Hereditary xerocytosis revisited

Abstract

A 21 year old male student presented in 1980 as an Olympic athlete with a 12 year history of jaundice, pallor, and darkened urine induced by the atraumatic exercise of swimming (1). Physical examination at that time was remarkable only for moderate scleral icterus without hepatosplenomegaly. Hematological examination revealed moderate macrocytosis (MCV 102 fL) without anemia (Hct 50%, Hb 17 g/dL, 9% reticulocytes). The peripheral blood smear showed occasional target cells. Red cell osmotic fragility was decreased. Red cell Na content was increased and K content was decreased, with reduced total monovalent ion content. Passive red cell permeability of both Na and K were increased. A supervised 2.5 hr swimming workout increased free plasma Hb from <5 to 45 mg/dL and decreased serum haptoglobin from 25 to 6 mg/dL. The post-exercise urine sediment was remarkable for hemosiderin-laden tubular epithelial cells, without frank hemoglobinuria. The circulating 15 day erythrocyte half-life measured after 6 days without exercise was further shortened to 12 days after resumption of twice-per-day swimming workouts for 1 week. The patient’s red cells were hypersensitive to in vitro shear stress applied by cone-plate viscometer.

Figure 1. HX osmotic Fragility and mutation detection

Osmotic fragility curve of proband’s red blood cells. Gray lines represent normal limits. B. Sanger DNA sequence of the proband’s PIEZO1 cDNA (sense strand) showing the heterozygous mutation (asterisk) encoding missense substitution R2488Q. C. Partial pedigree of the family, with proband (I.1) and his three sons (II.1, II.2, and II.3). Genotypes are shown for PIEZO1 aa2488, UGT1A1 TA6/7 polymorphism, and HFE aa282.

Figure 2. HX red cell channel activity

A. Representative trace from cell-attached patch on the proband’s erythrocyte. The negative of the command potential applied to the pipette (−Vp) = −100mV. “C” indicates closed level. “O” indicates open channel. B. Composite single channel current voltage relationship from 4 patches. Mean slope conductance was 25.6 pS (r² = 0.99). C. NPo values from the proband’s erythrocyte cell-attached patches in the absence (left open bar) and presence (right filled bar) of 1 μM GsMTx-4 (*, p<0.05).

Figure 3. The effect of PIEZO1 mutation R2488Q on mechanically activated whole-cell currents

A. Whole-cell currents were elicited by applying indenting stimuli to the surface of HEK-293 cells transiently transfected with cDNA encoding wild-type hPiezo1. The stimuli (waveforms above current traces) consisted of indentation steps of 500 ms duration, applied in successive, 1 μm depth increments from 1–6 μm. Shown in black are mean whole-cell current traces elicited from cells transfected with wild-type PIEZO1 (n=15). The inactivation phase of the mean trace at 6 μm indentation depth was curve-fitted (red) using a mono-exponential equation ( $\mathrm{y}={\text{Ae}}^{\mathrm{x}/\mathrm{t}}+\mathrm{y}0$), yielding the time constant of inactivation (τ_{6 μm}). B. Mean whole cell current traces elicited from cells transfected with PIEZO1 mutant R2488Q (n=10), and the curve-fit of the mean data at 6 μm indentation (red). C. Superposed, normalized mean current traces elicited from indentations of 4, 5, and 6 μm (taken from panels A and B) with R2488Q currents shown in red. The inactivation half-times of whole-cell currents in cells expressing the PIEZO1 R2488Q mutant were longer than in cells expressing wild-type PIEZO1 protein.