Evaluation of Phenotype-Genotype Correlation in Two Common PIEZO1 Mutations p.R2456H and p.L2495_E2495dup

Hereditary xerocytosis (HX) is a rare autosomal dominant hemolytic anemia caused by mutations in the mechanosensitive cation channel PIEZO1 or, less commonly, in the Ca²⁺-gated K⁺ channel KCNN4 (Gardos channel). It is a clinically heterogeneous condition, characterized by erythrocyte dehydration. As erythrocytes traverse narrow capillaries and sinusoids, PIEZO1 is thought to be activated by mechanical stimuli, leading to increased intracellular Ca²⁺ which then may activate KCNN4, causing K⁺ efflux and water loss. The p.R2456H and p.L2495_E2496dup PIEZO1 mutations are likely the most common of the PIEZO1 disease-causing mutations and both result in delayed channel inactivation (Glogowska et al., 2017, Blood 130:1845). The prolonged channel activity in response to mechanical stimulation is likely to contribute to erythrocyte dehydration.

Ten patients with PIEZO1-associated HX (5 with p.R2456H and 5 with p.L2495_E2496dup PIEZO1 heterozygous mutations) had their diagnosis established utilizing a Next Generation sequencing panel for hereditary hemolytic and congenital dyserythropoietic anemias (HHA/CDA panel). These patients, along with family members who had similar clinical characteristics (a total of 8 patients with p.R2456H and 6 patients with p.L2495_E2496dup) ranging from 2 - 63 years of age, were enrolled in an IRB-approved study to explore the phenotype-genotype correlation of the disease. We collected patient and family histories and laboratory data and performed phenotypic testing including ektacytometry and erythrocyte cation determinations by atomic spectroscopy.

Most of the patients in our cohort had compensated hemolysis without anemia at the time of evaluation and had not required any transfusions. All of the patients had elevated reticulocyte counts ranging from 5.7 to 15.7 %. Reticulocyte counts of individuals with the p.R2456H mutation were significantly higher than those with the p.L2495_E2496dup mutation (Student's t test, p=0.01). Some of the patients had splenomegaly, but it was not a universal finding. Although elevated MCHC is often considered a hallmark of HX, most of the individuals in this cohort had increased MCV and MCH but only high-normal MCHC. Varying percentages of hyper dense cells were detected in blood samples analyzed using an Advia Hematology System and stomatocytes were notable in most peripheral blood smears.

Several complications have been associated with HX including hydrops fetalis, hemolytic episodes, thrombosis (especially after splenectomy), gallstones, and iron overload. One patient in our cohort had experienced ascites and a pleural effusion in the perinatal period and one was diagnosed with gallstones at age 11. Nearly all the patients had an increase in total bilirubin. Ferritin levels were increased in the majority of the patients tested after 15 years of age. The three patients older than 40 years of age had iron overload verified by T2* MRI imaging of the liver.

HX presents several diagnostic challenges due to its heterogeneity. Two of the subjects in our study had been previously diagnosed with hereditary spherocytosis (HS) and one had the diagnosis of congenital dyserythropoietic anemia (CDA) prior to genetic evaluation. Misdiagnosis of individuals with HX as having HS is especially problematic since splenectomy is contraindicated in HX due to a greatly increased incidence of thromboembolic complications. Another diagnostic consideration concerns the use of osmotic fragility tests. Although osmotic fragility tests are frequently used to diagnose HX, ektacytometry provides greater accuracy. In the present study two patients who had ektacytometry results characteristic of HX had reportedly normal osmotic fragility tests in previous work-up. All individuals in our cohort with confirmed disease-causing mutations in PIEZO1 demonstrated the classic left shifted ektacytometry osmoscan indicative of erythrocyte dehydration. In addition, all individuals with these mutations demonstrated the expected decrease in erythrocyte potassium content not associated with a proportional increase in sodium. This illustrates the importance of specialized testing (ektacytometry and/or intracellular cation determinations) and the use of appropriately designed genetic panels to establish an accurate diagnosis in patients with hemolytic anemia of ambiguous phenotype, especially when splenectomy is being contemplated.