RHD Zygosity Determination from Whole Genome Sequencing Data

John Baronas,Connie M Westhoff,Helen Mah,Robin Smeland Wagman,Maria Aguad,Sunitha Vege

doi:10.4172/2155-9864.1000365

John Baronas, Connie M Westhoff + Show 4 more

Open Access

https://doi.org/10.4172/2155-9864.1000365

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Abstract

In the Rh blood group system, the RHD gene is bordered by two homologous DNA sequences called the upstream and downstream Rhesus boxes. The most common cause of the D− phenotype in people of European descent is a deletion of the RHD gene region, which results in a hybrid combination of the two Rhesus boxes. PCRbased testing can detect the presence or absence of the hybrid box to determine RHD zygosity. PCR hybrid box testing on fathers can stratify risk for haemolytic disease of the fetus and new born in mothers with anti-D antibodies. Red blood cells and genomic DNA were isolated from 37 individuals of European descent undergoing whole genome sequencing as part of the MedSeq Project. A whole genome sequence-based RHD sequence read depth analysis was used to determine RHD zygosity (homozygous, hemizygous, or null states) with 100% agreement (n=37) when compared to conventional RhD serology and PCR-based hybrid box assay.

Highlights

The Rh blood group system consists of the two homologous genes RHD and RHCE
The most common cause of the D− phenotype in people of European descent is a deletion of the RHD gene region, which results in a hybrid combination of the two Rhesus boxes
Committee (IRB), samples for RBC and genomic DNA isolation were collected from 37 individuals of European descent who had Whole Genome Sequencing (WGS) through participation in the MedSeq Project [10]

Summary

Introduction

The Rh blood group system consists of the two homologous genes RHD and RHCE. The RHD gene controls the expression of the RhD protein, which contains numerous extracellular epitopes comprising the D antigen [1]. The most common cause of the D− phenotype in persons of European descent is deletion of the RHD gene due to a recombination event between of the upstream and downstream Rhesus boxes. Several different PCR-based assays are currently used to detect the hybrid box Most of these methods depend on small differences between sequences of the 5’ upstream region and 3’ downstream region of the Rhesus boxes. The hybrid box PCR assay is not always reliable and requires that the hybrid box region does not subsequently mutate or recombine again This assay can be falsely negative due to ethnic variations in the hybrid box sequences, or falsely positive due to other gene conversion events involving only one of the Rhesus boxes without RHD deletion (e.g. DAU-1; DIII type 4) [3]. We report that it was not possible to find Rhesus box split reads in WGS of D− individuals, it was possible to use read depth analysis from WGS data to correctly determine RHD homozygous, hemizygous, and null states

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