Plasma Mir Let-7c Is a Marker of Previous Acute Chest Syndrome in Sickle Cell Disease

Acute chest syndrome (ACS) is the leading cause of death among children with sickle cell disease (SCD). Identification of a biomarker associated with ACS has the potential to improve our ability to predict and prevent this terrible complication of SCD. Aberrant cell-cell interactions involving the endothelium are central to the pathophysiology of sickle cell disease (SCD), including ACS. We previously demonstrated that the plasma of SCD patients contains circulating small extracellular vesicles (EVs) and that those vesicles can disrupt endothelial integrity in vitro. The extent of endothelial damage in vitro was related to the clinical history of ACS, with small EVs isolated from patients with a history of ACS causing more disruption in vitro. The current study was designed to start to interrogate the miRNA contents of those exosomes to determine if any are associated with a history of ACS.

Methods; We initially performed miRNA sequencing on the exosomal contents of 29 baseline samples from our SCD biobank; 12 without a history of ACS and 19 with a history of ACS. Using a fold-change cut-off of 1.5 and FDR < 0.1 we identified 15 miRNA that were differentially expressed based on a history of ACS. Of those 15 miRNA we selected miR-let-7c to perform an initial replication cohort. Since sample material is limited this initial replication was performed with plasma. Within the replication cohort, 9 had a history of ACS (ACS(+)) and 7 did not (ACS(-)). For all subjects, we reviewed clinical data relevant to their SCD and laboratory data (including hemoglobin, absolute reticulocyte count, white blood cell count) obtained at the same time as the baseline samples.

Results; Subjects were similar clinically, except those with a history of ACS were more likely to be on hydroxyurea (p<0.05) and to have obstructive sleep apnea (p<0.05). Hematologic laboratory values were similar irrespective of ACS history. The mean plasma miR-let-7c level was 2-fold less in subjects with a history of ACS than in those who had never had ACS (p<0.05). A plasma miR let-7c level < 1 (normalized to the control subjects) had a positive predictive value of 0.78 for history of ACS and a sensitivity of 78% and specificity of 71%. Among subjects with a history of ACS, the let7c levels did not correlate with time since the last ACS event. However, among subjects who developed ACS following the baseline samples, higher miR let-7c levels correlated with increased length of time to next ACS event (R=0.8).

Discussion; Our results highlight that continued work should be done to identify biomarkers of disease severity in SCD. We were able to use miRNA sequencing of exosomes to identify potential targets and then validated them at the plasma level. Further work needs to be done to understand how miR-let-7c is altered throughout the variety of complications in SCD. For example, can it distinguish a painful vaso-occlusive episode from an ACS episode? Does it change with age? In addition, this work suggests that there are increasing opportunites to identify patients that are at higher risk of complications. We hope to expand are analysis to additional miRNA and identify a signature that is distinct to ACS.

No relevant conflicts of interest to declare.