Hereditary spherocytosis (HS) is a common type of hereditary hemolytic anemia, with primary clinical manifestations including hemolytic anemia, jaundice, and splenomegaly. It is currently believed that the disease is primarily genetically related, with genetic polymorphisms potentially involved in the pathogenesis. HS is a disease reported worldwide and is the most common type of hereditary anemia in populations of Nordic descent, with a prevalence of approximately 1 in 2,000 in Nordic populations. There are currently no definitive diagnostic criteria for the disease. The clinical severity of HS varies among patients, with some presenting with mild anemia and jaundice, while others have no clinical manifestations. However, some patients exhibit severe hemolytic anemia and require regular blood transfusions. Among these genes, five genes are associated with abnormalities in the encoding of red blood cell membrane skeleton proteins: the ANK1 gene encodes the anchor protein, the SPTB gene encodes the β-hemoglobin protein, SPTA encodes α-spectrin, SLC4A1 encodes band 3 protein, and EPB42 encodes protein 4.2]. These gene mutations result in abnormal membrane anchoring on the red blood cell surface, altering the normal biconcave disc shape of red blood cells and causing spherocytosis. Spherical red blood cells have poor deformability and are easily destroyed when passing through the splenic sinuses, leading to hemolytic anemia. Due to increased red blood cell destruction, the bone marrow undergoes stimulatory hematopoiesis as a compensatory mechanism. At this stage, bone marrow smears often show active erythroid proliferation, while peripheral blood may exhibit increased reticulocyte counts, hyperbilirubinemia, and imaging studies may indicate gallstones and splenomegaly.

This study aims to further clarify the consistency between HS gene diagnosis and clinical diagnosis, explore the feasibility of routinely applying gene diagnosis to HS diagnosis, and analyze the characteristics of HS-related gene mutations in southwestern China.

Among 35 pediatric patients with hereditary spherocytosis, the patients were grouped based on two different gene mutation types, ANK1-HS and SPTB-HS, and intergroup comparisons were conducted to compare blood routine and liver function indicators between the two groups. It was found that children with ANK1 mutations had significantly lower RBC (p = 0.021) and HGB (p < 0.01) levels compared to those with SPTB gene mutations, but there were no statistically significant differences between the two groups in other indicators, suggesting that children with ANK1-HS have more severe anemia than those with SPTB-HS (Figures 2, 3).

To further clarify whether different types of gene mutations affect the clinical phenotype of HS, 35 children with hereditary spherocytosis were grouped based on nonsense mutations and frameshift mutations. Blood counts and liver function indicators were compared between the two groups, and no significant statistical differences were found between the two groups (p > 0.05, HGB Cohen d = 0.67). This suggests that different types of gene mutations do not affect the severity of clinical symptoms in children with HS.

HS exhibits significant variability in clinical phenotypes and genotypes. The incidence of HS varies across different regions and ethnic groups. This highlights the significant role of biological factors and genetic background in the onset and progression of HS. The mutation profiles of disease-associated genes also differ across regions. There are no so-called hotspot mutations in the gene mutations in HS. Most of the mutations are sporadic and unique to individual patients or their families (6).For example, in Brazil, the prevalence of SPTB mutations is 34.6%, making it the key variant gene for HS in Brazil (7). The mutation rate of ANK1 ranges from 40%–65% in the United States and Europe, but only 5%–10% in Japan. In a cohort of 95 Dutch HS patients, the positive rate of HS genetic testing was 89% (85/95). In the ranking of pathogenic genes, the top three were SPTA1 (36%, 31/85), ANK1 (27%, 23/85) and SPTB (20%, 17/85) (8).In a cohort of 166 HS patients from Canada, the positive detection rate reached 97% (160/166), and the top three pathogenic genes were ANK1 (49%, 79/160), SPTB (33%, 52/160) and SLC4A1 (13%, 21/166) (9).These findings suggest genetic differences exist among different countries and ethnic groups.

In this study (with cases primarily from southwestern China), 19 cases (54.29%) had ANK1 mutations, and 9 cases (25.71%) had SPTB mutations. To date, reports of HS cases caused by EPB42 gene mutations leading to defects in the encoded 4.2 protein are extremely rare, whether in adult or pediatric settings (10, 11). Additionally, a review (12) of all relevant literature published between 2000 and 2020 on Chinese HS patients, which covered genetic findings and clinical data, listed a total of 158 variants, with pathogenic gene mutation frequencies as follows: ANK1 (46%), SPTB (42%), SLC4A1 (11%), and SPTA1 (1%). This finding revealed differences in the distribution patterns of HS gene mutations across regions and further indicated that ANK1 and SPTB are the key genes driving the onset of HS in Chinese patients.

Among the 35 children with HS included in this study, 16 had nonsense mutations (45.71%), 8 had frameshift mutations (22.86%), 7 had missense mutations (20.00%), and 2 had splicing mutations (5.71%), among others. This suggests that the primary mutation types in the HS gene include nonsense mutations, frameshift mutations, and missense mutations, which is consistent with most reported findings (11, 13, 14). However, in a study of 28 HS patients in Hubei Province, missense mutations were the most common mutation type (9/28) (14), while in Wang D's (12) study, missense mutations were relatively rare. This phenomenon may be related to geographical differences, and the current studies have limited sample sizes. This conclusion requires further multi-center, large-scale sample studies for confirmation.

Currently, there is no consensus on the relationship between different types of gene mutations and their phenotypes. Studies by Aggarwal (15) and Tole (9) et al. indicate that the clinical manifestations of ANK1-HS patients are very similar to those of SPTB-HS, and that the type or location of a single gene mutation cannot be used as a basis for predicting the severity of clinical manifestations (such as anemia, jaundice, etc.) in these patients. Therefore, it is necessary to classify different genetic populations for analysis to guide the development of subsequent treatment plans. Data from Park (16) revealed that SPTA1-HS patients exhibit the most severe clinical symptoms, while SLC4A1-HS patients exhibit the mildest clinical manifestations. In Park's study, although there was no statistically significant difference in disease severity between patients with ANK1 gene mutations and those with SPTB gene mutations, the proportion of children who underwent splenectomy due to SPTB gene mutations (3/31) was significantly lower than that of patients with ANK1 gene mutations (14/44) (p = 0.028) (16), indicating that the impact of blood transfusions and splenectomy should be excluded when comparing disease severity between groups. According to the findings of Qin (17) and his team, the mean red blood cell volume and mean hemoglobin content in ANK1-HS patients were higher than those in patients with SPTB mutations. The conflicting conclusions across different regions and populations may stem from the fact that anchor protein 1 (encoded by the ANK1 gene) has three main structural domains: an N-terminal membrane-binding domain, a haptoglobin-binding domain, and a C-terminal regulatory domain (18). Ankyrin is a major protein in the red blood cell membrane, anchoring transmembrane proteins to the cellular membrane skeleton via band 3 protein, haptoglobin, and protein 4.2. Most ANK1 mutations are pathogenic (9, 19–21). Previous studies have shown that patients with hereditary spherocytosis caused by mutations in the spectrin-binding domain exhibit the most severe anemia symptoms compared to those with mutations in other domains (22, 23). Spectrin is a major component of the erythrocyte membrane scaffold network, consisting of α and β subunits, and plays a key role in regulating membrane deformability and mechanical stability (23). The SPTA1 gene encodes α-spectrin, while the SPTB gene encodes β-spectrin, a structural protein of the red blood cell membrane (24). β-Spectrin consists of an N-terminal actin-binding domain and 17 spectrin repeat sequences [including a dimerization domain, partial spectrin repeat sequences, an anchor protein-binding domain, and a tetramerization domain (16)]. Compared to mutations in the SPTA1 gene encoding the α subunit, mutations in the SPTB gene encoding the β subunit are more common. In normal red blood cells, the quantity of α-hemoglobin is three to four times that of β-hemoglobin. Therefore, a mutation in a single β-hemoglobin allele is sufficient to cause HS, while both α-hemoglobin alleles must be mutated to cause the disease (25, 26), Therefore, HS caused by SPTA1 gene mutations accounts for a smaller proportion (5%), and most SPTA1-HS cases only exhibit typical HS-related clinical manifestations when α-hemoglobin expression is reduced to less than 25% of normal levels (27, 28). The data we included suggest that children with ANK1 gene mutation have more severe anemia than those with SPTB gene mutation, which may lead to earlier treatment of children with ANK1 gene mutation and younger age of onset. There is literature (9) showing that HS anemia caused by SLC4A1 gene mutation is mild, and these patients will not see a doctor in childhood, but if it is a study in an adult center, these patients need further analysis, so we did not compare the association between the age of onset of children and the type of gene mutation.The sample size in this study was relatively small, and further expansion of the sample size and collection of patient data prior to blood transfusion and splenectomy are needed for statistical analysis.

This study classified the 35 HS patients included in the study into two major categories based on the type of mutation: nonsense mutations and frameshift mutations, and analyzed the clinical indicators of these two groups. The results showed that the differences between the two groups were not statistically significant (p > 0.05), consistent with the findings of Wang D and his team (12). However, due to the relatively small number of HS cases included in this study and the fact that the cases primarily originated from the southwestern region, further research is needed to explore the association between the types of gene mutations and their phenotypes, requiring the inclusion of additional sample resources.

At present, there is no clear disease reporting system for hereditary spherocytosis in China, so there is little epidemiological data on HS. This study is the first large-scale clinical retrospective study on HS patients in Southwest China.This study investigated how different types of pathogenic gene mutations affect disease phenotypes and further explored the association between different genotypes of HS and their clinical phenotypes. The study found that children with ANK1-HS had significantly more severe anemia than those with SPTB-HS. Additionally, mutation types did not influence disease severity in this study.

This study has several limitations. First, this is a retrospective study. Many newly diagnosed patients received emergency treatment at local hospitals, and the number of cases treated with blood transfusions may deviate from the actual number. Second, determining baseline hemoglobin levels was challenging when patients were undergoing treatment, as repeated red blood cell transfusions may have been administered during hemolytic crises. This was addressed by calculating the average hemoglobin levels over multiple measurements within 24 months, while excluding samples taken after transfusions. Third, the included patients were young, and the follow-up period was short, making it impossible to assess the necessity and efficacy of splenectomy.