A novel variant in the SPTB gene underlying hereditary spherocytosis and a literature review of previous variants

Abstract

Background: Hereditary spherocytosis (HS, MIM#612641) is one of the most common hereditary hemolytic disorders. This study aimed to confirm a novel variant's pathogenicity and reveal a patient's genetic etiology.

Methods: The clinical data of a patient with HS who underwent genetic sequencing at the Children's Hospital of Chongqing Medical University were reviewed retrospectively. In silico prediction and in vitro minigene splicing reporter system were then conducted on the detected variant to analyze its intramolecular impact. A summary of the literature related to HS due to SPTB gene variants was also presented.

Results: A novel variant (c.301-2 A > G) in the SPTB gene (NM_001024858.4) was identified in the proband. Using Sanger sequencing, we conclusively confirmed that the inheritance of the variant could not be traced to the biological parents. The in vitro minigene assay revealed three different transcripts derived from the c.301-2 A > G variant: r.301_474del, r.301_306delCCAAAG, and r.301-1_301-57ins. Through a literature review, patients with HS who had been genotypically validated were summarized and the SPTB gene variant profile was mapped.

Conclusion: We identified a splicing variant of the SPTB gene, thus confirming its aberrant translation. The novel variant was the probable genetic etiology of the proband with HS. Our findings expanded the variant spectrum of the SPTB gene, thus improving the understanding of the associated hereditary hemolytic disorders from a clinical and molecular perspective and contributing to the foundation of genetic counseling and diagnosis.

Keywords: SPTB gene; Hereditary spherocytosis; Minigene; Novel variant.

Fig. 1

Genetic variant in the HS family. The figure shows the genetic segregation of variants and the details of the SPTB variants. (A) Family pedigree. Circles represent female family members, and squares represent males. Filled symbols designate the proband. (B) Sanger sequencing showed that the c.301–2 A > G variant carried by the proband was likely de novo and was not inherited from the parents. (C) Multiple sequence alignment in the c.301-2 site variant of SPTB among different species, indicating that this variant was highly conserved. Conserved nucleotide is marked in red boxes

Fig. 2

Schematic diagram of variant of the c.301–2 A > G variant affecting splicing. The online RNA Splicer tool predicted two splice patterns: splice pattern 1, a 6-bp deletion and a new acceptor, and splice pattern 2, exon 3 (containing 174 bp) skipping resulting in the loss of 58 codons

Fig. 3

In vitro minigene assay of the c.301–2 A > G variant. (A) Plasmid construction flow chart. The WT minigene (pMini-Cop GFP-WT) contains exons 2–4 and full-length intron 2 and intron 3 of the SPTB gene. (B) Agarose gel electrophoresis of the RT-PCR fragments from HEK 293 T cells. Original pictures of the amplified bands from the gel are available in Supplementary Material 3. (C) The schematic representation of the different transcripts. (D) Sanger sequencing of the RT- PCR products. WT, wild-type; MT, mutant-type

Fig. 4

Pie diagram and Lollipop chart illustrating the distribution of variants reported in the literature. (A) Distribution of de novo variants. (B) Distribution of inherited variant. (C) Lollipop chart of all variants. Variant diagram circles are colored with respect to the corresponding variant types. De novo: build up from nothing; CNV: copy number variation