Genetic Variant RNF213 c.14576G>A in Various Phenotypes of Intracranial Major Artery Stenosis/Occlusion
Background and Purpose—Recently, we reported a common genetic variant, ring finger protein 213 (RNF213) c.14576G>A variant, a susceptibility gene for moyamoya disease (MMD), among patients with intracranial major artery stenosis/occlusion (ICASO) in a selected Japanese population. The aim of this 2-center–based case–control study was to confirm our previous finding in a larger population.
Methods—Study participants were recruited from The University of Tokyo Hospital and Kanto Neurosurgical Hospital. The occurrence rate of c.14576G>A variant was investigated in 323 patients, 22 with definite MMD, 8 with unilateral MMD, 84 with ICASO in the absence of MMD (non-MMD ICASO), 34 with extracranial carotid atherosclerosis, 44 with cerebral aneurysm, 21 with intracerebral hemorrhage, and 110 control subjects.
Results—RNF213 c.14576G>A variant was found in 1.8% (2/110) of the normal control group and had significant associations with definite MMD (P<0.0001; odds ratio, 144.0; 95% confidence interval, 26.7–775.9), unilateral MMD (P=0.0001; odds ratio, 54.0; 95% confidence interval, 7.5–386.8), and non-MMD ICASO (P<0.0001; odds ratio, 16.8; 95% confidence interval, 3.81–74.5). There was no significant association with extracranial carotid atherosclerosis, cerebral aneurysm, or intracerebral hemorrhage. This result replicated our previous findings.
Conclusions—A particular subset of patients with various phenotypes of ICASO has a common genetic variant, RNF213 c.14576G>A, indicating that RNF213 c.14576G>A variant is a high-risk allele for ICASO.
Stroke, one of the leading causes of death and a major cause of acquired disability in adults, affects ≈15 million people worldwide every year.1 Clinical stroke risk factors are well established.2 However, conventional clinical risk factors explain only a small part of all stroke risks.3 Many studies supported a role for genetic risk factors for stroke.4 Recent progress in genetic analytical methods, such as genome-wide association studies, has discovered several risk alleles for some specific stroke subtypes. For instance, paired-like homeodomain transcription factor 2 (PITX2) and zinc finger homeobox 3 (ZFHX3) have an association with cardioembolic stroke and histone deacetylase 9 (HDAC9), 9p21 locus and 6p21.1 locus have an association with large artery stroke.5 However, the odds ratios (OR) associated with these risk alleles are relatively low and account for only a small proportion of estimated heritability.
Intracranial major artery stenosis/occlusion (ICASO) is one of the most common causes of stroke. The pathogenesis usually involves atherosclerosis or cardiac embolism, occasionally moyamoya disease (MMD), vasculitis, or dissection and rarely autoimmune diseases and others. The most common cause of ICASO is atherosclerosis, caused by acquired factors, such as hypertension, diabetes mellitus, dyslipidemia, and smoking. However, ICASO is known to be common among nonwhite populations, particularly Asian populations, indicating a potential involvement of genetic factors.6 However, no common genetic factor for ICASO has yet been identified.
Recently, we reported a common genetic variant among patients with ICASO without signs of MMD (non-MMD ICASO) in the Japanese population.7 This variant gene, ring finger protein 213 (RNF213; a gene located in chromosome 17q; on the basis of National Center for Biotechnology Information Reference sequence, NP_065965.4), has been identified as a susceptibility gene for MMD in East Asian populations.8–11 Therefore, particular subset of patients with ICASO, generally thought to be caused by acquired factors such as hypertension, has a native genetic factor common to MMD among East Asian populations.
The aim of the present study was to verify the generalizability of our previous findings, investigating the occurrence of c.14576G>A variant in patients with ICASO not diagnosed as MMD compared with the occurrence in patients with MMD and other cerebrovascular diseases associated with extracranial carotid atherosclerosis (ECAS), cerebral aneurysm, and intracerebral hemorrhage (ICH) as control groups.
Materials and Methods
This 2-center–based case–control study was conducted at The University of Tokyo Hospital (university medical center) and Kanto Neurosurgical Hospital (regional stroke center with responsibility for the primary care of most stroke patients in the northern region of Saitama Prefecture). A total of 323 Japanese patients, new and revisiting outpatients from the 2 hospitals, were enrolled from October 2011 to March 2013. This cohort did not include the 196 patients reported previously.7
Diagnosis of Cerebrovascular Diseases
Diagnosis of cerebrovascular disease was based on magnetic resonance (MR) angiography findings interpreted by ≥2 physicians, including ≥1 radiologist and 1 neurosurgeon.
The phenotypes of the stenosed or occluded intracranial major artery were categorized as: definite MMD, unilateral MMD, and non-MMD ICASO. Other cardiovascular diseases included in this study were ECAS, cerebral aneurysm, and ICH. Only patients with hypertensive ICH, who had no abnormal lesion in the intracranial major arteries, were recruited. A control group was included to check the natural occurrence rate of the c.14576G>A variant. Blood samples for DNA analysis were collected from patients visiting the hospital for brain trauma or screening studies and who had no history of cerebrovascular disease. Most of control group had MRI scans to confirm an absence of cerebrovascular lesions.
Diagnostic criteria for definite MMD were based on the criteria of the Research Committee on the Pathology and Treatment of Spontaneous Occlusion of the Circle of Willis (Moyamoya disease), Health Labour Sciences Research Grant for Research on Measures for Intractable Diseases, Japan.12 Briefly, these criteria require all of the following findings: (1) steno-occlusive lesions around the terminal portions of the internal carotid arteries (including proximal portions of the anterior and middle cerebral arteries); (2) moyamoya vessels at the base of the brain appearing as abnormal vascular networks on conventional angiography or MR angiography; (3) findings 1 and 2 are present bilaterally (unilateral finding is defined as unilateral MMD, which is discriminated from definite MMD according to the diagnostic criteria); and (4) exclusion of known diseases with similar angiographic findings (arteriosclerosis, autoimmune disease, meningitis, brain neoplasm, Down syndrome, neurofibromatosis type 1, head trauma, irradiation to the head, protein C deficiency, protein S deficiency, etc).
Unilateral MMD has similar angiographic findings to definite MMD (bilateral MMD), but whether unilateral and bilateral MMD have a common cause and genetic background remains under discussion.13
MR angiography findings of ICASO that did not satisfy MMD criteria (either definite or unilateral MMD) were categorized as non-MMD ICASO. This group showed partial stenosis or occlusion of the major intracranial artery without abnormal vascular networks in the basal ganglia. Most lesions were unilateral, located in middle cerebral artery or internal carotid artery. Atherosclerotic features within this group were associated with a history of hypertension, dyslipidemia, and diabetes mellitus in relatively elderly patients (Table I in the online-only Data Supplement). No patient had any signs of cardiac embolism, dissection, vasculitis, or any of the other syndromes that cause ICASO.
The algorithm and profile of the differential diagnosis of these phenotypes are shown in Figure 1. Representative MR angiograms of each phenotype of ICASO are shown in Figure I in the online-only Data Supplement.
Identification of RNF213 Variants
Peripheral blood samples were obtained from all enrolled patients. Genomic DNA was obtained from the peripheral blood leukocytes at SRL, Inc (Tachikawa, Tokyo, Japan) using a DNA Extraction Kit (Talent Srl, Trieste, Italy). Variant analysis of exon 61, which includes the c.14576G>A variant of RNF213 (GenBank accession number, NM_020914.4), was performed by direct sequencing at FASMAC Co, Ltd. (Atsugi, Kanagawa, Japan) using an ABI Genetic Analyzer 3130XL or ABI DNA Analyzer 3730xL (Applied Biosystems, Foster City, CA) and analyzed with Sequence Scanner version 1.0 (Applied Biosystems). The same primer sequences and polymerase chain reaction conditions were used as described previously.10 The investigators involved in genotyping were blinded from the phenotypic information. All analyses of the sequenced data were performed at the Department of Neurosurgery, The University of Tokyo.
Fisher exact test was used to compare the rates of occurrence of the c.14576G>A variant in each phenotype with the rate in the control group. All analyses were performed with JMP Pro version 10.0.2 (SAS Institute, Inc, Cary, NC). A P value <0.05 was considered statistically significant. The statistical methods used in the tables in the online-only Data Supplement are described in the Method in the online-only Data Supplement.
This study was approved by the Human Genome, Gene Analysis Research Ethics Committee of the Faculty of Medicine, The University of Tokyo (approval number, 3516; approval date, September 12, 2011). This study was also approved by the Ethics Committee of Kanto Neurosurgical Hospital (approval date, June 23, 2012). Written informed consent was obtained from all study participants.
The 323 participants were divided among definite MMD (22), unilateral MMD (8), non-MMD ICASO (84), ECAS (34), cerebral aneurysm (44), and ICH (22). Control subjects without cerebrovascular disease (110) were also recruited. Clinical characteristics and conventional risk factors of each phenotype are shown in Table I in the online-only Data Supplement.
The c.14576G>A variant was present in 16/22 in the definite MMD group (15 heterozygotes and 1 homozygotes), 4/8 patients in the unilateral MMD group (4 heterozygotes), and 20/84 in the non-MMD ICASO group (20 heterozygotes). In contrast, only 1 patient (1 heterozygotes) with ECAS and no patient with cerebral aneurysm or ICH had the c.14576G>A variant. Two of 110 patients in the control group had the c.14576G>A variant (2 heterozygotes). The RNF213c.14576G>A genotypes were in Hardy Weinberg equilibrium in each group and control (Table I in the online-only Data Supplement).
The c.14576G>A variant had significant associations with definite MMD (P<0.0001; OR, 144.0; 95% confidence interval, 26.7–775.9), unilateral MMD (P=0.0001; OR, 54.0; 95% confidence interval, 7.5–386.8), and non-MMD ICASO (P<0.0001; OR, 16.8; 95% confidence interval, 3.81–74.5) but no association with ECAS, cerebral aneurysm, or ICH (Table). Figure 2 shows MR angiograms of 4 non-MMD ICASO with the c.14576G>A variant. The 2 control subjects with the c.14576G>A variant had a normal MR angiogram and no asymptomatic disease (Figure II in the online-only Data Supplement).
Previous studies have shown an association between c.14576G>A gene dosage and age of onset of MMD. In our cases, there was no such association (Table II in the online-only Data Supplement). However, among all ICASO phenotypes, onset age of cases with c.14576G>A (both A/A and G/A) was significantly earlier than cases without c.14576G>A (G/G).
Combined data (from the present and previous study) are shown in the online supplement (Figure III and Tables III–V in the online-only Data Supplement). The combined data confirmed that the RNF213 c.14576G>A variant is associated with not only definite MMD, but also unilateral MMD and non-MMD ICASO.
This study confirms the strong association of the genetic variant RNF213 c.14576G>A with various phenotypes of ICASO lesions: definite MMD, unilateral MMD, and non-MMD ICASO but no association with other cerebrovascular diseases, such as ECAS, cerebral aneurysm, and ICH.
The strong association of genetic variant RNF213 c.14576G>A with definite MMD among the East Asian populations has been reported recently.8–11 Whether unilateral MMD has a common cause or genetic background with definite MMD, despite the angiographic similarity, remains unclear.13 The association of RNF213 c.14576G>A with unilateral MMD was not as strong as with definite MMD but indicates that a high proportion of unilateral MMD cases have a common genetic variant with definite MMD.
Furthermore, a particular subset of non-MMD ICASO also had RNF213 c.14576G>A variant, in common with definite MMD, consistent with our previous report from a smaller cohort and single facility.7 Several previous reports have shown that Asians experience development of atherosclerotic ICASO with significantly higher rates than whites,6 suggesting that a genetic factor peculiar to Asians might be responsible. The c.14576G>A variant in RNF213 could be the candidate genetic variant for this epidemiological difference because the RNF213 c.14576G>A variant is present in ≈2% of East Asian populations, a relatively higher rate compared with whites.8–11,14 In the present study, 2 of 110 (1.8%) of the control group and 20 of 84 (23.8%) patients with non-MMD ICASO had the c.14576G>A variant in RNF213 (P<0.0001; OR, 16.8; 95% confidence interval, 3.81–74.5).
This identification of a common genetic variant among ICASO lesions may lead to a new genetic diagnosis system in the field of stroke, conventionally thought to be caused by acquired risk factors. In terms of stroke prevention, management of conventional acquired risk factors, such as hypertension, diabetes mellitus, and dyslipidemia, has been considered important. However, the findings of our present study indicate that a proportion of the general population has a congenital or genetic risk factor (RNF213 c.14576G>A) for ICASO, a major cause of ischemic stroke. In addition, the presence of RNF213 c.14576G>A may associate with high blood pressure.15 Thus, there are strong indications that RNF213 c.14576G>A is a robust genetic risk indicator for ischemic stroke. Preventive treatment for stroke, such as blood pressure management, and image screening, such as MR angiography, for intracranial cerebral arterial lesion would be very important, especially for individuals with this specific allele, which is present in ≈2% of the Japanese population.14
One limitation of this study is that analysis of the RNF213 variant was exclusively focused on the c.14576G>A variant. Some patients with MMD and typical angiographic features have a RNF213 variant other than the RNF213 c.14576G>A variant.8–11 Therefore, some of our MMD or even non-MMD ICASO patients without the RNF213 c.14576G>A variant might have had another genetic variant, such as the c.13342G>A (p.A4448T) variant. Further comprehensive genetic analysis of RNF213, such as whole genome analysis, is necessary to determine whether patients with MMD or non-MMD ICASO without the c.14576G>A variant have another RNF213 variant.
The present study strongly indicates that a particular subset of patients with various ICASO phenotypes has a common genetic variant, RNF213 c.14576G>A, indicating that RNF213 c.14576G>A variant is a high-risk allele for ICASO. Genetic screening for the RNF213 c.14576G>A variant would enable appropriate preventive treatment for stroke.
This work was supported by a Grant-in-Aid for Scientific Research (B) (No. 25293304) to Dr Saito and a Grant-in-Aid for Scientific Research (C) (No. 25462206) to Dr Imai from the Japan Society for the Promotion of Science, and by grants from SENSHIN Medical Research Foundation to Dr Miyawaki. The other authors report no conflict.
The online-only Data Supplement is available with this article at http://stroke.ahajournals.org/lookup/suppl/doi:10.1161/STROKEAHA.113.002477/-/DC1.
- Received June 28, 2013.
- Accepted July 8, 2013.
- © 2013 American Heart Association, Inc.
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