Efficient Screening Tests and Gene Mutation Spectrum for Hereditary Stomatocytosis in Japan

Background: Hereditary stomatocytosis (HSt) is a group of congenital hemolytic anemia caused by abnormally increased cation permeability of erythrocyte membranes. The most common subtype is dehydrated HSt (DHSt), and heterozygous mutations of the mechanosensitive calcium channel gene PIEZO1 have been associated with it most frequently. DHSt is suspected by screening tests such as erythrocyte morphology, cation concentration measurements inside and outside the erythrocyte membrane, or osmotic gradient ektacytometry; target-captured sequencing (TCS) is used for definitive diagnosis. We have shown that an increase in the residual red cells (%RRC) in a quantitative osmotic fragility test using a flow cytometer (FCM-OF) is useful as a screening test for DHSt.

Purpose: We report the clinical findings and mutation spectrum of Japanese patients with DHSt confirmed by genetic testing.

Methods: From April 2015 to June 2021, 27 patients who had a clinical diagnosis of DHSt and provided written consent were genetically tested. The clinical indications were hemolytic anemia with stomatocytes, accompanied by hemochromatosis, a family history, perinatal edema, and severe jaundice. Laboratory tests showed increased MCV, and subjects with an increased %RRC in FCM-OF were analyzed. TCS was performed using a hemolytic anemia-related gene panel.

Results: Of the 27 patients, 14 had PIEZO1 variants, 3 had KCNN4 variants, and 2 had ABCB6 variants, for a total of 19 cases diagnosed as HSt. There was 1 SPTB mutation, 1 GCLC mutation, and 6 cases without mutations in genes known to be related to hemolytic anemia. There were 12 previously reported mutations (KCNN4: R352H, PIEZO1: V598M, T2014I, R2488Q, E2496ELE) and 5 novel mutations (KCNN4: P204R, A279T, PIEZO1: 427_428ins9AA, A1457V, K2323T).Notably, 5 E2496ELE mutations were found in unrelated individuals. There were no differences in age at diagnosis and severity of anemia between the E2496ELE mutation and other PIEZO1 mutations, but jaundice was more severe in patients with the E2496ELE mutation (p=0.007).The median age at diagnosis of the DHSt patients was 28 years (range: 2 months to 89 years); there were 6 men and 11 women. Three patients underwent splenectomy, and 2 patients with PIEZO1 mutations had postoperative thrombosis; 1 KCNN4 mutation had no complications, but no improvement in hemolytic anemia. Six patients had gallstones, 3 had fetal ascites, and 11 received red blood cell transfusions. Laboratory test results showed median Hb 10.4 g/dL (6.9-15.6), median MCV 99.7 fL (85-127.4), median MCHC 35.6% (33-39), and median T-Bil 3.4 mg/dL (0.5-37.9). The median ferritin level was 569.3 ng/mL (87.1-3895); of the 14 patients whose ferritin was measured at the time of diagnosis, 6 had already exceeded 1,000 ng/mL. The FCM-OF showed high values in all 16 cases tested.

Discussion: Genetic testing was performed on cases in which DHSt was suspected based on clinical findings, smears, and FCM-OF; the diagnosis of DHSt was confirmed at a high percentage. As previously reported, the severity of hemolytic anemia was wide-ranging, and many cases of hemochromatosis were observed. The PIEZO1 mutation is the most common in the Japanese population, and the number of E2496ELE mutations is particularly conspicuous. Patients carrying E2496ELE mutations are reported to have a younger age at diagnosis and more severe hematological findings than other mutations; however, our results showed no significant differences in age at diagnosis or degree of anemia. Since the correlation between PIEZO1 gene mutation and phenotype has not yet been clarified, further research is considered necessary.