Diagnosis of Hereditary Spherocytosis and Secondary Hemochromatosis in a Patient with Jaundice

Hereditary spherocytosis (HS) is a type of hemolytic anemia resulting from hereditary defects in the erythrocyte membrane. The main clinical features are anemia, jaundice, splenomegaly, gallstones, visible spherical red blood cells (RBC) in the peripheral blood, and increased RBC osmotic fragility. The pathological cause of HS is a genetic mutation that leads to an RBC membrane protein deficiency, increased cell permeability, decreased deformability, and increased damage to the spleen. The genes involved are ANK1, SLC4A1, SPTA1, SPTB, EPB42, and EPB41; ANK1 mutations are the most common [1, 2]. Splenectomy is an effective and safe treatment for HS symptom relief, especially in children [3, 4]. With sporadic treatment, the misdiagnosis and missed diagnosis rates are high [5]. The incidence of HS is reportedly much higher than the clinical diagnosis rate [6]. Morton et al. [7] studied the genetic characteristics of HS and found that while HS can occur in all ethnic groups, its genetic frequency remains unknown. Approximately 75% of cases are autosomal dominant, and the ANK1 gene is most commonly mutated. The other 25% cases are autosomal recessive; in these cases, although there may be no family history, other relatives may also have gene mutations, phenotypic variations, and autosomal recessive inheritance [8].

According to the 2011 guidelines for HS diagnosis and management of the General Haematology Task Force of the British Committee for Standards in Haematology, HS can be diagnosed without further tests if a patient has the combination of a family history of HS and typical HS clinical manifestations and blood parameters. Currently, there are no laboratory tests for an HS diagnosis alone; the combination of the eosin-maleimide test (EMA) with the acidified glycerol lysis test (AGLT) is the first choice, and it has a sensitivity of almost 100%. The sensitivity of the combination of the erythrocyte osmotic fragility test (OFT) with AGLT is 97%, while that of the combination of the EMA and the OFT is 95%. If the patient’s blood smear results are positive, the family members will be recommended to undergo the OFT and EMA, too [9]. Patients with normal blood smears from the parents will be screened for molecular defects, which may indicate recessive inheritance resulting from new mutations in the SPTB and ANK1 genes. Further analysis of the SPTB and ANK1 genes of patients and their families may reveal new single-allele gene expression as the cause of the disease [10].

Here, we found a novel pathogenic mutation in ANK1. We present the case of a 51-year-old man repeatedly experiencing fatigue and yellow urine for 10 years. The patient had undergone cholecystectomy. He had suffered hyperbilirubinemia and had undergone a bone marrow puncture when he was an infant (data not available). There is no similar history in his family. Laboratory tested revealed the following: white blood cell count, 6.14 × 10⁹/L; RBC count, 3.39 × 10¹²/L; hemoglobin, 99 g/L; mean corpuscular volume, 83.8 fL; platelet count, 145 × 10⁹/L; reticulocyte count, 9.41%; total bilirubin, 96.2 μmol/L (reference value: 1.7–25.6 μmol/L); indirect bilirubin, 79.7 μmol/L (reference value: 0.1–21 μmol/L); total iron binding capacity, 51.4 μmol/L; iron, 12.5 μmol/L; transferrin saturation, 24.3%; ferritin, 523.41 μg/L; copper protein, 282 mg/L. B ultrasound revealed hepatomegaly and splenomegaly. Peripheral blood smear showed 40% spherical RBCs by microscopy. The erythrocyte OFT was normal. For differential diagnosis, the patient underwent the following tests: sucrose hemolysis, serum acidification hemolysis, Coombs, alkali denaturation, hemoglobin A₂, hemoglobin electrophoresis, hemoglobin H inclusion body, modified globin body, isopropyl alcohol, methemoglobin reduction, glucose 6-phosphate dehydrogenase fluorescent spot, and glucose 6-phosphate dehydrogenase activity tests, all of which yielded normal results. Therefore, the diagnosis of HS was confirmed. In addition, upper abdominal nuclear magnetic resonance (NMR) spectroscopy revealed iron deposition in the liver and spleen. A liver biopsy indicated liver hemosiderosis with mild fibrosis, and, in view of the normal transferrin saturation, he was diagnosed with secondary hemochromatosis.

Then, we detected 6 exons of HS-related genes through next-generation high-throughput sequencing. Analyzing the function of and variation in each gene, it was found that the ANK1, SPTA1, and SPTB genes were mutants (Table 1) without SLC4A1, EPB42, or EPB41 gene mutations. According to the classification of genetic variants published by the American College of Medical Genetics and Genomics (ACMG), ANK1 (MN_000037.3) exon 26: c.2803C→T was suspected pathogenic mutation, which was ultimately verified by Sanger sequencing (Fig. 1). His father had passed away and had no clinical manifes-tations while alive, and the blood smear results of the patient’s mother, son, and daughter were all normal. Therefore, it was considered a novel nonsense mutation.

In summary, as HS is prone to be misdiagnosed and missed altogether, relevant medical projects should be performed thoroughly in medical institutions. Clinicians should also raise the awareness of HS and take it into account, especially in cases of unexplained jaundice. Here, we diagnosed a patient with HS and secondary hemochromatosis, and we found a novel nonsense mutation. Moreover, we used a new technique, upper abdominal NMR spectroscopy, to assess the iron content of the liver, which could help predicting the prognosis. However, further clinical studies still need to be performed to explore the genetic types and hereditary characteristics of HS.