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.

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.

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.

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.