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Moussa H.,Sanguine | Moussa H.,Laboratoire Of Biochimie Et Biologie Moleculaire | Tsochandaridis M.,Laboratoire Of Biochimie Et Biologie Moleculaire | Chakroun T.,Sanguine | And 8 more authors.
Transfusion Medicine | Year: 2012

Background: Most studies of the molecular basis of Rhesus D-negative phenotype have been conducted in Caucasian and African populations. A comprehensive survey of RHD alleles was lacking in people from North Africa (Tunisians, Moroccans and Algerians) which could be very efficient for managing donors and patients carrying an RHD molecular variant. We analyse the molecular background of D-negative population in Tunisia in the present study. Materials and methods: Blood samples were collected from native Tunisians. A total of 448 D-negative donors from different regions of Tunisia were analysed by RHD genotyping according to an adopted strategy using real-time PCR, ASP-PCR and sequencing. Results: Among the 448 D-negative samples, 443 were phenotyped unequivocally as true D-negative including three molecular backgrounds which were RHD gene deletion (n = 437), RHDψ pseudogene (n = 2) and RHD-CE-D hybrid gene (n = 4) with the respective frequencies of 0·9900, 0·0023 and 0·0046. The remaining five samples, in discordance with the serological results, were identified as two weak D type 11, one weak D type 29, one weak D type 4·0 and one DBT-1 partial D. Conclusion: This study showed that the Tunisian population gets closer to Caucasians, given that the RHD gene deletion is the most prevalent cause of D-negative phenotype, but it is slightly different by the presence of the RHDψ pseudogene which was found with a very low frequency compared with that described in the African population. Nevertheless, the relative occurrence of weak D variants among studied serologically D-negative samples make necessary the adaptation of RHD genotyping strategy to the spectrum of prevalent alleles. © 2012 The Authors. Transfusion Medicine © 2012 British Blood Transfusion Society.

Ba A.,Sanguine | Ba A.,Aix - Marseille University | Beley S.,Laboratoire dHematologie Moleculaire | Beley S.,Aix - Marseille University | And 6 more authors.
Transfusion | Year: 2015

BACKGROUND Knowledge of RH variants in African populations is critical to improving transfusion safety in countries with populations of African ancestry and to providing valuable information and direction for future development of transfusion in Africa. The purpose of this report is to describe RH diversity in individuals from Mali. STUDY DESIGN AND METHODS Blood samples collected from 147 individuals self-identified as Dogon and Fulani were analyzed for Rh antigens and alleles. RESULTS The most common RHD allele variant was RHD∗DAU0. Five predicted partial-D phenotypes were attributed to RHD∗DAU3 or RHD∗DIVa. Neither RHD∗DAR nor RHD∗DIIIa was found. Investigation of RHCE revealed three predicted partial-e antigens encoded by RHCE∗ce(254G) in trans to RHCE∗cE. Regarding C antigen, 28 Fulani typed as C+ and 16 of 28 harbored at least one RHCE∗Ce-D(4)-ce, two being homozygous and predicted to show a rare RH:32,-46 phenotype. A new RHCE∗ceTI with replacement of Exon 2 by RHD (RHCE∗ceTI(D2)) was identified in Dogon and was identified by inheritance study to be in cis to RHD∗DIVa. These samples typed C- with anti-C polyclonal antibody and monoclonal antibodies (MoAbs) MS24, P3X2551368+MS24, and MS273, but positive with anti-RhCe MoAb-BS58. The same pattern was observed in sample with RHD∗DIVa/RHCE∗ceTI. CONCLUSION Our survey indicated an uneven distribution of RH variant alleles between Dogon and Fulani, suggesting that study in well-documented cohorts is warranted. A high incidence of predicted partial-C phenotype encoded by RHCE∗Ce-D(4)-ce was found in Fulani. Further study will also be needed to clarify the clinical significance of the new DIVa/ceTI(D2) haplotype encoding partial D and variant ce antigens. © 2015 AABB.

Silvy M.,Laboratoire dhematologie moleculaire | Silvy M.,Aix - Marseille University | Filosa L.,Laboratoire dhematologie moleculaire | Chiaroni J.,Laboratoire dhematologie moleculaire | And 3 more authors.
Transfusion Clinique et Biologique | Year: 2014

Aim of the study: Current knowledge of the molecular basis of most blood groups enables genetic testing for blood groups to overcome the limitations of agglutination. A retrospective review was carried out on genotyping assays performed between 2011and 2013. Methods and patients: The Molecular Hematology Laboratory of the EFS Alpes-Méditerranée implements commercially available tools (BioArray, Gen-Probe) and other techniques (TaqMan, tetra-primer ARMS-PCR, sequencing). It provides a high-level of expertise in molecular biology, complying with regulatory requirements and standards. Results: A total of 2382genotyping assays was performed including 764extended typings and 115large extended typings essentially in cases involving multiple transfusion and suspected rare blood type. Phenotype discrepancies linked to the RH system accounted for 1501genotypings. Discrepancies linked to the D and E were mainly related to an allele coding for weak antigen (weak D type 1, 2, 3and EIV) while those linked to C, c and e antigens were related to an allele coding for a partial antigen (RN, ces(340), ceMo). A high prevalence of (C)ces haplotype in trans of a DAR allele was observed in Afro-Caribbean (54/62). Conclusion: In transfusion medicine, red-cell genotyping can overcome the limitations of hemagglutination. It must be used only in situations where it provides a benefit either for the patient or resource management. For implementation of appropriate transfusional practices, this technique requires a sound knowledge of the genetic characteristics of blood groups and clinically relevant variants. It also requires competency with molecular biology tools and continuously updated scientific data. © 2014.

Durousseau De Coulgeans C.,Laboratoire dHematologie Moleculaire | Durousseau De Coulgeans C.,Aix - Marseille University | Chiaroni J.,Laboratoire dHematologie Moleculaire | Chiaroni J.,Aix - Marseille University | And 4 more authors.
Transfusion | Year: 2015

BACKGROUND Given the high heterogeneity of sub-Saharan populations especially between nonpygmoids and pygmoids, differences are expected during investigation of the DO/ART4 gene. STUDY DESIGN AND METHODS Using genomic DNA extracted from blood samples collected from 77 Tswa pygmoids and 39 Teke and seven San nonpygmoids, DO coding regions were amplified and sequenced. A tetra-primer amplification refractory mutation system-polymerase chain reaction method was developed to specifically detect the DO∗B-SH-Gln149Lys variant. Membrane expression of newly identified variant alleles in K562-transduced cells was studied by flow cytometry. RESULTS Extensive polymorphism was confirmed in Teke or San nonpygmoids and Tswa pygmoids with, respectively, 12, zero, and 24 DO∗A; 54, 10, and 115 DO∗B or DO∗B-WL; five, zero, and 14 DO∗HY; and six DO∗JO alleles in Teke only. The DO∗B-SH-Gln149Lys variant was observed as the third most frequent after the DO∗HY and DO∗JO alleles. Two novel DO∗B alleles were identified in the San samples, that is, DO∗B-Ile5Thr and DO∗B-Trp266Arg. Study of K562-transduced cells showed that compared to the DO∗B allele, DO∗B-Ile5Thr was expressed more strongly while DO∗B-Trp266Arg variant was expressed to a lesser extent and was not recognized by MIMA-123 monoclonal antibodies. CONCLUSION Sequencing analysis showed more allelic combinations in nonpygmoids than in pygmoids with high frequencies of DO∗HY, DO∗JO, and DO∗B-SH-Gln149Lys variant alleles. This finding underlines the importance of including DO∗HY and DO∗JO single-nucleotide polymorphisms in genotyping tests to improve transfusion safety. Characterization of two novel DO∗B alleles highlights the value of testing selected ethnic groups in understanding DO allele diversity. © 2015 AABB.

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