STATEMENTThis report describes 22 new RHD variants whose identification was prompted by discrepancies between the results of targeted genotyping and serology.

No abstract

Three new alleles in the ABO, RHD, and RHCE genes consist of single-nucleotide changes within GATA motifs, emphasizing the key role of GATA transcription factors in the expression of blood group genes.

The prevalence of maternal RHD variants was significantly higher in alloimmunised cases. The causative mechanism for this needs further investigation. Mathematical modeling simplifies the detection of maternal RHD variants.

Background: Conventional sequencing uses gene-specific primers to determine the location of RH variants and permits a qualitative assessment of zygosity. Whole-genome and whole-exome sequencing determine the genetic location of variants and enable a quantitative assessment of zygosity. Nonspecific sequencing uses RH-consensus primers to detect variants and sequencing-read ratios to quantify their copy number. Study Design and Methods: Two hundred seventy eight samples with diverse genotypes were analyzed by next-generation sequencing with RHconsensus primers. Custom-developed data analysis software was used to detect individual variants and infer the RH genotype. The method was evaluated for its quantitative nature, its ability to discriminate similar genotypes, its accuracy to detect variants, and its accuracy to assign them to RHD or RHCE. Results: As a measure of balanced amplification of RHD and RHCE sequences, observed ratio medians deviate from expected ratios by 3% or less of the ratio range. As a measure of discriminatory power, contiguous RHCE / [RHD + RHCE] ratio averages are separated by 4 or more standard deviations of the mean. Variants are detected with a sensitivity and specificity greater than 99%, and variants at consensus positions are correctly assigned to RHD vs RHCE with a sensitivity greater than 72% and a specificity greater than 99%. The method is successful in the identification of genotypes with large conversion events and in the detection of copy number variation. Conclusion: Nonspecific sequencing of homologous gene sets combines detection and quantification of genetic variation in a single assay. Evidence is provided for the quantitative nature of the method, its sensitivity and specificity, and its ability to identify complex RH genotypes. Conventional sequencing of homologous blood group genes such as RHD and RHCE makes use of gene-specific primers, with data analysis limited to a qualitative determination of homozygosity for the common sequence,