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.

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

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 = 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 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 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.

Inter-rater reliability for witnessed collapse and presence of bystander CPR.

OBJECTIVE: Witnessed collapse and bystander CPR are the variables most frequently associated with good outcome from out-of-hospital cardiac arrest (OOHCA). The reliability of abstracting witnessed collapse and bystander CPR from prehospital Emergency Medical Services (EMS) patient care records (PCRs) is not known. We sought to determine the inter-rater reliability for different methods of ascertaining and defining witnessed collapse and performance of bystander CPR. METHODS: A sample of 100 PCRs for patients with OOHCA was selected at random from a pool of 325 PCRs between May 2003 and January 2005. Paramedics used a drop down menu to indicate witnessed collapse and bystander CPR, and completed a narrative description of the event. An on-scene EMS physician also completed a data sheet. The PCR was examined by two separate evaluators to determine the presence of witnessed collapse and bystander CPR. A consensus was reached by three other reviewers using all available data sources. Inter-rater agreement was quantified using the unweighted kappa statistic. RESULTS: For witnessed collapse, there is substantial agreement between the following: individual evaluators (kappa=0.76, S.D.=0.07), individual evaluators and consensus group (kappa=0.61, S.D.=0.07 and 0.66, S.D.=0.07), and physician and consensus group (kappa=0.68, S.D.=0.08). Agreement between individual evaluators and the physician was fair to moderate (kappa=0.38, S.D.=0.07 and 0.44, S.D.=0.07). Agreement between individual evaluators, physician, consensus group and the PCR drop down menu was fair to moderate (kappa range 0.33, S.D.=0.09 to 0.54, S.D.=0.09). For bystander CPR, there is substantial agreement between the individual evaluators and the consensus group (kappa=0.64, S.D.=0.07 and 0.63, S.D.=0.06) and between the physician and the consensus group (kappa=0.61, S.D.=0.08). Agreement between the two individual evaluators is moderate (kappa=0.59, S.D.=0.07). Agreement between the physician and individual evaluators is fair (kappa=0.36, S.D.=0.07 and 0.38, S.D.=0.07). The PCR drop down menu had moderate to substantial agreement with the individual evaluators, physician, and consensus group (kappa range 0.50, S.D.=0.09 to 0.75, S.D.=0.09). CONCLUSIONS: Determination of witnessed collapse and bystander CPR during OOHCA may be less reliable than previously thought, and differences between methods of rating could influence study results.