Simultaneous high throughput genotyping of 36 blood group systems using NGS based on probe capture technology.

Human blood group antigen has important biological functions, and transfusion of incompatible blood can cause alloimmunization and may lead to serious hemolytic reactions. Currently, serological methods are most commonly used in blood group typing. However, this technique has certain limitations and cannot fully meet the increasing demand for the identification of blood group antigens. This study describes a next-generation sequencing (NGS) technology platform based on exon and flanking region capture probes to detect full coding exon and flanking intron regions of the 36 blood group systems, providing a new high-throughput method for the identification of blood group antigens. The 871 capture probes were designed for the exon and flanking intron sequences of 36 blood group system genes, and synchronization analysis for 36 blood groups was developed. The library for NGS was tested using the MiSeq Sequencing Reagent Kit (v2, 300 cycles) by Illumina NovaSeq, and the data were analyzed by the CLC Genomics Workbench 21.0 software. A total of 199 blood specimens have been sequenced for the 41 genes from 36 blood groups. Among them, heterozygote genotypes were found in the ABO, Rh, MNS, Lewis, Duffy, Kidd, Diego, Gerbich, Dombrock, Globoside, JR, LAN, and Landsteiner-Wiene blood group systems. Only the homozygous genotype was found in the remaining 22 blood group systems. The obtained data in the NGS method shows a good correlation (99.98 %) with those of the polymerase chain reaction-sequence-based typing. An NGS technology platform for 36 blood group systems genotyping was successfully established, which has the characteristics of high accuracy, high throughput, and wide coverage.

Genetic and mechanistic evaluation of an individual with para-Bombay phenotype associated with a compound heterozygote comprising two novel FUT1 variants.

BACKGROUND: As is well documented, the para-Bombay phenotype is typically characterized by the reduction or absence of ABH antigens on red blood cells but the presence of corresponding antigens in saliva. Herein, the underlying molecular mechanism of an individual with para-Bombay AB phenotype combined with two novel variants of the FUT1 gene was investigated. MATERIALS AND METHODS: ABH antigens and antibodies were detected in the serum of the proband using conventional serological methods. The coding region nucleotides of the ABO, FUT1, and FUT2 genes were directly sequenced by polymerase chain reaction. Moreover, the FUT1 haploid type in the proband was analyzed by TA clone sequencing. The 3D structure of wild-type and mutant fucosyltransferases were simulated and analyzed using Phyre2 and Pymol software. Lastly, the effect of missense substitution on the function of fucosyltransferase was predicted by the Polymorphism Phenotyping algorithm (PolyPhen-2) and MutationTaster. RESULTS: ABH antigens were noted to be absent on the surface of red blood cells of the proband. The ABO genotype was ABO*A1.02/ABO*B.01, while the FUT2 genotype was FUT2*01/FUT2*c.357T. Interestingly, two novel missense variants (c.289G>A, p.Ala97Thr and c.575G>C, p.Arg192Pro) and one synonymous SNP (c.840G>A) were identified in the FUT1 gene. Furthermore, c.289G>A was detected in one haploid type, whereas c.575G>C and c.840G>A were discovered in another haploid type. Meanwhile, in silico analysis revealed that amino acid substitution caused by missense variants altered the partial spatial structure of the a-helices where residues 97 and 298 were located using 3D homology modeling software. Finally, both missense variants were defined as probably damaging based on PolyPhen-2 prediction. DISCUSSION: Two novel FUT1 variants were identified in a Chinese individual with para-Bombay AB phenotype, which can expand our understanding of the molecular mechanism underlying the para-Bombay phenotype and contribute to improving the safety of blood transfusion.

[Serological characteristics and molecular mechanism of an individual with p phenotype].

OBJECTIVE: To analyze the serological characteristics and molecular mechanism for an individual with p phenotype. METHODS: An individual with p phenotype upon blood group identification at Jiaxing Blood Center in May 2021 was analyzed. ABO, RhD and P1PK blood groups and irregular antibodies in her serum were identified using conventional serological methods. The encoding region of α1, 4-galactosyltransferase gene (A4GALT) encoding P1 and Pk antigens was analyzed by polymerase chain reaction-sequence-based typing (PCR-SBT). RESULTS: The individual was A group, RhD positive and had a p phenotype of the P1PK blood group system. Anti-PP1Pk was discovered in her serum. Sequencing analysis revealed that she has harbored a homozygous c.343A>T variant of the A4GALT gene. CONCLUSION: The homozygous c.343A>T variant of the A4GALT gene probably underlay the p phenotype in this individual.

A case of McLeod syndrome caused by a nonsense variation c.942G>A in the XK gene: A case report.

McLeod syndrome is a rare XK gene-related progressive, debilitating disease involving multiple systems. The blood group phenotypes in McLeod syndrome patients usually display the Kx antigen loss and a decrease in the Kell blood group system antigen expression. This paper describes a 41-year-old male Chinese patient with McLeod syndrome. He first attended a hospital in 2015 and developed progressively worsening symptoms 4 years ago. As the disease progressed, the patient exhibited memory loss, unresponsiveness, and chorea and displayed elevated creatine kinase levels. However, McLeod syndrome could not be diagnosed by these signs and laboratory results. The patient was readmitted to the hospital in 2020 and was suspected of having McLeod syndrome. Serological analysis of the Kell blood group system and genotyping for the XK blood group system were performed, revealing the weak expression of the K antigen and the negative K antigen. Sequencing of the coding region of the XK gene showed a hemizygous c.942G>A variation in the XK gene, which resulted in a premature stop codon at position 314 (p.Trp314Ter). Therefore, the patient was diagnosed with McLeod syndrome. In conclusion, this paper presents a case of McLeod syndrome caused by a nonsense variation c.942G>A in the XK gene. The analysis of the XK gene and blood group antigen is helpful for the diagnosis of McLeod syndrome and for distinguishing it from many other diseases.

[Study of the molecular characteristics of a Bweak phenotype due to a novel c.398T>C variant of the ABO gene].

OBJECTIVE: To explore the molecular mechanism for an individual with Bweak subtype. METHODS: Serological methods were used to identify the proband's phenotype. In vitro enzyme activity test was used to determine the activity of B-glycosyltransferase (GTB) in her serum. The genotype was determined by PCR amplification and direct sequencing of exons 5 to 7 and flanking sequences of the ABO gene. T-A cloning technology was used to isolate the haploids. The primary physical and chemical properties and secondary structure of the protein were analyzed with the ProtParam and PSIPRED software. Three software, including PolyPhen-2, SIFT, and PROVEAN, was used to analyze the effect of missense variant on the protein. RESULTS: Serological results showed that the proband's phenotype was Bweak subtype with anti-B antibodies presented in her serum. In vitro enzyme activity assay showed that the GTB activity of the subject was significantly reduced. Analysis of the haploid sequence revealed a c.398T>C missense variant on the B allele, which resulted in a novel B allele. The 398T>C variant has caused a p.Phe133S substitution at position 133 of the GTB protein. Based on bioinformatic analysis, the amino acid substitution had no obvious effect on the primary and secondary structure of the protein, but the thermodynamic energy of the variant protein has increased to 6.07 kcal/mol, which can severely reduce the protein stability. Meanwhile, bioinformatic analysis also predicted that the missense variant was harmful to the protein function. CONCLUSION: The weak expression of the Bweak subtype may be attributed to the novel allele of ABO*B.01-398C. Bioinformatic analysis is helpful for predicting the changes in protein structure and function.

Simultaneous genotyping for human platelet antigen systems and HLA-A and HLA-B loci by targeted next-generation sequencing.

In order to treat the alloimmunization platelet transfusion refractoriness (PTR), human leukocyte antigen (HLA)-type and/or human platelet antigen (HPA)-type matched platelets between donors and patients are usually used. Therefore, genotyping of HLA-A and HLA-B loci, as well as HPA systems, for donors and patients, is of great significance. However, there is a rare report of genotyping for HLA-A and HLA-B loci as well as HPA systems at the same time. In this study, a high-throughput method for simultaneous genotyping of HLA-A and HLA-B loci, as well as HPA genotyping, was developed. A RNA capture probe panel was designed covering all exon sequences of the GP1BA, GP1BB, ITGA2, CD109, ITGB3, and ITGA2B genes and HLA-A and HLA-B loci. The HLA-A, HLA-B, and 34 HPA systems were genotyped using a targeted next-generation sequencing (NGS) method. The genotypes of the HLA-A and HLA-B loci, as well as the HPA, were assigned based on the nucleotides in the polymorphism sites. Using the NGS method, 204 unrelated blood specimens were successfully genotyped for all 34 HPA systems as well as HLA-A and HLA-B loci. The accuracy of the NGS method was 100%. Only HPA-2, HPA-3, HPA-5, HPA-6w, HPA-15, and HPA-21w showed polymorphism with frequencies of 0.9412, 0.6863, 0.9853, 0.9779, 0.4314, and 0.9951 for a allele, respectively. Thirty-two single nucleotide variants (SNVs) were detected. Of them, 12 SNVs can lead to amino acid change. HLA-A*11:01 and HLA-B*46:01 are the most common alleles for HLA-A and HLA-B loci. A targeted next-generation sequencing method for simultaneously genotyping HPA systems and HLA-A and HLA-B loci was first established, which could be used to create a database of HLA-typed and/or HPA-typed unrelated donors.

Analysis of the Genomic Sequence of ABO Allele Using Next-Generation Sequencing Method.

Background: Although many molecular diagnostic methods have been used for ABO genotyping, there are few reports on the full-length genomic sequence analysis of the ABO gene. Recently, next-generation sequencing (NGS) has been shown to provide fast and high-throughput results and is widely used in the clinical laboratory. Here, we established an NGS method for analyzing the sequence of the start codon to the stop codon in the ABO gene. Study Design and Methods: Two pairs of primers covering the partial 5'-untranslated region (UTR) to 3'-UTR of the ABO gene were designed. The sequences covering from the start codon to the stop codon of the ABO gene were amplified using these primers, and an NGS method based on the overlap amplicon was developed. A total of 110 individuals, including 88 blood donors with normal phenotypes and 22 ABO subtypes, were recruited and analyzed. All these specimens were first detected by serological tests and then determined by polymerase chain reaction sequence-based typing (PCR-SBT) and NGS. The sequences, including all the intron regions for the specimens, were analyzed by bioinformatics software. Results: Among the 88 blood donors with a normal phenotype, 48 homozygous individuals, 39 heterozygous individuals, and one individual with a novel O allele were found according to the results of the PCR-SBT method. Some single-nucleotide variants (SNV) in intronic regions were found to be specific for different ABO alleles from 48 homozygous individuals using the NGS method. Sequences in the coding region of all specimens using the NGS method were the same as those of the PCR-SBT method. Three intronic SNVs were found to be associated with the ABO subtypes, including one novel intronic SNV (c.28+5956T>A). Moreover, six specimens were found to exhibit DNA recombination. Conclusion: An NGS method was established to analyze the sequence from the start codon to the stop codon of the ABO gene. Two novel ABO alleles were identified, and DNA recombination was found to exist in the ABO alleles.

The Significance of RHD Genotyping and Characteristic Analysis in Chinese RhD Variant Individuals.

Background: RhD is the most important and complex blood group system because of its highly polymorphic and immunogenic nature. RhD variants can induce immune response by allogeneic transfusion, organ transplantation, and fetal immunity. The transfusion strategies are different for RhD variants formed by various alleles. Therefore, extensive investigation of the molecular mechanism underlying RhD variants is critical for preventing immune-related blood transfusion reactions and fetal immunity. Methods: RhD variants were collected from donors and patients in Zhejiang Province, China. The phenotypes were classified using the serologic method. The full coding regions of RHD gene were analyzed using the PCR-SBT method. The multiplex ligation-dependent probe amplification (MLPA) assay was used to analyze the genotype and gene copy number. SWISS-MODLE and PyMOL software were used to analyze 3D structures of RhD caused by the variant alleles. The effect of non-synonymous substitutions was predicted using Polymorphism Phenotyping algorithm (PolyPhen-2), Sorting Intolerant From Tolerant (SIFT), and Protein Variation Effect Analyzer (PROVEAN) software. Results: In the collected RhD variants, 28 distinct RHD variant alleles were identified, including three novel variant alleles. RH-MLPA assay is advantageous for determining the copy number of RHD gene. 3D homology modeling predicted that protein conformation was disrupted and may explain RhD epitope differential expression. A total of 14 non-synonymous mutations were determined to be detrimental to the protein structure. Discussion: We revealed the diversity of RHD alleles present in eastern Chinese RhD variants. The bioinformatics of these variant alleles extended our knowledge of RhD variants, which was crucial for evaluating their impact to guide transfusion support and avoid immune-related blood transfusion reactions.

Six splice site variations, three of them novel, in the ABO gene occurring in nine individuals with ABO subtypes.

BACKGROUND: Nucleotide mutations in the ABO gene may reduce the activity of glycosyltransferase, resulting in lower levels of A or B antigen expression in red blood cells. Six known splice sites have been identified according to the database of red cell immunogenetics and the blood group terminology of the International Society of Blood Transfusion. Here, we describe six distinct splice site variants in individuals with ABO subtypes. METHODS: The ABO phenotype was examined using a conventional serological method. A polymerase chain reaction sequence-based typing method was used to examine the whole coding sequence of the ABO gene. The ABO gene haplotypes were studied using allele-specific primer amplification or cloning technology. In silico analytic tools were used to assess the functional effect of splice site variations. RESULTS: Six distinct variants in the ABO gene splice sites were identified in nine individuals with ABO subtypes, including c.28 + 1_2delGT, c.28 + 5G > A, c.28 + 5G > C, c.155 + 5G > A, c.204-1G > A and c.374 + 5G > A. c.28 + 1_2delGT was detected in an Aw individual, while c.28 + 5G > A, c.28 + 5G > C, and c.204-1G > A were detected in Bel individuals. c.155 + 5G > A was detected in one B3 and two AB3 individuals, whereas c.374 + 5G > A was identified in two Ael individuals. Three novel splice site variants (c.28 + 1_2delGT, c.28 + 5G > A and c.28 + 5G > C) in the ABO gene were discovered, all of which resulted in low antigen expression. In silico analysis revealed that all variants had the potential to alter splice transcripts. CONCLUSIONS: Three novel splice site variations in the ABO gene were identified in Chinese individuals, resulting in decreased A or B antigen expression and the formation of ABO subtypes.

[Identification of a glycosyltransferase allele associated with Bw subtype and analysis of the protein structure].

OBJECTIVE: To explore the molecular basis for an individual with Bw subtype. METHODS: Routine serological reactions were used to determine the surface antigens of erythrocytes and antibodies in serum. PCR-sequence-based typing (PCR-SBT) was used to analyze the coding regions of the ABO gene and erythroid-specific regulatory element in its intron 1. Amplicons for exons 5 to 7 containing the variant site were subjected to TA cloning for the isolation of the haploid and verification of the sequence. The 3D structure of mutant protein was predicted with Pymol software. Changes of amino acid residues and structural stability were also analyzed. RESULTS: Serological assay showed that the individual had weakened B antigen and anti-B antibody in his serum. His genotype was determined as ABO*B.01/ABO*O.01.01. Sequencing of the entire coding region of the ABO gene identified an additional heterozygous c.734C/T variant. No variant was found in the erythroid-specific regulatory element of intron 1. Haploid cloning and isolation has obtained an ABO*O.01.01 allele and a ABO*B.01 allele containing a c.734T variant, which has led to substitution of Thr by Ile at position 245 in the functional center of glycosyltransferase. Based on the 3D structure of the protein, the residues binding with the mutation were unchanged, but the bonding distance between the hydrogens was changed with the amino acid substitution. Meanwhile, the connections with water molecules were increased. CONCLUSION: The c.734C>T variant of the GTB gene can lead to an amino acid substitution in the functional center of the enzyme, which in turn may affect the stability of glycosyltransferase B protein and reduceits enzymatic activity.

[Molecular characterization of a recombination allele of ABO blood group].

OBJECTIVE: To analyze the molecular characteristics of a recombinant allele of the ABO blood group. METHODS: The ABO phenotype was determined with the tube method. The coding regions of the ABO and FUT1 genes were analyzed by PCR-sequence based typing. The ABO alleles of the proband were determined by allele-specific primer sequencing. The full sequences of the ABO gene of the proband and her mother were determined through next generation sequencing. RESULTS: The red blood cells of the proband did not agglutinate with anti-H, and the sequence of the FUT1 gene was homozygous for c.551_552delAG.The proband was thereby assigned as para-Bombay. Bi-directional sequencing also found that she was heterozygous for c.261G/del,467C>T,c.526C>G,c.657C>T,c.703G>A,c.796C>A,c.803G>C and c.930G>A of the coding regions of the ABO gene. Allele-specific primer sequencing also found her to carry a ABO*A1.02 allele and a recombinant allele from ABO*O.01.01 and ABO*B.01. The recombination site was located between nucleotide c.375-269 and c.526, and the allele was maternally derived. CONCLUSION: An recombinant allele of the ABO gene has been identified, which has originated from recombination of ABO*O.01.01 with the ABO*B.01 allele.

Mechanism evaluation for an amino acid substitution p.Y246C of B-glycosyltransferase enzyme with Bweak phenotype.

BACKGROUND: The amino acid substitutions caused by ABO gene variants are usually predicted to impact the glycosyltransferase function. Here, the effect of an amino acid substitution in the vicinity of the catalytic active region of the B-glycosyltransferase was explored in vitro and in silico study, which is important for further recognizing the ABO subgroup. METHODS: The ABO serological tests were performed by the routine methods. The ABO genotype was analyzed by polymerase chain reaction and sequenced bidirectionally. The haplotype of the variant allele was separated using single-strand amplification and sequencing with allele-specific primers. Stably expression cell lines with variant were constructed for study in vitro. 3D structure of the B-glycosyltransferase (GTB) variant was simulated by PyMOL software. The free energy change (ΔΔG) was calculated by FoldX. RESULTS: A variant c.737A > G was identified in a Chinese individual with Bweak phenotype, which led to an amino acid substitution p.Y246C in the vicinity of the catalytic active region of GTB enzyme. The stably expression cell lines with variant and wild type were successfully established and showed that the variant caused a decrease in protein levels and/or enzyme activity. The 3D structural of the GTB modelling found the amino acid substitution p.Y246C caused the hydrogen bond of the protein changes. Meanwhile, the free energy change (ΔΔG) value predicted the destabilizing effect on the variant GTB. DISCUSSION: The p.Y246C variant in the vicinity of the enzyme active centre reduced the antigen expression because of greatly destabilizing effect on the GTB variant.