[Analysis of a Chinese pedigree with Bw subtype due to a novel variant of α-1,3-N-acetylgalactosaminyltransferase gene].

OBJECTIVE: To explore the serological characteristics and genetic variant in a Chinese pedigree with Bw subtype. METHODS: A 32-year-old female proband who had undergone prenatal examination on December 10, 2020 at the 960th Hospital of the PLA Joint Logistics Support Force and five members from her pedigree were selected as the study subjects. Peripheral blood samples were collected and subjected to ABO blood group phenotyping with serological methods and ABO blood group genotyping with fluorescent PCR. Genetic testing and haplotype analysis were carried out by direct sequencing of the entire coding region of the ABO gene in the proband and cloned sequencing of exons 1-7. RESULTS: The blood type serology of the proband showed Bw, and her ABO blood type genotype determined by fluorescence PCR was B/O. The direct sequencing results showed that the proband had matched the ABO*O.01.01/ABO*B.01 genotype and carried a c.1A>G variant. Cloned sequencing has confirmed the c.1A>G variant to have occurred in the ABO*B.01 allele. Family analysis revealed that the mother of the proband had an O blood type, her husband had a B phenotype, and her three children had a normal B blood type. DNA sequencing showed that the two sons of the proband had a genotype of ABO*B.01 and c.1A>G/ABO*B.01. The daughter of the proband was ABO*O.01.01/ABO*B.01, whilst her mother was ABO*O.01.01/ABO *O.01.02. The novel c.1A>G variant sequence has been registered with the database with a number MZ076785 1. CONCLUSION: The novel c.1A>G variant of exon 1 of α- 1,3 galactose aminotransferase gene probably underlay the reduced expression of B antigen in this pedigree.

Non-O blood types are associated with a greater risk of large artery atherosclerosis stroke and dysregulation of cholesterol metabolism: an observational study.

BACKGROUND: Previous research on ABO blood types and stroke has been controversial, predominantly suggesting heightened risk of stroke in non-O blood types. Nonetheless, investigations into the correlation and underlying mechanisms between ABO blood groups and stroke subtypes, especially within Chinese cohorts, remain limited. METHODS: The ABO blood types of 9,542 ischaemic stroke (IS) patients were inferred using two ABO gene loci (c.261G > del; c.802G > A). The healthy population was derived from the 1000 Genomes Project. Patients were classified by the causative classification system (CCS). Volcano plot and gene ontology (GO) analysis were employed to explore protein differential expression among blood types. Additionally, HT29 and SW480 cell lines with downregulated ABO expression were generated to evaluate its impact on cholesterol uptake and efflux. RESULTS: A greater proportion of stroke patients had non-O blood types (70.46%) than did healthy individuals (61.54%). Notable differences in blood type distributions were observed among stroke subtypes, with non-O blood type patients mainly classified as having large artery atherosclerosis (LAA). Clinical baseline characteristics, such as the low-density lipoprotein cholesterol level, activated partial thromboplastin time and thrombin time, varied significantly among blood types. A volcano plot revealed 17 upregulated and 42 downregulated proteins in the O blood type. GO term analysis indicated that downregulated proteins were primarily associated with lipid metabolism pathways. In vitro experiments revealed that reducing ABO gene expression decreased cholesterol uptake and increased cholesterol efflux. CONCLUSIONS: This study revealed that the non-O blood type increased the risk of LAA stroke through cholesterol metabolism.

[Genetic analysis of an individual with A3 phenotype due to variant of A-glycosyltransferase enzyme gene].

OBJECTIVE: To explore the serological characteristics and molecular mechanism underlying an individual with A3 phenotype. METHODS: A 27-year-old ethnic Han Chinese woman presented at the Fourth Affiliated Hospital of China Medical University on May 12, 2022 was selected as the study subject. ABO blood type was determined with standard serological techniques. The ABO gene was subjected to direct sequencing of PCR products. Exons 6 and 7 of the ABO gene were sequenced using specific primers to determine the haplotypes. Bioinformatic software was used to analyze the structure of the mutant protein. RESULTS: Serological typing of the ABO blood group has suggested a rare A3 phenotype. The proband was found to harbor heterozygous c.261delG, c.467C>T and c.745C>T variants by direct sequencing. Single strand sequencing revealed that she has harbored ABO*A3.07 and ABO*O.01.01 alleles. The ABO*A3.07 allele has contained a c.745C>T (p.R249W) variant on the background of an ABO*A1.02 allele. The p.R249W substitution was predicted to be probably damaging by the PolyPhen2 software. The free energy change (ΔΔG) value predicted it to have a destabilizing effect on the GTA protein. Meanwhile, modeling of the 3D structure has predicted that the p.R249W amino acid substitution may alter the hydrogen bond network of the GTA protein. CONCLUSION: The p.R249W substitution of the α-1,3-N-acetylgalactosaminyltransferase gene may reduce the antigen expression owing to a great destabilizing effect on the structure and function of the GTA protein.

Association between blood groups and myocardial injury after non-cardiac surgery: a retrospective cohort study.

Blood group is a potential genetic element in coronary artery disease. Nevertheless, the relationship between different ABO blood groups and myocardial injury after non-cardiac surgery (MINS) is poorly understood. This study verified whether ABO blood group is a potential MINS influencing factor. This retrospective cohort study included 1201 patients who underwent elective non-cardiac surgery and a mandatory troponin test on postoperative days 1 and 2 from 2019 to 2020 at a university-affiliated tertiary hospital. The primary outcome was associations between ABO blood groups and MINS, assessed using univariate and multivariate logistic-regression analyses. Path analysis was used to investigate direct and indirect effects between blood group and MINS. MINS incidence (102/1201, 8.5%) was higher in blood-type B patients than in non-B patients [blood-type B: 44/400 (11.0%) vs. non-B: 58/801 (7.2%); adjusted odds ratio = 1.57 (1.03-2.38); p = 0.036]. In the confounding factor model, preoperative hypertension and coronary artery disease medical history were associated with MINS risk [adjusted odds ratio: 2.00 (1.30-3.06), p = 0.002; 2.81 (1.71-4.61), p < 0.001, respectively]. Path analysis did not uncover any mediating role for hypertension, diabetes, or coronary artery disease between blood type and MINS. Therefore, blood-type B is associated with higher MINS risk; potential mediators of this association need to be investigated.

Association between plasma proteome and pulmonary heart disease: A two-stage Mendelian randomization analysis.

Pulmonary heart disease (PHD) involves altered structure and function of the right ventricle caused by an abnormal respiratory system that causes pulmonary hypertension. However, the association between changes in plasma proteomics and PHD remains unclear. Hence, we aimed to identify causal associations between genetically predicted plasma protein levels and PHD. Mendelian randomization was performed to test the target proteins associated with PHD. Summary statistics for the human plasma proteome and pulmonary heart disease were acquired from the UK Biobank (6038 cases and 426 977 controls) and the FinnGen study (6753 cases and 302 401 controls). Publicly available pQTLs datasets for human plasma proteins were obtained from a largescale genome-wide association study in the INTERVAL study. The results were validated using a case-control cohort. We first enrolled 3622 plasma proteins with conditionally independent genetic variants; three proteins (histo-blood group ABO system transferase, activating signal cointegration 1 complex subunit 1, and calcium/calmodulin-dependent protein kinase I [CAMK1]) were significantly associated with the risk of pulmonary heart disease in the UK Biobank cohort. Only CAMK1 was successfully replicated (odds ratio: 1.1056, 95% confidence interval: 1.019-1.095, p = 0.0029) in the FinnGen population. In addition, the level of CAMK1 in 40 patients with PHD was significantly higher (p = 0.023) than that in the control group. This work proposes that CAMK1 is associated with PHD, underscoring the importance of the calcium signaling pathway in the pathophysiology to improve therapies for PHD.

Potential therapeutic targets for membranous nephropathy: proteome-wide Mendelian randomization and colocalization analysis.

Background: The currently available medications for treating membranous nephropathy (MN) still have unsatisfactory efficacy in inhibiting disease recurrence, slowing down its progression, and even halting the development of end-stage renal disease. There is still a need to develop novel drugs targeting MN. Methods: We utilized summary statistics of MN from the Kiryluk Lab and obtained plasma protein data from Zheng et al. We performed a Bidirectional Mendelian randomization analysis, HEIDI test, mediation analysis, Bayesian colocalization, phenotype scanning, drug bank analysis, and protein-protein interaction network. Results: The Mendelian randomization analysis uncovered 8 distinct proteins associated with MN after multiple false discovery rate corrections. Proteins related to an increased risk of MN in plasma include ABO [(Histo-Blood Group Abo System Transferase) (WR OR = 1.12, 95%CI:1.05-1.19, FDR=0.09, PPH4 = 0.79)], VWF [(Von Willebrand Factor) (WR OR = 1.41, 95%CI:1.16-1.72, FDR=0.02, PPH4 = 0.81)] and CD209 [(Cd209 Antigen) (WR OR = 1.19, 95%CI:1.07-1.31, FDR=0.09, PPH4 = 0.78)], and proteins that have a protective effect on MN: HRG [(Histidine-Rich Glycoprotein) (WR OR = 0.84, 95%CI:0.76-0.93, FDR=0.02, PPH4 = 0.80)], CD27 [(Cd27 Antigen) (WR OR = 0.78, 95%CI:0.68-0.90, FDR=0.02, PPH4 = 0.80)], LRPPRC [(Leucine-Rich Ppr Motif-Containing Protein, Mitochondrial) (WR OR = 0.79, 95%CI:0.69-0.91, FDR=0.09, PPH4 = 0.80)], TIMP4 [(Metalloproteinase Inhibitor 4) (WR OR = 0.67, 95%CI:0.53-0.84, FDR=0.09, PPH4 = 0.79)] and MAP2K4 [(Dual Specificity Mitogen-Activated Protein Kinase Kinase 4) (WR OR = 0.82, 95%CI:0.72-0.92, FDR=0.09, PPH4 = 0.80)]. ABO, HRG, and TIMP4 successfully passed the HEIDI test. None of these proteins exhibited a reverse causal relationship. Bayesian colocalization analysis provided evidence that all of them share variants with MN. We identified type 1 diabetes, trunk fat, and asthma as having intermediate effects in these pathways. Conclusions: Our comprehensive analysis indicates a causal effect of ABO, CD27, VWF, HRG, CD209, LRPPRC, MAP2K4, and TIMP4 at the genetically determined circulating levels on the risk of MN. These proteins can potentially be a promising therapeutic target for the treatment of MN.

Gene-Gene Interaction Between Factor-XI and ABO Genes in Cerebral Venous Thrombosis: The BEAST Study.

BACKGROUND AND OBJECTIVES: Gene-gene interactions likely contribute to the etiology of multifactorial diseases such as cerebral venous thrombosis (CVT) and could be one of the main sources of known missing heritability. We explored Factor XI (F11) and ABO gene interactions among patients with CVT. METHODS: Patients with CVT of European ancestry from the large Bio-Repository to Establish the Aetiology of Sinovenous Thrombosis (BEAST) international collaboration were recruited. Codominant modelling was used to determine interactions between genome-wide identified F11 and ABO genes with CVT status. RESULTS: We studied 882 patients with CVT and 1,205 ethnically matched control participants (age: 42 ± 15 vs 43 ± 12 years, p = 0.08: sex: 71% male vs 68% female, p = 0.09, respectively). Individuals heterozygous (AT) for the risk allele (T) at both loci (rs56810541/F11 and rs8176645/ABO) had a 3.9 (95% CI 2.74-5.71, p = 2.75e-13) increase in risk of CVT. Individuals homozygous (TT) for the risk allele at both loci had a 13.9 (95% CI 7.64-26.17, p = 2.0e-15) increase in risk of CVT. The presence of a non-O blood group (A, B, AB) combined with TT/rs56810541/F11 increased CVT risk by OR = 6.8 (95% CI 4.54-10.33, p = 2.00e15), compared with blood group-O combined with AA. DISCUSSION: Interactions between factor XI and ABO genes increase risk of CVT by 4- to 14-fold.

[Genetic analysis of two patients with a rare Ael subtype].

OBJECTIVE: To analyze the genetic sequences of two patients with a rare Ael blood subgroup. METHODS: Two female patients undergoing treatment respectively for adenomyoma of the uterus and gastritis at the Second Affiliated Hospital, Yuying Children's Hospital of Wenzhou Medical University in June 2019 and September 2020 were selected as the study subjects. Their Ael subtypes were identified with a saline tube agglutination assay and absorption-emission assay. Sequence of the ABO gene Ael subtypes was determined by the Sanger method. The impact of genetic variants on the structural stability of N-acetylgalactosaminyl transferase (GTA) was analyzed with PyMOL software by constructing a structure predicted model. RESULTS: Both patients were determined as Ael blood subgroup. Sequencing result of patient 1 was ABO*O.01.02/ABO*O.01.02, which has resulted in a p.Thr88Profs*31 amino acid substitution. The sequencing result of patient 2 was ABO*Ael.06/ABO*O.01.02, in which c.425C>T and c.467C>T variants in exon 7 have led to p.Met142Thr and p.Pro156Leu substitutions. Prediction of the protein model speculated that the p.Met142Thr not only can change the binding of GTA protein with water molecules, but also the local hydrogen bond network of GTA, which may lead to decreased enzymatic activity. By contrast, the p.Pro156Leu variant has trivial effect on the structural stability of GTA. CONCLUSION: The molecular structure of Ael subtypes can be diverse. The genotypes of the two patients have been respectively determined as ABO*O.01.02/ABO*O.01.02 with a G261 deletion and ABO*Ael.06/ABO*O.01.02.

[Molecular study of an individual with Bel subtype due to a novel c.620T>C variant].

OBJECTIVE: To explore the molecular basis for an individual with Bel subtype of the ABO blood type due to a novel c.620T>C variant gene, and assess its impact on the structure of GTB transferase. METHODS: An individual who had visited the First Affiliated Hospital of Zhengzhou University on February 11, 2023 was selected as the study subject. ABO phenotyping was initially conducted with serological methods, which was followed by direct sequencing of 7 exons of the ABO gene. Subsequently, single-strand sequencing was carried out by using allele-specific primers, and the variant in the B transferase was homology-modeled using the Modeller software. The impact of the variant on the transferase's spatial structure was analyzed with the PyMOL software. RESULTS: The serological phenotype of the patient was identified as the Bel subtype. Direct sequencing revealed that she has harbored a novel c.620T>C variant, resulting in a p.Leu207Pro substitution in the polypeptide chain. Combined with single-strand sequencing, her genotype was ultimately determined as ABO*BELnew/ABO*O.01.02. Three-dimensional protein structure modeling showed that, compared with the wild type, the distance of one hydrogen bond between Proline and Glycine at position 272 has increased, along with disappearance of another hydrogen bond. CONCLUSION: The novel c.620T>C (p.Leu207Pro) variant of B allele may affect the structural stability of the glycosyltransferase. The weakened enzyme activity in turn may lead to reduced B antigen expression, manifesting as the Bel subtype by serological analysis.

Serology and molecular genetic analysis of two unrelated individuals with the same novel CisAB blood type.

OBJECTIVE: This study aims to report two unrelated individuals with the same novel CisAB blood type and confirm this rare blood type using a comprehensive approach that combines serological and molecular biology techniques. METHODS: Peripheral blood samples were collected from two patients and their family members. ABO blood typing and antibody detection were performed using conventional tube methods. Molecular biology techniques were employed to amplify and sequence the 6th and 7th exons of the ABO gene, with reference to gene mutation databases provided by NCBI and ISBT. RESULTS: The genotypes of the two unrelated individuals were identical and were confirmed as a new genotype through ISBT gene database comparison. Serological testing results showed different antigen reaction patterns, especially in terms of reverse typing. Gene sequencing identified a series of mutation points, and both unrelated individuals and one of their daughters had mutations at 297 A>G, 526 C>G, 657 C>T, 703 G>A, 803 G>C, and 930 G>A. According to the comprehensive results from The Blood Group Antigen Gene Mutation Database provided by NCBI, the genotype was determined as Bw37. However, based on the results from Names for ABO (ISBT 001) blood group alleles v1.1 171023, the sequencing results indicated a novel mutation combination not found in the ISBT database. Considering the serological reactions of all three individuals, the final determination was CisAB. CONCLUSIONS: This study confirmed the novel CisAB blood type in two individuals through the comprehensive application of serology and molecular biology techniques. The identified gene mutation points were not recorded in known databases, emphasizing the uniqueness of CisAB blood types. This research provides important insights into the genetic basis of ABO subtypes and the characteristics of CisAB blood types, and the relevant results have been submitted to the ISBT website for further research.

Prevalence of ABO and Rhesus (D) Blood Group and Allelic Frequency at Blood Bank of Nigist Eleni Mohammed Hospital, Ethiopia.

Background: The purpose of this cross-sectional study was to determine the pattern of the ABO and rhesus D (RhD) blood group distribution among voluntary blood donors attending five blood donation centers at Nigist Eleni Mohammed General Hospital in Hossana, Ethiopia. Methods: A total of 1,120 participants who fulfilled the "who can give blood" criteria of the World Health Organization were selected randomly. Blood samples were collected, transported to the laboratory, and analyzed for ABO and RhD typing. The data was analyzed using descriptive statistics and chi-square correlation analysis. Results: The study found that the O blood group was the most prevalent (39.0%), followed by A (32.2%), B (22.5%), and AB (6.4%). When considering both the ABO and Rh blood groups together, 92.9% of blood donors were RhD positive, while only 7.1% were RhD negative. The distribution pattern of the ABO blood groups in Gurage Zone, Hadiya Zone, Kembata Zone, and Silte Zone showed that the O blood group was the most prevalent, followed by A, B, and AB, in that order. Conversely, the ABO blood group distribution pattern in Halaba Zone was A > O > B > AB. Civil servants from different occupational statuses were the most dominant voluntary blood donors, accounting for 53.2%, followed by students from different high schools and universities (41.9%), self-employed individuals (4.1%), and others (0.7%). The ABO blood group system had observed allele frequencies significantly different from the expected frequencies (p = 0.007), while the RhD system did not (p = 0.037). Allele frequencies for A, B, and O in the ABO system were 0.3531, 0.2576, and 0.3893, respectively. Observed frequencies for RhD-positive and RhD-negative alleles were 0.9647 and 0.0531, respectively. Conclusion: This study highlights the regional ABO and RhD blood group variations in Ethiopia, noting disparities from expected ABO allele frequencies, and identifies the O blood group predominance among donors with a high RhD-positive prevalence.

Identification and Blood Transfusion of a Rare A1.02/BA.02 Genotype.

BACKGROUND: The aim was to analyze the serological and molecular genetic characteristics of a rare B(A) subtype pedigree, explore its pathogenesis, and discuss transfusion strategies. METHODS: ABO blood typing serological tests were conducted on a female subject and her family member using standard serological methods. Sequencing analysis of the ABO gene exons 6 and 7 was performed using PCR technique for the female subject and her family member to examine the blood types of the participants. RESULTS: The serological test results showed a discrepancy between the forward and reverse typings of the female subject. The forward typing was similar to that of AB subtype serological forward typing, while the reverse typing indicated AB blood type. Based on the sequencing results, it is inferred that the female subject and her son have 8 mutations on one BA.02 chain: 297A>G, 526C>G, 657C>T, 700C>G, 703G>A, 796C>A, 803G>C, and 930G>A. Comparing these eight mutation sites with the Blood Group Antigen Gene Mutation Database (BGMUT), it was found that the female subject had a heterozygous mutation at c.700C>G in the 7th exon of the B.01 gene, consistent with the characteristics of the BA.02 allele. The genotype of the female subject was determined as A1.02/ BA.02, while the genotype of her son was determined as O.01.01/BA.02. CONCLUSIONS: The serological presentation of the B(A) subtype for the female subject reported in this study was unique. It differed from previously reported cases, indicating that the determination of B(A) subtypes cannot solely rely on serological testing. It requires a comprehensive analysis combining the results of genetic testing and pedigree investigation.

CRISPR/Cas13a-based single-nucleotide polymorphism detection for reliable determination of ABO blood group genotypes.

The ABO blood group plays an important role in blood transfusion, linkage analysis, individual identification, etc. Serologic methods of blood typing are gold standards for the time being, which require stable typing antisera and fresh blood samples and are labor intensive. At present, reliable determination of ABO blood group genotypes based on single-nucleotide polymorphisms (SNPs) among A, B, and O alleles remains necessary. Thus, in this work, CRISPR/Cas13a-mediated genotyping for the ABO blood group by detecting SNPs between different alleles was proposed. The ABO*O.01.01(c.261delG) allele (G for the A/B allele and del for the O allele) and ABO*B.01(c.796C > A) allele (C for the A/O allele and A for the B allele) were selected to determine the six genotypes (AA, AO, BB, BO, OO, and AB) of the ABO blood group. Multiplex PCR was adapted to simultaneously amplify the two loci. CRISPR/Cas13a was then used to specifically differentiate ABO*O.01.01(c.261delG) and ABO*B.01(c.796C > A) of A, B, and O alleles. Highly accurate determination of different genotypes was achieved with a limit of detection of 50 pg per reaction within 60 min. The reliability of this method was further validated based on its applicability in detecting buccal swab samples with six genotypes. The results were compared with those of serological and sequencing methods, with 100% accuracy. Thus, the CRISPR/Cas13a-mediated assay shows great application potential in the reliable identification of ABO blood group genotypes in a wide range of samples, eliminating the need to collect fresh blood samples in the traditional method.

Transfusing children with sickle cell disease using blood group genotyping when the pool of Black donors is limited.

BACKGROUND: Red blood cell transfusion is an effective treatment for patients with sickle cell disease (SCD). Alloimmunization can occur after a single transfusion, limiting further usage of blood transfusion. It is recommended to match for the ABO, D, C, E, and K antigens to reduce risks of alloimmunization. However, availability of compatible blood units can be challenging for blood providers with a limited number of Black donors. STUDY DESIGN AND METHODS: A prospective cohort of 205 pediatric patients with SCD was genotyped for the RH and FY genes. Transfusion and alloimmunization history were collected. Our capacity to find RhCE-matched donors was evaluated using a database of genotyped donors. RESULTS: Nearly 9.8% of patients carried a partial D variant and 5.9% were D-. Only 45.9% of RHCE alleles were normal, with the majority of variants affecting the RH5 (e) antigen. We found an alloimmunization prevalence of 20.7% and a Rh alloimmunization prevalence of 7.1%. Since Black donors represented only 1.40% of all blood donors in our province, D- Caucasian donors were mostly used to provide phenotype matched products. Compatible blood for patients with rare Rh variants was found only in Black donors. A donor with compatible RhCE could be identified for all patients. CONCLUSION: Although Rh-compatible donors were identified, blood units might not be available when needed and/or the extended phenotype or ABO group might not match the patient. A greater effort has to be made for the recruitment of Black donors to accommodate patients with SCD.

The increasing need for ABO blood group genotyping and quality assurance implications for laboratory implementation.

ABO blood group antigens are critical determinants of immunologic self and non-self and are ubiquitously expressed on all cellular tissues. Antibodies against non-self ABO antigens are naturally present and can mediate pathologic reactions against incompatible transfused blood cells and transplanted tissues. Laboratory testing for ABO antigens and isoagglutinins is essential for safe and effective transfusion and transplantation. Testing for ABO antigens has traditionally depended on serologic testing. However, there is increasing need for evaluation of genetic analysis of ABO antigens, to enable evaluation of ABO blood group in cases where serologic testing may be ambiguous or impossible to accurately perform. The clinical need for ABO genotyping is being addressed by the development of multiple molecular diagnostic approaches. Recent data have clearly demonstrated the potential utility of ABO genotyping in solid organ transplantation, yet widespread implementation has been slow. We propose that this lag is related to practical considerations in laboratory testing, including limited regulatory guidance on the performance and reporting of these assays and the absence of widely available external proficiency testing programs for quality assurance. Here we describe approaches to ABO genotyping, current initiatives in developing ABO genotyping proficiency testing programs, and laboratory quality assurance considerations for ABO genotyping.