Significant miRNAs as Potential Biomarkers to Differentiate Moyamoya Disease From Intracranial Atherosclerotic Disease

Dear Editor,

Moyamoya disease (MMD) is characterized by chronic progressive steno-occlusive arteriopathy that leads to the spontaneous occlusion of the circle of Willis with abnormal basal collaterals called moyamoya vessels.¹ The prevalence of MMD is higher in East Asian countries, such as Japan, South Korea, and China than in other countries.²

Both MMD and intracranial atherosclerotic disease (ICAD) are associated with stroke, transient ischemic attack (TIA), seizures, headache, cognitive decline, and other neurologic symptoms.¹,³ MMD and ICAD should be distinguished since they have different pathomechanisms and treatment options. However, differentiating MMD from ICAD based solely on angiographic findings is often challenging. In this study we aimed to differentiate MMD and ICAD using plasma microribonucleic acids (miRNAs) as biomarkers.

This multicenter, case-control study was conducted at two hospitals in South Korea. From January 2020 to August 2022, five patients with MMD confirmed by angiographic findings (MMD group) were enrolled. During the same period, five age- and sex-matched controls diagnosed with ICAD were recruited as control participants (ICAD group). None of the participants had been diagnosed with ischemic stroke or TIA within the 5 years before enrollment.

All patients in the MMD group were diagnosed based on the 2021 Diagnostic Criteria by the Research Committee on Moyamoya Disease by measuring ring finger 213 gene (RNF213) polymorphisms as follows¹,⁴: 1) cerebral angiography with bilateral progressive stenosis or occlusion of the terminal portion of the internal carotid artery and compensatory capillary collaterals, 2) abnormal vascular networks in the vicinity of the occlusive or stenotic lesions in the arterial phase, and 3) presence of an RNF213 polymorphism.

The study protocol was approved by the Institutional Review Board of Hanyang University (IRB No. 2019-11-034) and adhered to the principles outlined in the Declaration of Helsinki. All participants provided written informed consents before blood samples were collected. The experimental methods are described in detail in the Supplementary Material (in the online-only Data Supplement).

The 10 participants had an age of 56.2±9.4 years (mean±standard deviation). Age and sex were matched between the MMD and ICAD groups, with ages of 55.8±9.3 years versus 56.6±10.6 years and four females in each group. Vascular risk factors did not differ significantly between the two groups. All patients in the MMD group and none in the ICAD group had RNF213 polymorphisms (Supplementary Table 1 in the online-only Data Supplement). Magnetic resonance angiography findings for all participants are shown in Supplementary Fig. 1 (in the online-only Data Supplement), and one additional transfemoral cerebral angiography image is shown in Supplementary Fig. 2 (in the online-only Data Supplement).

Table 1 comprehensively lists p values and fold changes for the 84 genes from a cardiovascular diseases (CVD) panel, excluding the 12 internal controls. The expression patterns of the 84 miRNAs in the MMD and ICAD groups were visualized using cluster graphs, scatter plots, and volcano plots (Supplementary Fig. 3 in the online-only Data Supplement). Five of the 84 miRNAs examined (hsa-miR-100-5p, hsa-miR-10b-5p, hsa-miR-150-5p, hsa-miR-30a-5p, and hsa-miR-7-5p) were significantly downregulated in the MMD group (p<0.05). However, only three of these miRNAs (hsa-miR-100-5p, hsa-miR-10b-5p, and hsa-miR-150-5p) displayed fold changes larger than 2. The associations between these three downregulated miRNAs in the plasma of patients with MMD and ICAD are shown in Supplementary Fig. 4 (in the online-only Data Supplement). These three miRNAs in the patients with MMD were strongly correlated with the adenylate cyclase (ADCY)-inhibiting adrenergic receptor signaling pathway. ADCYs are known to be related to memory, learning, and movement,⁵ and many neurologic disorders are associated with abnormal ADCYs: ADCY1, -5, and -7 are associated with sleep deprivation or autism, familial dyskinesia and movement disorders, and autoimmune diseases, respectively.⁵ To our best knowledge, the present study is the first to identify associations between MMD and ADCYs.

Table 1. Probability values and fold changes for comparisons between the MMD and ICAD groups in an miRNA PCR array.

miRNA	p	Fold change
hsa-miR-100-5p	0.048	-2.66
hsa-miR-10b-5p	0.007	-2.24
hsa-miR-150-5p	0.032	-2.74
hsa-miR-30a-5p	0.005	-1.80
hsa-miR-7-5p	0.032	-1.88
hsa-let-7d-5p	0.089	2.13
hsa-miR-103a-3p	0.080	2.91
hsa-miR-107	0.069	2.53
hsa-miR-122-5p	0.321	-3.61
hsa-miR-124-3p	0.317	-4.96
hsa-miR-126-3p	0.180	2.05
hsa-miR-130a-3p	0.113	2.07
hsa-miR-142-3p	0.093	3.13
hsa-miR-146a-5p	0.105	2.38
hsa-miR-149-5p	0.067	-2.41
hsa-miR-15b-5p	0.100	2.70
hsa-miR-17-5p	0.109	2.20
hsa-miR-181b-5p	0.073	-2.11
hsa-miR-182-5p	0.067	-2.41
hsa-miR-183-5p	0.067	-2.41
hsa-miR-199a-5p	0.118	2.04
hsa-miR-206	0.067	-2.41
hsa-miR-208a-3p	0.067	-2.41
hsa-miR-208b-3p	0.067	-2.41
hsa-miR-21-5p	0.141	2.71
hsa-miR-214-3p	0.067	-2.41
hsa-miR-221-3p	0.073	4.15
hsa-miR-223-3p	0.084	3.57
hsa-miR-23a-3p	0.148	2.20
hsa-miR-23b-3p	0.099	2.56
hsa-miR-24-3p	0.146	2.16
hsa-miR-26a-5p	0.092	2.91
hsa-miR-26b-5p	0.090	2.52
hsa-miR-27a-3p	0.091	2.71
hsa-miR-27b-3p	0.132	2.74
hsa-miR-302a-3p	0.067	-2.41
hsa-miR-302b-3p	0.067	-2.41
hsa-miR-31-5p	0.067	-2.41
hsa-miR-365a-3p	0.067	-2.41
hsa-miR-423-3p	0.135	2.20
hsa-miR-486-5p	0.131	-2.23
hsa-miR-499a-5p	0.067	-2.41
hsa-let-7a-5p	0.230	1.55
hsa-let-7b-5p	0.859	1.18
hsa-let-7c-5p	0.172	1.57
hsa-let-7e-5p	0.763	-1.05
hsa-let-7f-5p	0.230	1.54
hsa-miR-1-3p	0.081	-1.70
hsa-miR-125a-5p	0.988	-1.11
hsa-miR-125b-5p	0.131	-1.54
hsa-miR-133a-3p	0.212	-1.64
hsa-miR-133b	0.184	-1.42
hsa-miR-140-5p	0.731	-1.05
hsa-miR-143-3p	0.892	-1.03
hsa-miR-144-3p	0.812	1.10
hsa-miR-145-5p	0.609	1.11
hsa-miR-155-5p	0.072	-1.95
hsa-miR-16-5p	0.323	-1.20
hsa-miR-181a-5p	0.354	1.26
hsa-miR-185-5p	0.558	1.56
hsa-miR-18b-5p	0.408	1.14
hsa-miR-195-5p	0.187	-1.49
hsa-miR-210-3p	0.193	-1.43
hsa-miR-22-3p	0.299	1.67
hsa-miR-222-3p	0.651	-1.03
hsa-miR-224-5p	0.073	-1.95
hsa-miR-25-3p	0.400	-1.12
hsa-miR-29a-3p	0.323	-1.46
hsa-miR-29b-3p	0.114	-1.50
hsa-miR-29c-3p	0.306	-1.38
hsa-miR-30c-5p	0.265	1.26
hsa-miR-30d-5p	0.161	1.79
hsa-miR-30e-5p	0.572	1.35
hsa-miR-320a-3p	0.743	1.35
hsa-miR-328-3p	0.226	1.44
hsa-miR-342-3p	0.709	-1.36
hsa-miR-378a-3p	0.054	-1.52
hsa-miR-424-5p	0.920	1.02
hsa-miR-451a	0.437	-1.38
hsa-miR-494-3p	0.110	-1.83
hsa-miR-92a-3p	0.292	-1.20
hsa-miR-93-5p	0.576	1.56
hsa-miR-98-5p	0.081	-1.71
hsa-miR-99a-5p	0.093	-1.59

ICAD, intracranial atherosclerotic disease; MMD, moyamoya disease.

Molecular biomarkers associated with moyamoya angiopathy, including proteins, genes, and signaling pathways, have recently been suggested,⁶ and some miRNAs in patients with MMD or ICAD have been investigated.⁷,⁸,⁹ However, to our knowledge there have been no reports on (hsa-miR-100-5p, hsa-miR-10b-5p, and hsa-miR-150-5p), which are thought to significantly differentiate MMD from ICAD, and also the roles of these miRNAs in MMD are not well understood. Our study has demonstrated that these three miRNAs are promising biomarkers.

This study had some notable limitations. First, no samples were obtained from healthy controls, and so it was unclear whether the levels of the three miRNAs (hsa-miR-100-5p, hsa-miR-10b-5p, and hsa-miR-150-5p) were significantly downregulated in MMD relative to healthy subjects. Second, the sample was small. Third, no racial differences were investigated since the study only included individuals of one race, among which the prevalence of MMD is relatively high. Fourth, the targets for the miRNA PCR array kit used were CVD, and not stroke, MMD, or ICAD. Fifth, considering that all patients in the MMD group were positive for RNF213, the presence of this gene might have affected the miRNAs. Sixth, vascular and brain tissues were not analyzed. The definition of MMD continues to change, and differentiating between ICAD and MMD has not been fully characterized. This suggests that the classification of patients with ICAD or MMD is unclear and that the diagnostic criteria for these diseases could change in the future.

In conclusion, the findings of this study suggest that plasma miRNAs can be used to distinguish patients with MMD from those with ICAD. Future research should include follow-up samples from earlier stages of the disease, multicenter studies with larger numbers of participants, and additional analyses such as real-time PCR or small RNA sequencing.

Availability of Data and Material

The datasets generated or analyzed during the study are available from the corresponding author on reasonable request.

Supplementary Materials

The online-only Data Supplement is available with this article.

Supplementary Table 1

Baseline characteristics and prevalence of RNF213 variant

Supplementary Fig. 1

Magnetic resonance angiography images of patients. Representative images of patients with moyamoya disease (A-E) and age–sex matched intracranial atherosclerotic disease (F-J).

Supplementary Fig. 2

Transfemoral cerebral angiography of the patient with moyamoya disease of Supplementary Fig. 1E.

Supplementary Fig. 3

Clustergram, scatter plot, and volcano plot images representing miRCURY miRNA PCR array results using human plasma samples from patients with MMD and ICAD. A: The clustergram shows significant expression levels of 84 miRNAs in both MMD and ICAD samples, with colors representing the magnitude of gene expression. B: The scatter plot shows comparative log10 values among MMD and ICAD groups. C: The volcano plot compares log2 values of genes in the MMD group to the ICAD group, enabling the identification of differential gene expression patterns. ICAD, intracranial atherosclerotic disease; MMD, moyamoya disease.

Supplementary Fig. 4

Associated genes and pathways between differentially expressed miRNAs and MMD and ICAD using bioinformatics tools. miRNA PCR array analysis revealed that hsa-miR-100-5p, hsa-miR-10b-5p, and hsa-miR-150-5p were significantly downregulated in the MMD group compared to the ICAD group. A: The interaction network between these three genes, MMD, and ICAD, was constructed using Novus Biologicals bioinformatics analysis. Grey lines represent the connections established through relevant references, whereas blue and orange circles depict the associated genes and pathways, respectively. B and C: Biological processes (B) and molecular functions (C) of these three genes were investigated using Gene Ontology analysis. ICAD, intracranial atherosclerotic disease; MMD, moyamoya disease.