Overhydrated hereditary stomatocytosis: A rare cause of familiar persistent macrocytosis due to SLC4A1 variants.

To the Editor: Defects of red blood cell (RBC) membrane include a heterogeneous group of hemolytic anemias caused by impaired membrane structural organization1 or altered ion membrane transport, resulting in unbalanced RBC hydration.2 The most common congenital hemolytic anemia is hereditary spherocytosis (HS), due to defects in genes encoding for key proteins of RBC cytoskeleton, in particular α- and β-spectrin (SPTA1, SPTB), ankyrin (ANK1), protein 4.2 (EBP42), and band-3 (SLC4A1).1 Band-3 protein (also known as anion exchanger 1, AE1) has multiple structural functions and is an important chloride/bicarbonate anion exchanger in RBCs.3 Although band-3 defects are usually associated to HS, mutations in different domains of AE1 protein may result in different phenotypes, as South-East Asian ovalocytosis, or rare forms of hereditary stomatocytosis (HSt).4 HSt includes a group of hemolytic anemias characterized by increased cation permeability of RBC membrane, resulting in deregulated cellular volume. A particular form, characterized by increased cellular hydration, is known as hydrocytosis or overhydrated stomatocytosis (OHSt), mainly caused by mutations in the RHAG gene,6 even if milder forms have been associated with mutations in SLC4A1 gene.4 Being a hemolytic condition, common clinical signs are jaundice, pallor, fatigue, splenomegaly, and gallstones. Due to difficulties in diagnosis, the prevalence of these disorders remains uncertain.7 However, because of the described high risk of severe thromboembolic complications,9 the differential diagnosis is particularly important to avoid splenectomy in these patients. Here, we describe a 17-year-old female who presented to the pediatric hematology outpatient clinic due to persistent unexplained macrocytosis. Born at term, she was hospitalized in neonatal intensive care unit due to severe jaundice, and treated with intravenous (IV) hydration and phototherapy. Macrocytosis was occasionally detected during hospitalization for viral gastroenteritis at the age of 6 years. Nutritional deficiency of folates and vitamin B12 were excluded. Annual routine blood tests did not show any relevant alteration other than macrocytosis (mean cellular volume [MCV] range: 100.5–107.4 fL). Hemoglobin level ranged at the lower limit for age (10.7–12.6 g/dL), with normal total reticulocyte count, reduced immature reticulocyte fraction (IRF), and relative increase of mature fraction (low fluorescence ratio [LFR]). Further investigations confirmed modest hemolysis, with slightly increased bilirubin, lactate dehydrogenase (LDH) and free hemoglobin, and normal haptoglobin. Coombs test (direct/indirect) resulted negative, as well as screening of abnormal forms of hemoglobin and erythrocyte osmotic fragility test. Erythropoietin, pyruvate kinase (PK), and glucose-6-phosphate-dehydrogenase (G6PD) activity in RBC were normal. No splenomegaly or gallstones were detected at abdomen ultrasound scan. The patient was a second-born girl from non-consanguineous parents. Unexplained macrocytosis (MCV 99.7–106.7 fL) not associated with anemia was previously detected both in her mom and maternal grandmother, but never investigated. In the suspicion of HS, further highly specialized diagnostic investigations were performed in the patient, resulting normal except for acidified glycerol lysis test, which showed increased erythrocyte osmotic fragility (Table 1). Stomatoctytes (12%) were detected in peripheral blood (PB) smear (Figure 1A). Ektacytometric osmotic gradient analysis (LoRRca MaxSis, Mechatronics)10 displayed normal elongation index (EI), with a 15% Omin and 12% Ohyper increase compared to normal controls, resulting in a right-shifted curve suggestive for overhydration (Figure 1B). The eosin 5 maleimide (EMA)-binding test,11 typically altered in HS, was normal, raising the suspicion of a different form of anemia other than HS. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of RBC membrane did not show any abnormality.12 A 43-gene next-generation sequencing (NGS)-targeted panel13 was performed after obtaining patient informed consent, showing the heterozygous pathogenic variant c.2188C>T (p.R730C) of the SLC4A1 gene (NM_000342). The variant was also identified in the mother by Sanger sequencing, confirming the autosomal dominant inheritance of this defect (Figure 1C). Spherocytes (%) Stomatocytes (%) 0 12 0 3 The p.R730C mutation in the SLC4A1 gene, reported in literature in another single case,14 is located within the proposed re-entrant loop 1 (RL1) of the AE1 polypeptide, near a group of previously reported mutations associated with stomatocytosis (p.S731P, p.H734R, p.D705Y, p.E758K).14-16 RL1 has been proposed as an important determinant of anion selectivity and transport regulation, and mutations in this region may contribute to impair the band-3 function without altering membrane stability or band-3 content.16 All the patients described carrying mutations in the RL1 domain had an initial diagnosis of HS (or sphero-stomatocytosis due to the presence of stomatocytes).14, 15, 17 The clinical features were mild hemolyis, jaundice, splenomegaly, and gallstones in some cases, requiring cholecystectomy and splenectomy. Genotyping allowed to clarify laboratory discrepancies and to finally make the correct diagnosis of OHSt. Although rare, these forms may be underestimated in absence of molecular testing; however, presence of overhydration and abnormal erythrocyte ion content has to be taken into account for patients’ management. In this case, based on the confirmed diagnosis of OHSt, disease-specific recommendations were provided to the patient and relatives; in particular, they were informed about the risk of increased thrombotic complications in case of splenectomy.9 Concluding, OHSt is a rare form of inherited RBC membrane disorder, which should be suspected in case of persistent otherwise unexplained macrocytosis, especially if present in more members of the same family. Diagnosis of this condition can be difficult and requires third level analyses by a specialized center and genetic testing. The correct diagnosis is important for optimal clinical care and to avoid improper therapy. Anna Paola Marcello and Francesca Ferrua designed the study and prepared the manuscript. Elisa Fermo and Paola Bianchi performed genetic testing. Cristina Vercellati, Anna Zaninoni, and Anna Paola Marcello performed laboratory investigations on patients’ samples. Camilla Visconti, Wilma Barcellini, Alessandro Aiuti, and Francesca Ferrua were responsible for patient's clinical management and follow-up. All the authors provided a critical review of the manuscript. This research was funded by Italian Ministry of Health-Current research IRCCS, Fondazione IRCCS Ca’ Granda Policlinico Milano, project no. RC 175/05. This work is generated within the European Reference Network on Rare Hematological Diseases (ERN-EuroBloodNet). FPA 739541. Wilma Barcellini, Paola Bianchi, and Elisa Fermo are EuroBloodNet members. The authors declare they have no conflicts of interest.