Two distinct RHCE alleles in cis to weak D type 31 alleles in individuals from different ethnicities

In 2005, Körmöczi and colleagues reported a white blood donor with a serologic weak D phenotype (D+C+E-c+e+).¹ Her RHD gene showed a C to T nucleotide change at Position 17 (c.17C > T; rs555567027) in Exon 1, leading to the substitution of a proline for a leucine (p.Pro6Leu) in the intracellular amino-terminal domain of the RHD protein. Because the location of the mutation was compatible with the definition of a molecular weak D type,² this allele was assigned weak D type 31. The allele’s description covered 50 nucleotides of the 5^’-untranslated region, 148 nucleotides of Exon 1, and 47 nucleotides of Intron 1 (GenBank Accession Number AJ557803.1). Family analysis revealed a daughter typing D-C-E-c+e+ and another daughter with the weak D type 31 allele typing D+C+E-c+e+, unambiguously linking the Ce antigens in cis.¹ No further molecular variations of the involved RHCE allele were determined at the time.

We collected fresh weak D type 31 samples from the original donor¹ and her daughter in Germany and from a newly observed weak D type 31 donor with a D+C–c+E–e+ phenotype in India. We resolved the linkage of the involved distinct RHD and RHCE alleles in both index cases, establishing sequence information for three RH haplotypes. The data can be applied to develop, evaluate, and validate next-generation sequencing techniques.³ Their robustness will be increased when the allele and haplotype data conclusively include RHD intron polymorphisms and the linked RHCE allele in cis on the same chromosome.

BRIEF METHODS

DNA was extracted from ethylenediaminetetraacetate-anticoagulated whole blood samples, collected with consent from the individuals at the blood centers in Dresden, Germany, and Surat, Gujarat, India. The 6145 nucleotides of the RHD and 8015 of the RHCE gene were sequenced as described previously.³ RHD zygosity testing was performed by restriction fragment length polymorphism.

RESULTS AND DISCUSSION

All three samples were hemizygous for the RHD gene by restriction fragment length polymorphism zygosity testing. Using our previously described RH sequencing strategy,³ we determined 14,160 nucleotides to establish three extended RH haplotypes.

Indian index case

RHD gene sequencing in the Indian donor revealed one single-nucleotide polymorphism (SNP), specific for the weak D type 31 allele, in Exon 1 and five SNPs in the noncoding regions (Introns 1, 3, and 5; Table S1, available as supporting information in the online version of this paper). The RHCE gene sequencing identified eight homozygous SNPs in the noncoding regions. Hemizygosity at the RHD locus for the Indian weak D type 31 allele combined with homozygosity at the RHCE locus allowed us to unambiguously identify the common RHCE*ce allele linked in cis (Fig. 1; GenBank Accession Number MH260574). The other RH haplotype represented the RHD deletion (RHD*01N.01) in cis to the common RHCE*ce allele (Fig. 1; GenBank Accession Number MH260575).

Fig. 1.

RH haplotype of weak D type 31. Both RH genes with 10 exons each are represented by numbered boxes for the RHD (yellow) and RHCE genes (green). The c.17C>T SNP (rs555567027; red line) in RHD Exon 1 represents the nonsynonymous change causing the weak D type 31 phenotype; there is no SNP in any RHCE exon relative to the consensus sequence. Multiple SNPs are present in the noncoding regions (black lines). Only one insertion–deletion variation (802–46_802–43delTCTC) in RHD but eight SNPs in RHCE differ between the Indian and German donors (blue arrows). The other five RHD variations in the promoter (−368A>G) and introns (149–29G>C, 486+117T>C, 486+124G>A and 801+219G>T) are SNPs of high prevalence commonly observed in many different RHD alleles, not diagnostic for the RHD*weak D type 31 allele.

German family duo

The RH haplotype for the original donor AJ557803.1 and her daughter was determined at the molecular level. They both shared an RHD*weak D type 31 allele with one SNP in Exon 1. Additional RHD intronic variations were also present in the original donor (Table S1). The RHCE gene sequencing identified eight heterozygous SNPs, identical to the Indian sample, and confirmed an RHCE*Ce allele in cis to the European weak D type 31 allele (Fig. 1; GenBank Accession Number MH260576). The other RH haplotype was an RHD*01N.01 allele in cis to the RHCE*ce allele with eight SNPs (Fig. 1; GenBank Accession Number MH260575), identical to the Indian sample.

Serology

The peculiar serologic phenotype of weak D type 31 has been published in detail.¹ We confirmed two red cell (RBC) populations characterized by mixed-field agglutination in tube and gel in the three cases of this study; besides the German family duo and two granddaughters, seven additional cases from Switzerland were tested in Vienna, all with D+C+E-c+e and mixed-field agglutination (data not shown). The histogram documented a negative and an adjacent weakly positive RBC population in all cases, using flow cytometry⁴ with 11 monoclonal anti-Ds as described previously.¹

Phylogeny

The one extra insertion–deletion variation (802–46_802–43delTCTC) present in the European as compared to the Indian weak D type 31 alleles was located in RHD Intron 5 and has been shown to be population specific.⁵ Based on this extra SNP and the different RH haplotype, the Indian and European weak D type 31 alleles may have arisen in two independent events. The complete list of SNPs detected in the RHD and RHCE genes in the Indian and German donors are tabulated as typically reported in our clinical diagnostic routine (Table S1).

We have identified three haplotypes in the RH system that differ from the human reference genome assembly (GRCh38), which is currently the gold standard reference.

Supplementary Material

1

Table S1. RHD and RHCE alleles, regions sequenced, and SNPs detected. Data are shown as typically reported in our clinical diagnostic routine.

ABBREVIATION:

SNP

single-nucleotide polymorphism