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V. Picard et al.
spherocytic chronic hemolysis in 84% of all cases, hyper- ferritinemia (36%), thrombosis after splenectomy (11%), and perinatal edema in 17% of all cases (Figure 1B).
Cholelithiasis was noted in 52% of cases (n=67) with the incidence increasing with age: 22% before 20 years old (n=23), 62% between 20 and 40 years (n=29) and 80% after 40 years (n=15). Splenomegaly was noted at diagno- sis in 56% of cases (n=47): 67% before the age of 20 years vs. 51% after the age of 40 years . The mean bilirubin and haptoglobin levels were, respectively, 2.5±1.4 mg/dL (n=36) and 0.28±0.24 g/L (n=24), among whom only nine had a level <0.1 g/L). All other notable medical issues are listed in Online Supplementary Table S1.
Biological diagnosis and genetic testing in hereditary xerocytosis
Ektacytometry was performed for 115 patients (90%), including 62/64 index cases and 53/62 family members. Genetic analysis was available for 103 subjects (81%) including 55 index cases (86%). Overall, 56 probands (87%) were diagnosed thanks to a typical dehydrated osmotic gradient ektacytometry profile (Online Supplementary Figure S1) and six (6/56, 11%) following genetic testing. Two index cases (one fetus and one neonate) died and could not be tested; the diagnosis was made after family study. Out of the 56 patients with a typ- ical osmotic gradient ektacytometry profile, 49 subjects could be genotyped. Rare PIEZO1 coding sequence varia- tions were recorded for all these families whereas no vari- ation was detected in KCNN4 (Table 1 and Online Supplementary Table S2). These PIEZO1 sequence varia- tions co-segregated with the disease in 30/49 informative families.
In 19/49 families (39%), a known PIEZO1 recurrent mutation in exon 51 was identified: (i) p.Leu2495_Glu2496dup (10 probands, 20 subjects; in 1 subject, it was associated with a second substitution p.Val2474Met), (ii) p.Arg2456His (6 probands, 15 sub- jects), p.Arg2488Gln (3 probands, 6 subjects, associated with the polymorphisms p.Gly718Ser in cis in 1 family and (iii) p.Arg1925Trp in another family). The other 30 fami- lies (61%) carried rare private PIEZO1 missense sequence variations (n=34). Six families were described or carried known mutations considered pathogenic (in 1 case, 2 mutations in cis: p.Gly782Ser and p.Arg808Gln); 15 fami- lies carried new unique mutations that we scored as prob- ably pathogenic (n=11) or of unknown significance (n=4) as defined in Table 1. A probably pathogenic sequence variation in exon 16 (p.Asp669Tyr) was present in two apparently unrelated families. In seven families, we iden- tified two associated missense sequence variations; in two of them, one mutation was scored probably pathogenic (p.Val598Leu and p.Gly2433Arg) and was associated with the quite frequent p.Pro2510Leu substitution, the other mutations were scored as being of ‘unknown significance’ and their role needs to be further established. Overall, ten families (20%) carried two PIEZO1 rare sequence varia- tions, in cis in three families, and whose transmission could not be defined in seven (Online Supplementary Table S2). Finally, one subject carried an apparently homozy- gous mutation, possibly due to hemizygosity since from exon 5 to 51, all PIEZO1 variations were homozygous (data from the parents were not available). This patient had chronic compensated hemolysis with a hemoglobin concentration of 175 g/L, reticulocyte count of 500x109/L,
a moderate increase in ferritin level and a typical osmotic gradient ektacytometry profile. In one case we only found the PIEZO1 c.1013C>A (p.Ser 338Tyr) substitution scored as a polymorphism.
Overlapping features with lymphedema were identified in two families: one patient with a mild HX phenotype presented two sequence variations (p.Leu939Met, p.Phe2458Leu) previously described in cis in the parent of a patient with PIEZO1-related lymphedema;23 three out of four related patients carrying two substitutions (p.Gly782Ser and p.Arg808Gln) in cis in exon 18 co-segre- gating with the disease through three generations presen- ted with severe perinatal edema, including one with per- sistent lymphedema during adulthood.
In six families (12 patients), osmotic gradient ektacy- tometry did not lead to the diagnosis of HX, since the pro- files were either atypical (n=1) or normal (n=5) (Online Supplementary Figure S1). All index cases had chronic hemolytic anemia/hyperferritinemia, four of them had a family history of undiagnosed dominant hemolysis. Gene analysis identified a mutation in KCNN4 in all of them (with no PIEZO1 nucleotide variation). Five families, two
A
B
Figure 1. Main clinical and biological features at diagnosis of the probands and their family members. (A) Repartition of age at diagnosis for the 64 index cases. The mean age was 32±18 years (range, 0-88). (B) Biological and clinical fea- tures at the time of diagnosis of hereditary xerocytosis (HX) in the 64 index cases and 62 family members; only post-splenectomy thromboses are shown; other: B19 parvovirus infection (n=1), persistent isolated jaundice after birth (n=1), systematic exploration in a context of familial porphyria leading to an “incidental” diagnosis of PIEZO1-related HX (n=1). The figure does not show one case of extramedullar hematopoiesis. NSCH: non-spherocytic chronic hemolysis; PNE: perinatal edema; NA: data not available (4 patients).
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haematologica | 2019; 104(8)