Paternal RHD zygosity determination in Tunisians: evaluation of three molecular tests

International audienceBackground. The choice of a molecular test for first intention determination of paternal RHD zygosity, before entering into invasive diagnostics, is important for the management of pregnancies at risk of haemolytic disease of the foetus and newborn related to anti-RhD. Materials and methods. RHD zygosity was evaluated in 370 RH:1 Tunisian donors by polymerase chain reaction-sequence-specific polymorphism (PCR-SSP) analysis and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) amplification of hybrid Rhesus box and by real time quantitative polymerase chain reaction (RQ-PCR) specific for RHD exon 5. To evaluate the accuracy of molecular tests in the cases of discordant results, the ten exons of RHD and Rhesus boxes were amplified by PCR and sequenced. Results. Molecular investigations revealed that our 370 donors comprise 193 dizygous and 145 hemizygous individuals and 32 subjects whose zygosity remains unknown. Positive predictive values were higher than 99% for all the methods, reaching 100% for RQ-PCR. Negative predictive values were 83.24%, 87.27% and 98% for PCR-SSP, PCR-RFLP and RQ-PCR respectively. This study also revealed 19 novel Rhesus box polymorphisms and three novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile). Discussion. RQ-PCR is the most convenient method for first intention determination of paternal RHD zygosity in Tunisians. However, taking into account positive and negative predictive values, PCR-RFLP could be an alternative despite the heterogeneity of Rhesus boxes and the complexity of RHD

Molecular study of the perforin gene in familial hematological malignancies

Perforin gene (PRF1) mutations have been identified in some patients diagnosed with the familial form of hemophagocytic lymphohistiocytosis (HLH) and in patients with lymphoma. The aim of the present study was to determine whether patients with a familial aggregation of hematological malignancies harbor germline perforin gene mutations. For this purpose, 81 unrelated families from Tunisia and France with aggregated hematological malignancies were investigated. The variants detected in the PRF1 coding region amounted to 3.7% (3/81). Two of the three variants identified were previously described: the p.Ala91Val pathogenic mutation and the p.Asn252Ser polymorphism. A new p.Ala 211Val missense substitution was identified in two related Tunisian patients. In order to assess the pathogenicity of this new variation, bioinformatic tools were used to predict its effects on the perforin protein structure and at the mRNA level. The segregation of the mutant allele was studied in the family of interest and a control population was screened. The fact that this variant was not found to occur in 200 control chromosomes suggests that it may be pathogenic. However, overexpression of mutated PRF1 in rat basophilic leukemia cells did not affect the lytic function of perforin differently from the wild type protein

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

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Short duplication within the RHCE gene associated with an in cis deleted RHD causing a Rhnull amorph phenotype in an immunized pregnant woman with anti-Rh29.

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