TWI810386B - Prediction method for risk of ischemic stroke of large-artery atherosclerosis onset timing - Google Patents

Abstract

The invention provides a method for predicting the onset risk or onset period of cerebral infarction. A method for predicting the onset period of cerebral infarction in a subject without cerebral infarction, comprising: a detection step of detecting the presence or absence of the RNF213 p.R4810K gene polymorphism in a sample from a subject without cerebral infarction; and The judging step is to judge whether the onset of cerebral infarction of the subject is earlier than usual based on the presence or absence of the RNF213 p.R4810K gene polymorphism in the detecting step. A gene marker for predicting the risk of cerebral infarction, including RNF213 p.R4810K gene polymorphism. A biological marker for predicting the risk of cerebral infarction, comprising a polypeptide encoded by the RNF213 p.R4810K gene.

Description

Onset of ischemic stroke in large artery atherosclerosis Period Forecasting Method

The present invention relates to the prediction of the onset risk or onset period of cerebral infarction, and the method for predicting the onset risk of cerebral infarction belonging to these research fields, especially for normal people.

Non-Patent Document 1 discloses that the RNF213 gene polymorphism (c.14576 G>A, p.R4859K, rs112735431), which is a susceptibility gene for moyamoya disease, is not only related to moyamoya disease by chance It is also associated with unilateral moyamoya disease or intracranial main artery stenosis (intracranial stenosis) of atherosclerosis (atherosclerosis). On the other hand, it is revealed that the RNF213 gene has not been identified Non-casual association of attitude with neck carotid stenosis, cerebral aneurysm, and intracerebral hemorrhage.

Furthermore, the expression (c.14576 G>A, p.R4859K, rs112735431) related to the RNF213 gene polymorphism in Non-Patent Document 1 is based on the naming of the Northeastern University group. In this specification, the same RNF213 gene polymorphism is expressed as p.R4810K polymorphism (c.14429 G>A, rs112735431) or simply expressed as p.R4810K polymorphism. Both are differences based on amino acid calculation methods and are the same polymorphism

[Prior art literature]

[Non-patent literature]

[Non-Patent Document 1] "RNF213 and Moyamoya syndrome", Miyawaki Tetsu, 2016 Niche Neuro-Angiology Conference (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

The purpose of the present invention is to provide a method for predicting the onset risk or onset period of cerebral infarction.

Cerebral infarction is classified into three types: atherothrombotic brain infarction (cerebrovascular embolism), lacunar infarction, and cerebral embolism.

According to the studies of the present inventors, it was found that the polymorphism of the RNF213 p.R4810K gene and atherothrombotic cerebral infarction are not incidentally correlated. According to this situation, the following method is provided: by detecting the presence or absence of the RNF213 gene polymorphism in a sample from a subject without cerebral infarction, the risk of onset of cerebral infarction or the time of onset of the subject is predicted. method (or auxiliary method of making predictions). The present invention includes the following inventions.

(1) A method for predicting the period of onset of cerebral infarction in a subject without cerebral infarction, comprising: a detection step of whether there is RNF213 p.R4810K gene polymorphism in a sample from a subject without cerebral infarction detection; and a determination step of determining whether the subject's onset of cerebral infarction is earlier than usual based on the presence or absence of the RNF213 p.R4810K gene polymorphism in the detection step.

(2) The method described in (1) above, wherein in the case where the polymorphism of the RNF213 p.R4810K gene is detected in the detection step, in the determination step, it is determined that the subject is Compared with persons who do not have the RNF213 p.R4810K gene polymorphism, the onset of cerebral infarction is earlier.

(3) The method described in (1) or (2), wherein in the determination step, based on the presence or absence of the RNF213 p.R4810K gene polymorphism and the identification information of the subject, It is determined whether or not the onset of cerebral infarction of the subject is earlier than usual.

(4) A gene marker for predicting the risk of cerebral infarction, including RNF213 p.R4810K gene polymorphism.

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

According to the knowledge of the present invention, for the examinees who have not suffered from cerebral infarction, it is possible to Cerebral infarction is prevented or counteracted from an early stage.

FIG. 1 is a flowchart showing a patient selection procedure.

Fig. 2 is a forest plot of the odds ratio of ischemic stroke between RNF213 p.R4810K polymorphism carriers and non-carriers based on stroke subtype. SE means standard error; IV means inverse variance method; 95% CI means 95% confidence interval; FSR means Fukuoka Stroke registry; NCVC means Japan National Center for Circulatory Organ Diseases ( National Cerebral and Cardiovascular Center (Japanese National Cerebral and Cardiovascular Center).

Figure 3 is a forest plot of the odds ratio of ischemic stroke between carriers and non-carriers of the RNF213 p.R4810K polymorphism based on sex. SE means standard error; IV means inverse variance method; 95% CI means 95% confidence interval; FSR means Fukuoka Stroke registry; NCVC means Japan National Center for Circulatory Organ Diseases ( National Cerebral and Cardiovascular Center).

Fig. 4 is a region map of the RNF213 locus (coordinates 77358945-79358945 on chromosome 17) associated with ischemic stroke.

Figure 5 is a comparison of the onset age of cerebral apoplexy between RNF213 p.R4810K polymorphism carriers and non-carriers. SD means standard deviation; IV means inverse variance method; 95% CI means 95% confidence area FSR means Fukuoka Stroke registry; NCVC means National Cerebral and Cardiovascular Center.

RNF213 (Ring finger protein 213) (GenBank accession number NM_001256071.1) has been identified as a disease susceptibility gene for moyamoya disease in recent years, and exists in the human chromosome region 17q25.3.

RNF213 p.R4810K gene polymorphism is 73097 G>A single base polymorphism (SNP; Single Nucleotide Polymorphism (single nucleotide polymorphism) in the nucleotide (nucleotide) sequence represented by sequence number 2 ). RNF213 p.R4810K is known as a moyamoya disease susceptibility polymorphism as said prior literature.

According to studies by the inventors of the present invention, it has been found that the RNF213 p.R4810K polymorphism increases the risk of ischemic stroke (ie, cerebral infarction) caused by aortic atherosclerosis. In this specification, ischemic stroke and cerebral infarction have the same meaning.

Ischemic stroke is one of the causes of premature death in recent years, and it is the main cause of physical disability and premature death especially in Asia, but the relationship between ischemic stroke and specific genetic determination is still unknown. Moyamoya disease, a cerebrovascular disease mainly identified in East Asia, is associated with a susceptibility gene called ring finger protein 213 (RNF213), whose dysregulation impairs cerebral perfusion in the mouse brain. Therefore, the present inventors established the hypothesis that RNF213 plays a more common role in ischemic stroke, and investigated The p.R4810K polymorphism of the RNF213 gene, the most common risk for moyamoya disease, its association with ischemic stroke and its subtypes.

Based on three independent Japanese studies of ischemic stroke, the present inventors conducted a case-control study of 46,958 people (17,752 cases and 29,206 normal people (controls)) of East Asian ancestry. data) for analysis. The inventors of the present invention performed a meta-analysis of East Asians under a fixed effects model.

As a result, according to the combined integration analysis of East Asians, it was shown that in the entire region of the genome, the p.R4810K polymorphism was associated with all ischemic strokes (OR (odds ratio) 1.91, 95% CI (95% reliable Interval) 1.55-2.36, p=1.5×10 ^-9 ) and large artery atherosclerosis (OR 3.58, 95%CI 2.55-5.03, p=2.0×10 ^-13 ) were not accidentally associated. When stratified by sex, the association was stronger in women (OR 1.50, 95% CI 1.14-1.98, p=0.004 in men: OR 2.69, 95% CI 1.95-3.69, p= 1.2×10 ^-9 ). Regarding the average age of stroke onset, compared with non-holders of RNF213 p.R4810K polymorphism, the holders of RNF213 p.R4810K polymorphism were 4.1 years younger (p=1.1×10 ^-8 ). Here, the term "onset of cerebral infarction earlier than usual" in this specification means earlier than the average onset of cerebral infarction among non-holders of the RNF213 p. R4810K polymorphism holders and RNF213 p.R4810K polymorphism non-holders had earlier onset of cerebral infarction on average.

According to these, the following conclusions can be drawn: RNF213 p.R4810K polymorphism is the cause of ischemic stroke, especially ischemic stroke of large artery atherosclerosis genetic risk factors.

The RNF213 p.R4810K gene polymorphism is a single base polymorphism (SNP; Single Nucleotide Polymorphism (single nucleotide polymorphism)) of 73097 G>A in the nucleotide sequence represented by SEQ ID NO: 2, which can be It is a gene marker used to predict the risk of cerebral infarction, or to assist in prediction.

SEQ ID NO: 2 is the partial nucleotide sequence of human chromosome 17 deoxyribonucleic acid (deoxyribonucleic acid, DNA) comprising genes [FLJ3520, NPTX1, CARD14, and Raptor (KIAA1303)] of the mysterin gene and its surrounding regions, corresponding to Nucleotides No. 43560001~43795000 of Contig #NT010783.15 recorded in the National Center for Biotechnology Information (NCBI).

In addition to the nucleotide sequence represented by SEQ ID NO: 2, in addition to the SNP at position 73097 of G or A (in this specification, abbreviated as 73097 G>A), there may also be a SNP at position 4766 of T or C ( 4766 T>C), the 120764th SNP of G or A (120764 G>A), the 152917th SNP of G or A (152917 G>A), and the 232102nd of G or A SNP (232102 G>A).

In addition, in this specification, the position of SNP is described based on the position of the nucleotide in the nucleotide sequence shown by SEQ ID NO: 2. For example, "the SNP at position 73097" refers to the SNP at the 73097th nucleotide in the nucleotide sequence represented by SEQ ID NO: 2. When described as "73097 G>A", etc., the base of the major allele (in this case, G) is described before the ">" mark, The base of the minor allele (in this case, A) is described after the ">" mark.

In addition, in this specification, unless otherwise specified, the nucleotide sequence is described as a sequence of DNA, but when the polynucleotide (polynucleotide) is ribonucleic acid (Ribonucleic Acid, RNA), thymine (thymine) (T) is suitably replaced by uracil (uracil) (U).

In the present invention, the polynucleotide may include an arbitrary 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, polynucleotides are preferably isolated or purified.

In the detecting step of the method of the present invention, the SNP of 73097 G>A is detected in a sample derived from a living body collected from a subject.

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

Here, a race is a group that can be distinguished as a specific subgroup in the Homo sapiens species. A human race has a unique and distinguishable combination of genes, and is identified by the characteristics (mental characteristics, and physical characteristics) formed by using the combination of genes. Members of the same human race share a common genetic ancestry and, as a result, share a similar combination of genes and therefore share clearly distinguishable genetic characteristics.

For example, with regard to the major human groups in the world, genetic kinship is investigated based on 23 kinds of gene information, and the classification as African (Black race) (Negroid)), Caucasoid (Caucasian), Oceanian (Australoid), East Asian (Mongolian) and Native American.

The so-called East Asian refers to a person whose origin is any one of Japanese, Korean, Chinese, Taiwanese, and Mongolian. East Asians are preferably Japanese, Korean, or Chinese.

Those skilled in the art can easily determine the ethnicity of an individual based on the individual's physical characteristics, country of origin, information on ancestral origin, and the like.

As the organism-derived sample used in the method, any tissue, cell, body fluid, etc. from which genomic DNA can be collected can be used, and it is preferable to use Hair, nails, skin, mucous membranes, blood, plasma, serum, saliva, etc.

Methods for detecting SNPs are well known in the art. For example, Restriction Fragment Length Polymorphism (Restriction Fragment Length Polymorphism, RFLP) (Restriction Enzyme Cutting Fragment Length Polymorphism) method, Polymerase Chain Reaction-Single Strand Configuration Polymorphism (Polymerase Chain Reaction -Single Strand Conformation Polymorphism, PCR-SSCP) (single-stranded DNA high-order structural polymorphism analysis) method, allele-specific oligonucleotide (Allele Specific Oligonucleotide, ASO) hybridization method, sequence method, amplification refraction mutation system ( Amplification Refracting Mutation System, ARMS) method, denaturing gradient gel electrophoresis (Denaturing Gradient Gel Electrophoresis) method, RNAseA cutting method, dye-labeled oligonucleotide connection (Dye-labeled Oligonucleotide Ligation (DOL) method, TaqMan (TaqMan) PCR method, primer extension (primer extension) method, invader method (invader method), etc.

In addition, the polypeptide encoded by the RNF213 p.R4810K gene is a polypeptide comprising the amino acid sequence represented by sequence number 1 (here, the arginine at No. 4810 in the amino acid sequence represented by sequence number 1 is replaced by Lysine) can be used as a biomarker for predicting the risk of cerebral infarction.

That is, as described above, the variation of the SNP of RNF213 p.R4810K is accompanied by the 4810th amino acid substitution of human mysterin (arginine → lysine). Therefore, the mysterin polypeptide was isolated from the subject and analyzed The identification of the amino acid No. 4810 enables prediction of the onset risk of cerebral infarction or assistance for prediction.

[Example]

Examples are shown below to describe the present invention in detail, but the present invention is not limited by the examples.

Stroke mortality rates have decreased over the past few decades1 ⁾ , but worldwide, stroke remains the second leading cause of premature death and the leading cause of disability2 ⁾ . In Southeast Asia and East Asia, which account for 31% of the world's population, stroke is the main cause of premature death, and the incidence and attack rate of stroke are increasing steadily1,2 ⁾ . Epidemiological studies implicate substantial geographic and ethnic differences in stroke subtypes3,4 ⁾ . Psychogenic embolism is the usual etiological subtype of ischemic stroke in Western European countries ⁵⁾ . On the other hand, large artery atherosclerosis caused by intracranial artery stenosis is the main cause in most Asian countries ⁶⁾ . The difference between environmental risk factor and genetic background is considered to be the main reason for the high attack rate of aortic atherosclerosis in Asia. Recently, 32 loci associated with stroke and stroke subtypes were identified by a large-scale multi-ancestry genome-wide association analysis ⁷⁾ . In this study, about 8 million single base polymorphisms (SNPs) were examined. However, SNPs with a minor-allele frequency (MAF) of less than 1% (<0.01) were excluded from this analysis, and further, the Asian-specific genetic determinants of ischemic stroke remain unknown.

The RING finger protein 213 gene (RNF213) on 17q25.3 was identified as a susceptibility gene for moyamoya disease8,9 ⁾ . The RNF213 p.R4810K polymorphism (c.14429 G>A, rs112735431) was detected in more than 80% of moyamoya disease patients, but the carrier frequency of the allele (allele) in healthy subjects in East Asia was about 2% ^{8, 9)} . RNF213 encodes a 591 kDa protein ¹²⁾ that functions as both an AAA+ ATPase and an E3 ligase, and is associated with the onset of intracranial trunk occlusive lesions and compensatory responses in reduced cerebral blood flow ^13,14) . Recently, two individual studies reported a high frequency (20%-25%) of the RNF213 p.R4810K polymorphism in East Asian non-Moyamoya disease patients with intracranial carotid stenosis or proximal middle cerebral artery stenosis10 ^{, 15)} . Therefore, the inventors of the present invention hypothesized that this genetic variation might be associated with global ischemic stroke in Asia. Here, patients with acute ischemic stroke who participated in three independent Japanese studies were analyzed to investigate the association of RNF213 p.R4810K polymorphism with ischemic stroke and its subtypes.

(method)

(Study Design and Participants)

In this two-stage case-control study, the inventors et al. Ischemic stroke data from East Asian participants were studied in three case-control studies. The inventors of the present invention excluded patients and controls who were not of East Asian descent. In the primary study, a hospital-based single population (NCVC Biobank) of the National Cerebral and Cardiovascular Center (NCVC) in Japan including extensive clinical and radiological data of all patients was used. In the replication studies, the p.R4810K genotype was obtained from Biobank Japan (16,256 ischemic stroke cases and 27,294 controls), Hisayama and Fukuoka Stroke Registry (FSR) Genome-wide genotyping data from the trial (1,113 cases and 901 controls). The study obtained written consent from all trial participants and was approved by the responsible ethics committee and the ethics committee of the National Center for Circulatory Diseases (NCVC) in Japan.

(Primary study)

Participants for the main study were recruited by the National Center for Circulatory Organ Diseases (NCVC), a 600-bed third center specializing in stroke and cardiovascular diseases in the Osaka and Kansai regions. Non-cardiac cerebral embolism (large-artery atherosclerosis, small-vessel occlusive disease) who will be admitted to the hospital from June 2012 to May 2017 and signed by NCVC in the comprehensive consent form -vessel occlusion) etc.) 383 cases of Japanese patients were included in this study. Based on the criteria of the Moyamoya Disease Research Committee of the Japanese Ministry of Health, Labor and Welfare, patients diagnosed with cardioembolic stroke (cardioembolic stroke) and confirmed/suspected moyamoya disease were excluded17 ⁾ . A flowchart of the patient selection sequence is detailed in FIG. 1 .

Demographic data, atherosclerotic risk factors, radiographic observations, and medical records were obtained from a database collected through prospective studies of stroke patients (prospectively-collected database). Stroke subtypes are classified according to the Org 10172 criteria [the Trial of Org 10172 in Acute Stroke Treatment (TOAST) criteria (Org 10172 in Acute Stroke Treatment (TOAST) criteria)] related to the treatment of acute ^stroke18) . Cerebrovascular lesions are identified by magnetic resonance imaging (MRI) and MR angiography (MR angiography), or computed tomography (CT) and CT angiography (CT angiography). Anterior circulation stenosis (anterior circulation stenosis) is defined as ^intracranial carotid artery (intracranial A reduction in diameter of more than 50% (>50%) in the internal carotid artery, middle cerebral artery, or anterior cerebral artery. Likewise, posterior circulation stenosis is defined according to such references in the intracranial, posterior spinal, or posterior cerebral arteries.

Control subjects were recruited in the Kansai region of Japan between 2007 and 2015. Of the 1,027 potential controls, 16 subjects with medical history of cerebral infarction or moyamoya disease were excluded. Ultimately 1,011 control subjects were selected. Venous blood samples were collected and stored at -80°C until analysis. As described above ⁸⁾ , using TaqMan SNP Assays (Applied Biosystems, Foster City, California (California, CA)) and 7300/7500 Real-Time PCR System (7300/7500 Real-Time PCR System) (Applied Biosystems, Foster City, CA) for p.R4810K genotype determination.

(Replication studies)

Ischemic stroke (large-artery atherosclerosis, small-vessel occlusion, and cardiogenic cerebral embolism) were performed by retrieving data from previous reports7 ^). Confirmation of the association between (cardioembolism)) and the RNF213 p.R4810K polymorphism. Specifically, 16,256 ischemic stroke patients were recorded from the Biobank Japan project. Controls were from Iwate Medical Megabank (IMM), a multi-purpose cluster study based on Japanese health centers (Japan Public Health Center-based Prospective study (JPHC)), Japanese Obtained from four population-based studies of Japan Multi-institutional Collaborative Cohort Study (JMICC) and Tohoku Medical Megabank (ToMMo). Furthermore, 1,113 cases of ischemic stroke and 901 cases of controls were obtained from the Hisayama Study, which studies cardiovascular diseases in Japan, and the FSR test, which is based on the records of multi-institutional hospitals in patients with acute stroke in Japan.

SNP genotyping (genotyping) is a combined analysis using Illumina's Human OmniExpress BeadChip and Human Exome BeadChip, or Illumina ) analysis on Human OmniExpress Exome BeadChip (Human OmniExpress Exome BeadChip) and was carried out by RIKEN Center for Integrative Medical Sciences, RIKEN. In order to exclude subjects other than East Asians, standard quality control procedures including identity-by-descent analysis (Identity-by-Descent analysis) and principal component analysis for specifying samples were applied. Haplotype phasing and genotype imputation were performed using filtered samples with genotype data meeting quality control criteria including success rates and Hardy-Weinberg balance tests. A genome-wide association study (GWAS) was performed using the doses of the imputed alleles, and a logistic regression model with an additive genetic model was fitted. The present inventors took 10 principal components, age, and sex as covariates. Details are described in ⁷⁾ above.

Samples are excluded in the following cases.

1) When the proportion of missing variation is high (missing >0.05)

2) There is an inconsistency between the reported sex or ethnicity and the genetically determined sex or ethnicity

3) Circumstances showing potential association

(Statistical analysisStatistical analysis)

Continuous variables are expressed as mean ± SD and compared using Student's t-test. Categorical variables are expressed as numerical values and percentages, and are suitable for chi-square tests and Fisher's exact tests for two-sided tests (two-tailed Fisher's exact test) for comparison. The inventors of the present invention performed an experiment assuming a dominant model for the correlation between the RNF213 polymorphism and the risk of ischemic stroke in the main study because the number of homozygotes was insufficient. In addition, the inventors of the present invention compared the association under the log-additive model (log-additive model) used for meta-analysis in order to compare with verification studies (replication studies) using imputation methods. sexual investigation. Multiple logistic regression models were used to control the potential confounding factors at the same time, and then the odds ratio (OR Ratio) and 95% confidence interval CI were calculated for each stroke subtype. The variables considered in the model were age (continuous), sex, hypertension, dyslipidemia, diabetes and smoking. All analyzes were performed using JMP Pro 12.2 software (SAS Institute Inc., Cary, North Carolina, NC).

It is not by chance that the probability values are two-sided and p < 0.05. The integrated analysis is to use the inverse variance fixed effects model (inverse variance fixed effects model) that p<5×10 ^-8 is not accidental in the genome as a whole and is reviewed by the review management (Review Manager) (RevMan) 5.3 software (Nordic Cochrane Center (The Nordic Cochrane Center), Denmark (Denmark), Copenhagen (Copenhagen)).

(result)

(Primary study)

In the main study, especially non-psychogenic cerebral embolism stroke patients were recorded. Among the 1,775 stroke patients with non-psychoembolic cerebral embolism who met the comprehensive benchmark 383 (21.6%) agreed to participate in the NCVC Biobank and were analyzed in the main study. Table 1 shows the characteristics of both NCVC Biobank participants and non-participants. Participants were younger and fewer women than non-participants in 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 equal. The baseline (base line) characteristics of the participants in this main study are shown in Table 2. Stroke patients were older, less female, and had a greater number of atherosclerosis risk factors than controls.

It was found that the RNF213 p.R4810K polymorphism was found in 5.2% of non-cardioembolic stroke patients and 2.1% of normal controls (crude OR 2.60, 95%CI 1.39-4.85, p=0.0019). After adjusting for age, sex, and risk factors for atherosclerosis, the association between RNF213 p.R4810K polymorphism and non-psychoembolic stroke remained non-accidental (adjusted OR 3.90, 95% CI 1.62-9.24, p=0.0026). Compared with control subjects, only large artery atherosclerosis was associated non-casually with the polymorphism (crude OR 5.19, 95% CI 2.53-10.64, p=2.6×10 ^-6 ; adjusted OR 11.45, 95% CI 3.46-36.17, p=0.0001) (Table 3).

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

As can be seen from Table 2 and Table 4, the average (68.5 years old) age of onset of cerebral infarction among subjects with or without RNF213 polymorphism, and the relationship between those with and without RNF213 polymorphism Compared with the age of onset of cerebral infarction (in Table 4, the average is 58.1 years old) of subjects with RNF213 polymorphism, the age of onset of cerebral infarction was about 11 years younger in patients with RNF213 polymorphism.

Among stroke patients, RNF213 polymorphism carriers included more females than non-carriers (55.0% vs. 27.3%, p=0.011), and showed narrowing of the anterior intracranial circulation (60.0% vs. , p=0.004) and a higher frequency of large artery atherosclerosis (65.0%: 32.5%, p=0.012). Existing atherosclerotic risk factors such as the occurrence of extracranial internal carotid artery or posterior circulation stenosis, and the incidence of hypertension, diabetes, dyslipidemia, and smoking are associated with polymorphisms There is no difference between carriers and non-carriers.

(Replication studies)

Regarding the RNF213 p.R4810K polymorphism, the present inventors used 16,256 ischemic stroke cases (including cardiogenic cerebral embolism stroke) and 27,294 East Asian cases recorded from the Japan Biobank item ^7). descendants to search. The RNF213 p.R4810K polymorphism was identified in 2.3% of all ischemic stroke patients and 1.3% in the general population. A non-casual association (OR 1.77, 95% CI 1.40-2.24, p =1.6×10 ^-6 ). The average age of onset of stroke was lower in RNF213 p.R4810K polymorphism than in non-carriers (62.1±10.0 years vs. 66.0±10.0 years, p=8.7×10 ^-7 ).

Analyzing only patients with large artery atherosclerosis (ncase=1,256), 4.0% of patients had RNF213 p.R4810K polymorphism, and the association with RNF213 p.R4810K polymorphism was more significant (OR 3.10, 95 %CI 1.98-4.84, p=6.9×10 ^-7 ; age of stroke onset: 56.9±10.2 years: 65.0±9.7 years, p=6.3×10 ^-5 ). In addition, RNF213 p.R4810K polymorphism was not confirmed to be associated with small-vessel occlusion (ncase=4,613, OR 1.18, 95%CI 0.80-1.72, p=0.403) or cardiogenic cerebral embolism (ncase=710, OR 1.34, 95%CI 0.62-2.90, p=0.455; Figure 2) non-casual association. When stratified by sex, these associations were stronger in women (OR 1.40 for men, 95%CI 1.03-1.91, p=0.030, OR 2.42 for women, 95%CI 1.69-3.45, p= 1.3×10 ⁻⁶ ; FIG. 3 ).

Figure 2 is a forest plot of the odds ratio of ischemic stroke between carriers and non-carriers of RNF213 p.R4810K polymorphism based on stroke subtype. Figure 3 is a forest plot of the odds ratio of ischemic stroke between carriers and non-carriers of the RNF213 p.R4810K polymorphism based on sex. SE means standard error; IV means inverse variance method; 95% CI means 95% confidence interval; FSR means Fukuoka Stroke registry; NCVC means Japan National Center for Circulatory Organ Diseases ( National Cerebral and Cardiovascular Center).

Region map of the RNF213 locus (coordinates 77358945-79358945 on chromosome 17) associated with ischemic stroke, as a read polymorphism in this region (p=2.6×10 ^-7 ) was identified as rs112735431 (RNF213 p .R4810K polymorphism) (Figure 4).

The same association was also found in the Jiushan-FSR study participants. Compared with the control group, the carrier frequency of RNF213 polymorphism was especially higher in patients with large artery atherosclerosis (OR 4.20, 95%CI 1.90-9.28, p=3.8×10 ^-4 ; Figure 2 ), and in women ( OR 3.73, 95%CI 1.10-12.73, p=0.035; Fig. 3), was not incidentally high among all ischemic stroke cases (OR 2.90, 95%CI 1.39-6.04, p=0.0045). Regarding the average age of onset of stroke, there was a lower tendency in the RNF213 polymorphism than in the non-carriers (66.6±12.4 years: 69.8±10.7 years, p=0.058).

(Combined meta-analysis)

In a meta-analysis of East Asians in three Japanese studies, the RNF213 p.R4810K polymorphism was associated with ischemic stroke (OR 1.91, 95%CI 1.55-2.36, p=1.5×10 ^-9 ) and A non-casual association was shown across the genome between large artery atherosclerosis (OR 3.88, 95% CI 2.55-5.03, p=2.0×10 ⁻¹³ ; Figure 2 ). When stratified by sex, non-casual associations were found across the genome as a whole (OR 1.50, 95% CI 1.14-1.98, p=0.004 in males: OR 2.69, 95% CI 1.95-3.69 in females, p=1.2×10 ⁻⁹ ; FIG. 3 ). The average age of onset of stroke was 4.1 years lower (95%CI 2.7-5.5 years) compared with non-carriers for the RNF213 polymorphism (p=1.1×10 ^-8 ; Figure 5).

Figure 5 is a comparison of the onset age of cerebral apoplexy between RNF213 p.R4810K polymorphism carriers and non-carriers.

Regarding the main observation of the study, the carrier frequency of the RNF213 p.R4810K polymorphism was significantly higher in ischemic stroke patients of East Asian descent, especially in patients with large artery atherosclerosis than in normal control subjects Not accidentally high. When stratified by sex, the association was more pronounced in females. Validation studies in two independent samples fully support these insights.

Validation studies ensure that genotype-phenotype associations show reliable associations. These data show a considerable continuum between Moyamoya disease and large artery atherosclerosis, an RNF213-associated vasculopathy.

The results of the present inventors et al. showed a clear sex-specific difference in the prevalence of the RNF213 p.R4810K polymorphism in ischemic stroke patients. It is well known that moyamoya occurs more frequently in female patients, with a female to male ratio of about 1.8 ²⁴⁾ for moyamoya. In addition, women with intracranial arterial stenosis have a higher risk of developing ischemic stroke ²⁶⁾ . Therefore, female sex and genetic polymorphisms contribute additively or multiplicatively to the pathogenesis of ischemic stroke with large artery atherosclerosis.

The present invention is effective for subjects having the RNF213 p.R4810K polymorphism, but the subjects are not limited to East Asians described in Examples.

(references)

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<110> National Cerebral and Cardiovascular Center, a national research and development corporation, KYOTO UNIVERSITY, a national university corporation, and SHIMADZU CORPORATION

<120> Method for predicting the onset period of cerebral infarction in subjects without cerebral infarction, gene markers and biomarkers for predicting the risk of developing cerebral infarction

<150>PCT/JP2018/045915

<151> 2018-12-13

<160> 2

<210> 1

<211> 5207

<212> PRT

<213> Homo sapiens

<400> 1

<210> 2

<211> 235000

<212>DNA

<213> Homo sapiens

<400> 2

Claims (2)

A method for predicting the onset period of aortic atherosclerosis and ischemic stroke in a subject without cerebral infarction, comprising: a detection step, detecting the presence or absence of RNF213p in a sample from a subject without cerebral infarction .R4810K gene polymorphism is detected; and the determination step is to determine the ischemic cerebral atherosclerosis of the subject's aortic atherosclerosis according to the presence or absence of the RNF213 p.R4810K gene polymorphism in the detection step Whether the stroke onset period is earlier than usual, in the determination step, if the RNF213 p.R4810K gene polymorphism is detected in the detection step, it is determined that the subject does not have the Compared with persons with RNF213 p.R4810K polymorphism, the onset period of ischemic stroke with large artery atherosclerosis was earlier than usual. The method described in item 1 of the scope of the patent application, wherein in the case where the polymorphism of the RNF213 p.R4810K gene is detected in the detection step, in the determination step, it is determined that the examinee is related to Compared with persons who do not have the RNF213 p.R4810K gene polymorphism, the risk of onset of ischemic stroke due to large artery atherosclerosis is higher.