Evaluation of the Role of PIEZO1 Genetic Variants in Red Blood Cell Health in Sickle Cell Disease

Background

Sickle cell disease (SCD) is a disorder arising from genetic variation in β-globin. Homozygosity for the sickle variant (HbSS) most commonly causes SCD, though compound heterozygous forms, such as HbSC, also exist.The abnormal hemoglobin produced by HbS polymerizes when deoxygenated, creating fragile and adherent “sickled” red blood cells (RBCs), leading to hemolysis, anemia, painful vaso-occlusive events, and multi-organ damage.

PIEZO1 is a mechanosensitive Ca2+ channel in RBCs; a gain-of-function mutation in the gene leads to dehydrated xerocytosis (Bae et al., 2013). Nader et al. (2023) found that PIEZO1 activation decreased deformability in SCD RBCs, augmented adhesion to the extracellular matrix, and increased phosphatidylserine (PS) exposure. Another study found that RBCs from SCD patients carrying the PIEZO1-E756 allele exhibited increased RBC density and dehydration (Ilboudo et al., 2018). Recently, PIEZO1 has been associated with protection from malaria (Ngueste et al., 2023). Therefore, we hypothesize that PIEZO1 effects on cellular and membrane characteristics of SS RBCs contribute to SCD pathophysiology by altering intracellular and membrane characteristics.

Objective

We examined the association between genetic variants in PIEZO1 and four markers of RBC biological health, including deformability (Lorrca [Laser Optical Rotational Red Cell Analyzer]), hemoglobin concentration (Hb), mean corpuscular volume (MCV), and red cell distribution width (RDW) in a cohort of SCD patients. We also evaluated the role of PIEZO1 variants in inflammatory responses and cell membrane damage via associations with RBC PS exposure and plasma matrix metalloproteinase-9 (MMP-9).

Methods

We analyzed nine non-synonymous single nucleotide polymorphisms (SNPs) in PIEZO1 present in the whole genome sequence data previously generated by the TOPMed program on a cohort of 34 adult HbSS patients for whom RBC biological characteristics were available. Additionally, we examined the PIEZO1-E756 variant, which was previously associated with red cell xerocytosis and available from our previous work (Ilboudo et al., 2021) on 25 of the 34 patients. Therefore, our Bonferroni multiple testing correction was p= 0.005 (0.05/10). To evaluate whether PIEZO1 variants were associated with RBC measures, we performed linear regression tests with PLINK, adjusting for sex and age. Additionally, to determine the relationship among the six phenotypes that we examined, we ran Pearson correlations in SAS.

Results

We did not observe any associations in our small data set that withstood correction for multiple testing. However, three non-synonymous SNPs that potentially affect protein function were nominally associated with some of the phenotypes of interest. Most significantly, rs114034093 was associated with both RDW (β = -2.666, p = 0.0078) and Hb (β = 1.285, p = 0.0128). Additionally, two SNPs in complete linkage disequilibrium (rs11645197 and rs35159887) were associated with PS exposure (β = 0 .700 p = 0.01004). We did not identify any associations between our phenotypes and PIEZO1-E756. Among the six phenotypes that we tested, the strongest correlation was between Lorrca and PS, which were negatively correlated (r = -0.66; p < 0.0001). To a lesser degree, RDW was negatively correlated with Lorrca (r = -0.59; p = 0.0001), Hb (r = -.49, p = 0.0017), MCV (r = -0.34, p = 0.0354), and positively correlated with PS (r = 0.56; p = 0.0003).

Conclusions

Our results provide continued support for the influence of PIEZO1 genetic variation on RBC health in the context of SCD. The strong negative correlation between Lorrca and PS may suggest a relationship between reduced RBC deformability and inflammatory-mediated stress/early apoptosis. PIEZO1 genetic variants may influence PS exposure, which is involved in several important pathologic processes in SCD, including cell adhesion, polymorphonuclear (PMN) leukocyte activation, and activation of coagulation. The three non-synonymous SNPs identified require additional research to determine their specific functional effects on PIEZO1. Future directions also include using a larger sample to increase statistical power and investigating the role of PIEZO1 genetic variants on clinical outcomes such as hospitalizations and vaso-occlusive events.

Telen:Novo Nordisk: Research Funding; GBT/Pfizer: Research Funding; CSL Behring: Research Funding.