Micro-RNA in a sample of Egyptian patients with incidental cerebral small vessel disease

Over the past few years, the prevalence, mortality, and morbidity of different neurological disorders have increased, despite the implementation of intervention programs aiming to reduce their health care burden [1]. This has incited the scientific community to explore reliable and affordable biomarkers for early detection of these disorders [2].

Incidental cerebral small vessel disease (iCSVD) is among the neurological diseases known to reduce the quality of life in elderly. It has a strong role in age-related disabilities as cognitive impairment, gait apraxia, urinary incontinence, and depression as well as being a leading cause of vascular dementia (VaD) [3‐5].

Diagnosis of iCSVD is based on brain imaging biomarkers, in the form of recent small subcortical infarcts, white matter hyperintensities (WMH), lacunes, cerebral microbleeds (CMBs), enlarged perivascular spaces (EPVS) and cerebral atrophy [6].

Recently there is an increasing body of evidence that miRNA plays a critical role in the control of key biological processes including development, differentiation, growth, and metabolism as well as pathophysiology of neurodegenerative and cardiovascular diseases [7]. Living neurons and other central nervous system (CNS) cells secrete miRNA and other small non-coding RNA into the extracellular space packaged in exosomes, micro-vesicles, or lipoprotein complexes [8].

In an interesting study, Prabhakar and colleagues in India, reported that four miRNAs (miR-409-3p, miR-502-3p, miR-486- 5p and miR-451a) could be used to differentiate iCSVD patients from healthy controls and might serve as diagnostic biomarkers for this disease [9]. However, to the best of our knowledge, no other studies have further investigated the role of these four miRNAs in patients with iCSVD among other populations or ethnic groups.

In a previous study, we reported that 63.7% of Egyptian patients with acute stroke are due to large artery disease, in addition to radiological evidence of CSVD [10]. Accordingly, we conducted this research to explore if any of the previously studied miRNAs could act as biomarkers for iCSVD in a sample of Egyptian patients. In addition to investigating if they have any clinical–radiological correlates.

This is an observational cross-sectional case-controlled study. It aims to compare the data of 30 patients with iCSVD to 30 age and sex matched healthy controls with regard to the prevalence of a set of miRNAs and their correlates with clinical and radiological data.

The study was approved by the ethical committee of the university and a written informed consent was obtained from patients or the first of kin to participate in the study. Patients were enrolled from outpatient clinics of the University Hospitals if their age was above 45 years, presenting with insidious onset of cognitive impairment, gait abnormalities or urinary incontinence and Magnetic Resonance Imaging (MRI) brain showing evidence of small vessel disease. Patients were excluded if they presented with or gave a history of acute stroke or showed carotid stenosis more than 50% on imaging. Cases were excluded also if they suffered from any other metabolic or neurological disease affecting white matter as sarcoidosis and multiple sclerosis. Healthy controls were included according to having no history of neurological, or any chronic metabolic disorders with a normal general and neurological examination. Vascular risk factors were not among the exclusion criteria for healthy controls.

All participants were subjected to comprehensive neurological assessment including history taking (past medical history, vascular risk factors, detailed history of current illness and drug history) and neurological examination. Cognitive assessment was done using Arabic version of Montreal Cognitive Assessment Test (MoCA) in which a maximum score of 30 denotes no cognitive affection while scoring ≤ 26 denotes more cognitive affection [11]. Gait assessment scores were compared between cases and controls and included: 10-m walk test (10MWT), self-selected velocity and fast velocity [12] and Berg Balance Scale (BBS) with highest possible score of 56 [13]. Arabic version of Beck Depression Inventory (BDI) [14] was used to assess depressive symptoms where a higher score indicates severer depression, with a maximum score of 63. Urinary problems were assessed using The Arabic International Consultation on Incontinence Questionnaire-Short Form (ICIQ-SF) Scale [15] with range 0–21 with higher grade denoting incontinence. MRI brain was performed using a 1.5T MR scanner (Achieva and Ingenia, Philips medical system, Eindhoven, Netherlands) for visual analysis of lesion burden of CSVD using Fazekas scale [16]. In addition, small vessel disease score (SVD) [17] was calculated by counting the presence of 4 MRI markers (white matter hyperintensities, lacunes, cerebral microbleeds, and perivascular spaces; ranging from 0–4). Also, brain atrophy was assessed using global cortical atrophy scale [18] which has a range 0–39 with higher grade signifying severe atrophy.

For miRNA analysis, venous blood samples collected using EDTA tubes were kept at − 80 degrees Celsius for later use. miRNA extraction was carried out on the stored plasma samples using simple miRNA extraction kit (Qiagen, Germany), according to the manufacturer's instructions. MiScript SYBR Green PCR kit (Qiagen, Germany) was used to perform quantitative PCR (qPCR) amplification of the extracted miRNAs in triplicates using the universal primer and miRNA-specific primers. The extracted miRNAs were reverse transcribed using the miScript II RT Kit (Eurofins, Belgium).

As a normalizer, a spike-in internal control (C. elegans miR-39 miRNA mimic, Qiagen, Germany) was employed, and each run's negative controls included using no template and negative RT reactions. In order to determine the specificity of the reactions, the initial denaturation stage of the reactions was conducted for 10 min at 92 °C, followed by 45 cycles of 95 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s followed by a high-resolution melt (HRM) analysis. The DTlite 4L thermal cycler (Russia) was used for temperature cycling.

Changes in the circulating levels of miRNAs were calculated by Livak analysis (2-ΔΔCt) method with fold change values more than 1.5 interpreted as up regulation and values less than or equal to 0.5 (2-fold reduction) were interpreted as downregulation. Patients’ fold change (RQ values) was calculated by normalizing the control (calibrators) data and calculating patients’ data accordingly [19].

This study involved 30 patients with iCSVD and 30 healthy controls. Both groups of cases and controls were age and sex matched with median age 61 years for cases and 61.5 years for controls. The male prevalence was 19 (63.3%) in cases and 18 (60%) in controls.

There was no significant difference among the 2 groups regarding the educational level with almost half of the subjects achieving 12–14 years of education. Additionally, there was no statistical difference between cases and controls regarding vascular risk factors.

The median (IQR) of the age of onset of symptoms in cases group was 65 (11) years old while the duration of illness was 2 (1) years.

Patients with iCSVD showed significant impairment of mood (p = 0.003), cognition, gait, and sphincteric control as compared to controls (p < 0.001) (Table 1).

The commonest presenting symptom was cognitive impairment representing 17 (56.7%) of cases followed by gait abnormality in 11 (36.7%), with only 2 patients were accidentally diagnosed due to vertigo or headache.

Regarding MRI brain, 93.3% had Fazekas scale grades 2 and 3. Also, the majority laid in SVD score 2 (50%) and 3 (33.3%). Global cortical atrophy scale had a range from 4–32 with a median (IQR) of 17 (9) and atherosclerosis was observed in all patients (Additional file 1: Table S1).

Regarding miRNA expression, cases with significant up- or down-regulation are shown in Fig. 1. It was noticed that 3 miRNAs (409-3p, 486-5p, 502-3p) were significantly downregulated with 90% of cases showing less than 0.5 (twofold reduction) regarding miRNA 409-3p and 486-5p and 80% of cases in miRNA 502-3p as compared to controls, while only one miRNA (451a) showed upregulation as 70% of cases had more than 1.5-fold change compared to controls.

There was significant correlation between dysregulated miRNA-486-5p with age and age of onset and negative correlation with duration of illness while dysregulated miRNA-451a showed negative correlation with SVD score as shown in Table 2.

The incidence of iCSVD was found to reach up to 6–10 times that of stroke [20], in addition it is the commonest underlying cause of vascular dementia [21]. The mainstay of management is to control vascular risk factors [22], and this warrants early diagnosis prior to development of permanent disability.

In this case-controlled study, 30 patients with iCSVD were recruited and assessed using clinical scales, MRI brain and blood sampling to detect the expression of four miRNAs as disease biomarkers.

In concordance with a previous study [23], we found no sex difference in iCSVD prevalence. Unexpectedly, there was no significant difference between both cases and control groups with regard to vascular risk factors, while in a recent systematic review and meta-analysis conducted in 2021, it was found that hypertension, diabetes, hyperlipidemia and smoking were significantly related to CSVD [24]. The possible differences between populations, as well as the variability of pathogenesis and presentation of iCSVD [25], are all considerable causes for this disparity of results, and this could point to the importance of further research among different populations with larger sample sizes.

Regarding the clinical scales, patients with iCSVD showed worse scores for depression and cognition, with slower gait speed and impaired sphincteric control than healthy controls. These findings were confirmed by other studies [26, 27].

The key finding of this study was the significant difference in expression of the studied miRNAs between cases and controls, with upregulation of miR-451a and downregulation of the rest (miR-409-3p, miR-486-5p and miR-502-3p). This indicates the potential role of dysregulated miRNA in the pathogenesis of iCSVD or the reciprocal effect of this disease on the homeostasis of certain miRNAs. This, however, still remains unclear and requires further studies in different populations.

These findings are partially in concordance with the previous study conducted in India yet (miR486-5p and miR-502-3p), which were upregulated unlike the results in the Egyptian sample [9]. This could be related to different genotypes of the disease or racial differences.

MicroRNAs usually regulate gene expression at a post-transcriptional level through multiple messenger RNAs (mRNAs) and functional proteins [28] and can therefore, modulate diverse biological processes, including cell differentiation, cell cycle, proliferation, apoptosis and cellular response to stress [29, 30].

MicroRNA was investigated in different CNS diseases as migraine [31] and amyotrophic lateral sclerosis [32], post-stroke cognitive impairment [33]. Moreover, it was employed to differentiate vascular from Alzheimer’s dementia [34]. And in 2017, Prabhakar and colleagues, reported 44 miRNAs that were differentially expressed in VaD patients compared to controls [9].

The neuroprotective role of miRNA in different neurological disorders was investigated thoroughly in a previously published review [35]. Cerebral ischemia in animal models was among the studied disorders where miRNA was reported to be dysregulated and to change following ischemic preconditioning. This suggests the important role of miRNA and its target proteins in cerebral ischemia and reperfusion [36, 37].

The finding that dysregulated miRNA had either positive or negative correlations with some clinical variables as age, age of onset and duration of illness; in addition to radiological biomarker represented by SVD score implies the sensitivity of miRNA to disease progression as well as the possible high yield for early diagnosis. This may be later confirmed by studying the same miRNA in patients at high risk of developing iCSVD and following up this group both clinically and radiologically to confirm such hypothesis [37].

These findings in addition to the few studies investigating the role of miRNA as a biomarker for iCSVD warrant further research on a larger scale with comparison of different races and populations, since this might offer an early diagnostic tool for iCSVD and consequently initiation of primary prevention before disability is established.

Incidental cerebral small vessel disease has no apparent age or sex predilection with huge impact upon quality of life by affecting cognition and gait. The possibility of early detection through the studied miRNAs before clinical manifestation would greatly help in preventive treatment either with the current available medications or by developing new ones that may alter the genetic pathway itself. However, more studies are needed in different populations.