Extensive clinical, serologic and molecular studies lead to the first reported Rhmod phenotype in Argentina.

BACKGROUND: A highly reduced expression of Rh antigens in the erythrocyte membrane is the main feature of Rhmod , an extremely rare phenotype. Mutations within RHAG gene, which encodes RhAG glycoprotein and modulates Rh antigen expression and Rh complex formation, are the molecular events responsible for the Rhmod phenotype. Here we report a clinical, serologic, and molecular study of an Argentinean proband with Rh-deficiency syndrome. MATERIALS AND METHODS: Rh antigens, RhAG and CD47 glycoproteins were studied by serologic methods in the proband, her parents and sister. Osmotic fragility and viscoelastic parameters were also examined. RHD zygosity was analyzed by RFLP-PCR. RHD, RHCE, and RHAG genes were studied by Sanger sequencing. RESULTS: No Rh antigens were detected in the proband by standard techniques. However, adsorption-elution and anti-RhAG tests showed that the proposita was Rhmod . Reduced expression of CD47, enhanced osmotic fragility, and surface viscosity alterations giving rise to spherocytes were observed in the patient. Sequencing analysis showed that a c.920C>T mutation in RHAG Exon 6 was present in a homozygous state in the proband and in a heterozygous state in the rest of the family. This novel missense mutation caused the p.Ser307Phe amino acid substitution in Transmembrane Segment 10 of the RhAG glycoprotein. CONCLUSION: This comprehensive study determined the causes of the proband's anemia allowing the diagnosis of Rh-deficiency syndrome.

Characterization of RHD alleles present in serologically RHD-negative donors determined by a sensitive microplate technique.

BACKGROUND AND OBJECTIVES: Weak D phenotypes with very low antigen densities and DEL phenotype may not be detected in RhD typing routine and could be typed as D-negative, leading to D alloimmunization of D-negative recipients. The present study aimed to investigate the presence of RHD-positive genotypes in blood donors typed as D-negative by an automated system using the solid-phase methodology as a confirmatory test. METHODS: Two screenings were performed in different selected donor populations. For the first screening, we selected 1403 blood donor samples typed as D-negative regardless of the CE status, and in the second screening, we selected 517 donor samples typed as D-negative C+ and/or E+. RhD typing was performed by microplate in an automated equipment (Neo-Immucor®), and the confirmatory test was performed by solid-phase technique using Capture R® technology. A multiplex PCR specific to RHD and RHDψ was performed in a pool of 6 DNA samples. Sequencing of RHD exons was performed in all RHD-positive samples, and a specific PCR was used to identify the D-CE(4-7)-D hybrid gene. RESULTS AND CONCLUSION: No weak D type was found in either screening populations. Additionally, 353 (18·4%) D-negative samples presented previously reported non-functional RHD genes, 2 samples had a DEL allele, and 6 samples demonstrated new alleles, including one novel DEL allele. Our study identified six new RHD alleles and showed that the inclusion of a confirmatory test using serological methodology with high sensitivity can reduce the frequency of weak D samples typed as D-negative.

How do we identify RHD variants using a practical molecular approach?

Serologic resolution of Rh discrepancies due to partial D or weak D phenotypes is a frequent problem encountered during routine typing that can be solved by RHD genotyping because it provides better characterization of these variants. The objective of the current study was to develop algorithms for identification of D variants in multiethnic populations based on a logic sequence of molecular tests using a large number of atypical RhD specimens. Thus, a total of 360 blood samples with atypical D antigen expression were analyzed. A previously published multiplex polymerase chain reaction (PCR) procedure was performed and depending on multiplex PCR analysis, the associated RHCE allele, and D variant frequency in our population, an algorithm was developed composed of six flow charts using specific PCR-restriction fragment length polymorphism and/or specific exon sequencing. This strategy allowed the identification of 22 different variants with few assays and a much reduced cost. This study describes a simple and practical algorithm that we use to determine RHD genotypes in samples with unknown RHD. This strategy is relatively easy to implement and the algorithm can be adapted to populations with various ethnic backgrounds after an initial assessment of the type and frequency of D variants. Essentially, we demonstrate that sequencing of all RHD exons is not necessary for the identification of the majority of known D variants.

From the investigation of RHD-CE hybrid genes to the recognition of RHCE variants and RHD zygosity. Expanding the analysis by QMPSF in Brazilian donors and in patients with sickle cell disease.

BACKGROUND: Hybrid genes are responsible for the formation of Rh variants and are common in patients with sickle cell disease (SCD). However, it is not usually possible to detect them by conventional molecular protocols. In the present study, hybrid genes were investigated using the Quantitative Multiplex Polymerase chain reaction of Short Fluorescent Fragments (QMPSF), a molecular protocol that quantifies the copy number of RHD and RHCE exons. In addition, we explored additional relevant information obtained with QMPSF, such as recognition of variant RHCE and RHD zygosity. MATERIALS AND METHODS: Three groups of subjects were selected for the study: patients with SCD, self-declared African descent donors (SDA), and D-negative donors. RHD and RHCE hybrids genes were investigated by the QMPSF method. Real-time multiplex polymerase chain reaction (PCR) assay was used to confirm the copy number of the RHD in two samples. Cloning was performed to investigate the allele. Relative RhD antigen density was investigated by flow cytometry, and RhCE phenotyping was performed with both tube and gel methods. RESULTS: In the 507 samples analysed, hybrid allele frequencies were found in 20.08% of patients with SCD, in 18.22% of individuals in the SDA group, and 3.67% of D-negative donors. The SCD and SDA groups had a higher frequency of hybrid alleles, most commonly involving exon 8, with which we found an association with c.733C>G, a common polymorphism observed in individuals of African descent. Of note, two patients with SCD were shown to carry three gene copies, as confirmed by quantitative PCR; no increase in D expression was observed in these patients. In addition, the QMPSF guided the investigation of 144 RHCE variants and RHD zygosity, and two novel alleles were identified. DISCUSSION: The QMPSF was shown to identify hybrid alleles involved in altered Rh phenotypes in Brazilian donors and patients with SCD. The association of the hybrid RHCE-D(8)-CE allele with c.733C>G suggests this hybrid allele may be used as a marker to detect the most frequent variants found in patients with SCD.

SMIM1 polymorphisms in a donor population from southeast Brazil and their correlation with VEL expression.

BACKGROUND: Vel is a high frequency blood group antigen and its alloantibody is involved in haemolytic transfusion reactions. After elucidation of the molecular basis of the Vel-negative phenotype defined by a 17-base pair deletion in SMIM1, genotyping has been the technique of choice to identify the Vel-negative phenotype, and molecular investigations have contributed to explain Vel expression variability. The present study was aimed at screening for Vel negative blood donors and characterising the genetic changes found in Brazilian donors with altered Vel expression. MATERIALS AND METHODS: Molecular screening for the SMIM1*64_80del allele was performed in 1,595 blood donor samples using a SNaPshot protocol previously standardised in our laboratory. Four hundred donor samples were also submitted to serological screening using a polyclonal anti-Vel from our inventory. Samples with variability in antigen strength were selected for SMIM1 sequencing. RESULTS: No homozygous SMIM1*64_80del allele was found and the SMIM1*64_80del allele frequency was 1.01%. Different patterns of reactivity were observed in serological testing varying from negative to 3+. Through sequencing analysis we highlighted two polymorphisms: rs1175550 and rs6673829. The minor G allele of rs1175550 was found in 16/20 samples reacting 3+, while the major A allele was found in 21/23 samples reacting 2+. Regarding rs6673829, the minor A allele was present in 14/23 and 3/20 samples reacting 2+ and 3+ respectively. DISCUSSION: We included molecular VEL screening in a previously standardised SNaPshot protocol, which besides enabling detection of Vel-negative donors, also searches for eight other rare blood types. Additionally, the present study demonstrated that although the SMIM1*64_80del allele is responsible for some variation of Vel phenotype in this donor population, Vel expression is also controlled by molecular changes in SMIM1 intron 2.