Association of rare RNF213 variants and intracranial aneurysm risk in a Chinese population

Introduction

Intracranial aneurysm (IA) is a complex disease characterized by the dilation or ballooning of intracranial arteries (1). The rupture of IAs has various clinical consequences, the most devastating of which is subarachnoid hemorrhage (SAH) (2). Overall, the incidence of crude aneurysmal SAH (aSAH) has been reported to be 6.9–9.0 per 100,000 people per year worldwide (3). Clinically, aSAH patients experience a sudden, severe headache, and half suffer a sudden loss of consciousness (4). The aSAH occurs in relatively young patients (aged 40–60 years on average) and leaves many with lasting disabilities that prevent them from returning to their previous jobs (5). However, the mechanisms of IA formation, growth, and rupture remain largely unknown.

Studies have revealed that hypertension, cigarette smoking and excessive alcohol consumption increase the risk of IA, while regular physical activity and healthy lifestyles can reduce the risk (6,7). Some unmodifiable factors (such as increasing age, and females) also increase the risk of IA (7). Besides, genetic factors also play a key role in the development of IA (8,9). The risk of IA is increased among family members with a history of IA or certain genetic disorders, such as autosomal dominant polycystic kidney disease or Ehlers-Danlos syndrome (8). Additionally, people living in specific geographical locations, such as Finland and Japan, have a higher risk of IA rupture (10). Genome-wide and candidate gene association studies have identified several common variants associated with IA, including SOX17 (rs9298506 and rs10958409), EDNRA (rs6841581), CDKN2B-AS1 (rs10757278), COL1A2 (rs42524), COL3A1 (rs1800255), HSPG2 (rs3767137), SERPINA3 (rs4934), and VCAN (rs251124 and rs173686) (8,11). However, these common variants only explain a small fraction of IA, and the genetic predispositions of IA are largely unknown.

Rare and low-frequency variants, which are not usually detected by genome-wide genotyping arrays, affect phenotypes (12,13). An early study on rare variants also suggested a large phenotypic effect (14). RNF213 has been identified as a susceptibility gene for Moyamoya disease (MMD), a cerebrovascular disorder characterized by progressive occlusive lesions in the circle of Willis (15,16). The founder mutation p.R4810K (rs112735431) of RNF213 was found to be associated with intracranial artery stenosis/occlusion diseases, coronary artery stenosis, and pulmonary artery stenosis in East Asians (17). Our previous study also identified the association between rare RNF213 variants and cerebral arteriovenous malformation (cAVM) in a Chinese population (18). Considering the association of RNF213 with various vascular diseases, the results of studies focusing on the RNF213 and its role in vasculature implied this gene could be a key antimicrobial protein in response to the infectious or autoimmune signaling signals in the process of vascular disease (19). Till now, several RNF213 variants were identified their association with IA in the French-Canadian population (1), whereas the study in the German and Japanese populations failed to obtain a significant result, revealing the existence of racial diversity (20,21). It is not yet known whether rare RNF213 variants play a role in the pathogenesis of IA in the Chinese population. Thus, we performed exploratory RNF213 target exome sequencing in a group of Chinese patients with IA and explored the association between rare RNF213 variants and the risk of IA by conducting a single-variant association study and gene-based burden analysis. We present the following article in accordance with the MDAR reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-5166/rc) (22).

Methods

Study subjects

Patients diagnosed with IA at the Department of Neurology and Neurosurgery of Xiangya Hospital and Hunan People’s Hospital from December 2018 to December 2019 were included in the study. All the patients with IA were enrolled and their diagnoses were confirmed by computed tomography angiography, magnetic resonance imaging, or digital subtraction angiography. The imaging results were interpreted by more than two physicians, including more than one radiologist and one neurological physician, and any disagreements were resolved by consensus or by a third neurological physician. Patients with traumatic or infectious IA or IA concomitant with other vascular diseases (e.g., MMD and cAVM) or hereditary disorders (e.g., autosomal dominant polycystic kidney disease, Ehlers-Danlos syndrome, and Marfan syndrome) were excluded from the study. Demographic and lifestyle information (e.g., age, gender, and behavioral factors, such as smoking and alcohol consumption), and clinical phenotype data (e.g., clinical symptoms, IA location and shape, and disease history) were collected through clinical interviews, health examinations, or imaging checkups. The minor allele frequency (MAF) of the control subjects was obtained from the following two online databases: the 1,000 Genome Project database of the Chinese Han Population (a public genome sequencing database of 208 normal Chinese Han individuals, http://www.internationalgenome.org/home), and the Shanghai GeneSky company in-house database (a private exome sequencing database of 1,007 Chinese individuals without known diseases, http://www.geneskybiotech.com/index.html). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the Medical Ethics Committee of Central South University (No. CTXY-150002-1), and written informed consent was obtained from all the participants.

DNA extraction and targeted exome sequencing

Peripheral blood samples were collected from all the enrolled patients using 5 mL EDTA-K2 vacuum tubes (LiuyangSanli Medical Technology Development Co., Ltd., Liuyang, China) and stored at −80 ℃. We used TIANamp Blood DNA Extraction Kit (Tiangen Biotech Co., Ltd., Beijing, China) for Genomic deoxyribonucleic acid (DNA) extraction from the leukocytes in peripheral blood. To identify the rare RNF213 variants, protein coding sequences were selected, and custom polymerase chain reaction (PCR) primers were designed according to the primerXL pipeline. Ultimately, 100 oligonucleotide pairs were produced, encompassing all coding sequences and untranslated regions of the target RNF213 gene (https://cdn.amegroups.cn/static/public/atm-22-5166-1.xlsx). The capture target regions contained 25-base flanking sequences at each exon’s borders. The AB 2720 Thermal Cycler (Applied Biosystems, Waltham, MA, USA) was used for amplification reactions. The DNA-adapter-ligated and -indexed fragments were pooled and hybridized after PCR products were processed to build a library for further detection. After the sequencing primer was hybridized, targeted sequencing was conducted using an Illumina HiSeq high-throughput sequencing platform (Illumina, San Diego, CA, USA) in accordance with the manufacturer’s instructions.

Read mapping, variant calling, and annotation

The sequencing read quality was evaluated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were aligned to the human reference sequence [National Center for Biotechnology Information (NCBI) Genome build GRCh37, https://www.ncbi.nlm.nih.gov/genome/guide/human/#download] using the Burrows-Wheeler Aligner (http://bio-bwa.sourceforge.net/). The variants calling was performed using the HaplotypeCaller from the Genome Analysis Toolkit (GATK, https://software.broadinstitute.org/gatk/best-practices/) and VarScan (http://varscan.sourceforge.net/). The coverage analysis was performed using the Picard software CalculateHsMetrics tool. The reads matching the exonic regions, including the exon-intron boundaries, were analyzed. The potential effects on protein function by the variants were assessed using polymorphism phenotyping V2 (Polyphen2, genetics.bwh.harvard.edu/pph2) and Sorting Intolerant From Tolerant (SIFT, http://sift.bii.a-star.edu.sg/). The protein BLAST search engine (http://blast.ncbi.nlm.nih.gov/Blast.cgi) was used for determination of variant homology.

Variant filtration

Filtering criteria were used to prioritize the variants identified from targeted exome sequencing. The variants were regarded as potentially functional variants low-frequency variants (MAF <0.05) or rare variants (MAF <0.01) if they were: (I) located in the exonic or splicing region; (II) predicted to affect protein coding sequences (including missense, nonsense, read-through, frameshift insertion or deletion, and splice-site variants); (III) less common in reference databases (MAF <0.05 in 1,000 Genome Project Chinese Han population database and Shanghai GeneSky company in-house Database); (IV) predicted to have a functional effect on proteins evaluated by SIFT or PolyPhen-2 (damaging, possibly damaging, or unknown); (V) sequenced with a read depth ≥10×.

Sanger sequencing

We performed the Sanger sequencing to re-confirm the existence of filtered RNF213 variants. The primers for PCR amplification were designed based on human reference sequences (NCBI, http://www.ncbi.nlm.nih.gov/tools/Primer-blast/index.cgi?LINK_LOC=BlastHome). Sanger sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Buenos Aires, Argentina) on an ABI 3730XL DNA sequencer (Applied Biosystems, Buenos Aires, Argentina). The peak map was generated by comparing variant sequences with human reference sequences using Seqman Pro 11.

Statistical analysis

SPSS software (version 21.0; SPSS Inc., Chicago, IL, USA) was used for the statistical analysis. The normally distributed continuous variables are described as means and standard deviations (SDs), and the categorical variables are described as proportions. Odds ratio (ORs) and 95% confidence intervals (CIs) were used to evaluate the associations of IA with single variants by binary logistic regression analysis assumed in an autosomal dominant inheritance model. The aggregate effects of non-common variants (MAF <0.05) were assessed by the gene-based burden tests, including the weight-sum statistic (WSS), the sequence kernel association test (SKAT), and the SKAT-optimal test (SKAT-O) (23,24). The continuous weight function was used to analyze the non-common variants. The logistic weights of the RNF213 variants were calculated and then applied to the analysis as follows (23):

wj=1/[MAFj(1−MAFj)]1/2[1]

which allows non-common variants to be considered in the overall statistic. For the comparison of the clinical characteristics between carriers and non-carriers of rare RNF213 variants, the Student’s t-test was used for the continuous variables of normal distribution, and the chi-squared test or Fisher’s exact test was used for the categorical variables. P less than 0.05 showed a statistical significance.

Results

Characteristics of the population

In total, 174 IA patients were recruited for this study, of whom 105 (60.3%) were female. The patients had an average age of 56.8±10.9 years (range, 18.2–87.7 years). In total, 129 (74.2%) patients had a single aneurysm. The location of IAs were as follows: internal carotid artery (46.8%), middle cerebral artery (16.4%), anterior communicating artery (14.8%), and other locations (22.0%). Detailed demographic and clinical characteristics are summarized in Table 1.

Rare variant identification and gene-based association analysis

RNF213 exome sequencing was performed using the FastTarget technology. We obtained 3.68–53.44 Mb of targeted sequence suitable for mapping, with a mean sequencing depth between 92.8- and 1,292.9-fold. The average target sequencing depth was 520-fold. Of these, 99.87% of the reads had a Phred-like quality score (Q score) >20, and 87.07% had a Q score >30. The average proportions of target bases with read depths of 2-, 10-, 20- and 30-fold were 95.02%, 91.65%, 88.74%, and 85.91%, respectively.

After the variant annotation, we focused primarily on less common non-synonymous variants (missense and nonsense variants), splice-acceptor and donor-site variants, and coding indels based on the assumption that these variants were more likely to be pathogenic, compared to those synonymous ones which do not change amino acids of proteins. We filtered our data according to the MAF information in the 1,000 Genome Project Chinese Han Population and Shanghai GeneSky database (MAF <0.05). Overall, 14 RNF213 variants were identified in 18 IA patients, including 10 missense variants, 3 splice-site variants, and 1 frameshift insertion variant (Tables 2,3). All the missense variants were predicted as deleterious variants. Twenty alternative alleles were identified in the IA patients (n=174) and 94 in the control subjects (n=1,215, cumulative allele OR, 1.73; 95% CI: 1.05–2.83; P=0.029).

A significant association was not identified among the variants (Table 4). The bene-based association analysis, which used both the burden and variance component tests, revealed that less common RNF213 variants (MAF <0.05) were significantly associated with IA (WSS P=2.27×10^-6; SKAT P=4.97×10^-5; SKAT-O P=1.13×10^-5). The allelic distribution for rare variants carried in IA patients was significantly different from controls (WSS P=5.08×10^-9; SKAT P=2.96×10^-6; SKAT-O P=3.56×10^-8) (Table 2).

Clinical characteristics of patients with and without rare RNF213 variants

Among eighteen patients carrying RNF213 variant, there was no significant difference between carriers and non-carriers in terms of any clinical characteristic, including age, gender, IA risk factors (including smoking, drinking, hypertension, diabetes, hyperlipidemia, and coronary heart disease), and IA rupture status, and number, and location (Table 5).

Discussion

In this study, we conducted RNF213 exome sequencing in 174 IA patients and identified 14 variants in 18 IA patients, including 3 low-frequency variants and 11 rare variants. A gene-based association analysis revealed that the rare RNF213 variants were significantly associated with IA in this Chinese population. However, there was no significant difference between the carriers and non-carriers of RNF213 variants in terms of the clinical characteristics. Further comprehensive studies on the mechanisms how RNF213 plays a role in the disease need to be conducted to confirm these results.

There is considerable evidence that genetic variants are involved in the pathogenesis of IA (25-27). IA-related genes might play different roles in the blood vessel wall; for example, these genes may be involved in endothelial formation and maintenance (i.e., SOX17), or endothelial cell integrity maintenance (i.e., the ADAMTS gene family) (28). RNF213 is a ring-finger protein gene that encodes a 591-KDa protein that functions as both an AAA-type ATPase and an E3 ligase, which play a role in the construction of vascular walls (15,29). It was first identified as an important susceptible gene for MMD in East Asian populations (15,16). Subsequent studies showed that RNF213 variants are not only susceptibility factors for MMD, but also for intracranial atherosclerosis (30) and systemic vascular diseases, such as coronary, pulmonary, and renal artery stenosis (17,31,32). Notably, patients with the RNF213 rare variant (p.R4810K) may present successively with arterial dissection, SAH, intracranial artery stenosis, and MMD (31). From a functional perspective, Kobayashi et al. found that the p.R4810K mutation of RNF213 resulted in the decreased angiogenic activity of vascular endothelial cells and the inhibition of angiogenesis in the presence of interferon or hypoxia in patients with MMD (33). A recent study showed that RNF213 is critical in the ubiquitination of the bacterial lipopolysaccharide and that cells lacking RNF213 suffer from cellular autoimmune deficiency (34). The RNF213 variant causes reduced E3 ligase activity and ubiquitination, which enhances the stimulation of NFĸB and inflammatory signaling, finally leading to vasculopathy (19). However, the mechanistic details of RNF213 have not yet been elucidated.

RNF213 was first found to be associated with IA in a French-Canadian (FC) study in which the following 5 rare RNF213 variants were identified in 4 affected families: p.E3806Rfs*27 (c.11413del), p.R673W (c.2017C>T), p.I4526T (c.13577T>C), p.N2327S (c.6980A>G), (NM_001256071.2), and p.S1045L (c.3134C>T) (NM_020954.3). IA-affected individuals carried the RNF213 variants with significantly higher rates compared to the controls (1). In a subsequent validation study with an increased sample size, the authors confirmed significant differences in RNF213 functional variation between the patient group and control group in the FC population (1). An exome sequencing study in Germany identified another four variants in three patients with IA/SAH; that is, p.L133M (c.397C>A), p.I209N (c.626T>A), p.A2695V (c.8084C>T), and p.T4677L (c.14030G>T) (NM_001256071) (20). In the present study, we did not identify p.R4810K in the IA patients, consisted with a previous study in the Japanese population (35). Murai et al. found that patients with internal carotid artery cystic aneurysms had a similar risk of p.R4810K mutation compared to patients with internal carotid artery stenosis (21). Additionally, 14 RNF213 variants were found in 18 patients in our study. Among them, all the variants except p.M3666T were not identified in cerebrovascular diseases (31), and none of the variants had been previously identified in patients with IA. However, the variant p.M3666T (c.10997T>C) was previously identified in a patient with MMD (36), and we identified the same variant in two patients with cAVM in our previous study (18).

A total of seven novel variants [four missense variants (p.R759Q, p.R3168W, p.S3223L, and p.G4847S), a splice-site variant (c.6184-15C>T), and two frameshift variants (p.N4850fs and p.V5193fs)] were identified in our study, which had not previously been reported neither in the online databases of the 1,000 human genomes nor 1,007 in-house controls (Table 3). In our study, four variants (i.e., p.I2417S, p.A2914T, p.R3168W, and p.S3223L) were located in the AAA-ATPase domain, and four variants [i.e., p.M3666T, splice-site variant (c.13186-13T>C), p.P4576S, p.G4847S, and p.N4850fs] were located in the E3 ligase domain; the remaining five were located in the N-arm region. Zhou et al. also identified two deleterious rare variants [i.e., p.R2438C (c.7312C>T), and p.A2826T (c.8476G>A)] in the AAA-ATPase domain and suggested that the domain might be a risk region for IA (1). Cecchi et al. conjectured that rare mutations in the C-terminus have a significant effect on MMD (37). Thus, the relationship between the pathogenesis of cerebrovascular disorders and the functional domain of RNF213 needs to be explored.

Additionally, our gene-based association analysis showed that rare RNF213 variants were significantly associated with IA, suggesting that RNF213 may be a susceptible factor for IA, which was consistent with the results of the previous study (1). Among the 18 RNF213 carriers, two patients carried two different variants, both of whom were elderly women with multiple unruptured IAs. Sauvigny et al. found that an IA patient with two rare RNF213 variants had a giant aneurysm (20). However, the relationship between the number of RNF213 variants and IA severity has yet to be investigated. Among MMD patients, homozygotes of c.14576G>A (p.R4810K) variant present at an earlier onset age, with more severe clinical symptoms, and result in a poorer cognitive prognosis than heterozygotes and wild types c.14576G>A variant (p.R4810K) (38,39). In addition, patients with intracranial artery stenosis/occlusion disease who carried a rare RNF213 variant showed significant age differences compared to non-carriers (40). Based on these studies, we explored the association between genotype and phenotype in patients with IA. However, we did not find any significant differences between the carriers and non-carriers of RNF213 variants in terms of their clinical characteristics, including age, gender, risk factors for IA (smoking, drinking, hypertension, diabetes, hyperlipemia, and coronary heart disease), or IA rupture status, number and location. An assumption arose that IA, as a multi-factor-caused disorder, could be influenced by environmental factors or other genetic factors beyond the RNF213 variations. Thus, the effect of other risk factors on IA should also be explored in future studies.

This study had several limitations. Firstly, it was an exploratory study in which only 174 patients were enrolled. The relatively small sample size may have limited the statistical power in exploring the associations between rare RNF213 variants and the risk of IA. Thus, further studies with larger sample sizes need to be conducted to validate our findings. Secondly, this was a regional study, and there might be a selection bias. Thus, multiple-center studies are needed in the future to further verify the association between RNF213 and IA. Thirdly, due to the invasive examination and relatively expensive cost of cerebrovascular angiography, recruiting control subjects who have been clinically proven to be aneurysm-free is difficult. The rare variant information of the control group was obtained from the 1,000 Genome Project and Shanghai GeneSky in-house database rather than matched controls from the same area. This may have affected the results. However, these two databases consist of >1,000 exome sequencing data of Chinese individuals without known diseases, so the results may not have been significantly affected. Fourthly, we did not perform any functional studies, and the exact mechanism of action of these variants in IA remains unclear. Fifthly, IA is a multifactorial disease that is usually caused by multiple environmental and genetic factors. However, information on environmental factors, such as hypertension and smoking, were not available from the control groups; thus, we could not adjust for the effect of these environmental factors in the gene-based association study.

Conclusions

In our study, rare RNF213 variants were associated with IA in a Chinese population. Our findings suggest that these rare RNF213 variants might have potentially important roles in IA. In the future, more comprehensive studies on the etiology of IA need to be conducted.

Acknowledgments

We would like to thank all the participants who provided samples and those involved in the sample collection.

Funding: This work was supported by grant to JY from the Natural Science Foundation of Hunan Province, China (No. 2021JJ30911) and Central South University Case Database Construction Project for Graduate Students, China (No. 2020ALK24).

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-5166/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-5166/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-5166/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). This study was approved by the Medical Ethics Committee of Central South University (No. CTXY-150002-1) and written informed consent was obtained from all the participants.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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