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

Abstract

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.

The “next generation” reference laboratory?

“…Many researchers have now shown that NGS or MPS can provide adequate coverage of the genes that determine blood group extended antigen typing in patients and donors. Schoeman and colleagues describe the first example of the use of NGS or MPS as part of the routine reference laboratory workup. The strength of this approach lies in the ability to potentially detect the relevant polymorphisms including null alleles, novel mutations, and complex gene rearrangements in blood group genes simultaneously.…”

“…Cost is a consideration, with the latest NHGRI data from July 2017 indicating a cost of $1121 to sequence an entire human genome . Exome sequencing, which targets only the coding region (exons), reduces cost and produces a smaller and more manageable volume of bioinformatics data, but as demonstrated by Schoeman and coworkers, the commercial exome library preparation they used did not target exons of all blood groups.…”

“…Novel genetic changes of unknown significance will be encountered and will require evaluation. This task will not be simple, and current computational prediction algorithms, such as SIFT and Polyphen utilized by Schoeman and colleagues, often provide conflicting results and are not robust enough to make conclusive structural and antigenic predictions. Blood group antigens are defined as polymorphisms associated with production of a specific antibody, and this direct relationship must be proven.…”

“…NGS or MPS data require a robust bioinformatics analysis pipeline, tested and optimized for transfusion medicine that also incorporates RBC antigen prediction and HPA, HLA, and possibly relevant white blood cell antigens. Schoeman and coworkers provide a detailed description of the data filters used in their study (i.e., quality, alternate allele percent and coverage thresholds, strand bias detection), but work remains to be performed to determine if parameter selection and threshold values need to be optimized for blood group genes or for specific data sets. Blood group allele tables are referenced according to location in cDNA, but for NGS analysis the polymorphisms must be converted to genomic coordinates.…”

Modified expression of the KEL2 (k) blood group antigen attributed to p.Leu196Val amino acid change three residues from the K/k antigen polymorphism site: implications for donor screening

“…Genomic DNA was extracted from ethylenediaminetetraacetic acid–whole blood tubes (EZ1 DNA Blood, QIAGEN). Blood group genomic sequencing, which included KEL and XK genes, was performed using a gene sequencing kit (TruSight One Panel, Illumina), as previously described . RefSeq KEL NG_007492.1 and XK NG_007473.2 were used for data analysis.…”