Rapid RHD Zygosity Determination Using Digital PCR

Sillence, Kelly A.; Halawani, Amr J; Tounsi, Wajnat A; Clarke, Kirsty A; Kiernan, Michele; Madgett, Tracey E.; Avent, Neil D.

doi:10.1373/clinchem.2016.268698

Cited by 12 publications

(10 citation statements)

References 23 publications

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“…The RHD zygosity was determined for all samples using dPCR to determine whether a sample was hemizygous (Dd) or homozygous (DD). 12,39 Samples were tested for RHD exon 5 (RHD5) and RHD exon 7 (RHD7) against the reference gene AGO1 on chromosome 1. 12,39 The droplet reader in combination with QuantaSoft software v1.7.4 analyzed the droplet signals and differentiated between negative and positive ones, creating an absolute concentration of DNA.…”

Section: Genomic Dna Extraction and Zygosity Testingmentioning

confidence: 99%

“…Serological testing also leads to prediction of the Rh genotype based on the most common haplotype present in the population, which, for some cases, is incorrect. 12 Unlike serological testing, genotyping provides the freedom to analyze a wider range of blood antigens including low-frequency antigens, for instance: Go a , BARC, and Tar which can cause HDFN and alloimmunization. 1 Complete blood group genotyping (BGG) could be widely used in transfusion practice where serology fails to clarify issues or resolve discrepancies.…”

Section: Introductionmentioning

confidence: 99%

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Complete RHD next-generation sequencing: establishment of reference RHD alleles

2018

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The Rh blood group system (ISBT004) is the second most important blood group after ABO and is the most polymorphic one, with 55 antigens encoded by 2 genes, RHD and RHCE. This research uses next-generation sequencing (NGS) to sequence the complete RHD gene by amplifying the whole gene using overlapping long-range polymerase chain reaction (LR-PCR) amplicons. The aim was to study different RHD alleles present in the population to establish reference RHD allele sequences by using the analysis of intronic single-nucleotide polymorphisms (SNPs) and their correlation to a specific Rh haplotype. Genomic DNA samples (n = 69) from blood donors of different serologically predicted genotypes including R1R1 (DCe/DCe), R2R2 (DcE/DcE), R1R2 (DCe/DcE), R2RZ (DcE/DCE), R1r (DCe/dce), R2r (DcE/dce), and R0r (Dce/dce) were sequenced and data were then mapped to the human genome reference sequence hg38. We focused on the analysis of hemizygous samples, as these by definition will only have a single copy of RHD. For the 69 samples sequenced, different exonic SNPs were detected that correlate with known variants. Multiple intronic SNPs were found in all samples: 21 intronic SNPs were present in all samples indicating their specificity to the RHD*DAU0 (RHD*10.00) haplotype which the hg38 reference sequence encodes. Twenty-three intronic SNPs were found to be R2 haplotype specific, and 15 were linked to R1, R0, and RZ haplotypes. In conclusion, intronic SNPs may represent a novel diagnostic approach to investigate known and novel variants of the RHD and RHCE genes, while being a useful approach to establish reference RHD allele sequences.

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Section: Genomic Dna Extraction and Zygosity Testingmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Complete RHD next-generation sequencing: establishment of reference RHD alleles

2018

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“…Because the 1000 Genome Project data does not provide blood group phenotypes, the correlation of intronic SNPs to a particular blood group genotype can be amended to the Erythrogene database using other studies, such as a number of RHD intronic SNPs that correlate with the DcE haplotype described by Sillence et al . . The integration enhances practical usability of Erythrogene in routine diagnostics.…”

Section: Role Of Ngs In Blood Group Genomicsmentioning

confidence: 99%

Blood group genotyping goes next generation: featuring ABO, RH and MNS

Wu¹,

Pai²,

Chen

2018

ISBT Science Series

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There are currently 36 blood group systems comprising over 300 antigens. The genomic backgrounds of polymorphisms responsible for antigens of each blood group have been established. Current blood group genotyping platforms are focused on testing for antigen‐determine single nucleotide polymorphisms, which are the differences on DNA level for predicting the antigen phenotype, and thus are faced with challenges because of lack of ability to detect unknown variants, structural variants and copy number variations. With the advances in technology and bioinformatics analyses, next‐generation sequencing (NGS) is now an applicable platform for high‐throughput, accurate and extensive blood group genotyping. The widely used platforms and processing steps of NGS are illustrated in this article, as well as an overview of published works. NGS has been used in various applications involving blood group genomics, including location of genetic variants for blood group expression, antenatal care, allele frequency estimation and blood group antigen prediction. Even for some of the most complicated blood group systems, NGS provides insight to solve cis/trans associations and gene rearrangements occurring in homologous genes, as featured for the ABO, RH and MNS systems in this review. Although certain issues such as cost, turnaround time, secured data storage and verification remain challenges for routine blood group genotyping practices, NGS still holds great promise advancement in future perspectives.

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“…All 35 maternal samples containing unidentified RHD variants were tested for RHD zygosity using the QX100 droplet digital PCR (ddPCR) platform and QuantaSoft v1.7 software (Bio-Rad Laboratories), as described in Sillence et al, 15,16 to determine whether the women carried 1 (hemizygous Dd) or 2 copies (homozygous DD) of the RHD gene. Primers and probes used in ddPCR zygosity testing were adopted from Fan et al 17 and Finning et al 18 The samples were tested for RHD exons 5 and 7, against the reference gene AGO1 on chromosome 1.…”

Section: Rhd Zygosity Testing By Droplet Digital Pcrmentioning

confidence: 99%

Next-generation sequencing of 35 RHD variants in 16 253 serologically D− pregnant women in the Finnish population

et al. 2020

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Fetal RHD screening for targeted routine antenatal anti-D prophylaxis has been implemented in many countries, including Finland, since the 2010s. Comprehensive knowledge of the RHD polymorphism in the population is essential for the performance and safety of the anti-D prophylaxis program. During the first 3 years of the national screening program in Finland, over 16 000 samples from RhD− women were screened for fetal RHD; among them, 79 samples (0.5%) containing a maternal variant allele were detected. Of the detected maternal variants, 35 cases remained inconclusive using the traditional genotyping methods and required further analysis by next-generation sequencing (NGS) of the whole RHD gene to uncover the variant allele. In addition to the 13 RHD variants that have been previously reported in different populations, 8 novel variants were also detected, indicating that there is more variation of RHD in the RhD− Finnish population than has been previously known. Three of the novel alleles were identified in multiple samples; thus, they are likely specific to the original Finnish population. National screening has thus provided new information about the diversity of RHD variants in the Finnish population. The results show that NGS is a powerful method for genotyping the highly polymorphic RHD gene compared with traditional methods that rely on the detection of specific nucleotides by polymerase chain reaction amplification.

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Rapid RHD Zygosity Determination Using Digital PCR

Cited by 12 publications

References 23 publications

Complete RHD next-generation sequencing: establishment of reference RHD alleles

Complete RHD next-generation sequencing: establishment of reference RHD alleles

Blood group genotyping goes next generation: featuring ABO, RH and MNS

Next-generation sequencing of 35 RHD variants in 16 253 serologically D− pregnant women in the Finnish population

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