Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting.

Background: We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single-nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP-typing cases.

Study Design And Methods: The TruSight One sequencing panel and MiSeq platform was used for sequencing. CLC Genomics Workbench software was used for data analysis of the blood group genes implicated in the serology and SNP-typing problem. Sequence variants were compared to public databases listing blood group alleles. The effect of predicted amino acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2.

Results: Among 29 unresolved samples, sequencing defined SNPs in blood group genes consistent with serologic observation: 22 samples exhibited SNPs associated with varied but known blood group alleles and one sample exhibited a chimeric RH genotype. Three samples showed novel variants in the CROM, LAN, and RH systems, respectively, predicting respective amino acid changes with possible deleterious impact. Two samples harbored rare variants in the RH and FY systems, respectively, not previously associated with a blood group allele or phenotype. A final sample comprised a rare variant within the KLF1 transcription factor gene that may modulate DNA-binding activity.

Conclusion: Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118_1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases.