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A retrospective series of 126 HX cases
iron overload and inefficient splenectomy. They demonstrate the high variability in clinical expression as well as genetic bases of PIEZO1-hereditary xerocytosis. These results will help to improve the diagnosis of hereditary xerocytosis and to provide recommendations on the clinical management in terms of splenecto- my, iron overload and pregnancy follow-up.
Introduction
Hereditary stomatocytoses are dominant red cell mem- brane disorders characterized by an increased cation leak through the membrane and a subsequent alteration in cell hydration.1,2 Also called hereditary xerocytosis (HX) (MIM #194380), dehydrated stomatocytosis is the most frequent of these disorders, with an estimated incidence of 1:50000 births.3,4 It is characterized by chronic hemolysis5 as well as non-hematologic features including transient perinatal edema, pseudohyperkalemia,6 iron overload7 and post- splenectomy thromboembolic complications8 that often dominate the phenotype and delay the diagnosis. In addi- tion, diagnosis is complex because of the lack of an easily available biological test. Osmotic gradient ektacytometry is the most efficient diagnostic tool so far,9 showing a typ- ical left-shifted curve with increased osmotic resistance. However, it is only performed in a few specialized labora- tories.
HX is most often caused by gain-of-function mutations in PIEZO1 which encodes a mechanosensitive ion channel that translates a mechanic stimulus into an increased intra- cellular calcium concentration.10–12 Recent reports have shown that PIEZO1 mutants induce an inappropriate increase in intracellular Ca2+ which in turn activates the Gardos channel inducing potassium leak, water loss and consequently erythrocyte dehydration.13 Of note, erythro- cyte dehydration related to PIEZO1 gain-of-function mutations has also been associated with protection against malaria.14 In HX, it can be assumed that PIEZO1 mutations lead to a stronger dehydration and subsequent- ly to hemolysis. Besides PIEZO1 mutations, gain-of-func- tion mutations in KCNN4, encoding the Gardos channel, directly cause another subset of HX.15,16
Up to now, with the exception of a recent report,17 the clinical and biological features of HX have been mostly described in small series. Large-scale clinical and biological descriptions at diagnosis and comparisons with the under- lying genotypes are required. We report here a retrospec- tive study describing the clinical and biological features in the largest series to date including 126 patients from 64 families with HX diagnosed based on ektacytometry and/or genetics between 1993 and 2017.
Methods
Patients and data collection
This retrospective cohort included 126 patients from 64 appar- ently unrelated families who were diagnosed with HX when tes- ted for red cell membrane diseases in a context of chronic hemol- ysis after elimination of other causes such as hemoglobinopathies except otherwise mentioned, at the hematology laboratory of Bicêtre hospital, between 1993 and 2017. The study was conduct- ed according to French ethics regulations regarding retrospective, non-interventional studies. Data were collected directly from clini-
cal records and through the French Cohort of Hereditary
Stomatocytosis, declared to the French National Commission on
Informatics and Liberty. Twenty families have been previously
reported and are presented here with complementary phenotypic or genotypic data.10,11,13,15,18,19
Phenotypic red cell analysis
Red blood cell analyses were performed in the same laboratory and were processed within 24 h after blood drawing and transport at 4°C. Cell counts, red cell and reticulocyte indices were meas- ured using an ADVIA2120 analyzer (Siemens®). Blood smears were observed after May-Grünwald-Giemsa staining using stan- dard methods. Screening for red cell membrane disorders was per- formed by osmotic gradient ektacytometry, as previously described.20–22
Gene analysis
Genetic analysis was performed with informed consent accord- ing to the Declaration of Helsinki. Genomic DNA was extracted from blood samples collected on EDTA using the QIAamp or QIASymphony DSP Midi blood DNA extraction kit (Quiagen®). The PIEZO1 and KCNN4 coding exons and exon-intron bound- aries were sequenced as described in the Online Supplementary Data. Each sequence variation with an allele frequency under 5% in the GnomAD v2.0.1 database was recorded. Family studies were performed when possible and pathogenicity scores were cal- culated using various bioinformatic software (SIFT 6.2.0, Mutation Taster 2013, Polyphen-2 2.2.6). The predicted pathogenicity is described in Table 1.
Statistical analysis
Statistical analyses were performed using parametric tests with two-tailed P values. Statistical significance used was α=0.05. We used the Student t-test for quantitative variables. All numerical val- ues are expressed as mean values ± standard error of mean.
Results
Patients and clinical features at diagnosis
We identified 126 patients with HX, including 64 probands and 62 family members, between 1993 and 2017. The diagnosis of HX was based on clinical pheno- type, red cell parameters and morphology, a normal eosin- 5-maleimide (EMA) binding test and a typical osmotic gra- dient ektacytometry curve for 103 patients, and genetic testing with non-typical or normal ektacytometry for 12 patients. Eleven patients presenting typical clinical or bio- logical features and belonging to families with genetically proven HX were also included although they had no ekta- cytometry or genetic analysis.
For probands, the mean age at diagnosis was 32±18 years (n=64), with the range extending from the perinatal period to more than 88 years old (Figure 1A). Clinical and biological features at diagnosis were comparable between probands and family members including mainly non-
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