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Letters to the Editor
found on the other allele was detectable in both cDNA and gDNA. Four HS patients exhibited novel, heterozy- gous ANK1 loss-of-function mutations, including ANK1 E883Gfs32X in HS7 (accompanied by the known, rare SPTA1 R1074H variant of uncertain significance), ANK1 A1110del2 in HS8 (mutating a splice acceptor site), ANK1 K1140Gfs86X in HS9 (accompanied by the SLC4A1/Band 3 Memphis II polymorphism) and ANK1 E1289Gfs86X in HS10. Combined cDNA and gDNA sequencing indicated that mRNA encoding both ANK1 mutations E883Gfs32X and E1289Gfs76X were substrates of nonsense-mediated decay, whereas the other two ANK1 mutations under- went partial nonsense-mediated decay (Table 1).
Two HS patients were found to have novel heterozy- gous loss-of-function mutations in the SPTB gene encod- ing b-spectrin, SPTB G1450Rfs41X in HS12 and SPTB E1815Pfs90X in HS13 (Table 1). Both of these frameshift termination mutations encoded substrates of nonsense- mediated decay. Patient HS11 exhibited compound het- erozygosity for the novel, “probably damaging” missense variant SPTB R1255G (Polyphen-2 score 0.999) and the known non-pathogenic ANK1 R619H variant. The likely pathogenic SPTB R1255G substitution is located in the ninth of b-spectrin’s 17 repeat domains, portions of which comprise a dimerization domain, a tetrameriza- tion domain, and the ankyrin-binding domain. Remarkably, the purified recombinant ninth b-spectrin repeat generated in E. coli was found to be more unstable (with a melting temperature of 20°C) than any other recombinant b-spectrin repeat polypeptide, each of which had melting temperatures ≥37°C,8 demonstrating increased mutation-associated susceptibility to dysfunc- tional conformational change.
We assessed some of the HS mutants shown in Table 1 for cation channel activity in on-cell patches, preserving any regulatory components of the red cell cytosol and membrane cytoskeleton. Red cells from patients carrying the known SLC4 HS mutants R150X, R490C, and M663del each exhibited channel activity. Red cells from the patients carrying the novel HS-associated mutations ANK1 A1110del2 and ANK1 K1140Gfs86X also exhibit- ed channel activity. In addition, red cells from the patient carrying the novel, predicted pathogenic VUS SPTB R1255G exhibited channel activity. The representative current trace from patient HS4 in Figure 1A with reversal potential of ~0 mV and unitary conductance of 21 pS (Figure 1B) is consistent with cation channel activity. On- cell patch recordings of red cells from patients HS2, HS4, HS5, HS8, HS9 and HS11, representing mutations in SLC4A1, ANK1, and SPTB, exhibited a mean unitary conductance of 26±2.1 pS.
In on-cell patch recordings of red cells from patients with the previously known SLC4A1 HS mutation R150X (HS2), the novel ANK1 mutation delA1110Q1111 (HS8), and the novel, rare predicted pathogenic SPTB variant R1255G (HS11), channel activity was also monitored under conditions in which the micropipette fluid includ- ed the mechanosensitive cation channel blocker, GsMTx4 (1 mM). Mean NPo of channel activity was 1.44±0.44 as measured in 16 cells representing six geno- types (Figure 1C, Table 1). The presence of 1 μM GsMTx4 in the recording pipette was associated with ~95% inhibition of channel activity, reducing mean NPo to 0.08±0.03 as measured in six cells representing three genotypes (Figure 1C, Table 1). The unitary conductance, reversal potential, and sensitivity of the current to inhibi- tion by GsMTx-4 are each consistent with PIEZO1 medi- ation of, or contribution to, the measured cation channel activity in HS red cells. The increased membrane tension
of the gigaseal inside the pipette9 may unmask increased cation current in on-cell patches which might be less readily detected in whole cell patch recordings.5 Interestingly, however, small increases in whole cell cur- rent were detected in some, if not all HS patients’ red cells haploinsufficient for SPTB or for SPTA1.5
Cation channel activity in the presence of pathogenic stomatocytosis mutations in transmembrane transporters such as SLC4A1, RHAG, GLUT1, and ABCB6 has been attributed either to direct cation permeation through the dysfunctional mutant membrane protein itself, or to direct or indirect modulation of PIEZO1 activity.10 However, the similar properties of the increased cation channel activities measured in the presence of pathogenic HS mutants of the cytoskeletal proteins b-spectrin and ankyrin very likely arise from direct or indirect modula- tion of PIEZO1 (and/or another unidentified cation chan- nel), possibly by perturbations transmitted through one of the SLC4A1/Band3-nucleated macro-complexes.11 This modulation might reflect PIEZO1 properties such as the lower hydrostatic pressure threshold for PIEZO1 acti- vation in on-cell patches of actin cytoskeleton-depleted cellular blebs than in on-cell patches with intact cell cor- tex, and/or the inhibition by cytochalasin D of pressure- activated PIEZO1 in on-cell patches of normal cultured cells, and by glass probe-mediated cell indentation as measured by whole cell currents.9
Our data suggest that PIEZO1 likely mediates or con- tributes a major fraction of the incremental cation perme- ability of HS red cells. Clarification of the relationships between apparent cytoskeletal modulation of erythroid PIEZO1 and PIEZO1 modulation by flow12 and by mod- ulation of lateral membrane tension via the ceramide- sphingomyelin balance of the red cell membrane13 will require further study.
David H. Vandorpe,1* Boris E. Shmukler,1* Yann Ilboudo,2 Swati Bhasin,3° Beena Thomas,3° Alicia Rivera,1
Jay G. Wohlgemuth,4 Jeffrey S. Dlott,4 L. Michael Snyder,4 Colin Sieff,5 Manoj Bhasin,3° Guillaume Lettre,2
Carlo Brugnara6 and Seth L. Alper1
1Division of Nephrology and Vascular Biology Research Center, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA, USA; 2Montreal Heart Institute and Université de Montréal, Montréal, Québec, Canada; 3Division of Integrative Medicine and Vascular Biology Research Center, Beth Israel Deaconess Medical Center and Department of Medicine, Harvard Medical School, Boston, MA, USA; 4Quest Diagnostics, Seacaucus, NJ, USA; 5Cancer and Blood Disorders Center, Dana-Farber Cancer Center and Boston Children’s Hospital, and Department of Pediatrics, Harvard Medical School, Boston, MA, USA and 6Department of Laboratory Medicine, Boston Children’s Hospital and Department of Pathology, Harvard Medical School, Boston, MA, USA
*DHV and BES contributed equally as co-first authors.
°Current address: Departments of Pediatrics and Biomedical Informatics, Emory University School of Medicine, Atlanta, GA, USA
Correspondence:
SETH L. ALPER - salper@bidmc.harvard.edu doi:10.3324/haematol.2021.278770 Received: March 12, 2021.
Accepted: May 28, 2021.
Pre-published: June 10, 2021.
Disclosures: JGW and JCD are employees of Quest Diagnostics. LMS and SLA are consultants to Quest Diagnostics. CB has received research funds from Quest Diagnostics after completion of this study.
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