RHD genotyping and its implication in transfusion practice.

BACKGROUND: The limitations of serology can be overcome by molecular typing. In order to evaluate the contribution of RH systematic genotyping and its implication in transfusion practice, a genotyping of D- blood donors was initiated. METHODS: Blood samples were collected from 400 unrelated D- individuals. All samples were tested by RHD exon 10 PCR. In order to clarify the molecular mechanisms of RHD gene carrier, we applied molecular tools using different techniques: PCR-multiplex, and PCR-SSPs. RESULTS: Among 400 D- subjects tested, 390 had RHD gene deletion; and 10 had RHD exon 10 of which seven were associated with the presence of the C or E antigens. Among D- carriers, we observed in five cases the presence of RHD-CE-Ds hybrid, in four cases the presence of pseudogene RHD ψ and in one case the presence of weak D type 4. CONCLUSION: Since the majority of aberrant alleles were associated with C or E antigens and the preliminary infrastructure for molecular diagnostic were absent in all Tunisia territory, we recommend to reinforce transfusion practice to consider D- donors but C+/E+ antigens as D+ donors and the application of RHD molecular typing only to solve serologic problems.

Weak D in the Tunisian population.

BACKGROUND: More than 90 weak D types have been discovered to date. As there are no published data on the frequencies of weak D types in the Tunisian population, the aim of this study was to determine the composition of weak D alleles in our population. MATERIAL AND METHODS: Blood samples from 1777 D+ and 223 D- blood donors were tested for markers 809G, 1154C, 8G, 602G, 667G, 446A, and 885T relative to translation start codon by polymerase chain reaction with sequence-specific primers to estimate the frequencies of weak D type 1, weak D type 2, weak D type 3, weak D type 4, weak D type 5 and weak D type 11 in our population. Twenty-three samples with positive reactions were re-evaluated by DNA sequencing of RHD exons 1-10 and adjacent intronic sequences. RESULTS: Among the D+ donor cohort, weak D type 4 was the most prevalent allele (n=33, 1.2%) followed by weak D type 2 (n=6, 0.17%), weak D type 1 (n=4, 0.11%), and weak D type 5 (n=1, 0.28%) and weak D type 11 (n=1, 0.28%). RHD sequencing identified a weak D type 4.0 allele in all 19 samples tested. Among the D- pool, comprising 223 samples, we detected one sample with weak D type 4.0 associated with a C+c+E-e+ phenotype which had been missed by routine serological methods. DISCUSSION: Weak D type 4.0 appears to be the most prevalent weak D in our population. However, all samples must be sequenced in order to determine the exact subtype of weak D type 4, since weak D type 4.2 has considerable clinical importance, being associated with anti-D alloimmunisation. One case of weak D type 4 associated with dCe in trans had been missed by serology, so quality control of serological tests should be developed in our country.

(C)ce(s) haplotype screening in Tunisian blood donors.

BACKGROUND: The (C)ce(s) haplotype, mainly found in black individuals, contains two altered genes: a hybrid RHD-CE-D(s) gene segregated with a ce(s) allele of RHCE with two single nucleotide polymorphisms, c. 733C>G (p.Leu245Val) in exon 5 and c. 1006G>T (Gly336Cys) in exon 7. This haplotype could be responsible for false positive genotyping results in RhD-negative individuals and at a homozygous level lead to the loss of a high incidence antigen RH34. The aim of this study was to screen for the (C)ce(s) haplotype in Tunisian blood donors, given its clinico-biological importance. MATERIAL AND METHODS: Blood samples were randomly collected from blood donors in the blood transfusion centre of Sousse (Tunisia). A total of 356 RhD-positive and 44 RhD-negative samples were tested for the (C)ce(s) haplotype using two allele-specific primer polymerase chain reactions that detect c. 733C>G (p.Leu245Val) and c. 1006G>T (p. Gly336Cys) substitutions in exon 5 and 7 of the RHCE gene. In addition, the presence of the D-CE hybrid exon 3 was evaluated using a sequence-specific primer polymerase chain reaction. RESULTS: Among the 400 individuals only five exhibited the (C)ce(s) haplotype in heterozygosity, for a frequency of 0.625%. On the basis of the allele-specific primer polymerase chain reaction results, the difference in (C)ce(s) haplotype frequency was not statistically significant between RhD-positive and RhD-negative blood donors. DISCUSSION: These data showed the presence of the (C)ce(s) haplotype at a low frequency (0.625%) compared to that among Africans in whom it is common. Nevertheless, the presence of RHD-CE-D(s) in Tunisians, even at a lower frequency, should be considered in the development of a molecular genotyping strategy for Rh genes, to ensure better management of the prevention of alloimmunisation.

RHD alleles in the Tunisian population.

BACKGROUND: A comprehensive survey of RHD alleles in Tunisia population was lacking. The aim of this study was to use a multiplex RHD typing assay for simultaneous detection of partial D especially with RHD/RHCE deoxyribonucleic acid (DNA) sequence exchange mechanism and some weak D alleles. MATERIALS AND METHODS: Six RHD specific primer sets were designed to amplify RHD exons 3, 4, 5, 6, 7 and 9. DNA from 2000 blood donors (1777 D+ and 223 D-) from several regions was selected for RHD genotyping using a PCR multiplex assay. Further molecular investigations were done to characterize the RHD variants that were identified by the PCR multiplex assay. RESULTS: In the 1777 D+ samples, only 10 individuals showed the absence of amplification of exons 4 and 5 that were subsequently identified by PCR-SSP as weak D type 4 variants. No hybrid allele was detected. In the 223 D-, RHD amplification of some exons was observed only in 5 samples: 4 individuals expressed only RHD exon 9, and one subject lacking exons 4 and 5. These samples were then screened by PCR-SSPs on d(C) ce(s) and weak D type 4, respectively. CONCLUSION: The weak D type 4 appears to be the most common D variant allele. We have not found any partial D variant. Findings also indicated that RHD gene deletion is the most prevalent cause of the D- phenotype in the Tunisian population.