Next‐generation sequencing: academic overkill or high‐resolution routine blood group genotyping?

Abstract

Blood group genotyping (BGG) has been in routine clinical practice ever since the molecular determination of blood groups was achieved, during the early to mid 1990s. These early methods were dependent on allele-specific PCR to detect simple single nucleotide polymorphisms (SNPs) responsible for blood group expression. As our knowledge regarding the molecular background of blood groups advanced, so did the numbers of SNPs requiring detection which necessitated the switch from allele-specific PCR to array-based technology, notably slide and bead-based approaches. All these methods thus described are totally dependent on the predefined genetic basis of each SNP. Hybrid blood group genes (as found in RH, ABO and MNS systems) are difficult to define by all of these aforementioned techniques. With the arrival of cheap next-generation sequencing (NGS) approaches in the past 5 years, we have conducted long-range PCR (LR-PCR) coupled with NGS determination of the major blood group genes. By analysis of these data using an Ion Torrent Personal Genome Machine TM (PGM TM ), it is readily apparent that NGS can highly effectively be applied with high resolution to blood grouping at costs no more than current array-based platforms. However, the complexity of the data obtained need careful filtering for effective clinical utilization, but provides useful insight on the evolution of blood groups and their environmental impacts which will be of undoubted value as an academic exercise, but of minimal cost (if any) to the original testing.

Duffy blood group phenotype–genotype correlations using high‐resolution melting analysis PCR and microarray reveal complex cases including a new null FY*A allele: the role for sequencing in genotyping algorithms

“…Avent et al . have signalled that this has the potential to supplant SNP‐based genotyping . In the meantime, our study shows that for a small percentage of samples, a combination of genetic approaches is required to complement serology .…”

Genomic coordinates and continental distribution of 120 blood group variants reported by the 1000 Genomes Project

“…Distinguishing neutral passenger variants from deleterious mutations has challenged genomics from its inception, and as NGS is applied to transfusion medicine, novel blood group variants have been a recurrent finding . Several prediction tools used widely in genomics have not been tested for immunohematology.…”

“…A number of successful clinical applications have been reported, and further efforts are under way in the areas of oncology, hemostasis, obstetrics, and pharmacology to identify the clinical benefits of genomic medicine . Transfusion medicine is an additional discipline with a strong genetics foundation that is exploring this approach …”

A D+ blood donor with a novel RHD*D‐CE(5‐6)‐D gene variant exhibits the low‐frequency antigen RH23 (DW) characteristic of the partial DVa phenotype

“…In this particular case, two SNP microarray typing platforms signaled an “indeterminate” or “not valid” genotype call which can now be attributed to the novel combination of SNPs, which did not fit the expected profile based on known RHD*D‐CE‐D hybrid alleles as analyzed by the software's algorithm. For both SNP microarray genotyping and MPS platforms, rapid updating of bioinformatics interpretative algorithms will be required to manage this genetic diversity …”