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.Ala2020Thr5,6 ) 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).

Figure 1.

Morphological description and functional characterization of PIEZO1 variants. Morphological description and functional characterization of cation channel activity in (A) PIEZO1:pIle2462Met RBCs and (B) PIEZO1:pVal1223Ile RBCs. In both panels: distribution of cell projected area (a) and cell circularity (b) from control (blue) and patient (red) RBCs, measured from peripheral blood smears and quantified using ImageJ (n > 1000 cells for both cell types). Circularity is defined as 4π.[area/perimeter2]. (c) Content in cell water (left), Na+ (middle) and K+ (right) of control (blue) and patient (red) RBCs. Averages ± SD from n = 3 experimental replicates. (d-e) Monitoring of membrane potential using the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) technique. (d) Control (blue) and patient (red) RBCs in a normal Ringer solution, followed by addition of A23187 (10 µM, arrows). (e) Control (blue) and patient (red) RBCs in an low ionic strength (LIS) solution, followed by addition of Ca2+ (1 mM arrows). At the end of experiment, zero is obtained by lysing the cells with Triton X100 in 3 M NaCl.

Figure 1.

Morphological description and functional characterization of PIEZO1 variants. Morphological description and functional characterization of cation channel activity in (A) PIEZO1:pIle2462Met RBCs and (B) PIEZO1:pVal1223Ile RBCs. In both panels: distribution of cell projected area (a) and cell circularity (b) from control (blue) and patient (red) RBCs, measured from peripheral blood smears and quantified using ImageJ (n > 1000 cells for both cell types). Circularity is defined as 4π.[area/perimeter2]. (c) Content in cell water (left), Na+ (middle) and K+ (right) of control (blue) and patient (red) RBCs. Averages ± SD from n = 3 experimental replicates. (d-e) Monitoring of membrane potential using the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) technique. (d) Control (blue) and patient (red) RBCs in a normal Ringer solution, followed by addition of A23187 (10 µM, arrows). (e) Control (blue) and patient (red) RBCs in an low ionic strength (LIS) solution, followed by addition of Ca2+ (1 mM arrows). At the end of experiment, zero is obtained by lysing the cells with Triton X100 in 3 M NaCl.

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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.

Table 1.

Clinical and laboratory data of the patients with idiopathic erythrocytosis and PIEZO1 pathogenic variants

PatientSexPIEZO1 variantRCM, %MCV, fLHb, g/LP50, mm HgMCHC, %Reticulo-cytes, G/LFerritinemia, µg/LTransferrin saturation, %Haptoglobin, g/LSerum bilirubin, µmol/LSpleno-megaly
Normal laboratory ranges/results — — — — 120-160 (F) 25-27 32-35 20-80 18-160 (F) 15-35 (F) 0.3-2 <17 — 
     130-170 (M)    18-270 (M) 20-40 (M)    
Arg2110Trp 119 87 192 22.4 36.3 119 120 NA >0.6 Between 50-80 No 
Thr2127Met 117 186 22 35.6 224 474 32 <0.1 13 Yes 
Ala2020Thr 97 161 33.8 323 1274 66 0.3 230 
Ile2462Met 123* 83.6 184 24.3 36.9 163 1674 40 0.62 19 
Val1223Ile 86.6 174 26.7 37 213 781 70 0.09 31 Yes 
PatientSexPIEZO1 variantRCM, %MCV, fLHb, g/LP50, mm HgMCHC, %Reticulo-cytes, G/LFerritinemia, µg/LTransferrin saturation, %Haptoglobin, g/LSerum bilirubin, µmol/LSpleno-megaly
Normal laboratory ranges/results — — — — 120-160 (F) 25-27 32-35 20-80 18-160 (F) 15-35 (F) 0.3-2 <17 — 
     130-170 (M)    18-270 (M) 20-40 (M)    
Arg2110Trp 119 87 192 22.4 36.3 119 120 NA >0.6 Between 50-80 No 
Thr2127Met 117 186 22 35.6 224 474 32 <0.1 13 Yes 
Ala2020Thr 97 161 33.8 323 1274 66 0.3 230 
Ile2462Met 123* 83.6 184 24.3 36.9 163 1674 40 0.62 19 
Val1223Ile 86.6 174 26.7 37 213 781 70 0.09 31 Yes 

No patient received a transfusion.

F, female; M, male; MCV, mean corpuscular volume; NA, not available; RCM, red cell mass; U, unknown.

*

RCM was determined after 3 phlebotomies.

Table 2.

Phlebotomy data

PatientAge at diagnosis, yPhlebotomy
23 Yes (late June and early July 2015) 
Yes 
31 
49 Yes (3 in November 2019) 
49 Yes (January 2019, August 2019, January 2020, August 2020) 
PatientAge at diagnosis, yPhlebotomy
23 Yes (late June and early July 2015) 
Yes 
31 
49 Yes (3 in November 2019) 
49 Yes (January 2019, August 2019, January 2020, August 2020) 

In all patients, ektacytometry showed a leftward shift.

U, unknown.

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.

Original data are available on e-mail request to francois.girodon@chu-dijon.fr.

The online version of this article contains a data supplement.

The authors thank Fabienne Danton for technical assistance.

This study was supported by grants from the Agence Nationale de la Recherche (ANR; PRTS 2015 “GenRED”) and the Labex GR-Ex, reference ANR-11-LABX-0051. D.M.A. was supported by the European Union’s Horizon 2020 Research and Innovation Program under grant 675115-Relevance-H2020-MSCA-ITN-2015/H2020-MSCA-ITN-2015.

Contribution: M.F., M.G., and F.G. wrote the manuscript; M.G.-B., B.A., F.A., and C.G. performed the genetic analysis; P.C., M.P., P.A.-M., and F.G. provided samples; S.E., D.M.A., L.P., G.B., B.A., L.D.C., V.P., and C.G. performed analyses; P.A.-M. and F.G. designed the study; and P.A.-M., M.G.-B., S.B., and B.G. revised the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: François Girodon, Service d’Hématologie Biologique, Hôpital du Bocage, CHU de Dijon, Dijon, France; e-mail: francois.girodon@chu-dijon.fr.

1.
Bento
C
,
Percy
MJ
,
Gardie
B
, et al;
ECE-Consortium
.
Genetic basis of congenital erythrocytosis: mutation update and online databases
.
Hum Mutat
.
2014
;
35
(
1
):
15
-
26
.
2.
Girodon
F
,
Airaud
F
,
Garrec
C
,
Bézieau
S
,
Gardie
B
.
Gene panel sequencing in idiopathic erythrocytosis
.
Haematologica
.
2017
;
102
(
1
):
e30
.
3.
Camps
C
,
Petousi
N
,
Bento
C
, et al;
WGS500 Consortium
.
Gene panel sequencing improves the diagnostic work-up of patients with idiopathic erythrocytosis and identifies new mutations
.
Haematologica
.
2016
;
101
(
11
):
1306
-
1318
.
4.
Oliveira
JL
,
Coon
LM
,
Frederick
LA
, et al
.
Genotype-Phenotype Correlation of Hereditary Erythrocytosis Mutations, a single center experience
.
Am J Hematol
.
2018
;
93
(
8
):
1029
-
1041
.
5.
Albuisson
J
,
Murthy
SE
,
Bandell
M
, et al
.
Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels [published correction appears in Nat Commun. 2013;4:2440]
.
Nat Commun
.
2013
;
4
(
1
):
1884
.
6.
Picard
V
,
Guitton
C
,
Thuret
I
, et al
.
Clinical and biological features in PIEZO1-hereditary xerocytosis and Gardos channelopathy: a retrospective series of 126 patients
.
Haematologica
.
2019
;
104
(
8
):
1554
-
1564
.
7.
Caulier
A
,
Jankovsky
N
,
Demont
Y
, et al
.
PIEZO1 activation delays erythroid differentiation of normal and Hereditary Xerocytosis-derived human progenitors
.
Haematologica
.
2020
;
105
(
3
):
610
-
622
.
8.
Moura
PL
,
Hawley
BR
,
Dobbe
JGG
, et al
.
PIEZO1 gain-of-function mutations delay reticulocyte maturation in hereditary xerocytosis
.
Haematologica
.
2020
;
105
(
6
):
e268
-
e271
.
9.
Knight
T
,
Zaidi
AU
,
Wu
S
,
Gadgeel
M
,
Buck
S
,
Ravindranath
Y
.
Mild erythrocytosis as a presenting manifestation of PIEZO1 associated erythrocyte volume disorders
.
Pediatr Hematol Oncol
.
2019
;
36
(
5
):
317
-
326
.
10.
Lew
VL
,
Tiffert
T
.
On the Mechanism of Human Red Blood Cell Longevity: Roles of Calcium, the Sodium Pump, PIEZO1, and Gardos Channels
.
Front Physiol
.
2017
;
8
:
977
.
11.
Zarychanski
R
,
Schulz
VP
,
Houston
BL
, et al
.
Mutations in the mechanotransduction protein PIEZO1 are associated with hereditary xerocytosis
.
Blood
.
2012
;
120
(
9
):
1908
-
1915
.
12.
Del Orbe Barreto
R
,
Arrizabalaga
B
,
De la Hoz Rastrollo
AB
, et al
.
Hereditary xerocytosis, a misleading anemia
.
Ann Hematol
.
2016
;
95
(
9
):
1545
-
1546
.
13.
Archer
NM
,
Shmukler
BE
,
Andolfo
I
, et al
.
Hereditary xerocytosis revisited
.
Am J Hematol
.
2014
;
89
(
12
):
1142
-
1146
.
14.
Orvain
C
,
Da Costa
L
,
Van Wijk
R
, et al
.
Inherited or acquired modifiers of iron status may dramatically affect the phenotype in dehydrated hereditary stomatocytosis
.
Eur J Haematol
.
2018
;
101
(
4
):
566
-
569
.
15.
Rotordam
MG
,
Fermo
E
,
Becker
N
, et al
.
A novel gain-of-function mutation of Piezo1 is functionally affirmed in red blood cells by high-throughput patch clamp
.
Haematologica
.
2019
;
104
(
5
):
e179
-
e183
.
16.
Russo
R
,
Andolfo
I
,
Manna
F
, et al
.
Multi-gene panel testing improves diagnosis and management of patients with hereditary anemias
.
Am J Hematol
.
2018
;
93
(
5
):
672
-
682
.
17.
Andolfo
I
,
Alper
SL
,
De Franceschi
L
, et al
.
Multiple clinical forms of dehydrated hereditary stomatocytosis arise from mutations in PIEZO1
.
Blood
.
2013
;
121
(
19
):
3925
-
3935, S1-S12
.
18.
Glogowska
E
,
Schneider
ER
,
Maksimova
Y
, et al
.
Novel mechanisms of PIEZO1 dysfunction in hereditary xerocytosis
.
Blood
.
2017
;
130
(
16
):
1845
-
1856
.
19.
Kiger
L
,
Guitton
C
,
Ghazal
K
, et al
.
Physiopathology of hereditary xerocytosis: Piezo1 gain-of-function mutations impact hemoglobin oxygen affinity [abstract]
.
Haematologica
.
2017
;
102
(
suppl 2
). Abstract E1082.
20.
Kiger
L
,
Oliveira
L
,
Guitton
L
.
Piezo1-xerocytosis red cell metabolome shows impaired glycolysis and increased hemoglobin oxygen affinity
.
Blood Advances
.
In press

Author notes

*

M.F., M.G.-B., B.G., and F.G. contributed equally to this study.

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