Impact of RNF213 c.14576G>A Variant on the Development of Direct and Indirect Revascularization in Pediatric Moyamoya Disease

Background: Superficial temporal artery (direct) and encephalomyosynangiosis (indirect) revascularization may develop after combined bypass surgery in pediatric patients with moyamoya disease (MMD). However, arterial development varies widely among patients, and the underlying mechanisms remain unknown. Objectives: We evaluated the relationship between the development of donor arteries after bypass surgery in pediatric patients with MMD and the MMD-susceptibility gene variant c.14576G>A of ring finger protein (RNF) 213. Methods: The data of pediatric patients with MMD (age <16 years at the time of surgery) treated with combined bypass surgery between September 2013 and April 2019 were consecutively analyzed. Quantitative measurements of the superficial temporal artery (STA), deep temporal artery (DTA), and middle meningeal artery (MMA) diameters with magnetic resonance angiography (MRA) source imaging were performed preoperatively and at 6–12 months postoperatively. The postoperative caliber change ratios (CCRs) were calculated. The relationship between CCRs and RNF213 c.14576G>A status was examined. Results: Forty-eight hemispheres from 28 pediatric patients with MMD were examined. Three hemispheres belonged to patients with the AA genotype; 33 to patients with the AG genotype (AA/AG group); and 12 to patients with the GG genotype (GG group; wild type). The CCRs for the DTA were significantly higher in patients with RNF213 variant (AA/AG group; 2.5 ± 0.1) than in the GG group (2.0 ± 0.2) (p = 0.03), whereas the CCRs for the STA were significantly higher in the GG (1.6 ± 0.1) than in the AA/AG group (1.3 ± 0.6) (p = 0.02). There was no significant difference in the CCRs for the MMA and basilar artery between the groups. Other factors, including sex, age, and MRA grading, were not associated with the development of specific bypass development. Conclusions: The extent of collateral development associated with direct or indirect bypass was found to differ between the genotypes of the RNF213 c.14576G>A associated with pediatric MMD. This genetic variant correlates with the development of the disease and affects revascularization after bypass surgery in pediatric patients with MMD.

© 2022 The Author(s). Published by S. Karger AG, Basel

Introduction

Moyamoya disease (MMD) is a cerebrovascular disease characterized by progressive occlusion of the supraclinoid segment of the internal carotid artery, which results in compensatory development of abnormal, fragile vascular networks at the base of the brain [1]. The etiology and pathogenesis of MMD have long been unknown; however, a single base substitution (nonsynonymous mutation), c.14576G>A (p.R4859K) in the ring finger protein 213 gene (RNF213 gene; chromosome 17q, NP_065965.4), has recently been identified as one of the major causes of MMD in Japanese and East Asian MMD populations [2, 3]. Transient ischemic attack and/or ischemic stroke are major clinical presentations in pediatric patients with MMD. Revascularization surgery, i.e., bypassing the superficial temporal artery (STA) to the middle cerebral artery (MCA) (direct bypass) and/or attaching the pediculate donor tissues to the brain surface (indirect bypass), has been shown to improve the cerebral hemodynamics and to reduce the risk of subsequent stroke [4].

However, neovascularization through direct and indirect bypass after surgery produces a heterogeneous arterial development pattern in pediatric patients with MMD, and the underlying mechanisms remain unknown. In this study, we sought to elucidate the relationship between the development of donor arteries after combined bypass surgery and the susceptibility variant in RNF213.

Materials and MethodsStudy Population

Pediatric patients with MMD (age <16 years at the time of surgery) treated with combined (direct and indirect) bypass surgery between September 2013 and April 2019, at Hokkaido University Hospital, were consecutively analyzed. All patients fulfilled the radiological criteria for the diagnosis of MMD using magnetic resonance (MR) imaging and MR angiography (MRA). We reviewed the medical records and radiological data of the patients in accordance with an Institutional-Review-Board-approved protocol (number 14-053). Although all procedures were completed at the time of study research, exposure measurement and the genomic backgrounds were not influenced by the disease outcome retrospectively; thus, this study can be classified as a prospective, observational, cohort study.

Combined Revascularization Surgical Procedures

Bypass surgery was selected when the hemispheres were considered to be the cause of the patients’ symptoms, most of which were hypoperfusion of blood supply to the brain. Asymptomatic hemispheres were also considered to indicate surgical intervention when the arterial stenosis progressed rapidly after contralateral surgical procedure since these patients are reported to be at high risk of ischemic symptoms in the near future [5]. Combined revascularization comprised a double or single STA-MCA anastomosis and an indirect bypass using the dura mater and temporal muscle, as previously described [6, 7]. Briefly, STAs were anastomosed to the cortical branches of the MCA as a direct bypass, followed by attaching the temporal muscle and the periosteal flap to the brain surface to create the indirect bypass.

Evaluation of Indirect and Direct Bypass Development with MRA

Preoperative MRA was performed within 1 week preoperatively, and postoperative MRA was performed between 6 and 12 months after the surgery, using a clinical MRI unit [7]. Bypass development was quantitatively evaluated using MRA source images by measuring the calibers of the relevant vessels, as previously described (online suppl. Fig. 1; for all online suppl. material, see www.karger.com/doi/10.1159/000526089) [8]. For direct bypass, we compared the calibers of the STA at the most proximal portion of the bifurcation of the frontal and parietal branches. Similarly, for indirect bypass, the caliber of the most proximal portion of the deep temporal artery (DTA) and MMA was evaluated. The caliber change ratios (CCRs) of the pre- and postoperative calibers were calculated using the following formula: CCRs = postoperative caliber diameter (mm)/preoperative caliber diameter (mm) (%). The caliber of the basilar artery was measured as an internal control.

Identification of RNF213 Mutations

RNF213 genotyping was performed as previously described, with minor modifications [9]. Briefly, white blood cells were collected from whole blood samples, and genomic DNA was extracted using the QIAsymphony DSP DNA Midi kit (Qiagen, Hilden, Germany). Then, the extracted DNA was amplified using the primer sequence of RNF213 (rs11273543 1 c.14576G>A [p.R4859K]) located in exon 61, based on the National Center for Biotechnology Information reference sequence number 1. NM_020914.4 as follows: forward, 5′-CTG CAT CAC AGG AAA TGA CAC TG-3′, reverse, 3′-TGA CGA GAA GAG CTT TCA GAC GA-5′. Sequence analyses were conducted using Applied Biosystems 3130 DNA Analyzers (Thermo Fisher Scientific, Waltham, MA, USA).

Data and Statistical Analysis

All values were treated in a blinded manner and are expressed as means ± standard deviations. Student’s t test was used to evaluate differences between the genotype groups. To assess the correlation between the specific bypass developments and multiple clinical and genetic variables, multivariate logistic regression analysis was performed. First, receiver operating characteristic analysis was performed to determine the cutoff value of indirect or direct superiority, and a CCR(DTA)/CCR(STA) ratio value of 1.72 was set from the maximum sensitivity and specificity. Then, the relationship between bypass development and clinical and genetic factors, which are selected from the clinical perspective, were compared using univariate analysis, followed by multivariate analysis. All statistical analyses were performed using JMP Pro 13 (Systat Software, San Jose, CA, USA), and a value of p < 0.05 was considered statistically significant.

ResultsPatient Characteristics

Forty-eight hemispheres from 28 pediatric patients with MMD were examined. There were three affected hemispheres in 2 patients with the AA genotype (homozygous mutation), 33 hemispheres in 19 patients with the AG genotype (heterozygous mutation), and 12 hemispheres in 7 patients with the GG genotype (the GG group; wild type). As the number of patients with the AA genotype was small, the patients with AA and AG genotypes were combined for subsequent analysis as the AA/AG group. There were no differences in the mean age at treatment, clinical types, and preoperative MRA grading between the AA/AG and GG groups (online suppl. Table 1). Forty-six hemispheres were treated with double bypass using frontal and parietal branches of the STA, whereas two hemispheres were treated with single bypass using the frontal branch. These 2 cases demonstrated hypoplastic parietal branch and were considered to be inappropriate for bypass procedure. Little to no blood supply was recognized at the postoperative MRA through this hypoplastic parietal branch, which confirmed that the majority of the blood supply was perfusing into the brain through the bypass.

Neovascularization

Neovascularization after bypass surgery showed distinctly different patterns between the two groups. The CCRs for the DTA significantly increased in the AA/AG group (2.5 ± 0.1) compared with those in the GG group (2.0 ± 0.2) (p = 0.03) (Fig. 1a), whereas the CCRs for the STA significantly increased in the GG group (1.6 ± 0.1) compared with those in the AA/AG group (1.3 ± 0.6) (p = 0.02). However, there was no statistically significant difference in the CCRs for the MMA and basilar artery between the groups. Specific bypass developments and confounding factors were further examined (online suppl. Table 2). Genotypes, sex, treated hemisphere, age (<6 years), and MRA grading (>3) were selected as clinical confounding factors. Univariate analysis revealed that genotype (AA/AG) and sex (female) were associated with better development of indirect bypass (p = 0.02 and 0.08, respectively), whereas treated hemisphere, younger age, and higher MRA grading were not. These factors are further evaluated with multivariate analysis, and only genotype (AA/AG) was found to be highly correlated with the development of indirect bypass (RR, 5.7; 95% confidence interval, 1.05–31.2; p = 0.04). Figure 2 shows the different vascular development patterns in the AA/AG (Fig. 2a, b) and GG groups (Fig. 2c, d). A patient in the AA/AG group (9-year-old female, AG variant) showed significant DTA development and minimal STA development. In contrast, a patient in the GG group (14-year-old female) displayed the opposite, where the development of the DTA was minimal and the STA was well developed.

Fig. 1.

Quantitative analysis of postoperative direct collateral development. The CCR values calculated by time-of-fight MRA with maximal intensity projection reconstruction before and after combined bypass surgery were compared between patients with RNF213 c.14576G>A AA/AG genotypes and those with the GG genotype. The DTA was well developed in the AA/AG group, while the STA was developed in the GG group. There was no significant difference between genotypes in terms of development of the MMA and basilar artery.

/WebMaterial/ShowPic/1454151Fig. 2.

Representative cases of revascularization in the RNF213 c.14576G>A AA/AG group and RNF213 c.14576G>A GG group A patient in the AA/AG group (9-year-old female, AG variant) showed significant development of the deep temporal artery (DTA) (a, b, arrowhead) and minimal development of the superficial temporal artery (STA), (a, b, arrow) when comparing the preoperative MMA by MRA (a) and postoperative MRA (b). In contrast, a patient in the GG group (14-year-old female) displayed the opposite, where development of the DTA (c, d, arrowhead) was minimal but the STA was well developed. (c, d, arrow) Comparing preoperative (c) and postoperative MRA (d) findings.

/WebMaterial/ShowPic/1454149Discussion/Conclusion

In this study, we were able to determine that genotypes of RNF213 c.14576G>A correlated with post-surgical neovascularization, in which pediatric patients with RNF213 variant (AA/AG genotypes) showed significant development in the indirect bypass through the temporal muscle, resulting in DTA enlargement, whereas the direct bypass was complementary and well developed in the patients with the GG genotype. Although the precise function of RNF213 remains unknown, the fact that a variant in RNF213 strongly correlates with development of MMD and other vascular diseases, including peripheral pulmonary artery stenosis and renal artery stenosis, it is strongly suspected to have an angiogenic function. In fact, RNF213 knockout resulted in angiogenic abnormalities during the embryonic period of zebrafish development [3] and in RNF213 knockout mice under ischemic conditions [10]. Genome-wide plasma/serum microRNA profiling revealed that inflammatory or angiogenetic molecular pathways are strongly correlated with MMD [9], and an M2 macrophage-related immune reaction is suspected in patients with MMD because of the upregulation of the serum CD163 and CXCL5 levels [11]. These reports strongly suggest that RNF213 has a positive/negative functional relationship with inflammation and neovascularization. We have recently reported that the postoperative revascularization status in adult patients with MMD with AA/AG genotypes showed a superior development of indirect collaterals, as compared with that of patients with MMD with the GG genotype, whereas direct collateral development was more prevalent in such patients with the GG genotype [12]. These data were in line with the data presented in this report and supported the hypothesis that postoperative angiogenesis correlates with genetic variants in patients with MMD. These findings suggest the complementarity of post-surgical bypass development, in which sufficient development of indirect bypass in the AA/AG group led to less direct bypass development, while poor development of indirect bypass in the GG group led to increased direct bypass development. In the previous report, preoperative advanced MRA findings were correlated with the development of an indirect bypass; however, we found that the severity of arterial stenosis estimated through MRA grading was similar between the AA/AG and GG groups and was not correlated with the development of indirect bypass. This result was in line with that of a previous work stating that the extent of hemodynamic compromise was not correlated with the development of postoperative STA and DTA in pediatric patients with MMD [8]. Dyslipidemia was also reported to be correlated with the development of indirect bypass in adult MMD patients, which may be related to the recent finding that RNF213 acts against intracellular lipid storage and triggers immune responses [11, 13]. However, our pediatric patients also did not show any dyslipidemia at the time of admission (data not shown). The reason for these differences between adult and pediatric MMD remains unclear; however, different mechanisms of RNF213 may be involved in disease progression even though the same results of revascularization are seen after surgery. Younger patient age and the size of cerebral hemodynamic compromise are reported to influence the degree of revascularization [14, 15]. However, we could not elucidate the influence of these factors in our series because of the limited number of pediatric patients with cerebral infarction. We also did not routinely perform preoperative cerebral hemodynamic evaluations using either single photon emission computed tomography or positron emission tomography. Larger series are necessary to elucidate the multivariate relationship between the genomic variant and other factors, including age and preoperative hemodynamic condition.

In conclusion, the degree of collateral development by direct or indirect bypass was found to differ between pediatric patients with MMD with different RNF213 c.14576G>A genotypes, with those with AA/AG and GG genotypes tending to develop an indirect and a direct bypass, respectively. Thus, RNF213 c.14576G>A genotypes correlate with the development of the disease and affect revascularization after bypass surgery.

Statement of Ethics

The trial protocols were reviewed and approved by the Institutional Review Boards of Hokkaido University Hospital (approval number 012-0317, 318, 153, and 1018), and the study was conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all study participants. Regarding the children under the age of 18 years, the written informed consent was obtained from the participants’ parent.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This work was partially supported by JSPS KAKENHI Grant 20K09362 (M.F.), JSPS KAKENHI Grant No. 18K08931 (M.K.), and Japan Brain Foundation research Grant 18&21 (M.K.).

Author Contributions

Conceptualization and study supervision: Masahito Kawabori and Miki Fujimura. Acquisition of data: Masahito Kawabori, Kikutaro Tokairin, and Ken Kazumata. Interpretation of data: Masaki Ito, Susumu Ishikawa, and Kanako C. Hatanaka. Drafting the article: Masahito Kawabori, Kiyohiro Houkin, and Miki Fujimura. Critically revising the article: Miki Fujimura. Statistical analysis: Masahito Kawabori.

Data Availability Statement

All data generated or analyzed during this study were included in this article or its online supplementary material. Further inquiries can be directed to the corresponding authors.

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