SMIM1 intron 2 gene variations leading to variability in Vel antigen expression among Brazilian blood donors.

BACKGROUND: There is a significant inter-individual heterogeneity of Vel antigen expression which can lead to inaccuracies on Vel phenotyping of blood donors and, potentially, to hemolytic post-transfusion reactions. Our aim was to evaluate the impact of genetic variants in the SMIM1 intron 2 on the expression of Vel antigen among Brazilian blood donors harboring the c.64_80del17 deletion in heterozygosity. METHODS: Donors presenting the SMIM1 c.64_80del17 in heterozygosity were included in the study and subjected to SMIM1 intron 2 direct sequencing aiming to genotype the following polymorphisms: rs143702418, rs1181893, rs191041962, rs6673829, rs1175550 and rs9424296. RESULTS: SMIM1 intron 2 sequencing was performed on two hundred donors presenting one c.64_80del17 allele. The rs1175550 polymorphism significantly impacted on Vel antigen expression. Variations in the strength of agglutination on Vel phenotyping were also observed according to the rs6673829 genotype, but this difference did not persist with statistical relevance after multivariate analysis. CONCLUSION: The presence of the rs1175550A allele of SMIM1 is significantly and independently associated with a decrease in Vel antigen expression. Even though the population in Brazil is intensely mixed, the allele frequencies obtained in the current study were very similar to that reported for Europeans.

Evaluation of the applicability and effectiveness of a molecular strategy for identifying weak D and DEL phenotype among D- blood donors of mixed origin exhibiting high frequency of RHD*Ψ.

BACKGROUND: Molecular tests designed to detect the presence of active RHD gene among D- donors have been successfully applied in people of European ancestry, but not in admixed populations with a considerable frequency of RHD*Ψ. Our goal was to evaluate the performance of a molecular screening tool for identifying active RHD alleles among Brazilian blood donors classified as D- C+ and/or E+. STUDY DESIGN AND METHODS: Pools of five DNA samples of serologically D- C+ and/or E+ donors were checked by a RHD polymerase chain reaction (PCR) assay specific for RHD Intron 4 and Exon 7. When a pool result was positive, samples were genotyped individually for RHD Intron 4 and Exon 7, RHD*Ψ, RHCE*Cc, and RHD zygosity. Donors suspected of active RHD gene were further evaluated by whole-coding region and flanking intron direct sequencing. RESULTS: A total of 405 donors were included. Two percent exhibited active RHD gene, codifying D-weak (38 and 45) or DEL phenotype. The most prevalent DEL allele was RHD*DEL1 (c.1227G>A), which is proven to be immunogenic. A high frequency of RHD*Ψ was detected in the donors with nondeleted RHD alleles (31%), far superior to the frequency of RHD variant alleles (15.5%). The proposed approach presented sensitivity of 100% and specificity of 85.7% for identifying active RHD gene. CONCLUSION: The strategy of checking D- donors with RHD PCR followed by exclusion of RHD*Ψ allele has proved efficient in identifying weak-D and DEL phenotype in the Brazilian population.

RHD and RHCE genotyping by next-generation sequencing is an effective strategy to identify molecular variants within sickle cell disease patients.

BACKGROUND: The complexity of Rh genetic variation among sickle cell disease (SCD) patients is high. Conventional molecular assays cannot identify all genetic variants already described for the RH locus as well as foresee novel alleles. Sequencing RHD and RHCE is indicated to broaden the search for Rh genetic variants. AIMS: To standardize the Next Generation Sequencing (NGS) strategy to assertively identify Rh genetic variants among SCD patients with serologic suspicion of Rh variants and evaluate if it can improve the transfusion support. METHODS: Thirty-five SCD patients with unexplained Rh antibodies were enrolled. A NGS-based strategy was developed to genotype RHD and RHCE using gene-specific primers. Genotype and serological data were compared. RESULTS: Data obtained from the NGS-based assay were gene-specific. Ten and 25 variant RHD and RHCE alleles were identified, respectively. Among all cases of unexplained Rh antibodies, 62% had been inaccurately classified by serological analysis and, of these, 73.1% were considered as relevant, as were associated with increased risk of hemolytic reactions and shortage of units suitable for transfusion. CONCLUSION: The NGS assay designed to genotype RH coding regions was effective and accurate in identifying variants. The proposed strategy clarified the Rh phenotype of most patients, improving transfusion support.