Molecular bases of unexpressed RHD alleles in Chinese D− persons

BACKGROUND: The aim of this study was to use a systematic survey to analyze RHD alleles in Chinese D− donors who do not express D antigen or who lack functional RhD protein.
STUDY DESIGN AND METHODS: A total of 733 D− Chinese donors, not including Del phenotypes, were investigated by RHD polymorphism–specific polymerase chain reaction (PCR), Rhesus box PCR- Pst I digestion, and RHD sequencing. The frequencies of identified alleles were calculated.
RESULTS: Three genetic mechanisms and eight alleles were found associated with the Chinese D− phenotype. One new RHD/CE hybrid allele and one novel mutation were also found. The rates of total deletion and the most frequent hybrid allele RHD(1)-CE(2-9)-D(10) were similar to those found in previous studies. A previously reported mutation RHD(711delC) was found to be the predominant cause of aberrant RHD alleles.
CONCLUSION: Informative population-based data for improving molecular diagnostic strategies for Chinese D− persons are suggested by this study. This type of systematic knowledge is important for the development of typing and transfusion strategies for the Chinese population.     

Molecular analysis of inactive and active RHD alleles in native Congolese cohorts

BACKGROUND: In Africa, RHD alleles have not been fully characterized. The purpose of this study was to identify inactive and active RHD alleles at the molecular level in Congolese cohorts.
STUDY DESIGN AND METHODS: Blood samples were collected from people living in central Congo populated by Teke ethnic group. A total of 110 D− and 40 D+ samples from Congo-Brazzaville and Teke groups, respectively, were selected for RHD genotyping using allele-specific primer polymerase chain reaction and sequencing.
RESULTS: In the 110 D− samples, RHD exon amplifications were observed in 7 samples that were subsequently identified by sequencing as weak D type 4 variants. In the remaining 103 D− samples, the frequencies of RHD gene deletion, RHDψ pseudogene, and RHD-CE-D^s hybrid gene were 0.75685, 0.20560, and 0.04468, respectively. In the D+ samples, 26 individuals carried at least a regular RHD gene; 9 carried aberrant RHD alleles belonging to the African D clusters, that is, DAU, DIVa, and weak D type 4; 3 carried RHDψ in trans with a DAU allele including one novel RHD allele ( V279M, S333N, T379M ) named DAU-7 ; and 2 others were partially determined.
CONCLUSION: This study revealed a high frequency of weak D type 4 alleles that confirmed the need to use indirect antiglobulin test to improve transfusion safety in the Congo and in countries hosting Congolese people. Findings also indicated that there is a geographic variation in RHD allele distribution and showed that RHD gene deletion is the most prevalent cause of the D− phenotype in the Congolese population.     

Weak D and DEL alleles detected by routine SNaPshot genotyping: identification of four novel RHD alleles

BACKGROUND: Molecular RHD blood group typing is very efficient for managing donors and patients carrying any of the various molecular types of weak D and DEL. The purpose of the work was to develop a multiplex polymerase chain reaction (PCR) SNaPshot assay for simultaneous detection of weak D and DEL alleles that are prevalent in Europeans, Africans, and Asians. STUDY DESIGN AND METHODS: Preliminary profiling was carried out on single‐nucleotide polymorphisms (SNPs) associated with 13 prevalent RHD alleles, that is, weak D Types 1, 2, 3, 4.0, 4.0.1, 4.1, 4.2, 5, 11, 15, and 17; RHD(IVS3+1g>a); and RHD(K409K). Multiplex PCR was used to amplify six RHD regions encompassing 14 SNPs. Identification was obtained by incorporation of the complementary dye single base at the 3′‐end of each probe‐primer. A prospective analysis was then carried out on 152 blood samples from patients (n = 53) and donors (n = 88) with equivocal RhD serology and pregnant women (n = 11). RESULTS: After validation, our SNaPshot assay allowed direct genotyping of 82.9% of samples overall and 100% of samples harboring weak D Types 1, 2, 3, and 4.1 alleles. In the remaining 17.1% of samples overall, sequence investigation allowed accurate genotyping. In addition, four novel RHD alleles were identified, that is, RHD(S256P), RHD(L390L), RHD(F410V), and RHD(IVS4‐2a>g). CONCLUSION: The SNaPshot assay described herein is a helpful supplementary tool for resolving doubtful RhD serology. By allowing accurate identification of weak D and DEL alleles this assay should allow better management of the donors and the patients genotyped weak D Types 1, 2, 3, and 4.1 who can receive D+ blood units.     

Identification of 12 novel RHD alleles in western France by denaturing high-performance liquid chromatography analysis

BACKGROUND: Unlike the standard RHD+ or RHD- alleles, serologic determination of weak or partial D alleles is often not clear-cut. Most importantly, rare weak D alleles, not typed by serology, are prone to alloimmunization when transfused with D+ blood. Although more than 100 RHD variants have currently been reported, many more rare alleles probably remain to be identified. STUDY DESIGN AND METHODS: To identify novel unusual RHD alleles, genomic DNA samples were collected from 333 blood donors or recipients in western France. All displayed ambiguity for D phenotype as determined by routinely used serologic reagents and analyzed by means of denaturing high-performance liquid chromatography (DHPLC) analysis in parallel with direct sequencing. RESULTS: For the first time it has been established that a reliable DHPLC-based approach potentiates the rapid screening of the entire RHD gene-coding sequence. In so doing, a total of 12 novel RHD alleles were identified. Except for the null allele that is in trans with a Weak D type 4 allele, the predicted effects of the other new alleles on gene expression correlated well with the discrepant routine D phenotype results. In particular, the carrier of the p.Leu214Phe missense mutation developed alloanti-D antibodies after transfusion of D+ blood. CONCLUSION: The identification of 12 novel RHD alleles represents a significant addition to the known repertoire of unusual RHD variants and, at the same time, serves to deepen our understanding of the molecular basis of weak and partial D. The accurate molecular typing of RHD alleles would allow to reduce the alloimmunization risk.     

Comprehensive analysis of RHD alleles in Argentineans with variant D phenotypes

BACKGROUND: The serologic assignment of the RhD status may be hindered in patients with weak D expression. A comprehensive study of RHD alleles occurring in the mixed population of Argentina is necessary to evaluate the most suitable DNA typing strategy. STUDY DESIGN AND METHODS: A total of 18,379 patients from two stratified groups, Group 1 (G1; public hospital) and Group 2 (G2; private laboratory), were RhD phenotyped, and 88 samples with reduced D expression underwent molecular characterization. RESULTS: The frequencies of D+, D–, and variant D phenotypes differed significantly (p < 0.001) between G1 and G2 (94.49% vs. 87.66%, 5.15% vs. 11.58%, and 0.36% vs. 0.75%, respectively). Eleven alleles were responsible for the weak D expression. Approximately 60% of the variant D phenotypes from G1 and G2 were weak D Types 1 through 4.0/4.2 and 25% were DVII. RHD alleles associated with African ancestry were encountered in G1. A new ?282G>A mutation within the promoter region of DAU‐4 and DOL alleles was identified. Three weak D Type 1 samples on R₀ haplotypes were found in G1. CONCLUSIONS: The D phenotype distribution in G2 resembles that in Europeans while the frequencies in G1 account for the Amerindian and African genetic contribution. The genotyping strategy described here is suitable to study D variants in the overall population and could allow a better use of the few available D– units and a rational administration of anti‐D immunoprophylaxis. The results also show that weak D Type 1 alleles do not exclusively segregate with a Ce allele, as assumed until present.     

Random survey for RHD alleles among D+ European persons

BACKGROUND: RHD alleles are considered more variable in African persons than in European persons. A systematic survey, however, was lacking among D+ European persons at the molecular level, precluding any definite frequency estimate. STUDY DESIGN AND METHODS: A random survey was performed among 500 ccDee, 250 CcDee, and 250 ccDEe blood donors in southwestern Germany. They were tested by polymerase chain reaction with sequence-specific priming (PCR-SSP) for up to 12 single-nucleotide polymorphisms representative for the most frequent RHD alleles among European persons. The RHD exon 5 nucleotide sequence was also tested in all 1000 samples. The nucleotide sequence of the 10 RHD exons was checked in all samples with aberrant exon 5 or positive PCR-SSP procedures. RESULTS: By PCR-SSP, 15 aberrant RHD alleles were found among the 500 ccDee, 2 among the 250 CcDee, and none among the ccDEe samples. One of these was the novel RHD(F223V, E233Q, T379M) allele dubbed DAU-5. Weak D type 4 was detected more frequently than expected, whereas the population frequencies of the other RHD alleles conformed to published estimates. Nucleotide sequencing of RHD exon 5 further revealed three novel alleles RHD(G212G), RHD(R234W), and RHD(V245L), dubbed DUC-1, DQC, and DUC-2. CONCLUSION: In a limited screen at the molecular level among 1000 random D+ donors in southwestern Germany, 20 donors were found carrying aberrant RHD alleles. Four of these alleles were new and likely sporadic. An estimate was derived of the variety that may be encountered in genotyping approaches, and it was concluded that even within the European population the variety of RHD alleles may be larger than anticipated.