PIEZO1 Hypomorphic Variants in Congenital Lymphatic Dysplasia Cause Shape and Hydration Alterations of Red Blood Cells

Abstract

PIEZO1 is a cation channel activated by mechanical force. It plays an important physiological role in several biological processes such as cardiovascular, renal, endothelial and hematopoietic systems. Two different diseases are associated with alteration in the DNA sequence of PIEZO1: (i) dehydrated hereditary stomatocytosis (DHS1, #194380), an autosomal dominant hemolytic anemia caused by gain-of-function mutations; (ii) lymphatic dysplasia with non-immune fetal hydrops (LMPH3, #616843), an autosomal recessive condition caused by biallelic loss-of-function mutations. We analyzed a 14-year-old boy affected by severe lymphatic dysplasia already present prenatally, with peripheral edema, hydrocele, and chylothoraces. By whole exome sequencing, we identified compound heterozygosity for PIEZO1, with one splicing and one deletion mutation, the latter causing the formation of a premature stop codon that leads to mRNA decay. The functional analysis of the erythrocytes of the patient highlighted altered hydration with the intracellular loss of the potassium content and structural abnormalities with anisopoikolocytosis and presence of both spherocytes and stomatocytes. This novel erythrocyte trait, sharing features with both hereditary spherocytosis and overhydrated hereditary stomatocytosis, complements the clinical features associated with loss-of-function mutations of PIEZO1 in the context of the generalized lymphatic dysplasia of LMPH3 type.

Keywords: PIEZO1; lymphedema; overhydration; red blood cell alterations; spherocytosis; stomatocytosis.

FIGURE 1

Genetic study and clinical findings. (A) The inheritance pattern of c.6165-7G>A and c.5725delA variants in PIEZO1 in the family here analyzed. The proband (II.1) is a compound heterozygous for these variants. The red arrow indicates the proband. (B) Magnetic resonance image of the proband (II.1) showing chylothoraces and hydrocele.

FIGURE 2

Characterization of PIEZO1 mutations: cDNA study and membrane proteins expression analysis. (A) Electropherograms showing sequencing analysis of the PIEZO1 variant c.5725delA in the proband. Genomic DNA (gDNA) and cDNA sequences are shown. (B) DNA electrophoresis profile of the PIEZO1 cDNA fragment encompassing exons 42–44 of the proband (green line), the father (orange line), and the control (blu line) by 4200 TapeStation system. The electropherogram shows the size distribution and the intensity of the detected bands of RT-PCR. (C) PIEZO1 mRNA level normalized to GAPDH in the proband II.1 compared to his parents, I.1 and I.2 and the HCs (n = 30). Data are presented as a mean ± SD. ^∗p-value < 0.05. (D) Immunoblot showing PIEZO1 protein expression normalized to β-actin in the proband II.1 compared to his parents, I.1 and I.2 and the HCs (pool of n = 3). Densitometric analysis of one representative western blotting is shown. (E) Immunoblot analysis of RBCs membrane proteins, Band 3 (Anion Exchanger 1) and Stomatin (Erythrocyte Membrane Protein 7.2), in the proband II.1 compared to the HCs (pool of n = 3). Protein levels are normalized to β-actin. Densitometric analysis of two separate western blotting is shown. Data are presented as a mean ± SD.

FIGURE 3

Characterization of the hematological phenotype. (A) The red cell deformability index was measured as a function of increasing osmolarity of RBCs from proband II.1 (red line), from mother I.2 (black line), from father I.1 (orange dotted line), and internal HCs (light blue lines). Values are means +/– SE of two independent experiments. Elongation index (EI) (B) intracellular K+ content (expressed as mmol/Kg/Hb) of blood from II.1, I.1, I.2 subjects, and from HC (the graph with gray bars). Plasma K+ content (expressed as mmol/L of whole blood) of blood from II.1, I.1, I.2 subjects, and from HC (the graph with black bars) ^∗p-value < 0.05. (C) Peripheral blood smear (May-Grünwald Giemsa stain 40×) examination of the proband II.1 showing marked anisopoikolocytosis. Blue arrows indicate fragmented cells; red arrows indicate stomatocytes; green arrows indicate spherocytes; black arrows indicate ovalocytes; orange arrows indicate mushroom cells.