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V. Picard et al.
mined the disease severity. Considering this heterogene- ity, we investigated whether genetics could discriminate patients who would require a more intensive manage- ment. We identified 49 families with PIEZO1-HX, and six with KCNN4-HX, this ratio being in agreement with those in other reports.5 One recurrent mutation (p.Arg352His) accounted for most cases of KCNN4-HX (5/6 families). We also identified a new 18-base pair KCNN4 deletion, while only missense mutations have been reported so far, indicating a new genetic basis for this disorder. In the Gardos channelopathies reported so far, the single residue substitution increased the activity of the Gardos channel through an altered calmodulin binding.15,26 In this family, we observed the 18-base pair deletion over- lapped the exon-intron 7 junction. We hypothesized that the use of a potential upstream splice site at c.G1104(TG/GT) might lead to an in-frame deletion of five residues (p.369_373VDISKdel), creating a Gardos channel missing five amino acids in the calmodulin-bind- ing domain. The consequences on Gardos channel sequence and function remain however unclear and need to be tested.
In contrast to Gardos channelopathy, recurrent muta- tions accounted for only one-third of PIEZO1-HX cases. The three recurrent mutations are located in exon 51, the main mutation hotspot that encodes the canal pore and the C-terminal region. Regions including exons 14 to 18 and exons 42 to 45 appeared as secondary ‘hotspots’, the for- mer region affects peripheral helices forming the extracel- lular ‘blade’, the latter region encodes distinct α helices close to the canal pore. Indeed, we identified a new recur- rent mutation, p.Asp669Tyr in exon 16, present in two unrelated families and recently reported in one other patient.17 In other families, one or two rare or undescribed private sequence variations were present, indicating very heterogeneous genetic backgrounds in PIEZO1-HX. Indeed, a PIEZO1 sequence variation, not scored as a poly- morphism, was identified in all tested families except one. For the large majority of patients, the diagnosis was made based on phenotypic data before the genetic test was avail- able. So far, no clear genotype-phenotype correlation could be drawn. Given the high number of PIEZO1 polymor- phisms described in databases, the effect of these private, newly described mutations cannot be ascertained. From a practical point of view, these data underline the require- ment for phenotype-based methods and functional experi- ments in addition to genetics to confirm PIEZO1-HX diag- nosis, when unreported mutations are identified. If osmot- ic gradient ektacytometry can be seen as an indirect phe- notypic test reflecting red cell dehydration, functional elec- trophysiological tests would represent a major advance in the characterization of new mutations. Some of the muta- tions involved here have been tested functionally, but not in a systematic manner because of the lack of available tools.10,13,26 The recent characterization of a PIEZO1 gain-of- function mutation through high-throughput patch clamp- ing on red cells is promising in this respect.27
Recent in vitro studies showed that both PIEZO1-HX and KCNN4-HX share a common pathophysiology lead- ing to red cell dehydration.13,26 Indeed, both disorders share hemolytic features and frequent hyperferritinemia. Hyperferritinemia was not related to transfusions and was frequently at the front line of the diagnosis. Hyperferritinemia is well described in chronic hemolytic diseases,28 but is notably much more frequent in HX than
in hereditary spherocytosis.29 Although associated HFE mutations may worsen iron overload in HX,7 they could not be associated with more severe iron overload in our series. Alternative mechanisms of increased iron uptake may be involved, including chronic hypoxia, increased erythroferrone secretion, and erythroblast proliferation possibly associated with some inefficient erythropoiesis. Alternatively, expression of a mutated PIEZO1 or Gardos protein at the cell surface could directly deregulate hep- cidin expression in liver cells or drive iron entry through the gut. From a practical point of view, we observed a weak correlation between ferritin and liver iron content, particularly for ferritin levels below 1000 ng/mL. In terms of clinical management, these data highlight the require- ment for an annual iron status evaluation and for measure- ment of liver iron content by magnetic resonance imaging when ferritin increases above normal values.
On the other hand, PIEZO1-HX and KCNN4-HX dif- fered in several ways. First, in terms of severity, patients with PIEZO1-HX had a milder hematologic phenotype: 27% had a hemoglobin level below 120 g/L vs. 75% of patients with KCNN4-HX. It is worth noting that some patients with PIEZO1-HX had a hemoglobin level in the upper normal range or above it. Therefore, PIEZO1 gain- of-function mutations may stimulate erythropoiesis by itself, explaining the ‘compensated hemolysis’ phenotype as a balance between hemolysis and increased erythro- poiesis. Of note, red cell dehydration was not the main cause of hemolysis since it was predominant in PIEZO1- HX, but discreet or absent in KCNN4-HX despite a more severe anemia. This difference has a practical conse- quence: ektacytometry, which responds to red cell hydra- tion, identified PIEZO1-HX but not KCNN4-HX. Therefore, genetic testing should be performed to rule out this subset of HX in the case of undiagnosed hemolysis, even when ektacytometry is normal. It has been recently suggested to use the term "Gardos channelopathy”, instead of xerocytosis,30 for this variant and we agree with this proposal.
Another difference between PIEZO-HX and Gardos channelopathy was the rate of post-splenectomy throm- bosis. Thombosis occurred in 100% of PIEZO1-HX cases (8/8), in agreement with other reports,8,24,30–34 but in 0/4 Gardos-mutated patients. However, these data must be interpreted carefully because: (i) the number of Gardos- splenectomized patients was low; (ii) four patients with thrombotic complications were not genotyped; and (iii) the persistence in all cases of hemolysis after splenectomy represents a risk by itself. Indeed, thrombosis is a well- described complication after splenectomy, particularly when removal of the spleen does not abrogate hemolysis. Several factors may be involved, including platelet aggre- gation, decrease in nitric oxide level, high level of circulat- ing microparticles, high rate of phosphatidylserine- expressing red cells and increased reticulocyte adher- ence.35–37 However, PIEZO1 seems to have a specific role that may involve endothelial dysfunction.38,39 Thus, a mutated PIEZO1 at the cell surface could alter interactions between endothelial cells and dehydrated erythrocytes and favor thrombosis. Considering the type of thrombo- sis, we made two interesting findings. There was a high rate of portal thrombosis (40% of patients), which may even be underestimated since asymptomatic portal thrombosis was not systematically evaluated after splenectomy as recently suggested.40 Secondly, we
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haematologica | 2019; 104(8)