This article is mentioned in:

Introduction

Hereditary spherocytosis (HS), an inherited condition characterized by the presence of spherocytes in peripheral blood smears, is the most prevalent cause of haemolytic anaemia (1,2). Although HS is a global disease, its incidence has increased in Northern Europe to ~1 in 2,000 cases (3). In East Asia, research from Korea and Japan has revealed that HS is the most common congenital haemolytic anaemia (4,5). The predicted prevalence in China is 1.27 per 100,000 for men and 1.49 per 100,000 for women (6). Currently, known mutant genes include ankyrin 1 (ANK1), spectrin α erythrocytic 1 (SPTA1), spectrin β erythrocytic (SPTB), solute carrier family 4 member 1 (SLC4A1) and erythrocyte membrane protein band 4.2 (EPB42), which encode the erythrocyte membrane proteins ankyrin, spectrin, band 3 and band 4.2, respectively. Mutations in these genes lead to abnormalities or malfunctions of their respective proteins (1,7-9). Approximately 90% of the cases of HS are inherited as autosomal dominant due to mutations in ANK1, SPTB and SLC4A1. A further 10% of the cases of HS are inherited as autosomal recessive due to mutations in SPTA1 and EPB42(10).

HS has a heterogeneous clinical manifestation, ranging from asymptomatic compensated haemolysis to transfusion dependence (1,11-13). The diagnostic foundation for this condition is a negative Coombs test, the presence of spherocytes and anaemia with a positive family history. Therapy is directed at preventing or minimizing the effects of chronic haemolysis and anaemia. Although there is no treatment for erythrocyte membrane abnormalities, available options include folic acid and erythropoietin supplements, as well as blood transfusions, splenectomy and cholecystectomy, depending on individual disease severity (7,14,15).

As a diagnostic tool, whole-exome sequencing based on next-generation sequencing technology has considerably contributed to the discovery of novel mutations that cause Mendelian diseases (16-18). A previous study reported a novel ANK1 c.4276C>T (p.R1426*) nonsense mutation identified by NGS in a patient with HS. However, Sanger sequencing confirmed that neither the parents or younger brother carried this mutation (19). Thus, to the best of our knowledge, reports of such mutations, particularly mutations within the ANK1 gene, and their clinical characteristics in families with HS, are rare. The present case report describes the identification of an HS pedigree with a novel ANK1 mutation using next-generation sequencing technology, thereby expanding the spectrum of ANK1 mutations. Moreover, the related literature is reviewed.

Case report

A 24-year-old male patient (proband) was hospitalized in July 2021 at the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University (Luzhou, China), with a 12-year history of unexplained anaemia and recurrent jaundice. A physical examination showed pale conjunctiva and an enlarged spleen. The patient had no history of any other diseases. Moreover, no history of smoking, drinking or drug use was reported, and the parents were not consanguineous. However, the patient's father, paternal aunt and paternal grandmother reported a similar medical history of anaemia and jaundice.

For investigating the cause of anaemia, tests for whole blood cell count, liver function, serum iron, serum ferritin, transferrin saturation, red blood cell osmotic fragility and eosin-5'-maleimide (EMA) binding, as well as the Coombs test, were performed on the patient, the patient's father and the paternal grandmother, while tests for whole blood cell count, liver function and EMA binding were performed on the paternal aunt (Table I). The patient, father and paternal aunt had compensated anaemia with an elevated reticulocyte ratio. The test results of the patient and family members showed a negative Coombs test, increased red blood cell osmotic fragility and decreased mean channel fluorescence levels in the EMA binding test.

Table I Blood test results of the patient and the family members.

Table I

Blood test results of the patient and the family members.

Characteristic	Reference range	Patient	Father	Aunt	Grandmother
Red blood cells, n (x1012/l)	4.30-5.80	4.03	3.56	3.67	1.90
Hemoglobin, g/l	130-175	139	124	117	64
Mean corpuscular volume, fl	82-100	101.20	103.90	91.90	106.70
MHC, pg	27-34	34.40	34.70	31.90	33.60
MHC concentration, g/l	316-354	340	334	347	315
Reticulocytes, %	0.90-2.20	8.94	9.92	8.87	11.01
Osmotic fragility	(-)	↑	↑	NA	↑
Spherocytosis	(-)	(+)	(+)	NA	(+)
Eosin-5'-maleimide, %	100	87.91	85.94	87.59	96.30
Coombs test	(-)	(-)	(-)	NA	(-)
Iron, µmol/l	10.60-36.70	19	47.50	NA	33.60
Ferritin, ng/ml	25-280	322.43	>1,000	NA	891.42
Serum total bilirubin, µmol/l	0-23	96.10	69.90	26.80	29.40
Direct bilirubin, µmol/l	0-7	11.50	21	8.40	11.10
Indirect bilirubin, µmol/l	0-20	84.60	48.90	18.40	18.30
Unbound iron-binding capacity, µmol/l	34-48	29.80	6.70	NA	7.10
Total iron-binding capacity, µmol/l	50-77	48.80	54.20	NA	40.70

[i] MCH, mean corpuscular haemoglobin; NA, not applicable; ↑, increased.

The patient underwent a bone marrow aspiration and bone marrow smear. The patient and family members underwent a peripheral blood smear test. The bone marrow and peripheral blood smears were performed using Wright's staining. Buffered Wright stain was added for 5 min to fix and stain the specimen, and then the slides were left to stand for 5 min after adding an equal amount of ultrapure water, all at room temperature. The slides were then washed, blotted and assessed via light microscopy. The results of the bone marrow smear and peripheral blood smear were analysed by experienced physicians in the Department of Pathology of The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University. Spherocytes were found in the peripheral blood smears (Fig. 1). All other causes of anaemia were excluded via bone marrow aspiration.

Figure 1 Morphological analysis of erythrocytes. Spherocytes (arrows) are visible in the patient's peripheral blood smear (Wright's stain; x1,000 magnification).

Abdominal magnetic resonance imaging (MRI) of the patient (Fig. 2) and father revealed splenomegaly and iron deposition in the spleen. A blood sample from the patient was sent to Guangzhou KingMed Diagnostics Group Co., Ltd., for whole-exome sequencing to identify possible mutations. Genomic DNA collection and whole-exome sequencing were performed according to the manufacturer's instructions as previously described (20). A novel mutation in ANK1, c.4707G>A (p.Trp1569*), was identified in the patient. The genetic pedigree map and sequencing results for the analysed family members are shown in Fig. 3. The heterozygous mutation was verified by Sanger sequencing at Guangzhou KingMed Diagnostics Group Co., Ltd. (Fig. 3), which showed an overlap of signals for G/A and C/T at position 4707 of the ANK1 gene in the chromatograms of the forward and reverse sequencing results, respectively. Furthermore, Sanger sequencing also confirmed that the mutation was present in the patient's father, aunt and grandmother (data not shown). The c.4707G>A (p.Trp1569*) mutation produced a truncated ANK1 protein, thereby impairing ANK1 function. This variation was not found in four major databases [Human Gene Mutation Database (https://digitalinsights.qiagen.com/products-overview/clinical-insights-portfolio/human-gene-mutation-database/), ESP6500siv2 (https://esp.gs.washington.edu/drupal/), 1000 Genomes (https://www.internationalgenome.org/) and dbSNP147 (https://www.ncbi.nlm.nih.gov/snp/)] and was not identified in the patient's mother or grandfather upon Sanger sequencing (data not shown).

Figure 2 MRI scans of the proband patient. (A) MRI scan of the patient revealing an enlarged spleen. (B) T2 MRI scan and (C) selective partial inversion recovery sequences revealing reduced signal intensity in the spleen, suggesting iron deposition. MRI, magnetic resonance imaging.

Figure 3 Genetic pedigree map. (A) A dominant inheritance mode is noted in all three family generations (I, II and III) with similar clinical signs. (B) ANK1 gene sequencing results showing the c.4707G>A mutation (G at position 4707 of the ANK1 gene is replaced by A). ANK1, ankyrin 1.

All affected family members are currently undergoing regular follow-ups every 3-6 months. Folic acid supplementation and blood transfusion are administered when necessary. Neither the patient nor the affected family members have received a splenectomy.

Discussion

The present study describes an HS pedigree with four family members involved. According to related guidelines (21,22), whole-exome sequencing was performed in the patient and a novel ANK1 mutation, c.4707G>A (p.Trp1569*), was identified. In recent years, an increasing number of novel mutations related to HS have been identified with second-generation sequencing (23). Moreover, second-generation sequencing has become an affordable and accessible test for diagnosing HS, as genetic sequencing costs have decreased significantly.

The genotypes and protein phenotypes of HS vary widely, with ANK1 mutations accounting for 35-65% of cases in Northern Europe, which is similar to the prevalence of ANK1 mutations in the Korean population (ANK1 mutations result in 66.66% of cases) (24,25). By contrast, most cases of HS in the Japanese population are due to mutations in EPB42 (45%), whereas in China, ANK1 and SPTB have been reported as the most commonly mutated genes, each being present in 45% of patients (5,26). Thus, deficiencies in ankyrin alone or ankyrin and spectrin combined, caused by mutations in ANK1 or ANK1 and SPTB, respectively, are the major causes of HS in China. The present report identified a novel HS-related mutation in ANK1.

Ankyrin is the main protein responsible for coupling the erythrocyte plasma membrane and the underlying cytoskeleton; it interacts with spectrin to maintain the biconcave disc shape of these cells, allowing them to undergo reversible deformation while traversing the microvasculature without changing the surface area (27). ANK1 mutations are responsible for the change from the biconcave disc shape to the spherical shape. This limits erythrocyte deformability and triggers mechanical rupture haemolysis in capillary network-rich organs such as the liver and spleen. The ANK1 gene is localized to 8P11.21 and contains 49 exons that encode three major structural domains, including the N-terminal binding, central spectral binding and C-terminal regulatory domains, of which the C-terminal regulatory domain is the most mutable (28). Recent studies have found that ANK1 mutations in Chinese patients with HS are mainly observed in the N-terminal membrane protein-binding domain, the two ZU5 domains, the UPA domain and the death domain, with clinical manifestations of different types of ANK1 mutations appearing in other structural domains being random (29,30). The mutation in ANK1 identified in the current study was a rare nonsense mutation located in the gene sequence encoding the C-terminal regulatory domain, resulting in autosomal dominant inheritance, which led to the development of clinical symptoms in three generations of the family.

Studies have shown that clinical manifestations of HS might be related to the ANK1 mutation site. Mutations in the central spectral binding domain of ANK1 reportedly elicit more severe anaemia and higher rates of erythrocyte deformation. Variants affecting the death domain are associated with a low mean corpuscular volume (MCV) and a low mean corpuscular haemoglobin (MCH) level (25,29). However, even the same ANK1 mutations may result in heterogeneous disease severity in different pedigrees (7,10). As a novel truncated mutation discovered at the end of the C-terminal regulatory domain, to the best of our knowledge, there is almost no information about its related clinical manifestations. As for truncated mutations at the end of the C-terminal regulatory domain, Wang et al (19) reported an ANK1 c.4276C>T (p.R1426*) nonsense mutation at the C-terminal regulatory domain in a patient with HS. However, Sanger sequencing confirmed that none of the other family members carried this mutation. To date, the present study is the first pedigree report of an ANK1 truncated mutation at the end of the C-terminal regulatory domain with multiple patients involved. Most individuals in the present patient's family had compensated anaemia, indicating mild HS and potentially explaining their normal MCV, MCH, and MCH concentration (MCHC) levels, which are typically reduced in patients with HS. Additionally, several studies have shown normal or increased MCHC level in patients with HS (29,31,32).

Multiple episodes of haemolysis may cause excessive iron deposition in the spleen of adult patients with HS. Therefore, these events must be differentiated from other iron overload diseases, such as hereditary hemochromatosis (33). In the present case, abdominal MRI showed spleen iron deposition in the patient and the father, which is seldom reported in patients with HS. As bone marrow aspiration and whole exosome sequencing excluded other iron overload causes, it was considered that the iron deposition in the spleen of the patient and the father was caused by chronic haemolytic anaemia.

Osmotic gradient ektacytometry is a robust measurement for diagnosing HS (34). However, it is not widely available for the clinical diagnosis of HS in mainland China. Therefore, the combination of EMA testing and second-generation sequencing is recommended in the Chinese and South Korean guidelines for HS diagnosis (21,22). In the present case, EMA testing and second-generation sequencing helped to diagnose HS.

In conclusion, the present study reported an HS pedigree with a novel ANK1 mutation and unique clinical manifestation. Affordable and accessible second-generation sequencing helped to diagnose HS and discover a novel HS-related mutation. Individuals in this pedigree showed a mild clinical manifestation, which might be related to the novel truncated mutation at the end of the C-terminal regulatory domain of ANK1. This novel mutation pedigree expands the mutation spectrum of ANK1-related HS. Further studies are warranted to show the pathogenesis of this novel c.4707G>A (p.Trp1569*) mutation for HS.

Acknowledgements

Not applicable.

Funding

Funding: The whole-exome and Sanger sequencing analysis were supported by funding from the National Traditional Chinese Medicine Clinical Research Base Construction Unit of The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University [grant no. (2020) 33], the Luzhou ‘Jiucheng Talents-Scientific and Technological Innovation Team’ [grant no. (2021) 162] and the Scientific Research Team Cultivation Project of the Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University [grant no. (2022) 68, 2022-CXTD-04].

Availability of data and materials

The datasets generated and/or analysed during the current study are available in the NCBI Sequence Read Archive repository (https://dataview.ncbi.nlm.nih.gov/object/PRJNA884085?reviewer=5isvljlpqtfr33fnpmdl3bjcq0) under the accession no. SRR21700253.

Authors' contributions

JW, SY, JC and XZ designed the study. XZ and MP collected the data. XZ drafted the manuscript. YY, YZ and DZ analyzed and interpreted the genetic data. MP and ZP followed up with the patient and analyzed the clinical data. JW, SY and JC revised the manuscript. JW and XZ confirm the authenticity of all the raw data. All authors read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of The Affiliated Traditional Chinese Medicine Hospital of Southwest Medical University (approval no. KY2022010). The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all individuals involved in the study.

Patient consent for publication

Written informed consent was obtained for the publication of clinical data and genetic analysis data, including the addition of genetic information to the NCBI repository.

Competing interests

The authors declare that they have no competing interests.

References