JPWO2020121489A1 - Method for predicting the risk of developing cerebral infarction - Google Patents

Abstract

It provides a method for predicting the risk and time of onset of cerebral infarction. RNF213 p. In a sample derived from a subject who has not developed cerebral infarction. A detection step for detecting the presence or absence of the R4810K gene polymorphism, and RNF213 p. A method for predicting the onset time of cerebral infarction in a subject who has not developed cerebral infarction, including a determination step for determining whether or not the onset time of cerebral infarction of the subject is earlier than usual depending on the presence or absence of the R4810K gene polymorphism. .. RNF213 p. A genetic marker consisting of the R4810K gene polymorphism for predicting the risk of developing cerebral infarction. RNF213 p. A biomarker for predicting the risk of developing cerebral infarction, which comprises a polypeptide encoded by the R4810K gene.

Description

The present invention belongs to the prediction of the onset risk and the onset time of cerebral infarction, and the research fields thereof, and particularly relates to a method for predicting the onset risk of cerebral infarction for a healthy person.

In Non-Patent Document 1, the RNF213 gene polymorphism (c.14576G> A, p.R4859K, rs112735431), which is a susceptibility gene for moyamoya disease, is found not only in moyamoya disease but also in the intracranial region of unilateral moyamoya disease and atherosclerosis. It has been disclosed that it is also significantly associated with main artery stenosis (intracranial stenosis), while the RNF213 gene polymorphism was not significantly associated with cervical carotid artery stenosis, cerebral aneurysm, or intracerebral hemorrhage. Is disclosed.

The notation (c.14576G> A, p.R4859K, rs112735431) regarding the RNF213 gene polymorphism in Non-Patent Document 1 is based on the naming by the Tohoku University group. In the present specification, the same RNF213 gene polymorphism is referred to as p. R4810K polymorphism (c. 14429G> A, rs112735431) or simply p. Notated as R4810K polymorphism. Both are due to differences in the way amino acids are counted and are the same polymorphism.

"RNF213 and Moyamoya syndrome", Satoshi Miyawaki, Niche Neuro-Angiology Conference 2016, http://nnac.umin.jp/nnac/NNAC#2016#files/%E5%AE%AE%E8% 84% 87% E5% 85% 88% E7% 94% 9F.pdf

An object of the present invention is to provide a method for predicting the onset risk and time of onset of cerebral infarction.

Cerebral infarction is classified into three types: atherothrombotic brain infarction, lacunar infarction, and embolism.

According to the studies by the present inventors, RNF213 p. It was found that there was a significant correlation between the R4810K gene polymorphism and atherothrombotic cerebral infarction. From this, a method (or auxiliary) of predicting the onset risk and time of cerebral infarction of the subject by detecting the presence or absence of the RNF213 gene polymorphism in the sample derived from the subject who has not developed cerebral infarction. How to predict) is provided.

The present invention includes the following inventions.
(1) RNF213 p. In a sample derived from a subject who has not developed cerebral infarction. A detection step for detecting the presence or absence of the R4810K gene polymorphism,
The RNF 213 p.I. A determination step for determining whether or not the subject has an earlier onset of cerebral infarction depending on the presence or absence of the R4810K gene polymorphism.
A method for predicting the onset time of cerebral infarction in a subject who has not developed cerebral infarction.

(2) In the detection step, the RNF 213 p. When the R4810K gene polymorphism is detected, in the determination step, the RNF213 p. The method according to (1) above, wherein the subject is determined to have an earlier onset of cerebral infarction than a person who does not have the R4810K gene polymorphism.

(3) In the determination step, the RNF 213 p. The method according to (1) or (2) above, wherein the subject determines whether or not the onset of cerebral infarction is earlier than usual based on the presence or absence of the R4810K gene polymorphism and the identification information of the subject.

(4) RNF213 p. A genetic marker consisting of the R4810K gene polymorphism for predicting the risk of developing cerebral infarction.

(5) RNF213 p. A biomarker for predicting the risk of developing cerebral infarction, which comprises a polypeptide encoded by the R4810K gene.

Based on the findings of the present invention, it is possible to prevent and take measures against cerebral infarction from an early stage for a subject who has not developed cerebral infarction.

The flow chart of the patient selection procedure is shown. FIG. 2 is a Forest plot of the odds ratio of ischemic stroke between RNF213 p.R4810K polymorphic carriers and non-carriers by stroke subtype. SE is the standard error; IV is the inverse variance method; 95% CI is the 95% confidence interval; FSR is the Fukuoka Stroke registry; NCVC is the national cardiovascular disease. The centers (National Cerebral and Cardiovascular Center) are shown respectively. FIG. 3 is a forest plot of odds ratios for ischemic stroke between RNF213 pR4810K polymorphic carriers and non-carriers by gender. SE is the standard error; IV is the inverse variance method; 95% CI is the 95% confidence interval; FSR is the Fukuoka Stroke registry; NCVC is the national cardiovascular disease. The centers (National Cerebral and Cardiovascular Center) are shown respectively. It is a regional plot of the RNF213 locus (coordinates 77358945-79358945 of chromosome 17) for ischemic stroke. FIG. 5 is a comparison of stroke ages between RNF213 p.R4810K polymorphic carriers and non-carriers. SE is the standard error; IV is the inverse variance method; 95% CI is the 95% confidence interval; FSR is the Fukuoka Stroke registry; NCVC is the national cardiovascular disease. The centers (National Cerebral and Cardiovascular Center) are shown respectively.

RNF213 (Ring finger protein 213) (GenBank accession number NM_001255671.1.) Has been recently identified as a disease susceptibility gene for moyamoya disease and is present in the human chromosomal region 17q25.3.

RNF213 p. The R4810K gene polymorphism is a single nucleotide polymorphism (SNP; Single Nucleotide Polymorphism) of 73097 G> A in the nucleotide sequence represented by SEQ ID NO: 2. RNF213 p. R4810K is known as a moyamoya disease-sensitive polymorphism as described in the above-mentioned prior art.

According to the research of the present inventors, RNF213 p. The R4810K polymorphism was found to increase the risk of ischemic stroke (ie, cerebral infarction) due to aortic atherosclerosis. In the present specification, ischemic stroke is synonymous with cerebral infarction.

Although ischemic stroke is one cause of premature death in recent years and is a major cause of disability and premature death, especially in Asia, the unique genetic deterministic correlation with ischemic stroke remains unknown. Moyamoya disease, a cerebrovascular disease found primarily in East Asia, is associated with a susceptibility gene called RING finger protein 213 (RNF213), the dysregulation of which impairs cerebral perfusion in the mouse brain. We therefore hypothesize that RNF213 plays a more common role in ischemic stroke, and p. The association between R4810K polymorphisms and ischemic stroke and its subtypes was investigated.

From three independent Japanese studies of ischemic stroke, we present case-control data (case-control) of 46,958 East Asian ancestors (17,752 cases and 29,206 healthy cases (control)). control data) was analyzed. We performed a meta-analysis of East Asians under a fixed effects model.

As a result, a complex meta-analysis of East Asians revealed that p. R4810K polymorphisms are total ischemic stroke (OR (odds ratio) 1.91, 95% CI (95% confidence interval) 1.55-2.36, p = 1.5x10 ^-9 ) and aortic atherosclerosis. It was shown to be significantly associated with the disease (OR 3.58, 95% CI 2.55-5.03, p = 2.0x10 ^-13). When stratified by gender, this association was more pronounced in females (OR 1.50, 95% CI 1.14-1.98, p = 0.004 in males: OR 2.69 in females, 95% CI 1.95-3.69, p = 1.2x10 ^-9 ). The average age of stroke onset is RNF213 p. RBF213 p. R4810K polymorphic carriers were 4.1 years lower (p = 1.1x10 ^-8 ). Here, the term "the onset time of cerebral infarction is earlier than usual" in the present specification is referred to as RNF213 p. Earlier than the average time of onset of cerebral infarction among non-carrying R4810K polymorphisms, or RNF213 p. R4810K polymorphic holder and RNF213 p. It means that it is earlier than the average time of onset of cerebral infarction including those who do not have R4810K polymorphism.

From these, RNF213 p. It is concluded that the R4810K polymorphism is a genetic risk factor for ischemic stroke, especially ischemic stroke in aortic atherosclerosis.

RNF213 p. The R4810K gene polymorphism is a single nucleotide polymorphism (SNP) of 73097 G> A in the nucleotide sequence represented by SEQ ID NO: 2, and is used to predict or assist in predicting the risk of developing cerebral infarction. Can be a genetic marker for.

SEQ ID NO: 2 is a partial nucleotide sequence of human chromosome 17 DNA containing the mysterin gene and genes in the peripheral region [FLJ3520, NPTX1, CARD14, and Raptor (KIAA1303)], and is registered in NCBI with Contig # NT010783. Corresponds to the 43560001 to 43795000th nucleotides of .15.

In addition to the SNP at position 73097 (abbreviated as 73097 G> A in the present specification) which is G or A, the nucleotide sequence represented by SEQ ID NO: 2 includes
4766th SNP (4766 T> C), which is T or C,
The 120764th SNP (120764 G> A), which is G or A,
There may also be a SNP at position 152917 (152917 G> A) that is G or A, and an SNP at position 232102 that is G or A (232102 G> A).

In addition, in this specification, the position of SNP is described with reference to the position of a nucleotide in the nucleotide sequence represented by SEQ ID NO: 2. For example, "SNP at position 73097" means the SNP at the nucleotide at position 73097 in the nucleotide sequence represented by SEQ ID NO: 2. When describing "73097 G> A" or the like, the base of the major allele (G in this case) is described before the symbol ">", and the base of the minor allele (A in this case) is described after.

Further, in the present specification, the nucleotide sequence is described as a DNA sequence unless otherwise specified, but when the polynucleotide is RNA, thymine (T) shall be appropriately read as uracil (U).

In the present invention, the polynucleotide may contain any additional sequence in addition to the continuous partial sequence of the nucleotide sequence represented by SEQ ID NO: 2 or its complementary sequence.

In the present invention, the polynucleotide is preferably isolated or purified.

In the detection step of the above method of the present invention, an SNP of 73097 G> A is detected in a biological sample collected from a subject.

The human race is not particularly limited, but is preferably East Asian (East Asian / Mongoloid).

Here, race is a group of Homo sapiens species that can be distinguished as a specific subgroup. Races have unique, distinguishable combinations of genes, and are identified by the characteristics (both mental and physical) created by the combination of genes. Members of the same race share a common genetic ancestor and, as a result, share similar genetic combinations and thus share distinct genetic characteristics.

For example, genetic relationships have been investigated for the world's major human populations based on information from 23 different genes, including African (Negroid), Caucasian (White), Oceanian (Australoid), and East Asian (Mongoloid). And Native American are classified into 5 types.

An East Asian means a person whose origin is one of the people of Japan, Korea, China, Taiwan and Mongolia. East Asians are preferably Japanese, Korean, or Chinese.

A person skilled in the art can easily identify the race of an individual based on information on the physical characteristics of the individual, the country of origin, the origin of the ancestors, and the like.

As the biological sample used in the above method, any tissue, cell, body fluid or the like from which genomic DNA can be collected can be used, but from the viewpoint of easy availability and minimal invasiveness, hair, nails, etc. Skin, mucous membranes, blood, plasma, serum, saliva and the like are preferably used.

Methods for detecting SNPs are well known in the art. For example, RFLP (restricted enzyme fragment length polymorphism) method, PCR-SCSP (single-stranded DNA higher-order structure polymorphism analysis) method, ASO (Allele Specific Oligonucleotide) hybridization method, sequencing method, ARMS (Amplification Refracting Mutation System) ) Method, Denaturing Gradient Gel Electrophoresis method, RNAseA cleavage method, DOL (Dye-labeled Oligonucleotide Ligation) method, TaqMan PCR method, primer extension method, invader method and the like can be used.

In addition, RNF213 p. The polypeptide encoded by the R4810K gene is a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 (where, arginine No. 4810 in the amino acid sequence represented by SEQ ID NO: 1 is replaced with lysine). It can be a biomarker for predicting the risk of developing cerebral infarction.

That is, as described above, RNF213 p. Since the SNP mutation in R4810K is associated with amino acid substitution of human mysterin No. 4810 (arginine → lysine), the brain was identified by isolating the mysterin polypeptide from the subject and identifying the amino acid No. 4810. It is possible to predict or assist in predicting the risk of developing infarction.

Examples are shown below and the present invention will be specifically described, but the present invention is not limited to the examples.

Stroke mortality has declined over the last few decades ^1), but stroke is the second leading cause of premature death worldwide and the leading cause of disability ²⁾ . In Southeast Asia and East Asia, which make up 31% of the world's population, stroke is a major cause of premature death, and stroke incidence and morbidity are steadily increasing ^1,2) . Epidemiological studies suggest substantive geographical and racial differences in stroke subtypes ^3,4) . Cardiogenic embolism is a common etiological subtype of ischemic stroke in Western Europe ⁵⁾ . On the other hand, aortic atherosclerosis caused by intracranial artery stenosis is the main cause in most Asian countries ⁶⁾ . Differences in environmental risk factors and genetic background are believed to be the main reasons for the high prevalence of aortic atherosclerosis in Asia. Recently, a large multiancestral genome-wide association study identified 32 loci associated with stroke and stroke subtypes ⁷⁾ . In this study, about 8 million single nucleotide polymorphisms (SNPs) were tested. However, this analysis excludes SNPs with a minor-allele frequency (MAF) of less than 1% (<0.01), and the Asian-specific genetic determinants of ischemic stroke remain unknown. Is.

The ring finger protein 213 gene (RNF213) on 17q25.3 has been identified as a susceptibility gene for moyamoya disease ^8,9) . More than 80% of patients with moyamoya disease have RNF213 p. The R4810K polymorphism (c. 14429G> A, rs112735431) was detected, but the allele carrier frequency in healthy East Asian subjects was about 2% ^8,9) . ^{RNF213 encodes a 591 kDa protein12)} that functions as both AAA + AMPase and E3 ligase, and is associated with the development of intracranial main trunk obstruction lesions and the compensatory response to reduced cerebral blood ^flow13,14) . Recently, two individual studies have conducted RNF213 p. A high frequency (20-25%) of R4810K polymorphisms has been reported 10,15 ⁾ . Therefore, we hypothesized that this genetic variation may be associated with overall ischemic stroke in Asia. Here, we analyze patients with acute ischemic stroke who participated in three independent Japanese studies, and RNF213 p. The association between R4810K polymorphisms and ischemic stroke and its subtypes was investigated.

(Method)
(Research design and participants)
In this two-step case-control study, we examined ischemic stroke data from East Asian participants in three case-control studies from Japan. We excluded patients and controls that were not of East Asian progeny. The primary study included a single hospital-based population (NCVC Biobank) of the National Cerebral and Cardiovascular Center (NCVC) containing extensive clinical and radiological data for all patients. used. In replication studies, p. The R4810K genotype is from Biobank Japan (16,256 ischemic stroke cases and 27,294 controls), and Hisayama and Fukuoka Stroke Registry (FSR) studies (1,113 cases and controls 901). It is derived from genotype analysis data of the whole genome. Written consent was obtained from all study participants and the study was approved by the Responsible Ethics Board and the National Cardiovascular Research Center (NCVC) Institutional Review Board.

(Primary study)
Participants in the main study were recruited at the National Cardiovascular Research Center (NCVC), a 600-bed tertiary center specializing in stroke and cardiovascular disease in the Osaka and Kansai regions. Non-cardiogenic cerebral embolism (large-artery atherosclerosis, small-) who was hospitalized from June 2012 to May 2017 and signed a comprehensive consent form at NCVC. 383 Japanese patients (vessel occlusion), etc.) were included in this study. Based on the criteria of the Moyamoya Disease Research Committee of the Ministry of Health, Labor and Welfare of Japan, patients diagnosed with cardioembolic stroke, deterministic / probably moyamoya disease, were excluded17 ⁾ . The flow chart of the patient selection procedure is detailed in FIG.

Demographic data, atherosclerosis risk factors, radiological findings, and medical history were obtained from a prospectively-collected database of prospective stroke patients. Stroke subtypes were classified according to the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria ¹⁸⁾ . Cerebral vascular lesions were identified by magnetic resonance imaging (MRI) and MR angiography, or CT and CT angiography. Anterior circulation stenosis is an intracranial internal carotid artery, middle cerebral artery or anterior cerebral artery according to Warfarin-Aspirin Symptomatic Intracranial Disease (WASID) research criteria ^19). It was defined as a diameter reduction of more than 50% (> 50%) in the anterior cerebral artery. Similarly, posterior circulation stenosis was defined according to the above criteria in the intracranial, posterior, or posterior cerebral arteries.

Control subjects were recruited in the Kansai area of Japan between 2007 and 2015. Of the 1,027 potential controls, 16 subjects with a history of cerebral infarction or moyamoya disease were excluded. Finally, 1,011 control subjects were selected. Intravenous blood samples were collected and stored at −80 ° C. until analysis. As previously described ⁸⁾ , using TaqMan SNP Assays (Applied Biosystems, Foster City, CA) and 7300/7500 Real-Time PCR System (Applied Biosystems, Foster City, CA) p. Genotyping of R4810K was performed.

(Replication studies)
By retrieving the data from the previous report ^7), ischemic stroke (aortic atherosclerosis (large-artery atherosclerosis), small vessel occlusion (small-vessel occlusion), and cardiogenic cerebral embolism (Cardioembolism) ) And RNF213 p. Confirmation of the association between R4810K polymorphisms was made. Specifically, 16,256 patients with ischemic stroke were enrolled from the Biobank Japan project. Controls include Iwate Medical Megabank (IMM), Japan Public Health Center-based Prospective study (JPHC), and Japan Multi-institutional Collaborative Cohort. It was obtained from four population-based studies, Study (JMICC)) and Tohoku Medical Megabank (ToMMo). In addition, 1,113 ischemic strokes and 901 controls were obtained from the Hisayama study of cardiovascular disease in Japan and the FSR study, a multicenter hospital-based enrollment of patients with acute stroke in Japan. ..

SNP genotyping was performed at the RIKEN Center for Integrative Medical Sciences using a combination analysis of Illumina HumanOmniExpress BeadChips and HumanExome BeadChips or Illumina Human OmniExpressExome BeadChip analysis. To exclude subjects outside of East Asia, standard quality control procedures were applied, including Identity-by-Descent analysis and principal component analysis to ensure that the samples were irrelevant. Haplotype phasing and genotype imputation were performed using filtered samples with genotype data that passed quality control criteria, including success rates and Hardy-Weinberg equilibrium tests. A genome-wide association study (GWAS) was performed using the doses of the assigned alleles and fitted to a logistic regression model with an additive genetic model. We have incorporated 10 principal components, age, and gender as covariates. Details are given earlier ⁷⁾ .

Samples were excluded if:
1) When the rate of defect displacement is high (deficiency> 0.05)
2) If there is a discrepancy between the reported gender or ethnicity and the genetically determined gender or ethnicity.
3) When showing potential relevance

(Statistical analysis)
Continuous variables were expressed as mean ± SD and compared using Student's t-test. Category variables were expressed as numbers and percentages and compared using the chi-square test and the two-tailed Fisher's exact test as appropriate. We tested the association between RNF213 polymorphisms and the risk of ischemic stroke assuming a dominant model in the main study due to the inadequate number of homozygotes. We also investigated the relevance under a log-additive model for meta-analysis for comparison with replication studies using imputation methods. rice field. Odds ratios (OR Ratio) and 95% confidence interval CIs were calculated for each stroke subtype after simultaneous control of potential interrelated factors using multiple logistic regression models. The variables considered in the model were age (continuous), gender, hypertension, dyslipidemia, diabetes and smoking. All analyzes were performed using JMP Pro 12.2 software (SAS Institute Inc., Cary, NC).

The probability values were bilateral and p <0.05 was considered significant. Meta-analysis, p <5x10 ^-8 by using the inverse dispersion fixed effects model considered significant throughout the genome (inverse variance fixed effects model), Review Manager (RevMan) 5.3 software (The Nordic Cochrane Center, Denmark, Copenhagen).

(result)
(Primary study)
The main study enrolled patients with non-cardiogenic cerebral embolism stroke in particular. Of the 1,775 noncardiogenic cerebral embolism stroke patients who met the above comprehensive criteria, 383 (21.6%) agreed to participate in the NCVC Biobank and were analyzed in this main study. The characteristics of both participants and non-participants in the NCVC Biobank are shown in Table 1. Participants were younger and fewer females compared to non-participants at the NCVC Biobank. Dyslipidemia and smoking habits were higher in participants than in non-participants, but the distribution of other risk factors and stroke subtypes was similar. Table 2 shows the baseline characteristics of the main research participants. Stroke patients were older, had fewer females, and had more risk factors for atherosclerosis than controls.

RNF213 p. R4810K polymorphisms were found in 5.2% of patients with noncardiogenic cerebral embolism stroke and 2.1% of healthy controls (crude OR 2.60, 95% CI 1.39-4.85). , P = 0.0019). After adjusting for age, gender, and risk factors for atherosclerosis, RNF213 p. The association between R4810K polymorphisms and noncardiogenic cerebral embolism stroke remained significant (adjusted OR 3.90, 95% CI 1.62-9.24, p = 0.0026). Only aortic atherosclerosis was significantly associated with polymorphism compared to control subjects (coarse OR 5.19, 95% CI 2.53-10.64, p = 2.6x10 ^-6 , Adjusted OR 11.45, 95% CI 3.46-36.17, p = 0.0001) (Table 3).

RNF213 p. In a comparison between stroke patients with and without R4810K polymorphism, the average age of stroke onset was lower with RNF213 polymorphism than with non-carriers (58.1 ± 15.5 years vs. 69.1 ± 13.2 years, p = 0.0003) (Table 4).

As can be seen from Tables 2 and 4, the average age of onset of cerebral infarction among subjects with and without RNF213 polymorphism (68.5 years) and the average age of onset of cerebral infarction among subjects with RNF213 polymorphism (average in Table 4). Even when compared with 58.1 years old), the age of onset of cerebral infarction is about 11 years younger in patients with RNF213 polymorphism.

In stroke patients, RNF213 polymorphic carriers contained more females (55.0% vs. 27.3%, p = 0.011) and intracranial anterior circulatory stenosis (60.0% vs. 60.0%) than non-carriers. It showed a higher frequency of 27.3%, p = 0.004) and aortic atherosclerosis (65.0% vs. 32.5%, p = 0.012). Conventional atherosclerotic risk factors such as the development of extracranial internal carotid artery and posterior circulation stenosis, and the incidence of hypertension, diabetes, dyslipidemia and smoking are There was no difference between polymorphic carriers and non-carriers.

(Replication studies)
The present inventors have RNF213 p. For the R4810K polymorphism, 16,256 cases of ischemic stroke (including cardiogenic cerebral embolism stroke) and 27,294 cases of East Asian descent registered from BioBank ^{Japan Project 7) were used.} searched. RNF213 p. The R4810K polymorphism was found in 2.3% of all ischemic stroke patients and 1.3% of the general population. RNF213 p. A significant association was found between the R4810K polymorphism and all ischemic strokes (ncase = 16,256) for genotypic frequency (OR 1.77,95% CI 1.40-2.24,). p = 1.6x10 ^-6 ). The average age of stroke onset is RNF213 p. The R4810K polymorphism was low (62.1 ± 10.0 years vs. 66.0 ± 10.0 years, p = 8.7x10 ^-7 ).

Analysis of only patients with aortic atherosclerosis (ncase = 1,256) revealed that 4.0% of patients had RNF213 p. It has an R4810K polymorph and has RNF213 p. The association with the R4810K polymorphism was more pronounced (OR 3.10, 95% CI 1.98-4.84, p = 6.9x10 ^-7 ; stroke age 56.9 ± 10.2 years vs. 65). .0 ± 9.7 years old, p = 6.3x10 ^-5 ). On the other hand, RNF213 p. R4810K polymorphism and small-vessel occlusion (ncase = 4,613, OR 1.18,95% CI 0.80-1.72, p = 0.403) or cardiogenic cerebral embolism No significant association was found with the disease (ncase = 710, OR 1.34,95% CI 0.62-2.90, p = 0.455; FIG. 2). When stratified by gender, these associations were more pronounced in females (male OR 1.40, 95% CI 1.03-1.91, p = 0.030, female OR 2.42, 95%). CI 1.69-3.45, p = 1.3x10 ^-6 ; Fig. 3).

FIG. 2 is a Forest plot of the odds ratio of ischemic stroke between RNF213 p.R4810K polymorphic carriers and non-carriers by stroke subtype. FIG. 3 is a forest plot of odds ratios for ischemic stroke between RNF213 pR4810K polymorphic carriers and non-carriers by gender. SE is the standard error; IV is the inverse variance method; 95% CI is the 95% confidence interval; FSR is the Fukuoka Stroke registry; NCVC is the national cardiovascular disease. The centers (National Cerebral and Cardiovascular Center) are shown respectively.

Domain plot of RNF213 loci for ischemic stroke (coordinates 77358945-79358945 of chromosome 17), rs112735431 (RNF213 p.R4810K polymorphism) as the lead polymorphic in this region (p = 2.6 x 10 ^-7) and identified (Fig. 4).

A similar association was found in Hisayama-FSR study participants. Compared to controls, the carrier frequency of RNF213 polymorphism is particularly high in patients with aortic atherosclerosis (OR 4.20, 95% CI 1.90-9.28, p = 3.8x10 ^-4 ; In Figure 2) and in women (OR 3.73, 95% CI 1.10-12.73, p = 0.035; Figure 3), all ischemic stroke cases (OR 2.90, 95%). It was significantly higher in CI 1.39-6.04, p = 0.0045). The mean age of stroke onset tended to be lower with RNF213 polymorphisms than with non-carriers (66.6 ± 12.4 years vs. 69.8 ± 10.7 years, p = 0.058).

(Combined meta-analysis)
A comprehensive meta-analysis of East Asians in three Japanese studies can be found in this RNF 213 p. R4810K polymorphism and ischemic stroke (OR 1.91, 95% CI 1.55-2.36, p = 1.5x10 ^-9 ) and aortic atherosclerosis (OR 3.88, 95% CI 2. A significant genome-wide association with 55-5.03, p = 2.0x10 ^-13 ; FIG. 2) was shown. When stratified by gender, a significant association was found throughout the genome (OR 1.50, 95% CI 1.14-1.98, p = 0.004 for males: OR 2.69, 95 for females. % CI 1.95-3.69, p = 1.2x10 ^-9 ; Fig. 3). The average age of stroke onset, 4 1 year (95% CI 2.7-5.5 years) in RNF213 polymorphism than noncarriers lower. (P = 1.1x10 ^-8; Fig. 5).

FIG. 5 is a comparison of stroke ages between RNF213 p.R4810K polymorphic carriers and non-carriers.

The main findings of this study are RNF213 p. The carrier frequency of the R4810K polymorphism is significantly higher in patients with ischemic stroke of East Asian progeny, especially in patients with aortic atherosclerosis, than in healthy control subjects. When stratified by gender, the association was more pronounced in females. Verification studies in two independent samples fully support these findings.

Empirical studies ensure that the genotype-phenotype association shows a reliable association. These data show considerable continuity between moyamoya disease and aortic atherosclerosis, ie RNF213-related vasculopathy.

Our results show that RNF213 p.I. in patients with ischemic stroke. It shows a clear gender-specific difference in the prevalence of the R4810K polymorphism. It is well known that moyamoya disease occurs more frequently in female patients, and the ratio of females to males with moyamoya disease is about 1.8 ²⁴⁾ . Females with intracranial artery stenosis are also at increased risk of developing ischemic stroke26 ⁾ . Therefore, female gender and genetic polymorphisms can contribute additively or synergistically to the development of ischemic stroke with aortic atherosclerosis.

The present invention relates to RNF213 p. It is effective for subjects having the R4810K polymorphism, and the subjects are not limited to the East Asians described in the examples.

(Reference)
1) Feigin VL, Forouzanfar MH, Krishnamurthi R, Mensah GA, Connor M, Bennett DA, Moran AE, Sacco RL, Anderson L, Truelsen T, O'Donnell M, Venketasubramanian N, Barker-Collo S, Lawes CMMM, Wang W , Shinohara Y, Witt E, Ezzati M, Naghavi M, Murray C, Global Burden of Diseases, Injuries and RFS 2010 (GBD 2010) and the GSEG. Global and regional burden of stroke during 1990-2010: findings from the Global Burden of Disease Study 2010. Lancet. 2014; 383: 245-254.
2) GBD 2015 Mortality and Causes of Death Collaborators. Global, regional, and national life expectancy, all-cause mortality, and cause-specific mortality for 249 causes of death, 1980-2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016; 388: 1459-1544.
3) Bang OY. Considerations when subtyping ischemic stroke in Asian patients. J Clin Neurol. 2016; 12: 129-136.
4) Gulli G, Rutten-Jacobs LCA, Kalra L, Rudd AG, Wolfe CDA, Markus HS. Differences in the distribution of stroke subtypes in a UK black stroke population --final results from the South London Ethnicity and Stroke Study. BMC Med. 2016; 14:77.
5) Kolominsky-Rabas PL, Weber M, Gefeller O, Neundoerfer B, Heuschmann PU. Epidemiology of ischemic stroke subtypes according to TOAST criteria: incidence, recurrence, and long-term survival in ischemic stroke subtypes: a population-based study. Stroke . 2001; 32: 2735-2740.
6) Mehndiratta MM, Khan M, Mehndiratta P, Wasay M. Stroke in Asia: geographical variations and temporal trends. J Neurol Neurosurg Psychiatry. 2014; 85: 1308-1312.
7) Malik R, Chauhan G, Traylor M, Sargurupremraj M, Okada Y, Mishra A, Rutten-Jacobs L, Giese AK, van der Laan SW, Gretarsdottir S, Anderson CD, Chong M, Adams HHH, Ago T, Almgren P , Amouyel P, Ay H, Bartz TM, Benavente OR, Bevan S, Boncoraglio GB, Brown RD, Butterworth AS, Carrera C, Carty CL, Chasman DI, Chen WM, Cole JW, Correa A, Cotlarciuc I, Cruchaga C, Danesh J, de Bakker PIW, DeStefano AL, den Hoed M, Duan Q, Engelter ST, Falcone GJ, Gottesman RF, Grewal RP, Gudnason V, Gustafsson S, Haessler J, Harris TB, Hassan A, Havulinna AS, Heckbert SR, Holliday EG, Howard G, Hsu FC, Hyacinth HI, Ikram MA, Ingelsson E, Irvin MR, Jian X, Jimenez-Conde J, Johnson JA, Jukema JW, Kanai M, Keene KL, Kissela BM, Kleindorfer DO, Kooperberg C, Kubo M, Lange LA, Langefeld CD, Langenberg C, Launer LJ, Lee JM, Lemmens R, Leys D, Lewis CM, Lin WY, Lindgren AG, Lorentzen E, Magnusson PK, Maguire J, Manichaikul A, McArdle PF, Meschia JF, Mitchell BD, Mosley TH, Nalls MA, Ninomiya T, O'Donnell MJ, Psaty BM, Pulit SL, Rannikmae K, Reiner AP, Rexrode KM, Rice K, Rich SS, Ridker PM, Rost NS, Rothwell PM, Rotter JI, Rundek T, Sacco RL, et al. Multiancestry genome-wide association study of 520,000 subjects identifies 32 loci associated with stroke and stroke subtypes. Nat Genet. 2018; 50: 524-537.
8) Liu W, Morito D, Takashima S, Mineharu Y, Kobayashi H, Hitomi T, Hashikata H, Matsuura N, Yamazaki S, Toyoda A, Kikuta K, Takagi Y, Harada KH, Fujiyama A, Herzig R, Krischek B, Zou L, Kim JE, Kitakaze M, Miyamoto S, Nagata K, Hashimoto N, Koizumi A. Identification of RNF213 as a susceptibility gene for moyamoya disease and its possible role in vascular development. PLoS One. 2011; 6: e22542.
9) Kamada F, Aoki Y, Narisawa A, Abe Y, Komatsuzaki S, Kikuchi A, Kanno J, Niihori T, Ono M, Ishii N, Owada Y, Fujimura M, Mashimo Y, Suzuki Y, Hata A, Tsuchiya S, Tominaga T, Matsubara Y, Kure S. A genome-wide association study identifies RNF213 as the first Moyamoya disease gene. J Hum Genet. 2011; 56: 34-40.
10) Miyawaki S, Imai H, Takayanagi S, Mukasa A, Nakatomi H, Saito N. Identification of a genetic variant common to moyamoya disease and intracranial major artery stenosis / occlusion. Stroke. 2012; 43: 3371-3374.
11) Miyatake S, Miyake N, Touho H, Nishimura-Tadaki A, Kondo Y, Okada I, Tsurusaki Y, Doi H, Sakai H, Saitsu H, Shimojima K, Yamamoto T, Higurashi M, Kawahara N, Kawauchi H, Nagasaka K, Okamoto N, Mori T, Koyano S, Kuroiwa Y, Taguri M, Morita S, Matsubara Y, Kure S, Matsumoto N. Homozygous c.14576G> A variant of RNF213 predicts early-onset and severe form of moyamoya disease. Neurology . 2012; 78: 803-810.
12) Morito D, Nishikawa K, Hoseki J, Kitamura A, Kotani Y, Kiso K, Kinjo M, Fujiyoshi Y, Nagata K. Moyamoya disease-associated protein mysterin / RNF213 is a novel AAA + ATPase, which dynamically changes its oligomeric state. Sci Rep. 2014; 4: 4442.
13) Koizumi A, Kobayashi H, Hitomi T, Harada KH, Habu T, Youssefian S. A new horizon of moyamoya disease and associated health risks explored through RNF213. Environ Health Prev Med. 2016; 21: 55-70.
14) Morimoto T, Enmi J, Hattori Y, Iguchi S, Saito S, Harada KH, Okuda H, Mineharu Y, Takagi Y, Youssefian S, Iida H, Miyamoto S, Ihara M, Kobayashi H, Koizumi A. Dysregulation of RNF213 promotes cerebral hypoperfusion. Sci Rep. 2018; 8: 3607.
15) Bang OY, Chung JW, Cha J, Lee MJ, Yeon JY, Ki CS, Jeon P, Kim JS, Hong SC. A Polymorphism in RNF213 Is a Susceptibility Gene for Intracranial Atherosclerosis. PLoS One. 2016; 11: e0156607.
16) O'Donnell M, Xavier D, Diener C, Sacco R, Lisheng L, Zhang H, Pias P, Truelsen T, Chin SL, Rangarajan S, Devilliers L, Damasceno A, Mondo C, Lanas F, Avezum A, Diaz R, Varigos J, Hankey G, Teal P, Kapral M, Ryglewicz D, Czlonkowska A, Skowronska M, Lopez-Jaramillo P, Dans T, Langhorne P, Yusuf S, INTERSTROKE investigators. Rationale and design of INTERSTROKE: a global case- control study of risk factors for stroke. Neuroepidemiology. 2010; 35: 36-44.
17) Fukui M. Guidelines for the diagnosis and treatment of spontaneous occlusion of the circle of Willis ("moyamoya" disease). Research Committee on Spontaneous Occlusion of the Circle of Willis (Moyamoya Disease) of the Ministry of Health and Welfare, Japan. Clin Neurol Neurosurg. 1997; 99 Suppl 2: S238-240.
18) Adams HP, Bendixen BH, Kappelle LJ, Biller J, Love BB, Gordon DL, Marsh EE. Classification of subtype of acute ischemic stroke. Definitions for use in a multicenter clinical trial. TOAST. Trial of Org 10172 in Acute Stroke Treatment . Stroke. 1993; 24: 35-41.
19) Chimowitz MI, Kokkinos J, Strong J, Brown MB, Levine SR, Silliman S, Pessin MS, Weichel E, Sila CA, Furlan AJ. The Warfarin-Aspirin Symptomatic Intracranial Disease Study. Neurology. 1995; 45: 1488? 1493 ..
20) Kim BJ, Kim JS. Ischemic stroke subtype classification: an Asian viewpoint. J stroke. 2014; 16: 8-17.
21) Park MG, Shin JH, Lee SW, Park HR, Park KP. RNF213 rs112735431 polymorphism in intracranial artery steno-occlusive disease and moyamoya disease in Koreans. J Neurol Sci. 2017; 375: 331-334.
22) Miyawaki S, Imai H, Shimizu M, Yagi S, Ono H, Mukasa A, Nakatomi H, Shimizu T, Saito N. Genetic variant RNF213 c.14576G> A in various phenotypes of intracranial major artery stenosis / occlusion. Stroke. 2013; 44: 2894-2897.
23) Kuroda S, Houkin K. Moyamoya disease: current concepts and future perspectives. Lancet Neurol. 2008; 7: 1056-1066.
24) Kuriyama S, Kusaka Y, Fujimura M, Wakai K, Tamakoshi A, Hashimoto S, Tsuji I, Inaba Y, Yoshimoto T. Prevalence and clinicoepidemiological features of moyamoya disease in Japan: findings from a nationwide epidemiological survey. Stroke. 2008; 39: 42-47.
25) Roy-O'Reilly M, McCullough LD. Sex differences in stroke: the contribution of coagulation. Exp Neurol. 2014; 259: 16-27.
26) Williams JE, Chimowitz MI, Cotsonis GA, Lynn MJ, Waddy SP, WASID Investigators. Gender differences in outcomes among patients with symptomatic intracranial arterial stenosis. Stroke. 2007; 38: 2055-2062.
27) Liu W, Senevirathna STMLD, Hitomi T, Kobayashi H, Roder C, Herzig R, Kraemer M, Voormolen MHJ, Cahova P, Krischek B, Koizumi A. Genomewide association study identifies no major founder variant in Caucasian moyamoya disease. J Genet . 2013; 92: 605-609.

The flow chart of the patient selection procedure is shown. FIG. 2 is a Forest plot of the odds ratio of ischemic stroke between RNF213 p.R4810K polymorphic carriers and non-carriers by stroke subtype. SE is the standard error; IV is the inverse variance method; 95% CI is the 95% confidence interval; FSR is the Fukuoka Stroke registry; NCVC is the national cardiovascular disease. The centers (National Cerebral and Cardiovascular Center) are shown respectively. FIG. 3 is a forest plot of odds ratios for ischemic stroke between RNF213 pR4810K polymorphic carriers and non-carriers by gender. SE is the standard error; IV is the inverse variance method; 95% CI is the 95% confidence interval; FSR is the Fukuoka Stroke registry; NCVC is the national cardiovascular disease. The centers (National Cerebral and Cardiovascular Center) are shown respectively. It is a regional plot of the RNF213 locus (coordinates 77358945-79358945 of chromosome 17) for ischemic stroke. FIG. 5 is a comparison of stroke ages between RNF213 p.R4810K polymorphic carriers and non-carriers. SD is the standard deviation (standard deviation); IV is reverse dispersion method (inverse variance method); 95% CI 95% confidence interval; is FSR, Fukuoka stroke registry (Fukuoka Stroke registry); NCVC National Cardiovascular The centers (National Cerebral and Cardiovascular Center) are shown respectively.

Samples were excluded if:
1) a high percentage of deficient mutation (loss> 0.05) case
2) If there is a discrepancy between the reported gender or ethnicity and the genetically determined gender or ethnicity.
3) When showing potential relevance

Claims (4)

RNF213 p. In a sample derived from a subject who has not developed cerebral infarction. A detection step for detecting the presence or absence of the R4810K gene polymorphism,
RNF213 p. A determination step for determining whether or not the subject has an earlier onset of cerebral infarction depending on the presence or absence of the R4810K gene polymorphism.
A method for predicting the onset time of cerebral infarction in a subject who has not developed cerebral infarction. In the detection step, the RNF 213 p. When the R4810K gene polymorphism is detected, in the determination step, the RNF213 p. The method according to claim 1, wherein the subject is judged to have a higher risk of developing cerebral infarction than a person who does not have the R4810K gene polymorphism. RNF213 p. A genetic marker consisting of the R4810K gene polymorphism for predicting the risk of developing cerebral infarction. RNF213 p. A biomarker for predicting the risk of developing cerebral infarction, which comprises a polypeptide encoded by the R4810K gene.

Images