(P-IG-33) Predicting RHD Zygosity By Novel Application of Nonspecific Quantitative Sanger Sequencing (NoQSS)

Paternal RHD zygosity is critical for predicting fetal D status and managing HDFN risk. Existing assays such as the hybrid box PCR and PCR-RFLP, RHD BeadChip Array (Werfen), the multiplex ligation-dependent probe amplification assay (MLPA), or the qualitative and quantitative assay based on multiplex PCR of short fluorescent fragments (QMPSF), are labor/cost intensive and/or have limited accuracy. The proposed simple and novel application of a nonspecific quantitative Sanger sequencing (NoQSS) assay employs the simultaneous amplification of RHD and RHCE (RH) and detection of prevalent alleles encoding D‒ phenotypes for accurate D zygosity prediction.

Study

Design/Methods:

Sanger sequencing of RH exons 4 and 7 (for determining RHD/RHCE copy number), 6 (for c.807G for RHD*Ψ), and intron 3 (for c.486+3100G for RHD*DIIIa-ceVS.03-D (RHD*03N.02)) was done. BioEdit 7.2.5 software is used to extract numeric data from sequence signal intensity. A custom Excel Visual Basic algorithm calculates the ratio of RHD to consensus RHCE*ce signal, as well as detects D‒ encoding alleles RHD*Ψ and RHD*03N.02. The RHD to RHCE ratios are compared to reference data to predict the number of alleles encoding D+. NoQSS performance was tested on 38 samples with known genotypes.

Results/Findings:

NoQSS analysis had 100% success in quantifying RHD copy number of exons 4 and 7. Among the total 38 samples tested,12 had varying copy numbers of conventional RHD, and 24 had RHD*Ψ and/or RHD*03N.02. NoQSS also had 100% success in RHD Zygosity prediction with considerations to both RHD quantitation and detection of the alleles encoding D‒ phenotypes. For 2 challenge samples, an increased copy number of RHD exon 4 was detected in a sample heterozygous for RHCE*CeRN, and decreased copy number of RHD exon 4 in a sample hemizygous for RHD*DVI.

Conclusions:

The accurate prediction of RHD alleles encoding a D-positive phenotype by the NoQSS assay in this diverse set of reference samples serves as proof of concept for our novel method. Among these samples, 5 previously had inaccurate results by the hybrid box PCR: 1 sample with RHD deletion falsely detected, and 4 samples with RHD deletion falsely undetected; NoQSS made the correct prediction. The hybrid box assay is known to be discrepant in samples with RHD and RHCE variants such as DIVa and ceCF. The CeRN and DVI alleles were tested as evidence that NoQSS is also effective at detecting gene conversion events.

While Sanger sequencing is normally used for detection of RHD and RHCE variants, the results of this novel application of Sanger sequencing demonstrate its potential as a simpler and more efficient assay than many current zygosity testing protocols by simultaneous determination of RHD and RHCE copy number. Additionally, for cases with D-negative maternal sample with anti-D in plasma, accurate RHD zygostity prediction of paternal samples is critical for predicting the risk of HDFN.