The differential expression of circulating microRNAs in sickle cell trait compared with the normal hemoglobin phenotype

Sickle cell trait (HbAS) is a common genetic carrier state found in ~3 million individuals in the United States, who are predominantly from the Black race.¹ Although HbAS was long considered benign, recent evidence has demonstrated strong clinical associations between HbAS and an increased risk for chronic kidney disease (CKD) and venous thromboembolisms (VTE).^2,3 Consequently, the American Society of Hematology has called for further research to better understand these clinical associations in HbAS, including genetic research.⁴ MicroRNAs are a crucial component of genetic research due to their role in modulating gene expression via messenger RNA regulation.^5,6 By modulating gene expression, microRNAs regulate the activity of various signaling pathways involved in disease pathogenesis.⁵ Consequently, the study of circulating microRNAs may provide insights into various pathophysiologies. To better understand whether the presence of HbAS may be an important factor in the modulation of gene expression, we sought to compare the differential expression of circulating microRNAs in individuals with HbAS with the normal hemoglobin phenotype (HbAA). We hypothesized that individuals with HbAS would have a significant differential expression of several microRNAs compared with HbAA. This was a cross-sectional study using data and plasma samples from the Mass General Brigham (MGB; formerly called Partners Healthcare) Biobank collected between 2005 and 2018. The MGB Biobank comprises samples voluntarily obtained with informed consent from individuals at MGB-affiliated hospitals and has been described in previous studies.⁷ MGB Biobank specimens are linked to clinical data from the electronic medical records of affiliated hospitals. The MGB and University of Texas Southwestern institutional review boards approved this study (University of Texas Southwestern institutional review board number STU2020-1053). We applied the following inclusion criteria at baseline (the time of sample collection): self-identified Black race, age ≥18 years, an estimated glomerular filtration rate (eGFR) >60 mL/min, and the presence of urine protein studies. The following exclusion criteria were applied at baseline: age ≥65 years, the presence of any comorbidity (eg, diabetes mellitus, hypertension, cardiovascular disease, CKD, cancer, transplantation, and autoimmune disease), or proteinuria. Matching was performed (1:1) on age ±5 years, sex, and eGFR ±20 mL/min. HbAS and HbAA were confirmed by a hemoglobin electrophoresis test interpreted by a pathologist. Frozen plasma samples (0.5 mL per patient) were sent to LC Sciences, Houston,^8-10 for processing. Total RNA was extracted using TRIzol reagent, following the manufacturer’s procedure. The quality and quantity of total RNA were analyzed by Bioanalyzer 2100, with a RNA Integrity number >7.0. Approximately 1 μg of total RNA was used to prepare a small RNA library using TruSeq Small RNA Sample Prep Kits, following the manufacturer’s protocol. Subsequently, single-end sequencing (50 base pairs) was performed on an Illumina HiSeq 2500 at LC Sciences, following the vendor’s recommended protocol. Raw reads were subjected to an in-house program, ACGT101-miR, to remove adapter dimers, junk, low complexity, common RNA families (ribosomal RNA, transfer RNA, small nuclear RNA [snRNA], and small nucleolar RNA [snoRNA]), and repeats. Subsequently, unique sequences with lengths ranging from 18 to 26 nucleotides were mapped to specific species precursors in miRBase 22.0 by Basic Local Alignment Search Tool (BLAST) search to identify known microRNAs and novel 3p- and 5p-derived microRNAs.