Novel compound heterozygous SPTA1 mutations in a patient with hereditary elliptocytosis

Abstract

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.

Figure 1.

Peripheral blood smear showing erythrocyte morphology in a proband with hereditary elliptocytosis (magnification, ×1,000).

Figure 2.

SDS-PAGE analysis of erythrocyte membrane proteins using Coomassie Brilliant blue staining. Lanes 1, 2, 3, 4 and 5 represent erythrocyte membrane proteins from the proband, the proband's father, the proband's mother, the proband's grandmother and a healthy control individual, respectively. Lane M, marker.

Figure 3.

Pedigree of the hereditary elliptocytosis family enrolled in the present study. Striped shapes indicate members with the c.161A>C mutation in exon 2; shapes including a black circle indicate members with the c.5572C>G and 6531-12 C>T mutations and the shape outlined by dashed lines indicates a member of the family who was not available for study analysis. The arrow indicates the proband. Circles indicate female members, while squares indicate male members of the family.

Figure 4.

DNA sequences of, from top to bottom, the proband, the proband's father, the proband's mother and the proband's grandmother. (A) Proband heterozygous point mutations A→C, C→G and C→T were located in exon 2, exon 40 and intron 45, respectively. (B) Heterozygous point mutation A→C in the father was detected in exon 2. (C) Heterozygous point mutations C→G and C→T in the proband and their mother, were detected in exon 40 and intron 45, respectively. (D) Heterozygous point mutation A→C, was found in exon 2 of the proband's grandmother. Arrows indicate point mutations.

Figure 5.

Matrix-assisted laser desorption-ionization time of flightmass spectrometry spectrum of α-spectrin peptides from the proband. (A) Peaks of normal (1,378.55 kDa, LEDSYHLQVFK) and variant (1,338.52 kDa, LEDSYPLQVFK) peptides of the His54Pro mutation. (B) Peaks of normal (1,623.86 kDa, GDCGDTLAATQSLLMK) and variant (1,609.83 kDa, GDCGDTLAATQSLVMK) peptides of the Leu1858Val mutation.

Figure 6.

Flow cytometric evaluation of EMA-labeled RBCs. (A) The fluorescence of EMA-stained RBCsfrom the (a-1) proband and a (a-2) healthy control, respectively. (B) Overlay plot comparing the fluorescence of RBCs from the patient and healthy control. EMA, eosin-5-maleimide; RBCs, red blood cells.

Figure 7.

Melting curve of exon 2 of the spectrin α chain erythrocytic 1 gene. The red-colored curve represents the proband, while blue curves represent healthy controls.