Dehydrated hereditary stomatocytosis linked to gain-of-function mutations in mechanically activated PIEZO1 ion channels

Abstract

Dehydrated hereditary stomatocytosis is a genetic condition with defective red blood cell membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations in the mechanically activated PIEZO1 (FAM38A) ion channel were associated with dehydrated hereditary stomatocytosis. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated dehydrated hereditary stomatocytosis cases, we identify three novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for dehydrated hereditary stomatocytosis. All the dehydrated hereditary stomatocytosis-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in red blood cells of dehydrated hereditary stomatocytosis patients. Our findings also suggest a new role for mechanotransduction in red blood cell biology and pathophysiology.

Figure 1. Mutation screening results in PIEZO1 amongst patients and relatives

(a) Graphical representation of the 51 exons of human PIEZO1 gene, with evolutionary proteic conservation analysis of the mutants surrounding regions amongst vertebrates. Blue boxes: exons. Red lollipops: location of the identified mutations. Red boxes in aligned sequences: Amino acid residues concerned by the mutations. (b) Pedigrees of the three French DHS families with corresponding mutations. Black arrowheads: index-cases; asterisks: affected patients selected for exome sequencing; + and −, respectively: presence or absence of the described mutation.

Figure 2. Hydrophobicity plot of human PIEZO1 and position of reported mutations

Kyte-Doolittle hydrophobicity analysis (19 residues window) of human PIEZO1 prepared using the ProtScale program (Expasy). Triangles indicate the position of previously reported (green) and newly identified (red) mutations associated with DHS. Inset: Magnification of C-terminal region. Peaks with scores greater than 1.8 (red dashed line) indicate possible transmembrane regions.

Figure 3. Recombinant hPIEZO1 channels with DHS-associated mutations display slow inactivation kinetics

(a)Representative traces of mechanically activated inward currents recorded at −80 mV from HEK293T cells expressing either WT or indicated mutant hPIEZO1. Cells were stimulated by a series of mechanical steps (150 ms duration) in 1 μm increments. For each representative trace, the channel response corresponds to the indentation distance of the glass probe and is shown as a series of responses starting from the step which generates the first mechanically activated current(the initial steps that did not cause channel activation are not shown) till the step which generates the maximum response for that cell. The distance of the probe for the maximum response for each construct is indicated. (b) Representative WT (black) and mutant (colored) hPIEZO1 traces normalized to peak. Maximum current from each construct shown in panel (a) are overlaid to highlight difference in inactivation kinetics. Scale bar; 25 ms.(c) Average of inactivation time constant (tau, ms) for WT and mutant hPIEZO1 channels. Bars represent mean ± s.e.m. and numbers in parenthesis indicate number of cells tested for each condition. * p< 0.05, *** p < 0.001; relative to WT hPIEZO1 (Student’s t-test).