Elevated p16INK4a expression in sickle cell disease correlates with diminished social functioning and reflects accelerated aging

Abstract Introduction People living with sickle cell disease (SCD) experience complications indicative of an accelerated aging phenotype, including early decline in physical function, increased risk for age-related conditions such as diabetes, and show acceleration in biomarkers of aging such as DNA methylation and telomere lengths. Cellular senescence, measured by expression of p16INK4a (p16)in peripheral blood T lymphocytes (PBTL), is a validated biomarker of biological aging in the general population. Our prior study showed that adolescent and young adults with SCD have elevated p16 expression, but due to the small sample, we were unable to fully explore the association between p16 expression and clinical and patient reported outcomes of SCD severity (Wilson et al, Aging 2024). In this study, we examined p16 expression in a larger sample of mostly adults with SCD to determine whether p16 expression correlated with functional and disease outcomes in SCD.Methods We conducted a cross-sectional study of 41 individuals with SCD (HbSS or HbS/beta-zero thalassemia genotypes only) recruited from the University of North Carolina (UNC) Adult and Pediatric SCD clinics. Participants provided peripheral blood samples for p16 expression analysis and completed validated patient-reported outcome (PRO) measures, including ASCQ-Me and PROMIS-29. CD3+ T cells were isolated from whole blood, and p16 expression was quantified using TaqMan RT-PCR and reported in log₂ units, similar to our prior study. Clinical and laboratory data were abstracted from the electronic medical record. Expression of p16 was compared to a 250 person healthy non-SCD adult cohort (Natural Aging Study), adjusting for age. Within the SCD group, associations between p16 expression and clinical and PRO variables were assessed using linear regression with age added as a covariate.Results The SCD group comprised of 41 participants with a median age of 31 (IQR 23-44, range 15-67), with 21 (51%) on hydroxyurea and 15 (37%) on chronic transfusions. Median p16 expression in the SCD group was 10.92 (IQR 9.87–11.55) log2 units compared to 10.83 (IQR 10.13 – 11.40) log2 units in the natural aging group. After adjusting for age, SCD was associated with higher p16 expression when compared to the natural aging group (β=0.67, 95% CI 0.32–1.0, p<0.001). Within the SCD group, age (β=0.09, p < 0.01), total WBC (β=0.09, p = 0.038), history of acute chest syndrome (β=1.0, p = 0.011) were associated with higher p16 expression after adjusting for age, but no significant associations were observed with the other laboratory measures, hydroxyurea use (β=-0.01, p>0.9), or chronic transfusion therapy (β=–0.10, p=0.8). Higher p16 expression also correlated with worse PROMIS (β=–0.04, p=0.029) and ASCQ-Me (β=–0.02, p=0.046) Social Functioning Impact PROs, and borderline associated with worse PROMIS Physical Function (β=–0.03, p=0.066).Conclusion This study adds to the growing evidence for accelerated aging in SCD, as we demonstrate that across the lifespan, people living with SCD have higher PBTL p16 expression compared to general population. The significance of the higher burden of senescent cells is to be determined and could be related to chronic inflammation that is present in SCD given the association with WBC count. In non-SCD cohorts, p16 expression is linked to frailty and sarcopenia. While this was not directly measured in this study, we found that p16 expression is associated with PRO surveys that assess ability to participate in social activities, a plausible surrogate measure of frailty, and borderline associated with the PROMIS physical function PRO. Limitations of this study include its cross-sectional design, modest sample size, and reliance on peripheral blood T-cell expression, which may not fully capture organismal aging or senescence in other tissues. In future studies, we plan to explore longitudinal assessments of p16 expression coupled with measures of physical function to determine the utility of p16 as a predictive biomarker for frailty in SCD. Biomarkers such as p16 may be useful for targeting and tracking interventions to improve functional outcomes in the aging SCD population.