Colsan - Associação Beneficente de Coleta de Sangue, São Paulo, SP, Brazil.
Hemocentro - Unicamp, Campinas, SP, Brazil.
Blood Transfus. 2023 May;21(3):202-208. doi: 10.2450/2022.0028-22. Epub 2022 May 16.
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.
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.
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.
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.
杂合基因是形成 Rh 变异体的原因，在镰状细胞病（SCD）患者中很常见。然而，通常无法通过常规分子方案检测到它们。在本研究中，使用定量多重短荧光片段聚合酶链反应（QMPSF）研究了杂合基因，该分子方案可定量 RHD 和 RHCE 外显子的拷贝数。此外，我们还探索了 QMPSF 获得的其他相关信息，例如识别变体 RHCE 和 RHD 基因型。
选择三组研究对象：SCD 患者、自我宣称的非裔血统供体（SDA）和 D 阴性供体。通过 QMPSF 方法研究 RHD 和 RHCE 杂合基因。使用实时多重聚合酶链反应（PCR）检测来确认两个样本中 RHD 的拷贝数。进行克隆以研究等位基因。通过流式细胞术研究相对 RhD 抗原密度，并使用试管和凝胶方法进行 RhCE 表型分析。
在分析的 507 个样本中，在 20.08%的 SCD 患者、18.22%的 SDA 组个体和 3.67%的 D 阴性供体中发现了杂合等位基因频率。SCD 和 SDA 组具有更高的杂合等位基因频率，最常见的是涉及外显子 8，在此发现与常见于非裔个体的 c.733C>G 多态性有关。值得注意的是，两名 SCD 患者被证实携带三个基因拷贝，这通过定量 PCR 得到证实；这些患者的 D 表达没有增加。此外，QMPSF 指导了对 144 个 RHCE 变体和 RHD 基因型的研究，并鉴定了两个新的等位基因。
QMPSF 可鉴定参与巴西供体和 SCD 患者改变 Rh 表型的杂合等位基因。杂合 RHCE-D(8)-CE 等位基因与 c.733C>G 的关联表明，该杂合等位基因可作为标记物用于检测 SCD 患者中最常见的变体。
