A de novo ANK1 mutation associated to hereditary spherocytosis: a case report

Hereditary spherocytosis (HS) results from defects in erythrocyte membrane proteins characterized by hemolysis, anemia, jaundice, gallstones and splenomegaly [1, 2]. The severity depends on rate of hemolysis, degree of compensation of anemia by reticulocytosis. The clinical manifestations vary widely, ranging from nearly asymptomatic to transfusion-dependent or severe life-threatening anemia. In the neonatal period, the major clinical manifestations are jaundice and anemia. Splenomegaly and spherocytes are rarely observed [3]. Therefore, it’s difficult to diagnose in neonates. Even during the first year of life, approximately 34% affected infants are diagnosed [4].

Previous researches have shown that mutations in ANK1 (ankyrin 1), SPTB (spectrin, beta, erythrocytic), SPTA1 (spectrin alpha, erythrocytic 1), SLC4A1 (solute carrier family 4, member 1, or band 3), and EPB42 (erythrocyte membrane protein band 4.2) are associated with HS [5]. The mutations of these genes lead to the normally double-concave disc-shaped red cells become spherical, fragile red cells [6]. ANK1 located on 8p11.21, its mutations include nonsense, splicing or frameshift and affect about half of patients with HS [7].

In most cases, HS is usually diagnosed on the basis of a positive family history, increased osmotic fragility, hyperbilirubinemia, reticulocytosis, splenomegaly and spherocytes on peripheral blood smears [8]. The neonatal HS ratio which is calculated by dividing the mean corpuscular hemoglobin concentration (MCHC) by the mean corpuscular volume (MCV) provides valuable information for the physicians. In the index infant, the ratio was > 0.36, which points towards a diagnosis of HS (97% sensitivity, 99% specificity) [3]. However, mild or atypical cases are difficult to identify because of the limitations of the classical approaches. It has been reported that approximately 10% patients of HS may be misdiagnosed due to the lack of the typical sphere-shaped erythrocytes in the peripheral blood [9].

The patient came from a Chinese family in Yunnan province. He showed anemia and jaundice without other pathological symptoms or signs when he was born. Gallstones were identified by B-ultrasound scanning. The results of blood tests before transfusion were shown in the Table 1 which indicated that the child suffered from neonatal moderate hemolytic anemia and hyperbilirubinemia. He had received two blood transfusions in neonates. Autoimmune antibody tests were negative. The neonatal HS ratio is 3.67, only slightly up the threshold.

At the age of 3 months, the patient received transfusion because of anemia (Hb: 72 g/L). Before blood transfusion, the following tests were performed. A glucose-6-phosphate dehydrogenase (G-6-PD) screening test and Coombs’ test were negative. Hemoglobin electrophoresis, α and β globin genetic analysis excluded α and β thalassemia. Bone marrow aspiration smears indicated normoblastic hyperplasia. The erythrocyte osmotic fragility wasn’t increased (hemolysis begins: 4.8 g/L referencing 4.4–4.8 g/L; hemolysis completes: 3.2 g/L referencing 2.8–3.2 g/L). Hepatosplenomegaly and spherocytes in peripheral blood smear weren’t observed. The parents were devoid of anemia, jaundice, splenectomy, or early gallstones.

At the age of 6 months, the patient received transfusion again (Hb: 76 g/L). To identify the cause of unexplained hemolysis, we performed genetic analysis by next-generation sequencing according to the methods of He’s [1]. Blood samples were collected before transfusion. Written informed consent for genetic testing was obtained from the parents. DNA was extracted from peripheral blood and 566 genes associated with hematopathy diseases were selected to detect. We detected a mutation in ANK1 (NM_001142446: exon 25:c.2693dupC: p.A899Sfs*11) in the patient that could be implicated in the patient’s phenotype. The variation resulted in an amino acid change and affected protein function. The mutation was a heterozygous mutation. (Fig. 1a). According to the ESP6500 database, the human genome database and the dbSNP database, this mutation hasn’t been reported previously. However, his parents did not carry this mutation (Fig. 1a-b). Therefore, the patient has a de novo mutation in ANK1. In addition, the prevalence of this mutation is extremely low in the population.

At the age of 11 months, pre-transfusion values of the routine blood examination were shown in Table 2 which indicated that he suffered from moderate hemolytic anemia with hyperbilirubinemia. The spherocytes on peripheral blood smear were 10% (Fig. 1c). The neonatal HS ratio is 4.38, obviously up the threshold. He needed a regular transfusion therapy at an interval of 2–3 months with a hemoglobin level of 70–80 g/L before transfusion. The growth and development of the boy are normal. Partial splenic embolization will be planned (Additional file 1).

In this report, we described a Chinese family with a patient affected by HS. A de novo mutation (exon 25: c.2693dupC:p.A899Sfs*11) causing an amino acid change in exon 25 of ANK1 was found through next-generation sequencing followed by Sanger sequencing to verify the relationship between the ANK1 mutation and HS.

HS is an inherited disorder characterized by the presence of spherical-shaped blood cells [10, 11]. Approximately two-thirds of cases are autosomal dominant (AD), and the remaining cases represent autosomal recessive (AR) inheritance or de novo mutations in some sporadic cases [12]. Cases of HS are sporadic in China [1]. However, in some countries and continents, many HS patients have no family history [13].

ANK1 mutations are responsible for the majority of cases of HS. A heterozygous ANK1 IVS3-2A > C mutation that may lead to exon 4 skipping of the ANK1 gene and cause HS was recently identified in a 7-year-old girl [14]. A patient with HS who was diagnosed clinically with only 10% spherical-shaped erythrocytes in the peripheral blood was identified to have a novel de novo ANK1 c.4276C > T (p.R1426*) nonsense mutation, while neither of his parents or his young brother carried this mutation [15]. A 6-year-old girl who was clinically diagnosed with HS carried a de novo nonsense ANK1 mutation (c.796G > T, p.Glu266X), a single-nucleotide change from G to T, which caused a substitution from glutamic acid to a premature stop at codon 266 [16].

ANK1 is an important red cell membrane protein which plays a vital role in the maintenance of erythrocyte membrane integrity [17, 18]. ANK1 consists of three structural domains: a multiple repeats N-terminal domain, a spectrin-binding center region and a regulatory C-terminal domain [7, 19, 20]. Mutations in the spectrin-binding domain and regulatory C-terminal domains result in the most severe anemia compared with those located in the other domains [19, 21].

In our study, the patient had suffered from unexplained hemolysis and hyperbilirubinemia since the neonatal period. At the age of 3 months, hepatosplenomegaly and spherocytes which is critical to diagnose the HS weren’t observed [22]. Erythrocyte osmotic fragility was negative. It was difficult to diagnose HS which originates from mutations in the genes coding for RBC membrane components. Gene detection is the principle method for cases with no family history of HS, especially in some atypical cases. NGS is able to provide a thorough genetic analysis and identify which candidate gene is responsible for the disease [23‐25]. Therefore, with this patient we used an NGS panel for the analysis of 566 genes responsible for hematological disorders. The genetic tests showed a de novo ANK1 c.2693dupC (p.A899Sfs*11) frameshift mutation which was not found in the 1000G, ExAC, or HGMD databases. Moreover, this mutation was located in the spectrin-binding domain, which might cause HS. At the age of 11 months, spherocytes on a peripheral blood smear were 10% and the neonatal HS ratio was 4.38. Our report strongly suggests that in infants, it is important for the physicians to monitor the sphere-shaped erythrocytes and the neonatal HS ratio when the patients are at risk for HS. Regrettably, eosin-5′-maleimide binding assay with flow cytometry is the test of choice to diagnose HS but isn’t available in our laboratory.

In conclusion, this report suggests that genetic detecting should be considered for some unexplained hemolytic diseases. Meanwhile, we identified a novel de novo ANK1 c.2693dupC (p.A899Sfs*11) heterozygous frameshift mutation in a Yi nationality patient. However, the pathogenesis of this ANK1 mutation should be explored further to improve the diagnosis and treatment of HS.