Introduction
Hereditary spherocytosis (HS, MIM#612641) is a hereditary genetic disease caused by abnormalities in red blood cell membrane proteins resulting from congenital haemolytic anaemia [
1]. HS is mainly characterized by anaemia, jaundice and splenomegaly [
2]. Additional features comprise increased erythrocyte osmotic fragility and spherical red blood cell (RBC) count in peripheral blood smears [
3]. The symptomatology and clinical outcomes of HS are highly variable. Patients with mild HS might have no clinical manifestations related to anaemia due to the compensatory effect of the bone marrow, causing more erythropoiesis than destruction. Meanwhile, patients with severe HS might suffer from hemolytic crisis or even death [
4]. The major genetic mode of HS is autosomal dominant manner, accounting for approximately 75% of cases. In addition, approximately one-fourth of HS patients have no family history of the disease, and the inheritance patterns indicate that HS can be inherited as an autosomal recessive single-gene disorder or as a result of de novo mutations [
5]. To date, 5 associated genes associated with HS have been reported:
ANK1,
SLC4A1,
SPTA1,
SPTB and
EPB42, encoding ankyrin protein, band-3 protein, alpha-spectrin protein, beta-spectrin protein and erythrocyte membrane protein band 4.2, respectively [
6]. In summary, HS shows marked clinical and genetic heterogeneity [
7]. The prevalence of HS patients also exhibits a cosmopolitan distribution. Epidemiological studies have shown that the incidence of HS in Europe is approximately 1/2000–1/5000 [
8], whereas in China the estimated incidence is 1.39/100000 [
9]. Notably, typical symptoms are not present at the same time in most patients with HS, and various factors can easily influence laboratory findings. Thus, diagnosing HS is difficult, and patients are frequently misdiagnosed and underdiagnosed. The incidence of the disease may be much higher than the clinical detection rate. Thus, obtaining a definite diagnosis is challenging, even though HS is not very rare. To the best of our knowledge, unequivocal gene sequencing can be used to confirm the diagnosis of HS [
10]. Therefore, the discovery of pathogenic gene variants detected by genetic testing and the detailed and intensive functional analysis associated with these genes might shed further light on HS pathogenesis.
In this article, online prediction tools and minigene constructs were used to further reveal the pathogenicity and characterization of novel variants detected in two Chinese families with HS, more accurately characterizing splicing variants in ANK1. This work further explains the relationship between the genotype and phenotype associated with ANK1 variants in the Chinese population and provides the basis for prenatal diagnostics and genetic counselling.
Discussion
We enrolled two unrelated families with probands showing typical HS clinical presentations. The two children, both of whom experienced HS onset shortly after birth, demonstrated clinical features of chronic haemolytic anaemia, jaundice, hepatosplenomegaly, and blood transfusion dependence, consistent with previous reports on HS patients [
14]. In both patients, a low percentage of spherical red blood cells was shown in peripheral blood smears. Additionally, the parents of the patients carrying de novo mutations had normal clinical characteristics and haematological test results [
15,
16]. Comprehensive clinical and genetic analyses were performed to provide detailed characteristics of the patients’ genotypes and phenotypes. The de novo variants c.1305 + 2 T > A and c.1305 + 2del in the
ANK1 gene were identified via target sequence capture combined with high-throughput sequencing technology. The variants were predicted to have a deleterious effect by bioinformatics tools. Furthermore, in vitro minigene experimental validations demonstrated that these variants affected the splicing process of pre-mRNA.
Previous research has shown that erythrocyte membrane protein deficiency caused by pathogenic mutations in the
ANK1,
SLC4A1,
SPTA1,
SPTB, and
EPB42 genes is the molecular pathogenesis of HS [
17]. More importantly, it was previously reported that nonsense mutations account for the majority of known
ANK1 mutations [
18]. In our study, the de novo splice variants c.1305 + 2 T > A and c.1305 + 2del in two unrelated Chinese HS pedigrees were identified by high-throughput sequencing. The sequence is highly conserved among various species. Moreover, the two variants that occur at canonical ±1 or 2 splice sites were identified as de novo variants by pedigree analysis. The clinical presentations of the two
ANK1 mutation carriers did fit the criteria for HS. The two variants are extremely rare in normal populations and were assessed as pathogenic according to the ACMG guidelines. The insertions to the coding sequence are likely to lead to premature stop codons, thus affecting the structure and function of the protein. Mutated mRNAs could be degraded by nonsense-mediated decay (NMD) to protect the integrity of the transcriptome and normal mRNAs to control the quantities of unmutated transcripts [
19]. The variants were defined as the key pathogenetic events in our study patients. This is because the two unrelated Chinese children carrying the splice variants demonstrated the classic clinical phenotype of HS. Combined with the clinical phenotype and genetic information, we speculated that these two novel splice variations in the
ANK1 gene, escape from NMD or fail to trigger NMD, might affect the protein structure and function. Certainly, further experimental validation is needed to confirm this. Our results further document and expand the database of
ANK1-causing mutations. Specifically, most mutation analyses in genetic diseases are performed only at the genomic DNA level, and experimental verification of the effect of mutations on mRNA expression and the pre-mRNA splicing process is rare. Here, we evaluated two variants in
ANK1, c.1305 + 2 T > A and c.1305 + 2del, by in silico and in vitro minigene splicing assays, respectively. The in vitro minigene data were partially in line with the predicted results, as shown in the results section. Therefore, further studies are essential for confirming the functional relevance of these predictions. In addition, both patients in the different lineages carried c.1305 + 2 variants involving a single-base substitution and deletion, suggesting that this variant may be recurrent. These new perspectives need to be verified in larger sample sizes. Remarkably, splicing variants are the genetic cause of HS in several patients, but intronic regions are not commonly included in genetic testing, even if these variants are found in DNA. Genetic testing should be carried out whenever possible to confirm the diagnosis [
20]. Further RNA analysis is also recommended for assessing the pathogenicity of the variants to reduce the misdiagnosis and underdiagnosis of HS.
There are no effective pharmacological therapeutics available for HS. The patients in our study were treated with at least one blood transfusion during acute haemolytic episodes. However, it is known that excessive iron accumulation is a greater potential threat than anaemia, ultimately leading to multiorgan failure and death [
21]. For patients with profound haemolysis, splenectomy is currently the most effective treatment [
22]. Research has revealed that partial/subtotal splenectomy or therapeutic spleen embolization tends to be applied in individualized treatment for children younger than 6 years [
23]. Regrettably, some HS patients require regular blood transfusion or are transfusion dependent after splenectomy [
24]. Moreover, splenectomy can lead to an increased risk of fatal bacterial infections and venous thrombosis [
25]. After decades of intense research, gene therapy is beginning to show promise for treating a wide range of monogenic diseases, especially monogenic haematopoietic disorders [
26]. methods are currently the most prevailing and efficient tools in haematopoietic stem cell gene therapy. CRISPR/Cas9 technology can directly repair mutated β-globin protein genes and restore normal expression of β-globin [
27,
28]. Furthermore, a previous study (Sara Fañanas-Baquero et al., 2021) found that the CRISPR–Cas9 system and donor recombinant adeno-associated vector delivery reconstituted human haematopoiesis in primary and secondary immunodeficient mice, effectively treating pyruvate kinase deficiency [
29]. With advances in modern medicine and personalized therapy, gene therapy must be modified to optimize safety and efficacy profiles. Gene therapy is a promising method for permanently curing currently untreatable diseases [
30]. Therefore, identifying novel mutations is essential for understanding genotype–phenotype relationships comprehensively and designing targeted gene therapies in the future.
In conclusion, it is recommended that genetic testing should be performed as early as possible. Specifically, variants in intronic regions deserve more attention. To reduce the incidence of HS, prenatal diagnostics and genetic counselling are necessary for families with affected members. We believe that a cure for HS will be achieved with the continuous optimization of gene therapy.
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.