Identification and functional analysis of novel SPTB and ANK1 mutations in hereditary spherocytosis patients

Abstract

Hereditary spherocytosis (HS) is the most prevalent form of congenital hemolytic anemia, being caused by genetic mutations in genes encoding red blood cell cytoskeletal proteins. Mutations in the ANK1 and SPTB genes are the most common causes of HS.; however, pathogenicity analyses of these mutations remain limited. This study identified three novel heterozygous mutations in 3 HS patients: c.1994 C > A in ANK1, c.5692 C > T, and c.3823delG in SPTB by whole-exome sequencing (WES) and validated by Sanger sequencing. To investigate the functional consequences of these mutations, we studied their pathogenicity using in vitro culture erythroblast derived from CD34 + stem cells. All three mutations lead to the generation of a premature stop codon. Real-time PCR assay revealed that the two SPTB mutations resulted in reduced SPTB mRNA expression, suggesting a potential role for the nonsense-mediated mRNA degradation pathway. For the ANK1 mutation, gene expression was not reduced but was predicted to produce a truncated version of the ANK1 protein. Flow cytometry analysis of red blood cell-derived microparticles (MPs) revealed that HS patients had higher MP levels compared to normal subjects. This study contributes to the current understanding of the molecular mechanisms underlying mutations in the ANK1 and SPTB genes in HS.

Keywords: ANK1; DNA sequencing; Hereditary spherocytosis; Mutation; NGS; SPTB.

Fig. 1

Levels of red blood cell (RBC)-derived microparticles (MPs). (A) Gating strategy for MPs based on forward scatter (FSC) and side scatter (SSC) properties, with a size range of 80 nm–1.32 μm (the red rectangle in (A). The gated MPs were analyzed using Annexin V (y-axis) and CD235a-PE (x-axis), where double positivity identifies RBC-derived MPs (the upper right quadrant in (B) and (C). Representative data of RBC-derived MPs for normal subjects (0.3%) and HS patients (14.1%) are shown in figure B and C, respectively. As shown in (D), the absolute number of RBC-derived MPs was significantly higher in HS patients than in healthy subjects.

Fig. 2

Cell expansion rate of in vitro cultured erythroblasts. Expansion rate of erythroblasts calculated from day 8 to day 14 of culture from 3 normal controls and 3 HS patients established by direct counting. In comparison to normal erythroblasts, HS erythroblasts showed increased cell expansion.

Fig. 3

Sanger sequencing analysis of gDNA and cDNA for mutations in ANK1 (c.1994 C > A) and SPTB (c.5692 C > T and c.3823delG). Consistent heterozygous nucleotide substitutions causing nonsense mutations were observed in both gDNA and cDNA sequences for ANK1 (c.1994 C > A) and SPTB (c.5692 C > T). The variant c.3823delG exhibits a frameshift mutation pattern in both gDNA and cDNA sequences.

Fig. 4

Relative mRNA expression levels of the SPTB (A) and ANK1 (B) genes normalized to GAPDH. qRT-PCR analysis revealed significantly reduced SPTB expression in patients harboring c.5692 C > T and c.3823delG mutations (A). In contrast, the c.1994 C > A mutation in ANK1 did not affect gene expression (B).

Fig. 5

Predicted structural models of wild-type and mutant proteins for SPTB (A) and ANK1 (B). The identified ANK1 c.1994 C > A mutation lies within the N-terminal membrane-binding domain. This mutation is predicted to lead to a truncated protein lacking the spectrin-binding and C-terminal regulatory domains, potentially affecting interactions with β-spectrin and other regulatory functions.