Original article

Ahead-of-Print

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

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Key words: hybrid gene, Rh system, sickle cell disease, Rh genotyping
Publication Date: 2022-05-16

Abstract

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.

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Authors

Tatiane A. de Paula Vendrame - Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil; Hemocentro - Unicamp, Campinas, SP, Brazil

Carine P. Arnoni - Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil

Flavia R.M. Latini - Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil

Afonso J. Pereira Cortez - Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil

Caroline Bénech - University of Brest, Inserm, EFS, UMR1078, GGB, Brest, France; Laboratory of Excellence GR-Ex, Paris, France

Yann Fichou - University of Brest, Inserm, EFS, UMR1078, GGB, Brest, France; Laboratory of Excellence GR-Ex, Paris, France

Lilian Castilho - Hemocentro - Unicamp, Campinas, SP, Brazil

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