AIH is an inflammation of the liver parenchyma caused by an autoimmune response to hepatocytes. It is characterized by elevated transaminase serum levels, positive serum autoantibodies, high IgG and/or γ-globulinemia, and interstitial hepatitis upon histological examination . According to the pattern of autoantibodies detected, a subclassification into three disease subtypes has been proposed: AIH-1, AIH-2, and AIH-3. AIH-1 is characterized by the presence of ANA and/or SMA. AIH-2 is characterized by the specific anti-liver and kidney microsomal antibody type-1, infrequently anti-LKM type-3, and/or antibody against liver cytosol type-1 antigen. AIH-3 is characterized by soluble liver antigen/liver pancreas antibodies . The clinical manifestations of AIH are diverse, but most patients have an insidious onset, presenting as chronic liver disease . The most common symptoms include lethargy, fatigue, and intermittent jaundice, while physical examination may reveal hepatomegaly, splenomegaly, ascites, and, occasionally, peripheral edema [1, 17]. In our study, the patient presented with yellow sclerae; liver and spleen enlargement; elevated levels of ALT, AST, TBIL, IBIL, and DBIL; positive ANA and SMA; slightly increased IgG; and increased γ-globulin levels. Her liver biopsy showed diffuse swelling of the liver lobule cells, cytoplasmic loosening, spotty necrosis, and extensive lymphocyte infiltration in the portal area. Based on these findings, the patient was diagnosed with AIH. In addition, morphological examination of her peripheral red blood cells showed that 18 % were spherical; the MCV, MSCV, and MRV were decreased; RET was increased; G-6-PD activity was normal; and the Coombs test was negative. These findings indicated that the patient’s abnormal liver function did not result from AIH alone, but that she might also suffer from hemolytic anemia.
HS is a common hereditary hemolytic disease. Gene mutations lead to defects in erythrocyte membrane proteins, which weaken the vertical connection between the protein and lipid bilayer. This results in destabilization or loss of the membrane lipid bilayer and the formation of microvesicles, reducing the cell membrane surface area to volume ratio and changing the red blood cell morphology from a biconcave disc to a spherical shape. These changes reduce the deformability and increase the osmotic fragility of erythrocytes, resulting in their destruction when passing through the small splenic vessels and, ultimately, hemolytic anemia. Clinical manifestations vary widely among HS patients. Those with severe and moderate HS often have anemia, jaundice, and splenomegaly, whereas patients with mild HS have either atypical symptoms or are asymptomatic . The guidelines for the diagnosis and treatment of HS, developed by the British Committee for Standards in Hematology , recommend that patients who have a positive family history, typical clinical features, and increased MCHC, spherocytosis, and reticulocytes, compared to normal values, should be diagnosed with HS without further examination. Therefore, patients with mild HS and no symptoms are easily misdiagnosed, or the diagnosis is entirely missed. Furthermore, because of the similar clinical manifestations of HS and AIH, such as jaundice and splenomegaly, the diagnosis of HS is frequently missed even in symptomatic patients and misdiagnosed as AIH. Deng et al.  reported a patient with mild HS who had jaundice but no symptoms of anemia and was misdiagnosed with AIH. The type of jaundice can be classified by measuring TBIL, DBIL, and IBIL. Patients with hepatobiliary disorders frequently have increased TBIL, mainly attributable to an increase in DBIL. Patients with hemolytic anemia also have increased TBIL, but it is mainly attributable to an increase in IBIL. In our patient, TBIL was increased due to both DBIL and IBIL, each accounting for 50 %. The patient had hepatomegaly and splenomegaly but no anemia symptoms, which might have easily led to missing the diagnosis of HS.
Erythrocyte morphology, blood count, and assessment of reticulocyte-related variables are simple, rapid, and practical means of diagnosing HS, which can be assessed in routine laboratories. When spherocytes comprised more than 7.8 % of red blood cells, the sensitivity and specificity for diagnosing HS were 56.7 % and 84.8 %, respectively . Spherocytes are also present in peripheral blood smears of patients with G6PD deficiency and autoimmune hemolytic anemia . Liao et al.  reported that when using an MCHC ≥ 334.9 g/L as the threshold, the HS diagnosis sensitivity was 82.1 %, and the specificity was 94.5 %. An increase in serum bilirubin can induce a false increase in MCHC. MSCV is a specific spherocyte measure and, in combination with MCV, can be highly effective in diagnosing HS. For example, Chiron et al.  reported that when the MSCV < MCV, the sensitivity and specificity for diagnosing HS were 100 % and 93.3 %, respectively. Broséus et al.  found that when the Coombs test was negative and the MCV-MSCV was > 9.6 fl, the sensitivity and specificity for diagnosing HS were 100 % and 90.57 %, respectively. In turn, Xu et al.  reported that the MRV is ideal for diagnosing HS; when the MRV was ≤ 95.77 fl, sensitivity and specificity were 86.80 % and 91.20 %, respectively. Our patient met all these diagnostic criteria. Thus, her laboratory results were highly suggestive of HS, but she had no typical clinical symptoms or positive family history. As a consequence, genetic analysis became necessary for an accurate diagnosis.
Through high-throughput sequencing, we found two novel compound heterozygous mutations, one in exon 2 c.134G>A (p.R45K) and one in exon 46 c.6544G>C (p.D2182H) of the SPTA1 gene. PCR combined with Sanger sequencing confirmed that the first mutation was from the asymptomatic mother, and the second mutation was from the father, who had a history of splenomegaly and yellow sclerae. The mother’s laboratory test results were normal, whereas her father and paternal grandmother showed an similar clinical and blood analysis results to the proband. Therefore, we hypothesized that the heterozygous mutation c.134G>A (p.R45K) inherited from the mother was more likely to be benign, and the heterozygous missense mutation c.6544G>C (p.D2182H) inherited from the father was more likely to be pathogenic.
The 80 Kb SPTA1 gene is located in the q22-q23 region of chromosome 1. It contains 52 exons and encodes α-spectrin, which contains 2419 amino acids. α-spectrin is a cytoskeletal protein that combines with β-spectrin to form α-β heterodimers in a reverse parallel arrangement. The terminal end of the polymers formed by the two heterodimers combine with actin and transmembrane proteins forming a “junction complex.” The latter forms a grid structure in the cell membrane, maintaining lipid bilayers and the biconcave disc shape of the erythrocytes . The expression of α-spectrin is four times that of β-spectrin, and the formation of α-β heterodimers is not affected by relative decreases in α-spectrin expression . Therefore, homozygous or compound heterozygous mutations in the SPTA1 gene may be involved in the pathogenesis of HS with recessive α-spectrin deficiency; heterozygous individuals may still produce enough α-spectrin to balance β-spectrin production and maintain the erythrocyte cytoskeleton [27, 28]. The SPTA1 missense mutation of c.134G>A (p.R45K) resulted in the 45th amino acid being changed from arginine to lysine. Amino acid sequence analysis showed that the mutation site is highly conserved among different species. In the protein structure analysis, the mutant residue was smaller than the wild-type residue, and the wild-type residue was found to be involved in a multimer contact. It is possible that the mutant residue is too small to make multimer contacts and thereby negatively affects the function of α-spectrin. The missense mutation of c.6544G>C (p.D2182H) resulted in the 2182nd amino acid being changed from aspartic acid to histidine. This site is also highly conserved among different species. Protein structure analysis revealed that this mutation is located within a repeated stretch of residues in the protein, called spectrin 20. The mutation into another residue might disturb this repeat and consequently any related function. The mutated residue is located in a domain that is important for the binding of other molecules and thus, may disturb this function . Furthermore, the c.6544G>C (p.D2182H) mutation was predicted to have a pathogenic effect by Polyphen-2, Mutation Taster, SIFT, and PROVEAN. Therefore, the novel compound heterozygous mutations c.134G>A (p.R45K) and c.6544G>C (p.D2182H) in the SPTA1 gene might have caused HS in this patient with AIH.