Evaluation of targeted exome sequencing for 28 protein‐based blood group systems, including the homologous gene systems, for blood group genotyping

Abstract

BACKGROUND: Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. STUDY DESIGN AND METHODS: Donor samples(n 5 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. RESULTS:The average sequencing depth per target region was 66.2 6 39.8. Each sample harbored on average 43 6 9 variants, of which 10 6 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. CONCLUSION:The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting.H uman blood group antigens are of significance in transfusion medicine, because patients who have made antibodies to red blood cell antigens are at risk of being affected by hemolytic transfusion reactions after the transfusion of incompatible blood. The International Society of Blood Transfusion has defined 36 blood group systems and over 350 blood group antigens.1 Different blood group systems exhibit varying degrees of antigen polymorphism, and the clinical significance of red blood cell antibodies also varies. [2][3][4] As a minimum requirement in blood transfusion safety, all blood donors are screened for ABO and the D antigen as well as for blood group antibodies known to be clinically significant. 5The majority of antigens are missense mutations and a consequence of single nucleotide variants (SNVs); however, genetic variations, such as insertions/deletions and splice-site variants, have a qualitative and/or quantitative impact on antigen expression. Blood group systems, such as RH and MNS, exhibit an additional layer of genetic variation. These arise because each system comprises homologous genes in which gene crossover or g...

Whole-exome sequencing for RH genotyping and alloimmunization risk in children with sickle cell anemia

“…A recent study that designed targeted exome sequencing for blood group systems encountered similar limitations in coverage and sequence alignment for exons 1 and 8 of RHD and RHCE. 24 Approaches used here to improve accuracy included a slight relaxation of the Mercury pipeline reference:variant ratio threshold, consideration of RHCE exon 2 copy number to detect the C 1 phenotype associated with RHCE*Ce, and recognition of the common hybrid allele RHD*DIIIa-CE(4-7)-D as an RHD locus variant (Table 4). In the future, standardization of the human genome reference sequence and increased coverage of exon 1 for RHCE*ce48C and exon 8 for RHD*1138T detection would further enhance accuracy (Table 4).…”

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 …”

Anti‐D in a mother, hemizygous for the variant RHD*DNB gene, associated with hemolytic disease of the fetus and newborn

“…However, advanced molecular typing platforms such as SNP‐microarray and DNA sequencing, including massively parallel sequencing, have now become increasingly available in reference typing laboratories to detect and genetically classify RHD variants . For clinicians, early identification of individuals with partial D would help guide patient management in the obstetric population …”