Hereditary spherocytosis (HS) is a type of heterogeneous disorder that belongs to the common form of congenital hemolytic anemia (HA). It is characterized by the presence of sphere-shaped red blood cells (spherocytes) on the peripheral blood smear, anemia, jaundice and splenomegaly. HS is featured by a huge heterogeneity in disease severity among patients, whom may be virtually asymptomatic or require transfusions frequently in early childhood. Therefore, the diagnosis of HS is challenging, particularly for asymptomatic or atypical cases that only depend on clinical manifestations, family history and hematologic laboratory testing (Bolton-Maggs et al., 2012). Through literature review, the incidence of HS is 1/2,000 among individuals of northern European ancestry (Perrotta et al., 2008). To date, accurate epidemiological data in China is scant. Wang et al. (2015) estimated in 2015 that the prevalence of HS in Chinese population was 1.27/1,00,000 in males and 1.49/1,00,000 in females. Since HS is generally an inherited disorder caused by gene mutations, gene mutation spectrum responsible for the pathogenesis of HS is of great significance. Conventional laboratory testing, however, often fails to diagnose HS. With the advanced medical technologies, molecular genetic testing, especially next-generation sequencing (NGS), is becoming a powerful tool to the clinical diagnosis of HS in neonates or infants through accurately identifying genetic variants (Christensen et al., 2015).

HS is attributed to gene mutations in erythrocyte membrane proteins (ANK1, SPTA1, SPTB, SLC4A1, and EPB42), which is inherited in the autosomal dominant (AD) or autosomal recessive (AR) manner (Perrotta et al., 2008). Their mutations usually result in the decreased membrane surface area relative to the intracellular volume of erythrocytes or dysfunction of the erythrocyte membrane, leading to the detachment of the lipid bilayer from the spectrin-based cytoskeleton. As a result, abnormal erythrocytes transform into spherocytes with an increased osmotic fragility, which are prone to be trapped and destroyed in the spleen. Consequently, hemolytic anemia and increased erythropoiesis lead to reticulocytosis, hyperbilirubinemia, gallstones, and splenomegaly (Bolton-Maggs et al., 2004). Thus, the definitive diagnosis of HS relies on molecular testing. Among the five virulence genes, the majority of HS cases are caused by mutations of the ANK1 (He et al., 2018).

The ANK1 gene is located at 8p11.21 and consists of 42 exons. It encodes the ankyrin-1 protein of 1,881 amino acids with three main domains, including a N-terminal membrane-binding domain containing binding sites for the band 3 protein, a central spectrin-binding domain involving two ZU5 domains and one UPA domain for interacting with the actin-spectrin cytoskeleton, and a C-terminal regulatory domain responsible for modulating the affinity of the other domains (Lux et al., 1990). Mutations have been identified throughout the entire gene, resulting in the lack of one haploid set of ankyrin-1 (Delaunay, 2002). HS caused by mutations of the ANK1 is mainly inherited in the AD manner, with the most common types of non-sense, frameshift and splicing mutations. Most of patients with recessive HS are caused by mutations in the gene promoter and missense mutations. The frequency of spontaneous mutations of the ANK1 is high, and approximately 15%–20% of reported mutations of the ANK1 are de novo (Gallagher, 2005; Perrotta et al., 2008).

Genetic testing is recently popular in assisting the diagnosis of HS in many countries and regions, including China (Nakanishi et al., 2001; Wang et al., 2018). Wang et al. (2018) and Wu et al. (2021) have preliminarily expanded the mutation spectrum of genes responsible for HS, and clarified the mutational characteristics of causative genes, which should be further comprehensively elucidated.

In this case report, 17 children with anemia or spherocytosis caused by mutations of the ANK1 were retrospectively analyzed to explore the clinical and mutational features of HS patients with mutations of the ANK1, thus providing references for clinical treatment and genetic counseling.

HS is a kind of non-immune hemolytic anemia, which is mainly inherited in an AD manner (Iolascon et al., 2019). Typical clinical manifestations of HS are similar to those of other types of hemolytic anemia. The severity of HS varies widely, and usually, HS occurs in infancy presents a serious phenotype. Laboratory testing, such as blood cell counts and erythrocyte osmotic fragility test (OFT), lacks ideal sensitivity or specificity in the diagnosis of HS (Wu et al., 2021b). Due to the challenge in timely diagnosis of HS by conventional examinations, misdiagnosis and missed diagnosis result in the poor prognosis and enhance the medical expenses of affected patients and their families. More seriously, undiagnosed HS may lead to complications like kernicterus, hemolytic episodes, aplastic crisis and cholecystolithiasis. In our study, the mean onset age of 17 HS patients was 4.47 years, including 4 cases with the onset of younger than 1 year. Only one patient (5.8%) developed cholecystolithiasis, suggesting that HS patients could benefit from an early diagnosis. MCV, MCH, and MCHC levels in most of HS patients were within normal ranges, which were consistent with previous reports and suggested that conventional laboratory testing was not ideal for the diagnosis of HS (Aggarwal et al., 2020; Wang et al., 2020).

Mutations of the ANK1 are the most common cause of HS (He et al., 2018; Wu et al., 2021a). Ankyrin-1 protein, encoded by the ANK1 gene, links β spectrin to band 3, and plays a key role in membrane elasticity and mechanical stability. Deficiency of ankyrin-1 leads to decreased spectrin assembly on the membrane (Hanspal et al., 1991; Li et al., 2020). We identified a total of 17 different causative mutations in the ANK1, including 15 novel ones and two previously reported. According to the ACMG criteria, 12 novel mutations were considered pathogenic, while the pathogenicity of the other 2 (c.4648A>G and c.5254delTTC) were unknown (Richards et al., 2015).

As of October 2022, 281 mutations of the ANK1 associated with HS have been reported, including non-sense mutations, missense mutations, splicing mutations, small or gross deletions, insertions, regulatory mutations, and complex rearrangements (https://www.hgmd.cf.ac.uk/ac/all.php). Our data showed that among the 17 mutations of the ANK1, there were five non-sense mutations, five frameshift mutations, three splicing mutations, three missense mutations and one in-frame mutation. The three-dimensional (3D) model prediction showed that the protein structure of the 3 novel missense mutations changed, leading to the impaired stability of the erythrocyte membrane structure and erythrocyte destruction.

The genotype-phenotype correlation in HS has been analyzed (Tole et al., 2020; Wu et al., 2021a; Xie et al., 2021a). We compared clinical data in HS patients with different mutation types of the ANK1, including RBC, Hb, MCV, MCH, MCHC, RDW-SD, RDW-CV, HCT, Ret%, T-Bil, D-Bil, and LDH. Consistently with previous findings, no significant differences in clinical data were identified in HS patients with different mutation types of ANK1 probably due to the small sample size (Tole et al., 2020). The MCHC levels in all recruited HS patients were lower than 359 g/L, which was similar to the findings of Wang et al. (2020), further confirming that the conventional laboratory testing of MCHC lacks the specificity and sensitivity.

Structurally, ANK1 is composed of a N-terminal membrane binding domain, a central spectrin binding domain and a C-terminal regulatory domain. The spectrin proteins (α and β) and ankyrin are of great significance in maintaining cell membrane stability. In our cases, HS patients with variants of the ANK1 located in the regulatory domains presented significantly higher Hb levels (97.6 ± 15.63 g/L) compared to those located in the membrane binding domains (73.89 ± 14.22 g/L). Meanwhile, HS patients with variants of the ANK1 in regulatory domains had lower T-bil levels than those located in membrane binding domains. Our findings were inconsistent with a previous study that patients with mutations of the ANK1 in the spectrin binding domain present the most severe anemia (van Vuren et al., 2019). However, when we extended the Hb and T-Bil data to all reported HS probands with ANK1 mutations and our cases, only T-Bil levels of HS patients with ANK1 mutations in membrane binding domains seemed higher than those with mutations in spectrin binding domain and regulatory domains, although significant differences were not detectable. Wang et al. (2020) proposed that variants of the ANK1 death domain, rather than other structural domains, is correlated with low MCV and MCH levels, which was not observed in our study. Correlation analysis between such clinical phenotypes and genotypes need to be conducted in larger samples in the future.

In summary, we reported 17 unrelated HS children with 17 mutations of the ANK1, including 15 novel ones and two previously reported, and analyzed the clinical phenotypes stratified by mutation types and mutation regions. Our study expanded the ANK1 variant spectrum of Chinese people. Genetic testing is a useful tool to predict clinical phenotypes in childhood and guide family counselling of HS, especially in patients without family history.