Genetic analysis of RNF213 p.R4810K variant in non-moyamoya intracranial artery stenosis/occlusion disease in a Chinese population

Objectives RNF213 p.R4810K was identified as a susceptibility variant for moyamoya disease in Asia and non-moyamoya intracranial artery stenosis/occlusion disease in Japan and Korea recently. The occurrence of this variant was evaluated in patients with non-moyamoya intracranial artery stenosis/occlusion disease in China. Methods Two study populations were used in this study. One was recruited from the Second Hospital of Hebei Medical University from April 2015 to May 2016. The other was the archived DNA samples of intracranial artery stenosis/occlusion patients in XiangYa Hospital collected in 2014. The occurrence of RNF213 p.R4810K was investigated in a total of 715 patients with non-moyamoya intracranial artery stenosis/occlusion disease. The carrier rate of RNF213 p.R4810K in 507 normal individuals was used as control. Results Six of 715 patients (0.84%) with non-moyamoya intracranial artery stenosis/occlusion disease and 2 of the 507 normal controls (0.39%) had RNF213 p.R4810K variant. The carrier rate of RNF213 p.R4810K was higher in non-moyamoya intracranial artery stenosis/occlusion group than that in the normal group. However, no statistically significant association was observed (Odds ratio, 2.14; 95% confidence interval, 0.43–10.63; p = 0.56). Conclusions The carrier rate of RNF213 p.R4810K in Chinese non-moyamoya intracranial artery stenosis/occlusion disease patients was significantly lower than that in Korea or Japan. Genetic heterogeneity was highly indicated. Further systematic genetic epidemiology studies with emphasis on Chinese-specific genetic variants and environmental risk factors of intracranial artery stenosis/occlusion disease in larger population are needed.

RNF213 located in chromosome 17q25.3, encoding a 596 kDa protein which functions both as an AAA-type ATPase and an E3 ligase [20]. AAA-ATPases mediate various cell functions, including membrane fusion/transport, proteolysis, protein disaggregation/refolding, DNA recombination/repair and mitosis/meiosis [21]. AAA-ATPase dysfunction can cause several diseases, such as PEX1/PEX6 mutations cause multiple organ degeneration [22,23] and Cdc48 mutations cause amyotrophic lateral sclerosis [24,25]. E3 ligase activity may play a role in protein degradation or signaling processes [1]. Even the complete physiological functions of RNF213 are still unknown, knockdown of RNF213 in zebrafish leads to abnormal sprouting and irregular diameter of intracranial vessels, suggesting some contribution to vascular formation [1]. Previous studies revealed that a wide spectrum of phenotypes could occur within a family unit despite the members having the identical p.R4810K variant, with some individuals showing the typical phenotype of MMD such as bilateral stenosis/occlusion of the terminal portion of the internal carotid arteries, some showing only unilateral or middle cerebral artery stenosis/occlusion, and others with no abnormalities [1]. Miyawaki [13]. ICASO is an important and the most frequent cause of cerebral ischemic stroke among patients of Asian ancestry [26]. Liu et al. explored the association of RNF213 p.R4810K with MMD in Chinese population [3,5,[17][18][19], however, no association data was available about this variant with ICASO in China.
The aim of present study was to verify the generalizability of previous findings in Japan and Korea, investigating the association of RNF213 p.R4810K variant with ICASO not diagnosed as MMD in a Chinese population, compared with the occurrence of normal individuals as control group.

Study population
The study population was mainly recruited from the Department of Neurology of Hebei Medical University from October 2015 to May 2016. All the patients with ICASO in the absence of MMD who agreed to participate in this study in this period were enrolled (totally 615). In addition, another 100 ICASO patients in XiangYa Hospital of Central South University who had complete clinical information and archived DNA samples also included in this study. The carrier rate of RNF213 p.R4810K in 507 normal individuals published in other study was used as control [3]. This study was approved by the Medical Ethics Committee of Central South University and the survey participants gave informed consent before the interview and blood samples were taken.

Diagnosis of ICASO
Participants were diagnosed as non-moyamoya ICASO when they experienced focal or lateralizing symptoms and showed ≥50% stenosis or occlusion at terminal and/ or proximal portions of the intracranial major arteries without abnormal vascular networks in the basal ganglia on conventional angiography or MRA which was required by MMD diagnosed criteria [27]. The angiography images were interpreted by ≥2 physicians, including at least 1 radiologist and 1 neurological physician. Clinical information, including age, gender, vascular risk factors such as smoking, alcohol consuming, disease histories of hypertension, diabetes, hyperlipemia was collected (these diseases were defined as self-reported physician diagnosis or pharmaceutical treatment). Patients with potential sources of cardioaortic embolism, other stroke mechanisms such as coagulopathy, vasculitis, arterial dissection or incomplete evaluations were excluded.

Identification of RNF213 p.R4810K variant
Peripheral blood samples were obtained from all enrolled patients. Genomic DNA was extracted from the peripheral blood leukocytes using TIANamp Blood DNA Extraction Kit and following the manufacturer's instructions (TIANGEN BIOTECH CO., LTD, Beijing, China). Genotyping of RNF213 p.R4810K variant was performed by Taqman method (Assay ID: C_153120198_10; TaqMan SNP Genotyping Assays; Applied Biosystems) using a Roche LightCycler® 96 Real-Time PCR System (Roche, Switzerland) and analyzed with the LightCycler® 96 software. The investigators involved in genotyping were blinded from the phenotypic information. All analyses of the genotyped data were performed at the Department of Epidemiology and Health Statistics in Central South University.

Statistical analysis
All statistical analyses were performed using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA). Continuous variable (age) was presented as the mean ± standard deviation (SD). Categorical variables (hypertension, diabetes, hyperlipemia, coronary heart diseases, smoking, drinking) were presented as proportions. Categorical variables were compared using the χ2 test or Fisher exact test, and continuous variables were compared using Student t-tests. A p-value less than 0.05 was considered statistically significant.

Results
This study totally included 715 patients with non-MMD ICASO and 507 normal individuals without known cerebrovascular diseases. Clinical characteristics are shown in Table 1. Six of 715 ICASO patients (0.84%) and 2 of the 507 normal controls (0.39%) had the RNF213 p.R4810K variant (all heterozygotes). Even no statistically significant association was observed, the carrier rate of RNF213 p.R4810K was higher in ICASO group than that in the normal individuals (Odds ratio, 2.14; 95% confidence interval, 0.43-10.63; p = 0.56). Table 2 shows the clinical characteristics of 6 non-MMD ICASO patients with the RNF213 p.R4810K variant. The Fig. 1 shows the MRA images of the 4 patients with ICASO identified with the p.R4810K variant (the digital MRA images of the other 2 patients with the p.R4810K variant in the ICASO group was not available due to the fact that the patients were referrals from the other hospitals). These ICASO patients showed partial stenosis or occlusion of the intracranial major artery without abnormal vascular networks in the basal ganglia on MRA. All the patients had hypertension, 4 patients had diabetes and 2 elderly patients also had coronary heart disease for more than 20 years. Table 3 shows the distribution of RNF213 p.R4810K in MMD, Non-MMD ICASO and normal individuals in Japan, Korea and China. The carrier rate of RNF213 p.R4810K in Chinese MMD and non-MMD ICASO patients was significantly lower than that in Korea and Japan.

Discussion
In this study, only few of Chinese ICASO patients (6/715, 0.84%) carried RNF213 p.R4810K variant, which was significantly lower than that in Korea or Japan. Genetic heterogeneity of ICASO in different population was highly indicated.
RNF213 was a susceptibility gene for MMD [25]. Previous studies showed that in Japan and Korea, the founder variant RNF213 p.R4810K was much more frequent in MMD patients (~80%) than in the general population (~1.0%), significantly increased MMD risk (ORs > 100) [1,2,5,8,9,25]. However, as a susceptibility gene, Chinese MMD patients have significantly different genetic architecture. The carrier rate of RNF213 p.R4810K in Chinese MMD and general population is about 20~30% and 0.3% respectively, accounting for less part of MMD risk [1,3,5,[17][18][19]. The genetic result is consistent with the unique epidemiological and clinical characteristics of Chinese MMD. In China, no significant difference in sex distribution of MMD, a female predominance is not observed compared to Japan and South Korea. Moreover, the familial occurrence of MMD is lower and the symptoms at the onset are different from those in Japan and South Korea [28]. Genetic heterogeneity is proposed to be partially responsible for the different clinical features of MMD in different