Haplotype sequence collection of ABO blood group alleles by long-read sequencing reveals putative A1-diagnostic variants

Gueuning, Morgan; Thun, Gian Andri; Wittig, Michael; Galati, Anna-Lena; Meyer, Stefan; Trost, Nadine; Gourri, Elise; Fuß, Janina; Sigurdardottir, Sonja; Merki, Yvonne; Neuenschwander, Kathrin; Busch, Yannik; Trojok, Peter; Schäfer, Marco; Gottschalk, Jochen; Franke, André; Gassner, Christoph; Peter, Wolfgang; Frey, Beat M.; Mattle-Greminger, Maja P.

doi:10.1182/bloodadvances.2022007133

Cited by 13 publications

(17 citation statements)

References 70 publications

(99 reference statements)

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“…Recently, long-read sequencing technologies for generating continuous sequences of a single molecule have been developed 9 and their application to the blood group genes has been reported. [10][11][12][13][14] The long-read and accuracy of HiFi reads enabled us to determine the 20 Kb-long sequence, including the initiation and termination codons, of the JK gene. In addition, we obtained more than 750 HiFi-reads of the JK silencing alleles (data not shown); however, 30 randomly selected reads were sufficient to determine the sequences in the present study.…”

Section: Discussionmentioning

confidence: 99%

“…In terms of genomic DNA analysis using Sanger sequencing or short‐read‐based next generation sequencing, it is difficult to determine the cis or trans positions of SNVs, which are occasionally more than 1 Kb away from each other. Recently, long‐read sequencing technologies for generating continuous sequences of a single molecule have been developed 9 and their application to the blood group genes has been reported 10–14 . The long‐read and accuracy of HiFi reads enabled us to determine the 20 Kb‐long sequence, including the initiation and termination codons, of the JK gene.…”

Section: Discussionmentioning

confidence: 99%

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Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

et al. 2023

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BackgroundThe Kidd blood group gene SLC14A1 (JK) accounts for approximately 20 Kb from initiation codon to stop codon in the genome. In genomic DNA analysis using Sanger sequencing or short‐read‐based next generation sequencing, it is difficult to determine the cis or trans positions of single nucleotide variations (SNVs), which are occasionally more than 1 Kb away from each other. We aimed to determine the complete nucleotide sequence of a 20‐Kb genomic DNA amplicon to characterize the JK allelic variants associated with Kidd antigen silencing in a blood donor.Study Design and MethodsThe Jk(a–b–) phenotype was identified in this donor by standard serological typing. A DNA sample obtained from whole blood was amplified by long‐range PCR to obtain a 20‐Kb fragment of the SLC14A1 gene, including the initiation and stop codons. The fragment was then analyzed by Sanger sequencing and single‐molecule sequencing. Transfection and expression studies were performed in CHO cells using the expression vector construct of JK alleles.ResultsSanger sequencing and single‐molecule sequencing revealed that the donor was heterozygous with JK*01 having c.276G>A (rs763262711, p.Trp92Ter) and JK*02 having c.499A>G (rs2298719, p.Met167Val), c.588A>G (rs2298718, p.Pro196Pro), and c.743C>A (p.Ala248Asp). The two JK alleles identified have not been previously described. Transfection and expression studies indicated that the CHO cells transfected with JK*02 having c.743C>A did not express the Jkb and Jk3 antigens.ConclusionsWe identified new JK silencing alleles and their critical SNVs by single‐molecule sequencing and the findings were confirmed by transfection and expression studies.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

et al. 2023

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“…In the past, magnetic beads were used to separate alleles for ABO sequencing (Danzer et al, 2010) and, only recently, Oxford Nanopore third‐generation sequencing technology was applied for ABO haplotyping (Gueuning et al, 2022). As opposed to these methods and next‐generation sequencing technology, our approach requires only standard sequencing equipment.…”

Section: Discussionmentioning

confidence: 99%

Allele‐specific long‐range sequencing as a method for ABO haplotyping in clinical blood group diagnosis and immunohematology research

Matzhold,

Drexler‐Helmberg,

Helmberg

et al. 2023

Molec Gen & Gen Med

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BackgroundSafe transfusion therapy requires accurate testing of blood donors and recipients to determine their ABO blood group compatibility. Genotyping does not always clarify serological blood typing discrepancies and conventional PCR methods are not suitable to identify ABO haplotypes. Therefore, an allele‐specific long‐range sequencing‐based typing method was established.MethodsStudy samples (n = 100) and six patient samples were ABO phenotyped and screened for specific single nucleotide polymorphisms (SNP) in the ABO gene. Based on identified heterozygous SNPs in intron 1 (12897G>A), 2 (437C>T) or 4 (102C>A, 1511G>T) both ABO alleles were investigated separately using a high‐fidelity long‐range PCR system and Sanger sequencing. The alleles were correlated to the ABO phenotypes determined.ResultsDirect sequencing of allelic PCR products up to 6743 bases has been successful in discriminating common combinations of the ABO*A1.01, ABO*A2.01, ABO*B.01, ABO*O.01.01, ABO*O.01.02 and ABO*O.02.01 alleles. 10 out of 64 haplotypes were found to be not previously described. The uncommon ABO*AW.31.01 and the unusual O alleles ABO*O.05 and ABO*O.02.03 alleles were detected in patient samples, resolving the initial inconclusive serologic ABO typing results.ConclusionThis method is an effective tool for analyzing ABO haplotypes. Applicable for ABO molecular diagnostics and immunohematology research it may help to improve pre‐transfusion blood type testing.

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“…According to the ISBT allele table (version 1.2, released on October 21, 2017), 84 alleles are associated with the A 1 , A 2 , and weak A phenotypes [4]. Most In a recent study by Gueuning et al [6], long-read sequencing of the entire ABO gene led to the identification of four sequence variations in intron 1 significantly associated with the ABO*A1 allele. These variants represent promising diagnostic targets for the A 1 blood group.…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Single Nucleotide Variants in Intron 1 of the ABO Gene as Diagnostic Markers for the A1 Blood Group

Bugert

Rink

Klüter³

2023

Transfus Med Hemother

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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.

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Haplotype sequence collection of ABO blood group alleles by long-read sequencing reveals putative A1-diagnostic variants

Cited by 13 publications

References 70 publications

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

Two new JK silencing alleles identified by single molecule sequencing with 20‐Kb long‐reads

Allele‐specific long‐range sequencing as a method for ABO haplotyping in clinical blood group diagnosis and immunohematology research

Evaluation of Single Nucleotide Variants in Intron 1 of the ABO Gene as Diagnostic Markers for the A1 Blood Group

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