FIELD OF THE INVENTION:

The present invention relates to the treatment and the diagnosis of the hereditary xerocytosis.

BACKGROUND OF THE INVENTION:

Water and solute homeostasis is essential for the maintenance of erythrocyte integrity and is controlled via the regulation of monovalent cation content. Several primary disorders of erythrocytes hydration exist and are characterized by an abnormal permeability of the erythrocyte membrane to sodium and potassium, resulting either in swelling or shrinkage of red cells (Rinehart et al. , 2010). Clinically, these inherited disorders are associated with chronic hemolytic anemia and are due to defects in various transmembrane ion channels or transporters (Da Costa L, et al. , 2013).

Hereditary xerocytosis (HX), also known as dehydrated hereditary stomatocytosis

(DHSt) is an autosomal dominant hemolytic anemia characterized by primary erythrocyte (also named red blood cells) dehydration caused by an increase of their permeability to cations (Delaunay J., 2004). Thus HX erythrocytes exhibit decreased total cation and potassium content that are not accompanied by a proportional net gain of sodium and water. HX patients typically exhibit mild to moderate compensated hemolytic anemia, with an increased erythrocyte mean corpuscular hemoglobin concentration and a decreased osmotic fragility, both of which reflect cellular dehydration. Patients may also show perinatal edema and pseudohyperkalemia due to loss of potassium from red cells stored at room temperature. A minor proportion of red blood cells (RBCs) appear as stomatocytes on blood films. Complications such as splenomegaly and cholelithiasis, resulting from increased red cell trapping in the spleen and elevated bilirubin levels, respectively, may occur. The course of DHS is also frequently associated with iron overload, which may lead to hepatosiderosis.

Up to now, two different genes have been linked to this disease: KCNN4, coding for a calcium activated potassium channel also named Gardos channel in RBCs, and PIEZOl, coding for the non-selective cation channel PIEZOl, activated by mechanical forces.

The Gardos channel has been initially described in erythrocytes by G. Gardos (1958 BBA) but it is present in many cell types including pancreas cells where it is called KCa3.1 or KCNN4 (Maher and Kuchel, 2003). The locus of the gene encoding the Gardos channel (KCNN4 protein) is mapped 19ql3.2. The Gardos channel is made of 4 identical subunits; each subunit is encoded by a single gene, KCNN4, and comprises 6 transmembrane domains and a pore region between the 5 ^th and the 6 ^th transmembrane domains (Maher and Kuchel, 2003). In steady state conditions, the Gardos channel is inactive. Its function is not fully elucidated in mature normal erythrocytes. Under external stimulation, intracellular Ca ²⁺ increases and then interacts with Calmodulin molecules that are bound tightly on each of the four channel subunits of the Gardos channel. Ca ²⁺ binding to Calmodulin results in the opening of the channel and rapid K ⁺ and water efflux leading to erythrocyte dehydration and shrinkage, a mechanism referred to as the Gardos effect (Maher and Kuchel, 2003; Fanger et al, 1999).

Three different point mutations in KCNN4 have been linked to HX (Rapetti-Mauss R et al., 2015, Andolfo I et al. 2015, Glogowska E et al. 2015), these mutations are gain of function mutations leading to a more active channel even though they do not share a common mechanism in altering channel characteristics (Rapetti-Mauss R et al., 2016). The mutated Gardos channel is over-activated in patient red blood cells (RBCs), which leads to an increased potassium loss and hence water loss and cell dehydration.

The inventors have previously shown that Senicapoc, an inhibitor of the Gardos Channel was efficient in preventing RBC K ⁺ loss, in case of the R352H or the V282M mutations of the Gardos channel (WO2016/202854).

PIEZOs are newly identified mechanically activated (MA) cation channels, which responds to a wide array of mechanical forces, including poking, stretching, and shear stress, and is essential for proper vascular development in mice (Nilius and Honore, 2012; Li et al., 2014; Ranade et al., 2014). These ion channels are proposed homotrimers, with each subunit encompassing 14 transmembrane domains (Ge J. et al., 2015). They are expressed in many cell types including human erythroid progenitor cells (Zarychanski R et al., 2004; Coste B et al., 2010; Coste B et al., 2012). Some insights into the roles of PIEZOl have been obtained recently by knockdown experiments in mammalian epithelial cells from the kidney and the lung, and in lung cancer cells (Eisenhoffer G.T. et al., 2012; McHugh B. J., et al., 2012).

Up to now, a dozen of different mutations in PIEZOl have been linked to HX phenotype (Zarychanski R. et al., 2012; Andolfo I. et al., 2013; Albuisson J., et al., 2013). Some of them have been characterized by expression in HEK293 cells, where it was observed that they change the kinetic of channel gating (Albuisson J., et al., 2013; Bae C. et al., 2013). These PIEZOl mutations induce a sustained activation of the channel that stays open longer than the wild-type (WT) channel. This channel is permeable to Na ⁺ , K ⁺ and divalent cations such as Ca ²⁺ (Gnanasambandam R et al., 2015; Gottlied PA et al., 2012). In mouse red blood cells, PIEZOl was shown to contribute to cell volume homeostasis by controlling Ca uptake and a functional link between PIEZOl and KCNN4 has been proposed (Cahalan et al., 2015). The opening of PIEZOl leads to a calcium influx that in turns activates KCNN4 mediating a K ⁺ efflux accompanied by CI ^" and osmotically linked water effluxes. In another study, PIEZOl was also shown to regulate ATP release from human red blood cells by increasing calcium influx (Cinar E. et al., 2015). Thus, PIEZOl appears as a major contributor to red blood cell response to mechanical stress by controlling calcium influx.

SUMMARY OF THE INVENTION:

The inventors have now discovered for the first time that, in case of HX due to mutation(s) in PIEZOl, KCNN4 activation alone is responsible for the red blood cell dehydration. They further demonstrated that the blocking of KCNN4 by an inhibitor of the Gardos channel completely reverses the red blood cell dehydration associated with the mutation of the PIEZOl channel. Thus, KCNN4 appears as the major effector of red blood cell dehydration in HX even in case of PIEZOl mutation.

The present invention therefore provides an inhibitor of the Gardos channel (i.e.: KCNN4 protein) for use in the treatment of hereditary xerocytosis (HX) in a subject, which is the carrier of at least one mutation of the PIEZOl gene encoding a mutated PIEZOl channel.

Optionally the mutated PIEZOl channel is a human PIEZOl channel having at least one mutation as compared to the wild- type human PIEZOl channel, said mutation being selected from V598M, F681S, G718S/R2488Q, G782S/R808Q, S1117L/A2020V, R1358P, A2003D, A2020T, T2127M, DELK2166-2169, M2225R, R2456H, E2496ELE and R2488Q, preferably said least one mutation is selected from V598M and F681S.

Furthermore, HX is difficult to diagnose because of a highly variable expression, ranging from the absence of clinical symptoms to lethal perinatal oedema, but the most frequent HX condition is moderately symptomatic hemolysis. The only test for HX is ektacytometry, which is available in a very limited number of laboratories. The disease may be overlooked for years or decades and is sometimes confused with spherocytosis. The provision of a diagnostic test is therefore or high clinical relevance.

The inventors have identified two additional single point mutations of the human

PIEZOl gene, namely c.l792G>A or c.2042T>C resulting respectively in the mutation V598M or F681S in the PIEZOl channel, which participate in HX physiopathology and lead to a gain of function of the PIEZOl channel. The mutations V598M and F681S are located in the N-terminal part of the extracellular domain of the protein, supposed to sense membrane shear stress (Ge J. et al., 2015).

The present application therefore further provides an in vitro method for diagnosing the presence of, or the predisposition to, hereditary xerocytosis (HX) in a human subject, comprising the step of:

(i) providing a biological sample from said subject and

(ii) detecting in said biological sample the presence of any one of the missense mutation c. l792G>A, or c.2042T>C in the PIEZOl gene or any one of the corresponding mutation V598M or F681S in the PIEZOl channel,

the presence of any one of these mutations in the PIEZOl gene or in the PIEZOl channel constituting a marker of a hereditary xerocytosis or a predisposition to hereditary xerocytosis in said subject.

DETAILED DESCRIPTION OF THE INVENTION:

1. Inhibitor of the Gardos channel (KCNN4 protein) for use in the treatment of hereditary xerocytosis

In a first aspect the present invention relates to an inhibitor of the Gardos channel (KCNN4 protein) for use in the treatment of hereditary xerocytosis (HX) in a subject, characterized in that the subject is the carrier of at least one mutation of the PIEZOl gene encoding a PIEZOl channel having at least one mutation. Typically, said at least one mutation is a gain of function mutation.

The PIEZOl channel is a mechanically- activated no specific cation channel that links mechanical forces to biological signals. As mentioned previously, it is permeable to Na ⁺ , K ⁺ and divalent cations such as Ca ²⁺ and generates currents characterized by a linear current- voltage relationship that are sensitive to ruthenium red and gadolinium. PIEZOl opening leads to a calcium influx in the red blood cell.

In a preferred embodiment, the PIEZOl channel is from human origin. The amino acid sequence of the wild- type (WT) PIEZOl channel is available under accession number Q92508 in the UniProtKB database, and referred herein as SEQ ID NO: l. The human Piezol channel is encoded by the gene PIEZOl . The nucleic acid sequence of the wild-type gene (map: 16q24.3) encoding the human PIEZOl channel is available under ID number 9780 (NG_042229.1) in the NCBI GenBank database, and referred herein as SEQ ID NO:2. The nucleic acid sequence of the mRNA (cDNA) encoded by the wild-type human PIEZOl gene is available under the accession number NM_001142864 in the NCBI GenBank database, and referred herein as SEQ ID NO:3.

The mutation in the PIEZOl gene may be any type of mutation, addition, deletion, missense mutation, or duplication. Typically the mutation is a missense mutation or a duplication.

By "gain of function mutation", it is intended herein a mutation which produces an increased cation transport in erythroid cells. In a heterozygote, the new function will be expressed, and therefore the gain-of-function mutation will act like a dominant allele and produce the new phenotype mutated channel.

All HX-causing mutations characterized so far (Zarychinski et al., 2012; Albuisson J et al., 2013; Andolfo I et al., 2013) produce a gain-of-function phenotype characterized by increased cation transport in erythroid cells, and in particular an increased calcium influx in the cell. Typically the increased cation transport of mutated channel is due to a slower channel inactivation rate of the mutated channel compared to the wild-type protein. Increased cation transport may be electrophysiologically assessed as exemplified in the results provided herein (see figure 3B). As a matter of example, inactivation kinetic of mutated PIEZOl channels as compared to the WT PIEZOl channel may also be assessed as illustrated in figure 3 of Albuisson et al. (Nature Communication 2013).

Optionally, the subject is a human subject.

Optionally, the mutated PIEZOl channel is a human PIEZOl channel having at least one mutation as compared to the WT human PIEZOl channel. Typically said at least one mutation is selected from V598M, F681S, G718S, R2488Q, G782S, R808Q, S1117L, A2020V, R1358P, A2003D, A2020T T2127M, DELK2166-2169, M2225R, R2456H, E2496ELE and R2488Q, optionally said at least one mutation is selected from V598M, F681S, G718S/R2488Q, G782S/R808Q, S1117L/A2020V, R1358P, A2003D, A2020T T2127M, DELK2166-2169, M2225R, R2456H, E2496ELE and R2488Q, optionally said at least one mutation is selected from V598M and F681S.

V598M should be intended as an amino acid change from valine to methionine at position 598 of the WT human PIEZOl channel. The same nomenclature applies to all other mutations mentioned in the present application.

The mutation G782S/R808Q should be intended as the double mutation G782S and R808Q.

E2496ELE means that the amino acid leucine and glutamate respectively in position 2495 and 2496 are duplicated. Said mutations are described in the documents of Zarychinski et al., 2012; Albuisson J et al., 2013 and Andolfo I et al., 2013, the content of which is included herein by reference. The single mutations V598M and F681S have now been discovered by the inventors.

The Gardos channel is a Ca ²⁺ sensitive, intermediate conductance, potassium selective channel also referred to as KCa3.1 or KCNN4 (Maher and Kuchel, 2003).

In a preferred embodiment, the Gardos channel is from human origin. The amino acid sequence of the wild-type human Gardos channel (KCNN4 protein) is available under accession number 015554 (GI: 17366160) in the UniProtKB database, and referred herein to as SEQ ID NO: 4.

An inhibitor of the Gardos channel refers to a selective Gardos channel blocker that specifically inhibits the efflux of potassium from the erythrocytes.

An inhibitor of the Gardos channel can be identified by screening a collection of candidate compounds for their ability to specifically inhibit the efflux of potassium from the erythrocytes. Methods for measuring the inhibition of the efflux of potassium from the erythrocytes are known in themselves. Examples of such methods are described in Brugnara et al., 1993a and 1993b; Ellory et al., 1994. Both the percent inhibition of the Gardos channel and the ½ ₀ of an inhibitor of the Gardos channel can be assayed utilizing the methods described in Brugnara et al., 1993b.

The potency of an inhibitor of the Gardos channel can be assayed using erythrocytes by a method such as that disclosed by Brugnara et al., 1993a.

Inhibitors of the Gardos channel include organic molecules (e. g. a small organic molecule (natural or not)), amino acids, antibodies or aptamers.

Aptamers are a class of molecules that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consist of conformationally constrained antibody variable regions displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al, 1996). The antibodies can be polyclonal or monoclonal antibodies. The term "antibody" or "antibodies" as used herein also encompasses functional fragments of antibodies, including fragments of chimeric, humanized, single chain antibodies or fragments thereof (e.g., antigen- binding fragments of antibodies that specifically bind human the Gardos channel, Fv, Fab, Fab'and F(ab') 2 fragments). Suitable antibodies are those which are directed to KCNN4 protein (Gardos channel). Advantageously, said antibody is a monoclonal antibody, or fragment thereof.

In a preferred embodiment, the inhibitor of the Gardos channel is selected from the group consisting of imidazole antimycotics (Brugnara et al., 1996), such as clotrimazole (Brugnara et al., 1993a) metronidazole (Brugnara et al., 1993a), econazole (Brugnara et al., 1993a); arginine (Romero et al., 2002); Tram-34 (l-[(2-Chlorophenyl)diphenylmethyl]-lH- pyrazole) (Wulff et al, 2000); Charybdo toxin; Maurotoxin (Castle et al., 2002); nifedipine (Brugnara et al , 1993a); Nitrendipine (Brugnara et al, 1993a); inhibitors of calcium activated potassium flux that display selectivity and a potency towards the Gardos channel described in International Applications WO 00/50026, WO 2004/016221, WO 2005/113490 and WO 2006/084031 , including senicapoc (ICA- 17043; bis(4-fluorophenyl)phenylacetamide; Ataga et al. , 2008; 2009), 2,2-Bis(4-fluorophenyl)-N-methoxy-2-phenylacetamidine, 2-(2- Chlorophenyl)-2,2-diphenylacetaldehyde oxime, 2-(2-Chlorophenyl)-2,2-bis(4-fluorophenyl)- N-hydroxyacetamidine, 2,2,2-Tris(4-fluorophenyl)-N-hydroxyacetamidine, 2-(2- Fluorophenyl)-2-(4-fluorophenyl)-N-hydroxy-2-phenylacetamidi ne, phosphoric acid 3-(2- oxazolyl)-4-[3-(trifluoromethyl)phenylsulfonamido]phenyl monoester, N-[2-(4,5- Dihydrooxazol-2-yl)phenyl]-3-(trifluoromethyl)benzenesulfona mide, N-[4-Methoxy-2-(2- oxazolyl)phenyl]benzenesulfonamide, N-[4,5-Dimethoxy-2-(3-methyl-l,2,4-oxadiazol-5- yl)phenyl]-3-(trifluoromethyl)benzenesulfonamide, N-[2-(2-Furyl)phenyl]-3- (trifluoromethyl)benzenesulfonamide and N-[4-Methyl-2-(2-oxazolyl)phenyl]-3-

(trifluoromethyl)benzene sulfonamide, preferably senicapoc (see also Stocker et al., 2003).

Optionally, the inhibitor of the Gardos channel is senicapoc or TRAM-34.

The inhibitor of the Gardos channel can be administered by any suitable route, for example, intravenously, intranasally, peritoneally, intramuscularly, orally and other conventional methods.

Said inhibitor according to the invention can included in a composition. It can be mixed and/or carried with one or more liquid and/or solid pharmaceutically acceptable carriers, ingredients and/or excipients. As used herein, "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active compounds can also be incorporated into the composition.

As used herein, the terms "treatment" or "treating" includes the administration of an inhibitor of the Gardos channel as defined above to a subject who is the carrier of at least one mutation of the PIEZOl gene encoding the PIEZOl channel, with the purpose to alleviate, relieve, alter, remedy, ameliorate, improve or affect this disorder.

The present invention also provides a method for treating hereditary xerocytosis, in a subject who is the carrier of at least one mutation of the PIEZOl gene encoding the PIEZOl channel as previously identified. This method of treatment comprises administering to said subject an effective amount of an inhibitor of the Gardos channel (KCNN4 protein) as defined above. Optionally, the inhibitor of the Gardos channel is senicapoc or TRAM-34.

The present invention also provides the use of an inhibitor of the Gardos channel (KCNN4 protein) as defined above for the preparation of a medicament for treating hereditary xerocytosis in a subject who is the carrier of at least one mutation of the PIEZOl gene encoding the PIEZOl channel as previously identified.

2. In vitro method of diagnosing the presence of or predisposition to hereditary xerocytosis in a human subject

The present invention also provides a method of diagnosing and treating hereditary xerocytosis in a subject, comprising the steps of:

(i) providing a biological sample from said subject,

(ii) detecting in said biological sample whether a mutation selected from the group consisting of, V598M or F681S is present in the PIEZOl channel,

(iii) diagnosing the subject with a hereditary xerocytosis when the presence of a mutation as defined in step (ii) in the biological sample is detected.

Optionally the method comprises an additional step iv) when the subject is diagnosed with hereditary xerocytosis is diagnosed consisting in administering an effective amount of an inhibitor of the Gardos channel (KCNN4 protein). Typically said inhibitor is senicapoc, or TRAM-34 as defined previously.

Optionally step (ii) rather comprises the detection in the said biological sample of at least one mutation in the PIEZOl gene (map: 16q24.3) as compared to the wild-type PIEZOl gene, preferably at least one mutation in the human PIEZOl gene (map: 16q24.3) as compared to the wild-type human PIEZOl gene which is responsible for the mutation V598M or F681S in the corresponding PIEZOl channel. More particular, step (ii) can consist in detecting in the biological sample the presence of a missense mutation selected from c. l792G>A mutation in exon 14 or c.2042T>C mutation in exon 16 in the PIEZOl gene.

A used herein, the term "detecting the presence of the missense mutation c. l792G>A or c.2042T>C refers to detecting the presence of the mutation corresponding to mutation c. l792G>A or c.2042T>C in the PIEZOl gene, either in mRNA or genomic DNA from a nucleic acid sample obtained from the subject.

The presence of said mutation c.l792G>A or c.2042T>C in the PIEZOl gene or of the corresponding mutation V598M or F681S in the PIEZOl channel constitutes a marker of a hereditary xerocytosis or a predisposition to hereditary xerocytosis in said subject.

A biological sample can be for example blood, serum, lymph, or any biological tissue. The biological sample may also be pretreated, for example, by homogenization, extraction, enzymatic and/or chemical treatments as commonly used in the field.

FIGURES:

Figure 1: Red blood cell osmotic fragility test using osmotic gradient ranging from 0.1% to 1% of NaCl solution for healthy volunteers (A) and informed patients carrying the V598M mutation (B), the F681S mutation (C), or the G782S/R808Q double mutation (D) in the PIEZOl at TO and after 18 hours incubation at 37°C in presence or absence of Senicapoc at 0.4 or 4 μΜ. (black line and white losange: control condition tO, grey square: t=18h control condition, light grey losange: t=18h senicapoc 0.4 μΜ, dark grey triangle: t=18h senicapoc 4 μΜ).

Figure 2: Variation in the red blood cell Na ⁺ (A) and K ⁺ (B) contents measured in red blood cells from informed patients carrying the F681S mutation, the G782S/R808Q double mutation, the V598M mutation or from healthy volunteers at TO (control condition, black bar), or after 18h incubation with 10 μΜ Tram-34 (white bar), or 4 μΜ of Senicapoc (grey bar). C-D: Cell water content (expressed in g water per g of dry weight) in patient red blood cells carrying the double mutation G782S/R808G (C) or control red blood cells from an healthy volunteer (D) measured at TO (control condition, black bar) and after 18h incubation with Senicapoc 4μΜ (dark grey bar) or 0.4μΜ (light grey bar).

Figure 3: Electrical features of patient red blood cells with Piezol mutations. (A) Representative V curves of patient (G782S/R808Q mutations) or control red blood cells in whole cell configuration. Currents were recorded just after cell break-in. Bath solution containing 15 μΜ Yodal was prepared just before adding the cell suspension. Quantification: current recorded at -80 mV, n=5 (ctrl) 7 (patient) 2 (Ctrl+Yodal). (B) Time course of currents measured at -80 mV for control, control+15 μΜ Yodal or patient with G282S/R808Q Piezol. Data are median from currents recorded in A). (C) Representative V curves of patient (V598M mutation) or control red blood cells recorded after extinction of the large conductance. Inset median currents recorded at +20 mV, n=5 (ctrl) 7 (patient) 3 (patient + Senicapoc) 3 (ctrl+ Yodal).

EXAMPLE:

The results which are presented herein show that mutated Piezol in HX leads to over- activation of KCNN4 and the blocking of KCNN4 by Senicapoc completely reverse the red blood cell dehydration. The mutated Piezol are constitutively leaky to calcium, no need to activate Piezol to observe stimulation of KCNN4 in patient red blood cells. KCNN4 activation alone is responsible for the red blood cell dehydration in case of HX due to mutations in Piezol.

Thus, KCNN4 appears as the major effector of red blood cell dehydration in HX characterized so far and Senicapoc seems a promising therapeutic strategy to treat this condition whatever the mutated ion channel, Piezol or KCNN4.

Patients and Methods

Our study focused on three independent index-cases with a typical DHSt clinical and biological phenotype. These subjects were initially diagnosed using osmolar gradient ektacytometry at the Hematology Department of the Hospital Kremlin-Bicetre and their characteristics are summarized in Table 1. Informed consents for genetic analysis were obtained for all patients, according to local institutional ethical boards. PIEZOl and KCNN4 coding sequences and intron-exons junctions were analyzed by Sanger sequencing.

Our study focused on three families and 11 independent index-cases with a typical DHSt clinical and biological phenotype. Patients were from all regions of France, and were diagnosed through specialized consultation by a hematologist or a clinical geneticist. Some of the patients have been published elsewhere: Family 1, which was described by us and others (Family VA, Grootenboer S et al., 2000; Beaurain G et al., 2007) presented with mild, uncomplicated hematological signs of the disease, pseudohyperkalemia, and no history of perinatal edema. In Family 2 (Family VE in Grootenboer et al, 2000) patients were affected by a mild to moderate hemolysis and anemia, by a recurrence of perinatal edema (3 affected cases), and varying recurrence of pseudohyperkalemia. Case 4 (Syfuss et al., 2006) and Family 3 (Carli et al., 2007; Martinaud C et al., 2008) were case reports, these patients expressed a moderate form of anemia and hemolysis without initial evidence of perinatal edema or pseudohyperkalemia. In these last three cases, diagnosis of HX was done late after the onset of hematologic manifestations. The other cases were referred to the Hematology Department of the Hospital Kremlin-Bicetre. Informed consents for genetic analysis were obtained for all patients, according to local institutional ethical boards.

Osmotic resistance tests: Fresh venous blood was obtained by venipuncture from informed patients and healthy volunteers in EDTA collecting tubes kept at 4°C and sent overnight to Nice. At reception (24h after withdrawn) blood was washed 4 times at room temperature in medium containing in mM: NaCl 147, KC1 5, MgCl ₂ 1, CaCl ₂ 1, Hepes 10, buffered with NaOH pH7.4 (320 mOsm). Red blood cell suspension (40% hematocrit) was then incubated at 37°C for 18 hours in presence or absence of 10 μΜ TRAM-34 or Senicapoc at 0.4 or 4 μΜ. An osmotic resistance test in hypotonic saline solutions was performed on blood at reception and after 18 hours' incubation at 37°C.

Red blood cell cation content and volume measurements: Red blood cell suspension before and after osmotic resistance test was used to quantify intracellular Na ⁺ and K ⁺ contents as previously described. For experiments with Yodal to activate Piezol in control red blood cells, washed red blood cell suspension was set to 30% hematocrit and 0.5 μΜ ouabain (Sigma- Aldrich) was added. At time zero, 15 μΜ of Yodal (Sigma- Aldrich) was added to cell suspension either containing 10 μΜ TRAM-34 (Chemi, 10 μΜ GsMTx4 (Smartox) or DMSO. Samples were collected 15, 30 and 60 minutes after Yodal addition and cell water, Na ⁺ and K ⁺ contents were measured. Electrophysiology: All patch-clamp experiments were performed with a PC-controlled

EPC 9 patch-clamp amplifier (HEKA, Lambrecht/Pfalz, Germany). Currents were acquired and analyzed with Pulse and Pulsefit softwares (HEKA). Whole cell configuration was used and hematocrit was set at 10%. Glass pipettes (Brand, Wertheim, Germany) were made on a horizontal pipette puller (P-97; Sutter Instrument Co.; Navato, CA) to give a final resistance ranging from 18 to 20 ΜΩ. For whole cell experiments, the bath solution was in mM: NaCl 150, KC1 5, MgCl ₂ 1, CaCl ₂ 1, Hepes 10, buffered with NaOH pH7.4 (320 mOsm). The intracellular solution was in mM: KC1 140, NaCl 5, MgCl ₂ 1, Hepes 10 pH 7.2 adjusted with NaOH, 0.5 μΜ CaCl ₂ . Currents were measured at room temperature using a ramp protocol from -120 to +80 mV from a holding potential of -60 mV (sampling frequency 10 kHz; filtered 1 kHz) Red blood cell suspension at 10% hematocrit.

Results

Patient description and identification of 2 new PIEZOl missense mutations

One subject was previously described by us and others and carried two Piezol substitutions G782S/R808Q (Andolfo et al, 2013; Grootenboer S et al, 1998). The second subject was a 35 year-old man presenting with undiagnosed compensated hemolytic anemia and iron overload, he was investigated because of an unexplained fatal hydrops history of a second pregnancy, his first son was well and unaffected. The third subject was a 38 year old women investigated for undiagnosed compensated hemolytic anemia. Both patients presented with typical red cell parameters, a typical ektacytometric profile and normal EMA test. PIEZOl sequencing revealed two new missense mutations: a c.l792G>A mutation in exon 14, leading to pVal598Met (i.e.: V598M) (predicted as tolerated by SIFT, score 0.1, and disease causing by Mutation taster, p value 0.998) and substitution in patient 2 and a c.2042T>C mutation in exon 16 leading to p.Phe681Ser substitution (i.e.: F681S) (predicted as deleterious by SIFT, score 0), in patient 3. No mutation was identified in KCNN4. Interestingly, in these 3 cases, HX mutations are not in the C terminal part of the protein, where most gain of function Piezo mutation were identified.

New Piezol mutants: description

Osmotic resistance curves were done on washed red blood cells in Ca ²⁺ containing medium after 18 hours incubation at 37°C. Different drugs blocking KCNN4, TRAM-34 or Senicapoc at two different doses, where added in incubating medium and the spider toxin GsMTx4, known to block Piezol channel (Suchyna TM et al, 2000; Bae C et al, 2011), was also assessed in red blood cells with G782S/R808Q or V598M mutations in Piezol.

Control red blood cells showed a faint rightward shift in osmotic resistance after 18 hours incubation at 37 °C (Figure 1A). This shift is insensitive to 4 μΜ Senicapoc. In contrast, red blood cells with each of the Piezol mutations showed a leftward shift of the osmotic resistance curve after 18h incubation at 37°C (50% haemolysis for a relative osmolality between 0.3 and 0.4 for Piezol mutated red blood cells compared to 0.50 for control, Figure 1). This leftward shift was inhibited by Senicapoc in a dose dependent manner. TRAM-34 at 10 μΜ also prevented patient red blood cell dehydration. The GsMTx4 was able to slightly prevent dehydration in red blood cells from patients with G782S/R808Q as well as V598 M mutations; it was not assessed on F681S mutant.

In parallel, red blood cell Na ⁺ and K ⁺ contents were measured at time zero, before running the 18h incubation at 37°C and at the end of the incubation when osmotic resistance tests were done. The cation contents at t=0 are given in table 1 below:

Table 1: Na ⁺ and K ⁺ contents in red blood cells from patients and corresponding control at blood reception (t=0). Data are expressed in μιηοΐ per gram of dry cell weight. Data are means+sem, n=6 for V598M and G782S/R808Q and n=3 for F681S Piezol mutations. * p<0.05, Mann and Whitney test, comparison of mutant versus control. For patient red blood cells, there was a significant K ⁺ loss compared to control whereas the Na ⁺ content was not significantly modified. After 18h incubation at 37°C, an increase in Na ⁺ content equivalent to a decrease in K ⁺ content was observed in control red blood cells (Figure 2). None of these cation movements were sensitive to TRAM-34 or to Senicapoc. For patient red blood cells, whatever the mutations in Piezol, the Na ⁺ uptake was significantly increased compared to control. This Na ⁺ uptake was neither sensitive to Senicapoc nor to TRAM-34 (Figure 2A). Similarly, the K ⁺ loss was greatly enhanced in all red blood cells with Piezol mutations (Figure 2B). This increased K ⁺ loss was similarly sensitive to 10 μΜ TRAM-34 or 4 μΜ Senicapoc. There was no correlation between the Na ⁺ uptake and the K ⁺ loss for red blood cells with mutated Piezol, the K ⁺ loss exceeded Na ⁺ uptake in all patient red blood cells. However, in presence of Gardos channel inhibitors, the K ⁺ loss equilibrated the Na ⁺ uptake for all the studied Piezol mutations.

In order to assess the involvement of Piezol channel in red blood cell cation permeability, Yodal, a Piezol activator was used (Syeda R et ah, 2015). Control red blood cells were treated by 15 μΜ Yodal in presence of ouabain to prevent cation recirculation through the Na ⁺ /K ⁺ ATPase pump. Na ⁺ and K ⁺ contents were measured and the effects of TRAM-34, blocker of Gardos channel or GsMTx, blocker of Piezol channel, were assessed. Results are plotted in table 2 below:

Table 2: Cation movements in control red blood cells in presence of 15 μΜ Yodal, activator of Piezol channel, with or without a KCNN4 blocker (TRAM-34 10 μΜ) or a Piezol blocker (GsMTx4 10 μΜ). Data are means + sem of 4 experiments. ** p<0.001 comparison of control condition with the 3 conditions with Yodal. ⁺⁺ p<0.001 comparison between Yodal and Yodal +inhibitors. Mann and Whitney test.

It is observed that in 15 minutes, Yodal was able to reverse cation gradient leading to a large K ⁺ loss that slightly exceeded Na ⁺ uptake, which is corroborated by an 8% water loss. In presence of Tram-34, the Na ⁺ uptake induced by Yodal was not significantly affected whereas K ⁺ loss was diminished by a third. K ⁺ loss in presence of Tram-34 compensated the Na ⁺ uptake, there was no net cation movements. As a result, there was no water loss. In contrast, the spider toxin blocking Piezol channel, GsMTx4, inhibited similarly the Na ⁺ uptake and the K ⁺ loss. Noteworthy, the inhibition is not complete, only 45% inhibition was observed at 10 μΜ (the effect of a lower concentration, 4 μΜ, was not statistically significant). Piezol activation by Yodal induced a rapid change in cation contents with a steady state reached in 15 minutes (data not shown). The water loss following Piezol activation is very faint and only due to the subsequent activation of KCNN4 as it is not observed in presence of KCNN4 inhibitor Tram-34.

Electrophysiology on Piezol mutated red blood cells:

Currents in patient RBCs with the G782S/R808Q or V598M mutations on Piezol have been recorded in whole-cell configuration. The intracellular calcium was maintained at a concentration around the threshold for KCNN4 activation (0.5 μΜ) whereas extracellular calcium was present at 1 mM. Figure 3 illustrates the V curves for patient and control RBCs. Just after whole cell configuration was reached, patient erythrocytes with G782S/R808Q mutations showed a large current with reverse potential close to zero mV (-1.4+0.9 mV, n=6) (Figure 3A). In contrast the WT RBCs exhibited a smaller current with a -29+14 mV (n=5) reverse potential. However, when whole cell recording in control RBCs was done in presence of 15 μΜ Yodal in the bath, a large linear current was measured similar to the currents in RBCs with mutated Piezol (Figure 3A). Figure 3B illustrates the kinetic of currents recorded at -80 mV for WT erythrocytes with or without Yodal and erythrocytes with G782S/R808Q mutated Piezol. The large linear current observed in patient RBCs declined with a slower kinetic than the conductance induced by Yodal. It was possible to record currents in patient RBCs or control RBCs with Yodal when the large conductance was turned down. V curves are plotted on figure 3C. A rectified current with resting membrane potential around -50 mV was observed in cells with V598M mutated Piezol. This current exhibited KCNN4 current features and it was sensitive to 0.4 μΜ Senicapoc. In control RBCs, the activation by Yodal of the large transient current stimulated a hyperpolarized rectified current similar to the one observed with V598M mutated Piezol. Thus, it was possible to mimic the electrical features of patient RBCs by activating Piezol in control RBCs.

Discussion

These results show for the first time the effect of Piezol activation on red blood cell Na ⁺ and K ⁺ contents. This channel has been shown to drive a non-selective cation current (Gnanasambandam R. et al., 2015) It is permeable to monovalent cations as well as divalent cations (Wu J. et al., 2017). It is activated by mechanical stimuli such as pressure or shear stress. However, a chemical activator has been recently identified: Yodal (Syeda R. et al., 2015). This compound has been shown to modify Piezol gating, stabilizing the open state of the channel in reconstituted lipid bilayers or in HEK293 cells. In these later its effect was noticed when the channel was mechanically activated, the activating effect was almost absent without mechanical stress as if Yodal was not able per se to open the channel. However, Yodal effect on mouse red blood cell hydration suggested that it activates a calcium uptake via Piezol independently of any mechanical stress (Cahalan SM. Et al., 2015). Here we show that Yodal is also able to induce a rapid change in human red blood cell Na ⁺ and K ⁺ contents. A steady state is reached within about 15 minutes for cation content and cell volume. The Na ⁺ uptake is counter-balanced by the K ⁺ leak in presence of KCNN4 inhibitors. In the absence of KCNN4 inhibitors, the K ⁺ loss exceeded the Na ⁺ uptake by nearly 25% that correspond more or less to the part of K ⁺ movement mediated by KCNN4. Thus, in addition to Na ⁺ and K ⁺ movements, Piezol activation leads to a Ca uptake able to subsequently activate the Gardos channel as observed in mouse red blood cells by S. Cahalan and co-workers. This confirms that Yodal is able to activate Piezol without any mechanical stress on red blood cells. However, according to whole cell recordings in control red blood cells with Yodal in bath solution, Piezol activation is still transient. Moreover, the kinetic of K ⁺ loss is no more sensitive to KCNN4 inhibitor after 15 minutes incubation with Yodal, suggesting that Yodal effect on Piezol after a while is no more able to change intracellular calcium concentration. Remarkably the cell volume is only slightly affected by Piezol activation, only an 8% water loss is measured showing that calcium rises do not sustain a strong activity of the Gardos channel. The efficiency of the Ca ²⁺ pump to maintain a low level of intracellular Ca ²⁺ together with a transient activity of Piezol might explain this observation. The important changes in Na ⁺ and K ⁺ contents induced by Piezol activation are expected to stimulate Na/K pump activity and are therefore to modify red blood cell metabolism.

The new mutations in Piezol we have identified are in the N-terminal part of the channel, the extracellular domain lying on the plasma membrane, which is proposed to sense shear forces on the membrane. Our data show that these mutants are constitutively active leading to a change in Na ⁺ and K ⁺ contents of red blood cells that resembles the effect of Yodal on control red blood cells. Incubation at 37°C induced a K ⁺ loss that exceeded Na ⁺ uptake. In presence of Gardos channel inhibitors, this K ⁺ loss is reduced and compensated by the Na ⁺ uptake. As a result there is no change in cell volume. Dehydration of patient red blood cell could be explained by a hyper- activity of Gardos channel due to the constitutive Ca ²⁺ leak mediated by mutated Piezol. The Piezol toxin GsMTx4 is able to partially prevent red blood cell dehydration and Na ⁺ and K ⁺ movements. The effect on dehydration is likely due to a partial inhibition of Ca ²⁺ uptake and hence a lower activation of KCNN4. Metabolic differences could explain discrepancy between patients for intracellular Na ⁺ and K ⁺ levels, as well as the changes in Piezol conductance induced by the different mutations.

Finally, Senicapoc, the Gardos channel inhibitor (Stocker JW et al., 2003; Ataga KI et ah, 2009), is efficient to prevent dehydration of patient red blood cell with Piezol mutations.

Even though all the Piezol mutations linked to HX are not expected to modify similarly Piezol gating, the calcium leak mediated by Piezol activation is able to subsequently stimulate Gardos channel and without this calcium uptake there is no red blood cell dehydration. The severity of the HX phenotype with Piezol mutations could depend on the way the mutations alter Piezol gating. The Piezol mutations we have characterized are likely to generate a more severe phenotype by inducing a constitutive calcium leak as well as constitutive Na ⁺ and K ⁺ leak in red blood cells compared to mutations that only alter the inactivation kinetic of the channel. In this case, red blood cell dehydration occurring in blood flow each time Piezol is activated by cell stretching might be less severe than with a constitutively activated Piezol channel. However, all the mutations described in HX generate an inappropriate Piezol activity that is responsible for the higher Gardos channel activity leading to red blood cell dehydration. The ability of red blood cells to cope with a hyperactive Gardos channel will depend on many other issues such as metabolism and pump efficiency. Senicapoc has already been shown to prevent red blood cell dehydration due to gain of function mutations in Gardos channel (Rapetti-Mauss R. et ah, 2016), the other molecular cause of HX. Thus this drug could also be used to treat HX due to gain of function mutations in Piezol.

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