(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.

Fetal RhD genotyping by real time quantitative PCR in maternal plasma of RhD-negative pregnant women from the Sahel of Tunisia.

OBJECTIVE: The aim of this study was to evaluate the diagnostic value of RhD fetal genotyping from the plasma of RhD-negative pregnant women. METHODS: We analysed the plasma samples of 65 pregnant women. DNA quantification was done using real time quantitative PCR (RQ-PCR) in multiplex targeting multiple RhD exons 5, 7 and 10, with a standardized pool of plasmid calibrators. Results were compared with serological analysis of cord blood after delivery. RESULTS: Fetal RhD status was predicted with 95.38% accuracy from maternal plasma of pregnant women in the 11(th) to 40(th) weeks of gestation. One false positive but no false negative results were found. Thus the sensitivity of the assay was 100% and the specificity was 94.44 %. CONCLUSION: The present data demonstrates that the fetal RhD genotyping approach could be achieved efficiently with RQ-PCR for RhD-negative tunisian pregnant women.

RHD zygosity assignments based on most probable genotype and hybrid Rhesus box detection in Tunisia.

BACKGROUND: Determination of the RHD zygosity is important for genetic counselling and risk evaluation of hemolytic disease of the newborn HDN in women with D iso-immunisation. OBJECTIVES: We proposed to determine the genotype frequencies of the RHD locus using a PCR-SSP method and assignment of the most probable genotype (MPG) and analyse the concordance between the two methods. METHODS: The complete Rh phenotype and the frequencies of RH haplotypes were determined on 506 blood donors. RHD zygosity was determined by both assignment of the MPG and PCR-SSP specific for the hybrid Rhesus box. For RH:-1 samples, analysis of the RHD exon 10 was done to detect eventual RHD aberrant alleles. RESULTS: Among the 466 RH:1 samples, 54.08% were hemizygous and 45.92% homozygous by PCR-SSP, and 64.16% hemizygous and 35.84% homozygous by the MPG. The comparison between the methods showed discordant results in 135 RH:1 samples. For the 40 RH:-1 samples, hybrid Rhesus box was detected in all samples and RHD exon 10 was detected in three samples suggesting unequivocal alleles identified as one RHDψ, one (C)ce(s) and one weak D type 4. CONCLUSION: The PCR-SSP should replace the MPG. However, studying of aberrant RHD alleles and aberrant Rhesus boxes could confirm the accuracy of this method in Tunisian population.