Increased incidence of germline PIEZO1 mutations in individuals with idiopathic erythrocytosis

TO THE EDITOR:

Erythrocytosis and polycythemia are generic terms for red cell diseases characterized by an increase in hematocrit (Ht) and/or hemoglobin (Hb) concentrations. They can be acquired as primary polycythemia vera, as a secondary disease resulting from an adaptive response to hypoxia, or as certain erythropoietin (EPO)-secreting tumors. On the other hand, congenital secondary erythrocytosis can result from mutations in the genes encoding (1) high-oxygen-affinity Hb; (2) proteins involved in the oxygen-sensing pathway, or, rarely; (3) bisphosphoglycerate mutase leading to decreased 2,3-DPG levels.¹  However, despite the recent use of next-generation sequencing (NGS), the underlying cause is found in only ∼25% of cases of congenital erythrocytosis.^2-4 

Recently, patients with elevated Ht and Hb concentrations have been reported in hereditary xerocytosis (HX) associated with a PIEZO1/FAM38A mutation.^5-9 PIEZO1 is the gene encoding the transmembrane nonselective cationic PIEZO1 channel, which is activated by shear stress. One of its major roles is to adjust the erythrocyte volume to environmental constraints, thus allowing traversing of microcapillaries.¹⁰ 

Gain-of-function mutations in PIEZO1 have been reported in HX,¹¹  inducing a massive entry of calcium into the erythrocyte cytoplasm,⁵  with a subsequent excessive activation of the Gárdos channel, which leads to massive loss of potassium and water. Most patients with HX present with abnormal cation channel activity, leading occasionally to chronic hemolytic anemia¹²  or more frequently to well-compensated hemolysis,¹³  because patients present with normal Hb levels.

We studied a cohort of 110 patients with well-characterized idiopathic erythrocytosis that was confirmed by clinical and biological analyses: measurements of full blood count, blood electrolytes, serum EPO, and blood gases (including venous P₅₀), functional respiratory tests, abdominal ultrasound scan, screening for JAK2 mutations on exons 14 and 12, and red cell mass (RCM) measurement. In patients with a venous P₅₀ <24 mm Hg, sequencing analysis of the HBB, HBA1, and HBA2 genes was performed to rule out Hb variants with high oxygen affinity.

None of the patients had JAK2 mutations or acquired conditions associated with erythrocytosis. Using a dedicated NGS panel, no mutations were found in genes involved in (1) the regulation of the hypoxia pathway, (2) proliferation and differentiation of erythroid progenitors, or (3) mature cell function.² 

All patients were sequenced for the PIEZO1 and KCNN4 genes, as previously described.¹⁴  Patients gave informed consent for genetic analyses according to the Declaration of Helsinki.

In vitro functional studies were performed to assess new PIEZO1 variants and their possible pathological implication in the phenotype, as previously described.¹⁵ 

In our cohort, 24 rare PIEZO1 variants were observed in 27 (24%) patients, whereas only a single rare variant with no pathological criteria was noted within the KCNN4 gene. Using the American College of Medical Geneticists classification, most of the PIEZO1 variants were classified as polymorphisms (class 1) or variants of uncertain significance (classes 2-3), and 4, identified in 4 patients at the heterozygous state, were considered potentially pathogenic (classes 4-5; supplemental Table 1, available on the Blood Web site). Our results indicate PIEZO1 is a highly polymorphic gene, and several sequence variations can be found with genetic analyses. These findings underline the need for obtaining an electrophysiological description of the mutation to define pathogenicity or significance, and it highlights the vast phenotypic heterogeneity among red blood cell (RBC) populations.¹⁵  Among the 4 potentially pathogenic variants, 3 (p.Arg2110Trp,^15,16  p.Thr2127Met,^5,6  and p.Ala2020Thr^5,6 ) have been reported in HX. The p.Arg2110Trp missense mutation, found in patient 1, is a gain-of-function mutation recently reported as being associated with a PIEZO1 functional impairment, identified by an original patch clamp-based, high-throughput screening assay. Indeed, patient 1 displayed a typical bioclinical presentation of HX and a classic leftward shift in the osmotic gradient ektacytometry profile. This patient had a family history of RBC disorders; his father was a carrier of atypical hereditary spherocytosis and died of pancreatitis due to gallstones. His grandmother has also been diagnosed with polycythemia, the origin of which is being evaluated.

The PIEZO1:p.Thr2127Met variant, previously reported as probably pathogenic and disease associated,^6,17  was found in patient 2, who was referred for hyperhemolysis associated with high ferritin levels and muscle pain that was alleviated by phlebotomy.

Patient 3 harbored a p.Ala2020Thr variant previously described in HX.¹⁴  The proband’s mother and sister bear the same PIEZO1 mutation and were diagnosed with HX.

We also identified in patient 4 a new PIEZO1 variant, p.Ile2462Met, localized in a mutation hot spot where other PIEZO1 variants were reported.^13,16-18  This patient had clinical and biological features compatible with HX and was treated via 3 phlebotomies, then with iron chelation for 3 months. The RCM measurement was performed, unfortunately after 3 phlebotomies, and revealed an increase of 23% which is just at the limit for a reliable assertion of true erythrocytosis. The diagnosis of HX was confirmed by osmotic gradient ektacytometry, showing an abnormal curve compatible with HX, a reduced projected cell surface area, and reduced cell circularity (Figure 1).

Finally, patient 5 harbored a p.Val1223Ile variant, associated with a clinical-biological picture strongly suggestive of HX. He presented with iron overload, splenomegaly (15 cm), hemolysis, high mean corpuscular hemoglobin concentration (MCHC), and a left shifted osmotic gradient ektacytometry profile. According to results of functional studies, the cells of this patient presented a clear alteration of morphological parameters associated with an accelerated repolarization after stimulation of the Gárdos channels by A23187 (Figure 1). On the other hand, very rare stomatocytes were noticed on a blood smear, as observed in nearly one-third of patients with HX.⁶  Although the phenotype and the functional studies were in accordance with HX, the p.Val1223Ile variant has been classified as class 3, meaning that it is of uncertain significance (supplemental Table 1) and highlighting the need to confirm its pathogenicity through familial studies.

The laboratory results and dates of phlebotomies of the 5 patients reviewed are shown in Tables 1 and 2.

Our results show that a significant proportion of individuals with idiopathic erythrocytosis, specifically 4% of our population, carry pathogenic mutations in the PIEZO1 gene, associated with clinical or biological manifestations of HX. In a previous large series of patients with HX and PIEZO1 mutations, 68% did not show any sign of anemia, with a mean Hb level of 131 ± 20 g/L. Interestingly, 7 adults had an Hb concentration >160 g/L, similar to our patients, and 2 were known to have polycythemia.⁶ 

Our study found heterogeneous and nonexclusive characteristics for each patient. For example, biomarkers of hemolysis were often missing. As suggested in a recent study, this atypical presentation could be partially explained by a concomitant iron deficiency.¹⁴  Remarkably, it is worth noting that in all 5 of our patients, none or very few stomatocytes were observed in the blood smears.

Surprisingly, the venous P₅₀ measurement was frequently low (<25 mm Hg), which indicates a shift of the oxygen dissociation curve to the left and an increase in Hb oxygen affinity. The increase in Hb oxygen affinity had already been reported in patients with HX who had mutated PIEZO1, inducing hypoxia and thus intense glycolytic activity to maintain the high ATP concentration necessary for cell homeostasis. Glycolysis led to a decreased 2,3-DPG concentration, associated with low P₅₀ and erythrocytosis.¹⁹  This has very recently been confirmed in an independent study.²⁰ 

In conclusion, our data suggest that a significant proportion of cases of idiopathic erythrocytosis are ultimately HX with well-compensated hemolysis. Because of specific patient management, the search for a mutation in the PIEZO1 gene must be performed in children and young adults with idiopathic erythrocytosis and more generally when erythrocytosis appears before the age of 50 years, especially when associated with a set of suggestive clinical and biological parameters (ie, iron overload, splenomegaly, elevated MCHC, increased reticulocytes, hemolysis, or decreased P₅₀), after excluding a variant of Hb with high oxygen affinity.