Abstract
Abstract
Background.
The Rh blood group is encoded by the highly homologous paralogues RHD and RHCE (~ 97% identity). Partial-D phenotypes arise from RHD variants, including localised RHD–RHCE gene conversions. DFR (RHD*DFR) is a clinically important partial-D allele defined by a discrete RHCE-derived micro-conversion within RHD exon 4 (RHD-CE(4:505–514)-D), introducing the missense changes p.Met169Leu, p.Met170Arg and p.Ile172Phe. As whole-exome sequencing (WES) is increasingly used opportunistically for blood-group genotyping, its behavior at this locus warrants explicit documentation.
Case/Methods.
We analyzed WES data from an individual with a serologically confirmed DFR partial-D phenotype (BioRad ID-Partial RHD typing). RHD was interrogated by three independent approaches: (i) GATK4 best-practice germline calling with functional annotation; (ii) paralogue-aware targeted genotyping at diagnostic weak-D/partial-D/DEL positions with dual mapping-quality thresholds; and (iii) RHtyper, a published RH-genotyping algorithm, applied with the tool’s WES mode.
Results.
Standard variant calling returned a normal RHD coding sequence and did not detect DFR; the common weak-D types (1, 2, 3) and the Asian-type DEL allele were correctly excluded. Critically, RHD exon 4, which harbors the DFR-defining c.505–514 micro-conversion had zero read depth at any mapping quality, while the paralogous RHCE exon 4 was covered at 161×. Reads originating from RHD exon 4 were therefore mis-assigned to RHCE owing to the near-identity of the two genes in this region, leaving no reads at the RHD coordinates from which the DFR variants could be called. A true deletion was excluded both by this reciprocal coverage and by the D-expressing DFR phenotype, which requires exon 4 to be present. RHtyper reproduced the false-negative, defaulting to a deletion/reference call.
Conclusions.
In this case, RHD/RHCE paralogous misalignment at exon 4 rendered short-read WES structurally blind to the DFR-defining variants, producing a clinically consequential false-negative that a purpose-built RH genotyper could not rescue. Because the reads are mis-assigned to the paralogue rather than absent, the failure is intrinsic to short-read alignment at this locus and is not remediable by deeper sequencing. WES should not be relied upon to exclude partial-D alleles at the RH locus; definitive characterization requires RHD-specific serological or molecular methods, or long-read sequencing.