Epigenome-Wide Association Study Reveals Differential Methylation Sites and Association of Gene Expression Regulation with Ischemic Moyamoya Disease in Adults

Background The association of DNA methylation with the pathogenesis of adult ischemic moyamoya disease (MMD) is unknown. Here, we investigated the genome-wide DNA methylation profiles in patients with MMD and identified the genes related to the pathogenesis of MMD. Methods Whole blood samples were collected from 20 individuals, including 10 patients with ischemic moyamoya disease without any underlying disease and 10 healthy individuals. Genome-wide DNA methylation analysis was performed using Illumina 850K microarrays. Transcriptional correlation was verified using quantitative reverse transcription-polymerase chain reaction. In vitro experiments were used to analyze the association of functional defects with candidate epigenetic markers. Results The genome-wide methylation level in the whole blood of adults with ischemic MMD was higher than that in the healthy individuals. In total, 759 methylation probes differed significantly between the case and control. The hypermethylated regions were mostly concentrated in the gene spacer regions. Among genes with the highest degree of the differential expression, KCNMA1 and GALNT2 were upregulated, whereas SOX6 and RBM33 were downregulated. Conclusions This is the first study showing that the low expression of genes associated with epigenetic regulation, such as SOX6 and RBM33, may be related to vascular occlusion in MMD, whereas the overexpression of KCNMA1 and GALNT2 may be related to the vascular hyperplasia. The results suggest that DNA methylation was involved in the pathogenesis of MMD, and new pathogenic genes were proposed as biological markers.

group: the GALNT2 sequence in the overexpression vector was transfected into HBMECs, which was cultured for 48 h for later use.
All the overexpressed vectors were prepared according to laboratory standards, and the target gene was amplified according to the design of the transfection primers.

Figure S1. Target gene construction
After determining the structure of the transfection vector and the sequence to be transfected, the following were mixed in a total volume of 50 µL for restriction enzyme digestion: ddH2O, 41 µL; 10× CutSmart Buffer2, 5 µL; 1 µg/µL pure plasmid DNA; 1 µL Nhe I (10 U/μL) and 1 µL Xho I (10 U/μL). The solution was mixed via mild agitation, centrifuged at 3,000 rpm for 1 min, and incubated at 37 C for 3 h. Agarose gel electrophoresis was performed on the digestion products of the carrier, and the target bands were recovered. One microliter of the linearized vector (100 ng/µL) was mixed with 1 μL double-stranded DNA (100 ng/μL), 2 μL ligase buffer, and 20 μL ddH2O to set up the ligation reaction overnightat 16 C. After the reaction, various ligated products were added to 100 µL of different groups of experimental cells, mixed gently, placed on ice, and incubated for 30 min. Then, the cells were heated at 42 C for 90 s and incubated in ice-water for 2 min.
After the culture, 500 µL basic medium was added and the cells were shaken at 37 °C for 1 h.
An appropriate amount of bacterial liquid was evenly spread on a plate containing ampicillin and incubated for 16 h.

Identification, amplification, extraction, and transfection of plasmids
First, PCR was performed for identification of genetic material in a colony, followed by extraction of plasmid from a PCR-positive colony, and transfection of the plasmid in experimental cells. The PCR system of 20 µL consisted of 10 µL 2× Taq Plus master mix, 9.2 µL ddH2O, and 0.4 µL each of upstream downstream primers. The reaction system was mixed via mild agitation and centrifuged at 3,000 rpm for 1 min. Next, a single colony was selected and placed in the reaction system using the head of a sterile gun on a sterile working the experimental grouping, were inoculated in 10 mL basic medium containing ampicillin and cultured overnight at 37 C. After the culture, the bacterial liquid was collected in different 5 mL centrifuge tubes and centrifuged at 12,000 rpm for 2 min. After centrifugation, the supernatant was removed, 250 µL cell resuspension was added, and the mixture was evenly mixed via sufficient oscillation. Cell lysate (250 µL) and 10 µL proteinase K were mixed via oscillation for 5−6 times and then allowed to stand for 1−2 min. Next, 350 µL buffer solution was added, and the mixture was reversed and shaken again to completely precipitate the protein product. The solution was incubated in ice water for 5 min. After freezing, the proteins were centrifuged at 10,000 rpm for 10 min. After centrifugation, the protein products were removed and the supernatant was aspirated in another sterile 1.5 mL tube. After centrifugation at 2,000 rpm for 5 min, the supernatant was transferred to a sterile recovery column. After centrifugation at 12,000 rpm for 1 min, the lower waste liquid was removed, and 600 µL of the rinsing liquid was added. The lower waste liquid was removed after centrifugation for 1 min at 12,000 rpm (the residual rinsing liquid was removed again as before). After the rinsing solution was cleaned, the recovery column was transferred to a new 1.5 mL tube on the aseptic operating table and allowed to stand for 10 min. Next, the column was air-dried, followed by addition of nuclease-free water. After standing for 2 min, the column was centrifuged at 12,000 rpm for 2 min and numbered.