Hereditary spherocytosis (HS) is a common type of hemolytic anemia caused by a red cell membrane disorder. HS type 1 (HS1) is mostly caused by mutations in ankyrin (ANK1). Newborns with HS1 usually only exhibit anemia and mild jaundice. We herein report a case of HS1 and discuss its clinical characteristics.

A 2-d-old male full-term newborn was admitted to our hospital with severe, intractable neonatal jaundice. Laboratory investigations showed hemolytic anemia and hyperbilirubinemia and excluded immune-mediated hemolysis. The patient underwent two exchange transfusions and one plasmapheresis resulting in significantly reduced serum bilirubin. Hematologic analyses and genomic DNA sequencing studies were performed. The trio clinical exome sequencing revealed a de novo null heterozygous mutation in the patient's ANK1 gene: c.841C > T(p.Arg281Ter). This mutation results in the premature termination of the ANK1 protein.

Our case demonstrates that genetic analysis can be an essential method for diagnosing HS when a newborn has severe hyperbilirubinemia.

Hereditary spherocytosis (HS) is the most common monogenic hemolytic anemia disease and is characterized by spherical-shaped erythrocytes in patients' peripheral blood. Approximately 75% of HS cases are inherited in an autosomal dominant manner[1]. The clinical manifestations of HS vary widely, ranging from almost asymptomatic to dependence on blood transfusions or severe life-threatening anemia. HS occurs worldwide, particularly in Northern Europe and North America, where the incidence is 1 in 1000 to 2000 births[2]. In China, the epidemiology of HS is poorly understood. A systematic review from China estimated that the prevalence was 1.27 cases per 100000 people in males and 1.49 cases per 100000 people in females between January 1987 and December 2013[3]. However, only one-third of children are diagnosed with HS in the first year of life[4], and HS is rarely diagnosed in the neonatal period. HS is thought to have a high underdiagnosis rate due to limited clinical recognition during the neonatal period. The clinical manifestations of HS include anemia, jaundice, and splenomegaly. More than two-thirds of neonates with HS have near-normal hemoglobin (Hb) values at birth but then develop a dramatic decrease in Hb levels during the first weeks of life[5]. Physiological jaundice is the most common presenting feature of HS in neonates without any treatments[6], and splenomegaly is rarely detected[5]. The characteristic spherocytes are observed less often in the blood smear of neonates[7].

ANK1 mutations (approximately 50%) are the most common cause of HS type 1 (HS1)[8]. The functional defects of ankyrin protein, located in the red blood cell membrane, lead to the loss of membrane cohesion, a loss of surface area, and an increase in the number of peripheral blood cells[9]. ANK1 mutations are associated with both dominant and recessive HS and are frequently mutated de novo[10]. Using exome sequencing to detect pathogenic mutations in ANK1 and other genes would allow an early HS diagnosis.

This report describes a unique case of HS1 in a Chinese neonate with extreme hyperbilirubinemia and anemia, which was treated with two exchange transfusions and one plasmapheresis. Using the trio clinical exome sequencing method, we identified a de novo ANK1 null mutant causing HS1. This report will contribute to the understanding and diagnosis of HS in neonates.

Genetic analysis revealed a heterozygous null mutation in the patient's ANK1 gene: c.841C > T(p.Arg281Ter). Sanger sequencing results showed that the heterozygous nonsense mutation was not detected in the patient's parents, indicating a de novo mutation (Figure 2). Combined with the above results, the patient was diagnosed with HS1.

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Figure 2 Genomic sequence of the patient. Genetic analysis revealed a heterozygous null mutation in the patient’s ankyrin gene [c.841C > T (p. Arg281Ter)]. No genetic mutations were found in his parents. The arrows indicated the mutation site.

Considering that this child had a rare but severe hyperbilirubinemia and neglected hemolytic disease, an exchange transfusion was performed via a continuous arterio-venous exchange. The SBR began falling, with subsequent measurements of 232.6 µmol/L. One day later, the SBR elevated to 469.7 µmol/L (direct 27.6), with Hb of 11.7 g/dL. The second exchange transfusion was performed, and the SBR decreased to 448 µmol/L. The next day SBR was elevated to 465 µmol/L. After two exchange transfusions without any obvious improvement, therapeutic plasma exchange was provided, which decreased serum bilirubin. The patient experienced a subsequent decrease in total bilirubin from 465 µmol/L to a nadir value of 197.0 µmol/L. No significant increase in bilirubin was observed under phototherapy.

After 4 d without phototherapy, bilirubin fluctuated between 83-130 µmol/L, and Hb was stable between 10.5-11.4 g/dL (Figure 1A). The patient did not have any abnormal neurologic manifestations and was discharged after 11 d of hospitalization. This patient has been followed up regularly. The spherocytes were observed on the blood smear at 6 mo of age (Figure 1C). To date, he has shown mild anemia and jaundice, but he has not experienced splenomegaly. No visual, auditory, or extrapyramidal abnormalities have been observed in this patient.

In this report, we describe a Chinese male neonate with HS1, resulting in severe hyperbilirubinemia. A de novo mutation in exon 9: c.841C > T(p.Arg281Ter) caused the premature termination of peptide translation in exon 9 of ANK1, which was found through trio clinical exome sequencing followed by Sanger sequencing.

HS is an inherited membranopathy of red blood cells characterized by phenotypic and genotypic heterogeneity, making it difficult to diagnose during the first year of life[11]. During the perinatal period, the clinical manifestation of HS ranges from severe fetal anemia with hydrops fetalis to no clinical symptoms. HS is characterized by hemolytic anemia, jaundice, splenomegaly, cholelithiasis, and spherocytes, which are observed on the peripheral blood smear. The typical neonatal erythropoiesis response is slow, often making the reticulocyte count relatively low in comparison with anemia. Nevertheless, these symptoms are not typical in the newborn. More than one-half of neonates with HS are anemia-free in the first week of life. Rare cases exhibit splenomegaly, and spherocytes are observed in the blood smear of neonates[7]. Neonate jaundice is the most common and can be the only manifestation of neonatal HS. Our patient's serum total bilirubin was raised higher than 400 μmol/L within 24 h after birth and was prolonged, suggesting a high risk of kernicterus. Afterwards, his brain MRI results showed symmetrical hyperintensity of globus pallidus on T1-weighted sequence. Although the symmetric hyperintensity of globus pallidus on T1-weighted in neonates can be seen in "a transient hyperintensity" or other diseases including cerebral injury, hypoproteinemia, premature delivery, apnea, purulent meningitis, sepsis, etc., the patient did not appear to have any of these illnesses. In our case, he exhibited severe and delayed remission of jaundice and anemia without splenomegaly or spherocytes in the neonatal period. Therefore, we have reason to believe that patients with severe jaundice and anemia in the neonatal period should undergo genetic testing to confirm whether they have HS after excluding the possibility of other hemolytic diseases.

ANK1, located at 8p11.21[12], encodes erythroid ankyrin, and its mutations are the most common causes of HS1. ANK1 contains 43 exons with a complementary DNA length of 8300 bp. The erythroid ankyrin 1 protein consists of 1880 amino acids with three main structural domains, an N-terminal membrane-binding domain, a central spectrin binding domain, and a C-terminal regulatory domain[13]. All three domains of ANK1 are key to maintaining the shape of erythrocytes essential for proper function and assembly of the erythrocyte membranous-cytoskeletal network. Mutations in ANK1 can result in spherocytes with high osmotic fragility observed in most HS patients[14]. ANK1 mutations, mainly nonsense and frameshift mutations, can significantly affect the function of ANK1[15,16]. In this case, a de novo mutation at c.841C > T (p. Arg281Ter) in the ANK1 gene was identified as a nonsense mutation. This mutation led to premature termination of the protein at the spectrin binding domain, thereby forming a truncated protein without normal function, causing an early-onset and severe phenotype.

In the literature, we found a few cases diagnosed with HS1 during the neonatal period and compared their clinical features with our patient (Table 1). Case 2 was a male neonate who developed severe hemolytic anemia 4 wk after his birth and was identified to have a novel de novo nonsense E9X: c.25G > T(p.Glu9Ter) mutant in exon 1 of ANK1[17]. Case 3 was a 31-wk premature infant who received an RBC transfusion 3 d after birth because of severe anemia and jaundice; this patient was clinically diagnosed with HS1 and carried a de novo nonsense mutation (Q109X: c.325C > T(p.Gln109Ter) in exon 4 of ANK1[18]. In these cases, the early phenotype can be attributed to the severely impaired protein function by a nonsense mutation. It is worth highlighting a notable difference between our case and the other two cases, both of whom presented with severe anemia as the first manifestation. Our patient presented with intractable pathological neonatal jaundice resistant to phototherapy and exchange transfusion, explaining the different length of retained truncated proteins by other mutations. In short, HS1 caused by nonsense mutations in ANK1 tends to have an early onset and more severe phenotype. Although there are a few HS1 cases caused by other mutation types of ANK1 and have a certain degree of clinical phenotype in the neonatal and infancy period, the diagnosis time is often delayed[19,20]. In summary, the physicians should include HS1 in the differential diagnosis in patients who develop severe jaundice or anemia without any known reason in the neonatal period. Moreover, physicians should consider sequencing of relevant genes, especially for ANK1, which will provide beneficial help for the timely diagnosis of HS1.

In conclusion, we report a Chinese neonate diagnosed as HS1 with severe hyperbilirubinemia caused by the heterozygous null mutation in the ANK1 gene: c.841C > T(p.Arg281Ter). It is worth noting that early genetic analysis plays an important role in the diagnosis of HS. Prompt treatment and anticipatory guidance are essential to prevent neonates' adverse outcomes with HS, so physicians should be more aware of signs of HS in the newborn period.

We are thankful for the members of the family for their participation and help in the study.