Department of Clinical Laboratory, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China.
Mol Med Rep. 2018 Apr;17(4):5903-5911. doi: 10.3892/mmr.2018.8632. Epub 2018 Feb 26.
Hereditaryelliptocytosis (HE) is a hereditary hemolytic disease, characterized by the presence of many elliptical erythrocytes in the peripheral blood that is caused by abnormal cytoskeletal proteins in the erythrocyte membrane. In the present study, a novel, causal HE mutation was reported. Routine blood examinations were performed on the proband and their family, and the fluorescence intensity of eosin‑5‑maleimide (EMA)‑labeled erythrocytes was determined via flow cytometry. Subsequently, DNA was extracted from the peripheral blood of the proband and their family members, and amplified by quantitative polymerase chain reaction. The Sanger sequencing approach was used to determine and identify gene mutations, which were verified by matrix‑assisted laser desorption‑ionization time of flight (MALDI‑TOF) mass spectrometry. To exclude genetic polymorphisms, newly identified mutations were subjected to large‑scale gene screening using high‑resolution melt analysis. Protein expression levels in the erythrocyte membrane of the proband were determined via SDS‑PAGE, which demonstrated that, compared with healthy controls, the proband exhibited a reduction in EMA‑labeled erythrocytes. In addition, DNA analysis demonstrated that the proband carried three mutations in the spectrin α chain erythrocytic 1 (SPTA1) gene: c.161A>C, c.5572C>G and 6531‑12C>T. The corresponding mutant polypeptides were also analyzed by MALDI‑TOF mass spectroscopy. SDS‑PAGE analysis indicated that the proband exhibited normal levels of erythrocyte membrane proteins. In the present study, a novel HE case with a His54Pro mutation in the SPTA1 gene was reported. The results suggested that the His54Pro mutation influenced the role of erythrocyte membrane proteins without reducing its level of expression.
遗传性棘状红细胞增多症 (HE) 是一种遗传性溶血性疾病,其特征是外周血中存在许多椭圆形红细胞,这是由红细胞膜异常细胞骨架蛋白引起的。本研究报道了一种新的、因果性的 HE 突变。对先证者及其家系进行常规血液检查,并通过流式细胞术测定吖啶橙-5-马来酰亚胺(EMA)标记的红细胞荧光强度。随后,从先证者及其家系成员的外周血中提取 DNA,通过定量聚合酶链反应进行扩增。采用 Sanger 测序方法确定和鉴定基因突变,并通过基质辅助激光解吸电离飞行时间(MALDI-TOF)质谱进行验证。为排除遗传多态性,对新发现的突变进行了高通量基因筛选,采用高分辨率熔解分析。通过 SDS-PAGE 测定先证者红细胞膜蛋白的表达水平,结果表明,与健康对照组相比,先证者 EMA 标记的红细胞减少。此外,DNA 分析表明先证者携带 spectrin α 链红细胞 1 (SPTA1) 基因的三个突变:c.161A>C、c.5572C>G 和 6531-12C>T。相应的突变多肽也通过 MALDI-TOF 质谱进行了分析。SDS-PAGE 分析表明,先证者的红细胞膜蛋白水平正常。本研究报道了一例新型 HE 病例,其 SPTA1 基因发生 His54Pro 突变。结果表明,His54Pro 突变影响了红细胞膜蛋白的作用,而不降低其表达水平。