MicroRNA profiling and validation in mixed ancestry individuals in South Africa

Abstract

Background: There has been a gradual increase in the prevalence of type 2 diabetes mellitus (T2DM) in South Africa, and in 2019, the prevalence was reported to be the highest in comparison to all other countries in the African region. Concerns have arisen regarding the efficiency of current methods of diagnosis, such as the invasiveness and cumbersomeness of the oral glucose tolerance test (OGTT), diurnal variations in blood glucose levels limiting the capacity of blood glucose tests and increasing the chances of missing risk groups, as well as the influence of age, ethnicity and haemoglobinopathies on HbA1c levels. In light of this, studies have shifted to small, non-coding RNA transcripts called microRNAs (miRNAs), which are present at consistent and reproducible levels in peripheral blood. They have been touted as potential diagnostic and prognostic biomarkers for various diseases, such as type 2 diabetes mellitus (T2DM), and expression patterns in response to treatment may further promote their use as therapeutic targets in T2DM. Therefore, the objectives of this study were to investigate the expression of miR-30a-5p, -1299, -182-5p, -30e-3p and -126-3p, and their association with- and diagnostic capability for dysglycaemia in a South African population previously reported to have a high prevalence of undiagnosed diabetes, as well as to explore their expression patterns in newly diagnosed and known diabetic individuals on treatment. Methods: This was a cross-sectional study involving a total of 1273 individuals (men, n=345), including 207 prediabetes, 94 screen-detected diabetes and 972 normotolerant individuals, all aged >20 years, from the ongoing Vascular and Metabolic Health (VMH) study. Glycaemic status was assessed using the OGTT and blood pressure measurements were taken, all in accordance with World Health Organization (WHO) guidelines. In addition, HbA1c, insulin levels, obesity indices, lipid profile, C-reactive protein and serum cotinine levels were performed. Five miRNA (miR-30a-5p, -1299, -182-5p, -30e-3p and -126-3p) expression profiles were assessed by TaqMan-based RT-qPCR. These were based on data obtained from whole genome miRNA profiling from the whole blood of 12 individuals with known diabetes, 12 with screen-detected diabetes, 12 with prediabetes and 12 with normal glucose tolerance, matched for age, blood pressure, smoking and body mass index (BMI). Receiver operating characteristic (ROC) curves were used to assess the ability of each miRNA to discriminate dysglycaemia, and univariate as well as multivariable logistic regression analyses were performed to link expression with dysglycaemia. Results: Relative expression between subgroups revealed all miRNAs were significantly highly expressed in prediabetes compared to normotolerant (≥3.1-fold and p<0.001), with miR-30a-5p showing the highest significant expression between the two groups (3.5-fold and p<0.001). miR-30a-5p, -1299, 182-5p, and -126-3p were significantly overexpressed when compared to screen-detected diabetes and normotolerant (≥1.6-fold and p≤0.013), with the exception of -30e-3p (1.3-fold and p=0.145). All miRNAs demonstrated overexpression in known diabetics when compared to normotolerant individuals (≥5.6-fold and p<0.001). Comparisons between prediabetes and screen-detected diabetes revealed significantly reduced expression of miR-1299, -182-5p and 126-3p (≤0.56-fold and p≤0.020), whilst miR-30e-3p and -30a-5p did not demonstrate significantly reduced levels (≤0.58-fold and p≥0.097), additionally a significant elevation was observed in known diabetics versus screen-detected (≥3.1-fold and p<0.001). Multivariable logistic regressions, adjusted for age, sex, BMI, systolic blood pressure (SBP), 2-hour blood glucose, HbA1c, triglycerides, high-density lipoprotein cholesterol (HDL-cholesterol) and low-density lipoprotein cholesterol (LDL-cholesterol), revealed all miRNAs were consistently and continuously associated with dysglycaemia when compared to normotolerant, while only miR-126-3p and -182-5p showed associations with screen-detected diabetes versus prediabetes, (odds ratio (OR)≥0.89, 95% confidence interval (CI): 0.83-0.96, p≤0.003) and (OR≥0.70, CI: 0.60-0.81, p≤0.001) respectively. The diagnostic capabilities of the miRNAs in distinguishing dysglycaemia was assessed using receiver operating characteristic curve (ROC) analyses, whereby miRNAs miR-126-5p and -182-5p in particular, outperformed HbA1c in distinguishing prediabetes, area under the curve (AUC) = 0.76 for miR-126-3p versus 0.70 for HbA1c, (p=0.042), and 0.74 for miR-182-5p versus 0.69 for HbA1c (p=0.217). Moreover, univariate regression analysis was used to illustrate the relationships between the miRNAs and the duration of T2DM. miR-1299, -182-5p and -126-3p were associated with the duration of diabetes upon models adjusted for age and sex, (OR≥0.076, CI: 0.001 – 0.151, p<0.046) however after adjustment for type of treatment, only miR-182-5p remained significantly associated with the duration of the disease (OR: 0.127, CI: 0.018-0.236, p=0.023). Conclusions: These results demonstrated altered expression levels and significant associations of the miRNAs in question with dysglycaemia, as well as demonstrating greater diagnostic capabilities of miR-126-3p and -182-5p compared to HbA1c in differentiating prediabetics from normotolerant individuals, implicating their use in potentially contributing to diabetes risk screening strategies. Furthermore, we illustrated important associations and altered expression-patterns of the miRNAs in known diabetics on anti-diabetic treatment compared to newly diagnosed individuals, with miR-182-5p expression decreased with increasing duration of T2DM, indicating the potential value of the miRNA in diabetes management. Further studies are however recommended to shed light on the involvement of the miRNAs in glucose homeostasis, to endorse their use as a therapeutic targets in DM and its associated complications.