe cases versus 3875 mild or population controls, followed by trans-ethnic meta-analysis with summary statistics of 3199 hospitalized cases and 897,488 population controls from the COVID-19 Host Genetics Initiative. We identified three significant signals outside the well-established 3p21.31 locus: an intronic variant in FOXP4-AS1 (rs1853837, odds ratio OR = 1.28, P = 2.51 × 10−10, allele frequencies in Chinese/European AF = 0.345/0.105), a frameshift insertion in ABO (rs8176719, OR = 1.19, P = 8.98 × 10−9, AF = 0.422/0.395) and a Chinese-specific intronic variant in MEF2B (rs74490654, OR = 8.73, P = 1.22 × 10−8, AF = 0.004/0). These findings highlight an important role of the adaptive immunity and the ABO blood-group system in protection from developing severe COVID-19. Chaolong Wang and colleagues report a large genome-wide association study for COVID-19 severity in Chinese individuals. By meta-analysis with European data, they identify 3 loci associated with severe disease that suggest key roles for the adaptive immune system and the ABO blood group system in development of severe COVID-19.
... Another study in Germany using ABO genotyping by PCR-SSP and ABO grouping revealed that in 60 paternity trios with confirmed paternity of the alleged father based on STR analysis both paternity likelihood and power of exclusion of the ABO genotype was significantly higher than of the ABO phenotype. 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%) [25]. As demonstrated in this study, the results of paternity tests were better when using STR analysis compared to serological tests. ...
Background
In resource-limited countries, ABO, HLA, MNS, Kells, and hemoglobin electrophoresis are classic tests for the resolution of paternity disputes due to their affordable cost. The limitations of these tests in cases of disputed paternity require the use of Short Tandem Repeats (STR) for their certification. This study aimed to determine the biological fathers of children using ABO-rhesus/hemoglobin electrophoresis and STR assays in Burkina Faso, West Africa.
Results
Of the fourteen trios studied, the ABO-rhesus/hemoglobin electrophoresis analysis revealed ten probable inclusion cases, three exclusion cases, and one undetermined paternity. DNA STR analysis found five inclusions of paternity out of the ten probable inclusions with ABO-rhesus/hemoglobin electrophoresis assay versus nine exclusions of paternity.
Conclusion
This study showed that the implementation of the analysis of short tandem repeat is required to resolve increasing disputed filiation cases in Burkina Faso.
... The involvement of the ABO blood system in biology and health has been ever increasing. Historically, ABO blood-typing is widely applied in paternity testing in an "exclusion" basis before being replaced by the genotyping-based "confirmation" methods [24]. ABO blood system is also essential in daily medical operations of blood donation/transfusion and organ transplantation [25,26]. ...
ABO blood system is an inborn trait determined by the ABO gene. The genetic-phenotypic mechanism underneath the four mutually exclusive and collectively exhaustive types of O, A, B and AB could theoretically be elucidated. However, genetic polymorphisms in the human populations render the link elusive, and importantly, past studies using genetically determined rather than biochemically determined ABO types were not and could not be evaluated for the inference errors. Upon both blood-typing and genotyping a cohort of 1008 people of the Han Chinese population, we conducted a genome-wide association study in parallel with both binomial and multinomial log-linear models. Significant genetic variants are all mapped to the ABO gene, and are quantitatively evaluated for binary and multi-class classification performances. Three single nucleotide polymorphisms of rs8176719, rs635634 and rs7030248 would together be sufficient to establish a multinomial predictive model that achieves high accuracy (0.98) and F1 scores (micro 0.99 and macro 0.97). Using the set of identified ABO-associated genetic variants as instrumental variables, we demonstrate the application in causal analysis by Mendelian randomization (MR) studies on blood pressures (one-sample MR) and severe COVID-19 with respiratory failure (two-sample MR).
... Point mutation was detected in 7 samples of the B gene with 700C>G mutation, which was previously defined as B The ABO gene that encodes the glycosyltransferases and is responsible for the conversion of H substance to blood group A and B antigens is located on chromosome 9 [21] . It consists of seven exons ranging in size from 28 to 688 bp [22] . The last two exons (exons 6 and 7), comprising 823 bp of the transcribed 1,062 bp mRNA, encode for the catalytic domain of the ABO glycosyltransferases [23,24] . ...
... ABO*A 201 allele is either absent i.e. within the Chinese or rare as for the Japanese (0,1%) [25], [26]. Comparatively ABO*A 102 allele is absent at the Spanish and the German [27], [12]. ...
Introduction: Historically, Morocco has known many successive conquests and invasions that have induced genetic changes in its autochthons population. It's known that blood groups are among the most polymorphic systems. The study of ABO blood groups showed that their distribution varied in different populations. The aim of this study is to analyze the diversity and genetic differentiation of ABO system in the Moroccan population. Material and methods: Data of ABO system genetic polymorphism from previous study were analyzed using statistical approaches which are the classical and the Bayesian methods. The classical approach has been used to assess genetic differentiation by adopting multivariate analysis type: PCA (Principal Component Analysis) and the index of genetic differentiation F st. The Bayesian approach was used to assess the genetic structure of ABO system in the Moroccan population compared to other countries. Results: Within the studied Moroccan population, 10 ABO alleles and 21 genotypes were identified. The heterozygosis rate is about 0.74 and 0.72, respectively, for the expected and observed heterozygosis. PCA analysis shows that the studied population forms 4 groups. Data of genetic distances confirm the presence of Morocco within a group formed by Kuwait, Spain and Jordan with low genetic distances of 1%, 1.8% and 2%, respectively. The Bayesian analysis shows that all the countries, except Germany, present 5 genetic pools. Besides Morocco and Kuwait that have been found to present 5 genetic pools with similar frequencies. Conclusion: The Moroccan population studied exhibits similarity with the countries of the Middle East and the southwest of Europe.
... For its variety and stability, STRs are widely used for genetic fingerprinting [3,4]. In the field of forensics, STR screening provides a high degree of error-free data on personal genetic identity [5,6], while being robust enough to survive degradation under non-ideal conditions. Profiles of certain STR sets are potent and can be used to determine whether twins are monozygotic or dizygotic [7]. ...
Background:
The ABO blood group is closely related to clinical blood transfusion, transplantation, and neonatal hemolytic disease. It is also the most clinically significant blood group system in clinical blood transfusion.
Objective:
The purpose of this paper is to review and analyze the clinical application of the ABO blood group.
Methods:
The most common ABO blood group typing methods in clinical laboratories are hemagglutination test and microcolumn gel test, while genotype detection is mainly adopted in clinical identification of suspicious blood types. However, in some cases, the expression variation or absence of blood type antigens or antibodies, experimental techniques, physiology, disease, and other factors affect the accurate determination of blood types, which may lead to serious transfusion reactions.
Results:
The mistakes could be reduced or even eliminated by strengthening training, selecting reasonable identification methods, and optimizing processes, thereby improving the overall identification level of the ABO blood group. ABO blood groups are also correlated with many diseases, such as COVID-19 and malignant tumors. Rh blood groups are determined by the RHD and RHCE homologous genes on chromosome 1 and are classified as Rh negative or positive according to the D antigen., the agglutination method is often used in clinical settings, while genetic and sequencing methods are often used in scientific research.
Conclusion:
Accurate ABO blood typing is a critical requirement for the safety and effectiveness of blood transfusion in clinical practice. Most studies were designed for investigating rare Rh blood group family, and there is a lack of research on the relationship between Rh blood groups and common diseases.
Background. Respiratory failure is a key feature of severe Covid-19 and a critical driver of mortality, but for reasons poorly defined affects less than 10% of SARS-CoV-2 infected patients.
Methods. We included 1,980 patients with Covid-19 respiratory failure at seven centers in the Italian and Spanish epicenters of the SARS-CoV-2 pandemic in Europe (Milan, Monza, Madrid, San Sebastian and Barcelona) for a genome-wide association analysis. After quality control and exclusion of population outliers, 835 patients and 1,255 population-derived controls from Italy, and 775 patients and 950 controls from Spain were included in the final analysis. In total we analyzed 8,582,968 single-nucleotide polymorphisms (SNPs) and conducted a meta-analysis of both case-control panels.
Results. We detected cross-replicating associations with rs11385942 at chromosome 3p21.31 and rs657152 at 9q34, which were genome-wide significant (P<5x10-8) in the meta-analysis of both study panels, odds ratio [OR], 1.77; 95% confidence interval [CI], 1.48 to 2.11; P=1.14x10-10 and OR 1.32 (95% CI, 1.20 to 1.47; P=4.95x10-8), respectively. Among six genes at 3p21.31, SLC6A20 encodes a known interaction partner with angiotensin converting enzyme 2 (ACE2). The association signal at 9q34 was located at the ABO blood group locus and a blood-group-specific analysis showed higher risk for A-positive individuals (OR=1.45, 95% CI, 1.20 to 1.75, P=1.48x10-4) and a protective effect for blood group O (OR=0.65, 95% CI, 0.53 to 0.79, P=1.06x10-5).
Conclusions. We herein report the first robust genetic susceptibility loci for the development of respiratory failure in Covid-19. Identified variants may help guide targeted exploration of severe Covid-19 pathophysiology.
Background
ABO genotyping is an increasingly being used for accurate prediction of blood transfusion, organ transplantation and forensic application. Traditional methods require DNA extract and purification which is time-consuming labor-intensive and limit the trace samples detection.
Methods
Present study developed the multiplex allele-specific polymerase chain reaction (AS-PCR) method to identify the six major genotypes of ABO blood groups in two tubes reaction. The samples of whole blood, buccal cell and hair root from 30 volunteers were examined.
Results
All genotyping results were interpreted and agreed with the serologic findings. The modified AS-PCR method was able to determine ABO genotyping using direct blood and non-invasive samples including buccal cell and hair root.
Conclusions
The modified technique has a high specific and sensitive, rapid and cost-efficiency of genotyping method which might be valuable for transfusion medicine in a clinical laboratory and personal identification in forensic investigation.
Background:
ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells.
Methods:
We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification.
Results:
Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing.
Conclusion:
The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.
Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.
Serological typing for the classical ABO blood groups is routinely performed using anti-A and anti-B antisera of polyclonal or monoclonal origin, which are able to distinguish four phenotypes (A, B, AB, and O). Modern molecular biology methods offer the possibility of direct ABO genotyping without the need for family investigations. Typing can be done with small amounts of DNA and without detection of blood group molecules on the surface of red blood cells. We developed a system of eight polymerase chain reactions (PCR) to detect specific nucleotide sequence differences between the ABO alleles O1, O2, A1, A2, and B. PCR amplification using sequence-specific primers and detection of amplification products by agarose gel electrophoresis is one of the fastest genotyping methods and is easy to handle. With our method we tested the A1,2BO1,2 genotypes of 300 randomly chosen persons out of a pool of platelet donors and found the results to be consistent with ABO glycosyltransferase phenotypes. We also identified a presumably new ABO allele, which may be the result of a crossing-over event between alleles O1 and A2.
In this study we 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, 3 of which were new. This was done by phylogenetic analysis and correlating sequence data with the ABO phenotypes. The study revealed multiple polymorphisms in noncoding regions. The intron-based phylogenetic analysis revealed 5 main lineages: ABO*A, ABO*B, ABO*O01, ABO*O02, and ABO*O03. The genomic sequences of most rare ABO alleles differed slightly from those of the common alleles. Singular mutations or hybrid alleles were most common, but a few exhibited mosaic sequence pattern containing multiple exon and/or intron motifs from other ABO lineages. Thus, both an accumulation of mutations as well as an assortment of the mutations by recombination seems to be responsible for the ABO gene diversity. The prevalence of replacement mutations indicates positive selection for allelic diversity. Phenotype-genotype correlation showed that sequence variations within the complete coding sequence can 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. These findings are suggestive of the requirement of a comprehensive coding sequence database for sequence-based phenotype prediction.
PCR using sequence-specific primers (PCR-SSP) is widely employed for the genotyping of single nucleotide polymorphisms (SNPs) in both routine diagnosis and medical research. The human platelet alloantigens (HPAs) represent SNPs in platelet-specific glycoproteins, and HPA-1, -2, -3, and -5 are the most relevant in immunohematology. In most protocols, the respective HPA-SNPs are analyzed in allele-specific reactions, each with at least 100 ng DNA. In many cases, prenatal HPA typing in the diagnosis of neonatal alloimmune thrombocytopenia is often limited by the restricted amounts of fetal DNA that are obtainable. We developed a novel PCR-SSP technique to achieve accurate HPA genotypes using only 1 ng DNA per reaction. The concentration of HPA-specific primers was increased to 1 microM each and exhibited a higher sensitivity compared to a commercial PCR-SSP kit. The modified PCR-SSP technique enabled the identification of fetal HPA genotypes using only 0.5 mL amniotic fluid (from week 16 of gestation) and from a maternal plasma sample (from week 38 of gestation). The principle of the modified PCR-SSP technique may also be applied for the genotyping of other SNPs from limited amounts of DNA.
A total of 52 SNPs reported to be polymorphic in European, Asian and African populations were selected. Of these, 42 were from the distal regions of each autosome (except chromosome 19). Nearly all selected SNPs were located at least 100 kb distant from known genes and commonly used STRs. We established a highly sensitive and reproducible SNP-typing method with amplification of all 52 DNA fragments in one PCR reaction followed by detection of the SNPs with two single base extension reactions analysed using CE. The amplicons ranged from 59 to 115 bp in length. Complete SNP profiles were obtained from 500 pg DNA. The 52 loci were efficiently amplified from degraded samples where previously only partial STR profiles had been obtained. A total of 700 individuals from Denmark, Greenland, Somalia, Turkey, China, Germany, Taiwan, Thailand and Japan were typed, and the allele frequencies estimated. All 52 SNPs were polymorphic in the three major population groups. The mean match probability was at least 5.0 x 10(-19) in the populations studied. Typical paternity indices ranged from 336 000 in Asians to 549 000 in Europeans. Details of the 52 SNP loci and population data generated in this work are freely available at http://www.snpforid.org.
The Blood Group Antigen FactsBook has been an essential resource in the hematology, transfusion and immunogenetics fields since its first publication in the late 1990s.The third edition of The Blood Group Antigen FactsBook has been completely revised, updated and expanded to cover all 33 blood group systems. It blends scientific background and clinical applications and provides busy researchers and clinicians with at-a-glance information on over 330 blood group antigens, including history and information on terminology, expression, chromosomal assignment, carrier molecular description, functions, molecular bases of antigens and phenotypes, effect of enzymes/chemicals, clinical significance, disease associations and key references.
Single nucleotide polymorphism (SNP) analysis of human DNA for the purpose of identification has some promising attributes. The question of approach is critical to the eventual adoption of this technology. The use of a low-volume open array platform was tested with a small selected set of eight SNP primers that have a low F(ST) (the proportion of the total genetic variance contained in a subpopulation [S subscript] relative to the total genetic variance [T subscript]) in human populations. Because multiple SNPs must be interrogated, issues concerning DNA concentration, total DNA, and whole genome amplification were investigated. Excellent correlations were obtained for seven of the eight SNP assays on a set of DNA samples of known configuration over a broad concentration range spanning 25-150ng/microl in blind studies. These seven SNP assays were then applied to 39 DNA samples in a population from southern India. These SNPs were sufficient to individualize each member of this sample population. In a paternity study, these same SNPs showed clear parental relationships. For low amounts of genomic DNA, the use of a commercially available whole genome amplification kit showed promise for genotyping sub-nanogram samples. Discrimination against nonhuman DNA was also demonstrated successfully. Because of the very low quantities of reagents used in the assay, the cost per test becomes reasonably inexpensive. Overall, using commercially available SNP assays, the OpenArray platform showed excellent promise as a highly automated, low-volume, high-throughput system for SNP analysis with potential applications to relevant forensic analyses such as identification and paternity.
Using a 52 SNP marker set previously developed for forensic analysis, a novel 49plex assay has been developed based on the Genplex typing system, a modification of SNPlex chemistry (both Applied Biosystems) using oligo-ligation of pre-amplified DNA and dye-labeled, mobility modified detection probes. This gives highly predictable electrophoretic mobility of the allelic products generated from the assay to allow detection with standard capillary electrophoresis analyzers. The loci chosen comprise the 48 most informative autosomal SNPs from the SNPforID core discrimination set supplemented with the amelogenin gender marker. These SNPs are evenly distributed across all 22 autosomes, exhibit balanced polymorphisms in three major population groups and have been previously shown to be effective markers for forensic analysis. We tested the accuracy and reproducibility of the Genplex system in three SNPforID laboratories, each using a different Applied Biosystems Genetic Analyzer. Genotyping concordance was measured using replicates of 44 standardized DNA controls and by comparing genotypes for the same samples generated by the TaqMan, SNaPshot and Sequenom iPLEX SNP typing systems. The degree of informativeness of the 48 SNPs for forensic analysis was measured using previously estimated allele frequencies to derive the cumulative match probability and in paternity analysis using 24 trios previously typed with 18 STRs together with three CEPH families with extensive sibships typed with the 15 STRs in the Identifiler kit.
The histo-blood group ABO, the major human alloantigen system, involves three carbohydrate antigens (ABH). A, B and AB individuals express glycosyltransferase activities converting the H antigen into A or B antigens, whereas O(H) individuals lack such activity. Here we present a molecular basis for the ABO genotypes. The A and B genes differ in a few single-base substitutions, changing four amino-acid residues that may cause differences in A and B transferase specificity. A critical single-base deletion was found in the O gene, which results in an entirely different, inactive protein incapable of modifying the H antigen.
We have isolated human genomic DNA clones encompassing 30 kbp of the histo-blood group ABO locus. The locations of the exons have been mapped and the nucleotide sequences of the exon-intron boundaries have been determined. The human ABO genes consist of at least seven exons, and the coding sequence in the seven coding exons spans over 18 kb of the genomic DNA. The exons range in size from 28 to 688 bp, with most of the coding sequence lying in exon 7.
Blood group ABO polymorphism was analysed in genomic DNA isolated from 150 blood donors by restriction endonuclease digestion of three polymerase chain reaction-amplified exons in the ABO genes and by sequencing of randomly selected samples. An anomalous O1 allele first described in a cancer cell line is now shown to account for approximately 40% of the O alleles described to date. This is 10 times more frequent than the only other known variant O allele (O2). This variant O1 allele has at least seven point mutations when compared to the consensus gene, in addition to the deletion characterising the normal O1 allele.
The blood group ABO gene shows considerable polymorphism in most of the 7 exons. Introns examined so far have also shown blood group-related polymorphisms, as has an upstream enhancer region. Several polymorphisms affect the specificity of the gene product (glycosyltransferase) and explain the occurrence of blood group A and B. Various lethal mutations result in blood group O. Other mutations are presumed to alter the activity rather than the specificity of the enzyme and result in weaker A and B blood group phenotypes. In total, 27 A alleles, 15 B alleles, 26 O alleles, and 4 AB hybrid alleles are described and surely more will surface in the near future. Variation in geographic/ethnic distribution of allele frequencies is discussed, along with the confusing nomenclatures currently in use.
The ABO blood group is the most important blood group system in transfusion medicine. Since the ABO gene was cloned and the molecular basis of the three major alleles delineated about 10 years ago, the gene has increasingly been examined by a variety of DNA-based genotyping methods and analysed in detail by DNA sequencing. A few coherent observations emerge from these studies. First, there is extensive sequence heterogeneity underlying the major ABO alleles that produce normal blood groups A, B, AB and O when in correct combination with other alleles. Second, there is also extensive heterogeneity underlying the molecular basis of various alleles producing ABO subgroups such as A2, Ax and B3. There are over 70 ABO alleles reported to date and these alleles highlight the extensive sequence variation in the coding region of the gene. A unifying system of nomenclature is proposed to name these alleles. Third, extensive sequence variation is also found in the non-coding region of the gene, including variation in minisatellite repeats in the 5' untranslated region (UTR), 21 single nucleotide polymorphisms (SNPs) in intron 6 and one SNP in the 3' UTR. The haplotypes of these variations reveal a specific relationship with the major ABO alleles. Fourth, excluding the common alleles, about half of the remaining alleles are due to new mutations and the other half can better be explained by intragenic recombination (both crossover and gene conversion) between common alleles. In particular, the recombination sites in hybrid alleles can be quite precisely defined through haplotype analysis of the SNPs in intron 6. This indicates that recombination is equally as important as point mutations in generating the genetic diversity of the ABO locus. Finally, a large number of ABO genotyping methods are available and are based on restriction analysis, allele specific amplification, mutation screening techniques or their combinations.
In this report, we analyze data assembled in the Blood Group Antigen Gene Mutation Database (www.bioc.aecom.yu.edu/bgmut/index.htm), which describes sequence information on human genes associated with expression of the various serologically-determined blood group phenotypes. The database documents 38 genetic loci and a total of 624 alleles that together encode a large repertoire of proteins and constitute 27 serologically-defined blood group systems. Analysis of sequence variation patterns across alleles of a number of genes is focused on their molecular profiles, including mutational sites and recurrence, patterns of gene rearrangements in duplicated gene families, correlation of predicted location of epitopes in extracellular loops with sites of alterations, and effects of mutations on protein expression. That information, and the relative ease of identifying individuals bearing variant alleles, has led to the proposal that genes encoding blood group antigens are an important and unique resource for studies of human DNA variation. Another focus is on mutations in regions that encode the antigenic epitopes and on their occurrence in world populations. These mutations may be viewed as coding single nucleotide polymorphisms (cSNPs). We propose that one group of these cSNPs, which are known to occur with significant frequency in all world populations, could serve as well-validated genetic markers. In addition, specific mutations in a number of "low incidence" and rare alleles could serve as cSNPs specific for a given population. The allelic frequencies of these mutations and knowledge of their world-wide occurrence add a valuable dataset to the existing cSNP pools documented in SNP databases.
Duffy and ABO blood group genetic polymorphisms were studied by minisequencing analysis of single-nucleotide polymorphisms (SNPs) at nucleotide positions--33, 125, 265, and 298 of the Duffy gene and at nucleotide positions-261, 297, 467, 646, and 703 of the ABO gene. In an Italian population sample, we found four alleles and seven genotypes for the Duffy and six alleles and 16 genotypes for the ABO systems. The lower limit for reproducible results was 200 pg DNA, with a range of up to 10 ng and an optimum at 1 ng. All of the 16 analyzed inclusive paternity tests were also consistent with parentage and two out of four inconsistencies with parentage cases were excluded by one or more SNPs. Although Duffy and ABO SNP typing show lower informativeness than most current forensic tests, their robustness, the limited population distribution of FY* Fy type, and the sensitivity of the minisequencing technology suggest that these markers can be useful in selected forensic applications.
Molecular genetic basis of the histoblood group system
- F Yamamoto
- H Clausen
- T White
- J Marken
- S Hakamori
Yamamoto F, Clausen H, White T, Marken J,
Hakamori S: Molecular genetic basis of the histoblood group system. Nature 1990;345:229–233.