A novel SNV on a previously reported hybrid RHCE background is responsible for an Rh haplotype potentially involved in the generation of an Rh null phenotype

Abstract

BACKGROUNDMore than 180 RHCE alleles have been reported so far including approximately 30 null variants.1 In some cases, when one RHCE null allele is present, an incorrect antigen zygosity is assumed that may lead to unexpected familial inheritance of phenotypes. This report describes a novel RHCE null allele in cis with RHD*08N.01 giving rise to a Rh haplotype potentially involved in the generation of a Rh null phenotype.BRIEF METHODSBlood samples from a Caucasian newborn (proband), his parents and maternal grandmother were referred to our laboratory because of apparent inconsistencies in the inheritance of RhCE antigens. Serologic studies showed that both the baby and the father were D+C+E-c-e+ (R1R1), the mother was D-C-E-c+e+ (rr) and the maternal grandmother was D+C-E+c+e- (R2R2). To elucidate these unexpected familial phenotypes, molecular studies were performed. Quantitative Multiplex PCR of Short Fluorescent Fragments (QMPSF)2 was applied to determine copy number of all RHD and RHCE exons. Conventional PCR strategies were used to analyse the RHD zygosity and the suspected RHD variant inferred by QMPSF and to determine the RHCE genotype. Also, Sanger sequencing of all RHD and RHCE exons was carried out.RESULTSQMPSF studies suggested that the proband and grandmother harboured two copies of the RHD gene, carrying one RHD*08N.01 each. The results also showed that the mother was RHD hemizygous carrying an RHD*08N.01 and the father was RHD homozygous. The RHD*08N.01 was confirmed by the detection of the 37 bp insertion at the RHD intron 3 - exon 4 junction with an allele specific PCR. No mutations besides those of RHD*08N.01 were detected by sequencing indicating that the other allele was RHD*01 (Fig. 1).As regards RHCE, QMPSF analysis allowed the identification of an RHCE*01.16-like allele, characterized by a hybrid RHCE-D(9)-CE structure and a c.48G>C SNV in RHCE exon 1 -RHCE*01(48C,1170T,1193A)- in the proband, mother and grandmother, suggesting that this allele is in cis with RHD*08N.01, as previously reported.3 Two copies of c.307C (one from the RHCE*01.16-like allele) and one c.48C (also from the RHCE01.16-like allele) RHCE nucleotides were detected in the mother and grandmother. On the other hand, one c.307C (from the RHCE*01.16-like allele) and 2 copies of the c.48C RHCE nucleotides were observed in the proband, one attributed to the RHCE*01.16-like inherited from the mother and the other one to the RHCE*02 inherited from the father (Table S1). The presence of RHCE*02 in the proband and his father was confirmed by the detection of the 109 bp insertion in RHCE intron 2. Allele specific PCRs detected c.676C and c.676G RHCE nucleotides in the grandmother and only c.676G RHCE nucleotide in the rest of the members.So far, molecular studies suggested that the proband´s and the grandmother?s genotypes were consistent with RHD*08N.01-RHCE*01.16-like/RHD*01-RHCE*02 and RHD*08N.01-RHCE*ce.16-like/RHD*01-RHCE*03, respectively, but inconsistent with the phenotypes observed (R1R1 and R2R2). On the other hand, the mother´s and the father´s genotypes were consistent with RHD*08N.01-RHCE*01.16-like/RHD*01N.01-RHCE*01 and RHD*01-RHCE*02/RHD*01-RHCE*02, respectively, in agreement with the serologic status (rr and R1R1).RHCE sequencing analysis in the mother´s sample revealed a novel c.807T>G SNV in RHCE exon 6 (Fig. S1). This SNV is responsible for a premature stop codon (p.Tyr269Ter) and thus a truncated RhCE protein, leading to the non-expression of c and e antigens in the red blood cell membrane (GenBank Accession Number OL675675). The polymorphism had been annotated as rs780267740 in the GnomAD database and is present in only two Europeans with an allele frequency of 0.000007971. A SNV in the same position has already been reported on an RHCE*01 allele basis but the T was replaced by an A, also generating a premature stop codon.4 The 807T>G SNV found in this work was confirmed with a RFLP PCR approach using Dde I restriction enzyme that recognizes the G at position 807 and was detected in the proband and his grandmother as well (Fig. S2). Segregation analysis showed that c.807T>G SNV was carried by the RHCE*01.16 allele (Fig. 1).Altogether these findings clearly show that RHD*08N.01-RHCE*01.16(807G) is the molecular structure of a new Rh haplotype that can potentially give rise to an Rh null phenotype if homozygous or compound heterozygous. To note, both RHD and RHCE alleles in this haplotype are rendered null by the same c.807T>G SNV, possibly a consequence of homologous recombination between RHD and RHCE sequences.BRIEF SUMMARYThe molecular findings reveal that the unexpected inheritance of phenotypes observed in family members can be attributed to a nonsense mutation in RHCE*01.16, in cis with RHD*08N.01, silencing c and e antigens expression.