Impact of RNF213 founder polymorphism (p.R4810K) on the postoperative development of indirect pial synangiosis after direct/indirect combined revascularization surgery for adult Moyamoya disease

Direct superficial temporal artery (STA) to middle cerebral artery (MCA) anastomosis combined with indirect pial synangiosis provides favorable surgical collaterals for Moyamoya disease (MMD), especially in adults; however, factors leading to the development of each direct and indirect collateral are not well documented. We aimed to investigate the association between RNF213 founder polymorphism (p.R4810K) and each direct and indirect collateral development. By qualitative and quantitative evaluations of direct and indirect surgical collaterals using time-of-flight MR angiography, postoperative development of each type of bypass was evaluated independently into two categories. Multivariate logistic regression analysis was performed to study the contributing factors for the development of each surgical collateral. Excellent development of postoperative direct and indirect bypass was observed in 65 hemispheres (70%) by qualitative evaluation, which was confirmed by quantitative evaluation. Multivariate logistic regression analysis of excellent indirect bypass development revealed a significant positive correlation with the p.R4810K (odds ratio, OR4.0; 95%-confidence interval, CI 1.2–16), advanced MR angiographic stage (OR9.5; 95%CI 1.7–73), and preoperative middle meningeal artery caliber (OR6.8; 95%CI 1.8–35), but a significant negative correlation was found with the excellent direct bypass development (OR0.17; 95%CI 0.03–0.75). No significant correlation was observed between excellent direct bypass development and the p.R4810K (OR0.95; 95%CI 0.37–2.4). In conclusion, excellent development of indirect collaterals after STA-MCA anastomosis combined with indirect pial synangiosis occurs more frequently in adult MMD with the RNF213 founder polymorphism, suggesting a role of the p.R4810K variant for marked in-growth of indirect collaterals and the utility of preoperative genetic analysis.


Introduction
Moyamoya disease (MMD) is characterized by progressive stenosis of the terminal portion of the internal carotid arteries, accompanied by the formation of abnormally dilated, fragile perforators at the base of the brain [18,31,33]. Direct superficial temporal artery (STA) to middle cerebral artery (MCA) anastomosis is accepted worldwide as the primary treatment of choice aiming at improving cerebral blood flow and surgical revascularization for symptomatic ischemic and hemorrhagic presentations of MMD [17,26]. While direct STA-MCA anastomosis offers the advantage of immediate revascularization, indirect bypass effectively induces the ingrowth of new collaterals to the underlying cerebral cortex overtime [18,31]. In the combined setting using both direct and indirect bypass, the advantages of both bypass procedures are expected, with a perioperative stroke risk of 4.7% per surgery but a favorable long-term clinical outcome [15,20]. Several studies demonstrated a reciprocal relationship between the direct and indirect bypass with a wider extent of surgical revascularization in the context of a combined setting [1,2,36,37]. Very recently, the association between a RNF213 founder mutation for east Asian MMD (p.R4810K) [14,23] and postoperative development of surgical collaterals has been demonstrated [8]; however, it is still obscure for which type of surgical collateral (i.e., direct or indirect or both?) the RNF213 founder mutation is responsible.
The extent of revascularization after Moyamoya bypass surgery is traditionally examined by catheter carotid angiography [2,24]. In the early 2000s, Yoon et al. [38] and Honda et al. [9] reported the usefulness of magnetic resonance (MR) angiography to evaluate the development of the external carotid artery tributaries, including the STA, middle meningeal artery (MMA), and deep temporal artery (DTA) after direct and/or indirect bypass surgery for MMD. Recent studies using time-of-flight (TOF) MR angiography or its source image provided insights into the discernible and sequential roles in direct and/or indirect bypass in MMD [1,37]. To comprehensively investigate the clinical and genetic factors associated with the induction of each direct and indirect surgical collaterals, we retrospectively examined our institutional records and MR angiography for adult patients undergoing combined direct/indirect bypass for MMD.

Materials and methods
Below are the main methods necessary to comprehend the results. Details were provided in the online-only supplementary information.

Study population
This study included all adult MMD patients (> 16 years of age at the surgery) who consented to genetic analysis and underwent repeat MR imaging within 3 years after combined direct/indirect bypass [10,19] between 2005 and 2019 in our hospital. The diagnosis of MMD was confirmed by the criteria outlined in the Japanese guidelines for the diagnosis and treatment of MMD [30]. The Houkin MR angiographical stage/grading system was used to stratify the angiographical stage of MMD (ranging 1 to 4 [most advanced]) [11]. Cerebrovascular reactivity to acetazolamide was evaluated quantitatively as previously reported [36]. According to our surgical protocol [10,19], double-barrel STA-MCA anastomosis combined with EDAMS or EDMAPS was performed as the standard combined direct and indirect bypass procedure. Basically, we discontinued antiplatelet(s) before surgery if prescribed, except for 14 operations. Thus, single antiplatelet agent (cilostazol, aspirin, or clopidogrel) was used in 15% (14 out of 93) of the surgery. In accordance with an institutional review board-approved protocol (number 14-053), medical records were retrospectively reviewed to gather demographic information, age at the surgery, symptoms at presentation, comorbid conditions before surgery, and results of radiographic studies (Please see supplementary Table 1), as well as clinical outcome in terms of global disability and mortality rated by modified Rankin scale (mRS), and recurrence of ischemic or hemorrhagic stroke when evaluating the postoperative development of surgical collaterals by MR angiography as described below.

Evaluation of the postoperative development of direct and indirect surgical collaterals by MR angiography
Evaluation of the postoperative development of direct and indirect surgical collaterals was performed qualitatively and quantitatively using TOF MR angiography and its source images, respectively, by a previously reported protocol with minor modifications [36,37]. In brief, MR angiography at two time points acquired before and 6 to 36 months after surgery were reviewed and compared by neurosurgeons (M. I. and T.S.) blinded to the results of genetic testing. For qualitative evaluation [36], postoperative development of each direct and indirect surgical collaterals was dichotomized into excellent or not, respectively. Thus, the development of direct surgical collaterals was evaluated by the development of the STA, while that of indirect surgical collaterals was evaluated by the development of the MMA and DTA. For quantitative evaluation, we reviewed MR angiography source images and measured the calibers of the STA, MMA, and DTA as previously reported [37]. The caliber change ratios (CCRs) of post-to preoperative calibers were calculated for each artery.

Genetic Analysis of the RNF213 founder polymorphism (p.R4810K)
Written informed consent was received for genetic analysis from MMD patients or their guardians. In accordance with the institutional review board-approved protocol, genetic analysis was conducted at the Department of Neurosurgery of Hokkaido University by K.T. and R.T. who were blinded to clinical data. Taqman single nucleotide polymorphism genotyping assay was employed to determine the allele type for RNF213 founder mutation (p.R4810K).

Data analysis
Continuous or rank variables were described as the mean or median with standard deviation or range. Dichotomous or categorical variables were expressed as the ratio or frequency. "Continuous (age, donor artery diameter) and rank variables (MR angiographical stage, mRS) were compared between two groups by the unpaired-t test and Mann-Whitney test, respectively. Dichotomous or categorical variables were compared by Fisher's exact test. For multiple comparisons, two-way repeated measures or ordinary analysis of variance followed by a post-hoc test was used, as appropriate. To assess the correlation between the excellent development of each surgical collateral and multiple clinical and genetic variables, multivariate logistic regression analysis was performed using the stepwise forward parameter selection that achieved significance levels of P < 0.1 in the univariate analysis. All clinical and genetic factors for the multivariate analysis are listed in the main Tables. The level of significance was set at P < 0.05. GraphPad Prism (version 9.1.1, San Diego, CA, USA) was used for these analyses.

Study population
During the study period, 110 adult patients with 160 hemispheres underwent STA-MCA anastomosis combined with indirect pial synangiosis using vascularized tissue, including dura mater, temporal muscle, and pericranium. Of these, 48 were excluded from analysis due to lack of genetic testing, missing follow-up MR imaging within 6 to 36 months after surgery, or the diagnosis of quasi-MMD (n = 43, 2, and 3 patients, respectively). Consequently, 62 patients (56%) with 93 operated hemispheres (58%) were included in the analysis: 47 female and 15 male patients with an average age of 42 years. In this study, 79 (84.9%) and five (5.4%) hemispheres underwent double-and single-barrel STA-MCA anastomosis as the direct bypass procedure, respectively. The rest of the nine hemispheres (9.7%) underwent STA-MCA and ACA anastomosis. In this series, 99% (92/93 hemispheres) of the direct bypass surgeries were successfully completed, while, in one hemisphere, intraoperative thrombotic occlusion occurred repeatedly at the site of anastomosis where white clot formation was observed. This operation was done by indirect revascularization, since conventional methods were not effective, including a vessel massage with intravenous heparin administration or a takedown and reanastomosis. RNF213 founder mutation (p.R4810K) was detected in 40 patients (65%) with 59 hemispheres (63%) in this series (Fig. 1A). There was no significant difference in baseline characteristics between RNF213-mutant (MT) and -wild type (WT) groups, except in familial occurrence and a comorbid condition of hypertension (Supplementary  Table 2). A representative patient who was a heterozygote for the RNF213 founder mutation is shown in Fig. 1B

Clinical outcome
Overall clinical outcome was favorable in terms of global disability and mortality when evaluating the development of surgical collaterals by MRA at 319 ± 140 postoperative days. Thus, fifty-three patients (85.5%) showed favorable outcome (mRS 0-1) and there was no mortality among the 62 patients with 93 operated hemispheres. In terms of the recurrence of ischemic or hemorrhagic stroke or TIA, we observed stroke recurrence in the three operated hemispheres (3.2%) in the three patients (4.8%). There was no significant correlation between the excellent postoperative development of indirect or direct collaterals and stroke recurrence (Tables 1 and 2).

Qualitative and quantitative MR angiography evaluations
Of all 93 hemispheres enrolled in this study, excellent development of postoperative direct and indirect surgical collaterals was observed in 65 (70%), respectively, after the combined bypass with a mean follow-up period of 319 ± 140 days by qualitative evaluation. In terms of the relationship of the postoperative development between direct and indirect bypass, dual/equal development was most frequently observed (62% of the operated hemisphere). Thus, postoperative direct-(STA) or indirect-(MMA and/or DTA) dominant development was observed in 44 and 14 hemispheres, respectively, when evaluated in the above-mentioned followup period. By quantitative MR angiography evaluation of all 93 hemispheres, a significant increase was observed in the caliber of the STA after surgery (pre: 1.8 ± 0.35 mm; post: 2.5 ± 0.65 mm, P < 0.0001). The caliber of the MMA and DTA also significantly increased (pre: 1.6 ± 0.44 mm; post: 1.8 ± 0.50 mm, P < 0.0001 in MMA; pre: 0.98 ± 0.33 mm; post: 1.6 ± 0.60 mm, P < 0.0001, respectively). The CCR of post to preoperative-calibers for the STA was significantly higher (1.5 ± 0.42 vs 1.0 ± 0.28, P < 0.0001) in the excellent direct bypass development group than in the non-excellent group. The CCRs for the DTA (2.0 ± 0.89 vs 1.2 ± 0.53, P < 0.0001) and MMA (1.3 ± 0.31 vs 1.0 ± 0.29, P = 0.042) were also significantly higher in the excellent indirect bypass development group.
We further analyzed the association between direct or indirect bypass development and RNF213 founder polymorphism (p.R4810K). Of note, excellent indirect bypass development was observed more frequently in the RNF213-MT group (78%, 46/59 hemispheres) than in the RNF213-WT group (56%, 19/34 hemispheres), with a significant difference (P = 0.035) ( Fig. 2A). Excellent direct bypass development was observed in 40/59 hemispheres (68%) and 24/34 hemispheres (71%) of the RNF213-MT and -WT groups, respectively, with no significant difference (P = 0.82). Multiple comparisons of the CCRs demonstrated a significant difference in the DTA, but not in the STA or MMA, between the two groups (Fig. 2B). Thus, the CCR for the DTA was significantly higher in the RNF213-MT group (1.9 ± 1.0) than in the RNF213-WT group (1.4 ± 0.52, P = 0.0007).

Factors correlated with excellent development of indirect and direct collaterals after combined bypass
To identify clinical and genetic factors that may underlie excellent revascularization after combined bypass in adult MMD, we examined which factors correlated with the excellent development of indirect and direct collaterals (Tables 1  and 2). Multivariate logistic regression analysis for excellent indirect bypass development revealed a significant positive correlation with RNF213 founder mutation (adjusted odds ratio (OR), 4.0), advanced MR angiographic stage (adjusted OR, 13 in stage 3; 9.5 in stage 4), and preoperative caliber of the MMA (adjusted OR, 6.8), whereas a significant negative correlation was noted with excellent direct bypass development (adjusted OR, 0.17). On the other hand, no significant correlation was observed between excellent direct bypass development and RNF213 founder mutation. Multivariate logistic regression analysis for excellent direct bypass development revealed a significant negative correlation with excellent indirect bypass development (adjusted OR, 0.23) and the comorbid condition of dyslipidemia (adjusted OR, 0.27). Please see supplementary Figs. 1 and 2 supporting these results.

Discussion
To our knowledge, this is the first study to demonstrate that the RNF213 gene polymorphism (p.R4810K) plays a role in the postoperative development of indirect, but not direct surgical collaterals after direct STA-MCA anastomosis combined with indirect pial synangiosis for adult patients with MMD. We were able to document this relationship by qualitative and quantitative evaluation of repeated TOF MR angiography during the short to mid-term follow-up period (6 to 36 months after surgery). A growing number of studies demonstrated that indirect bypass for adult MMD is less effective in terms of angiographical outcome (postoperative development of indirect surgical collateral) compared to pediatric or young adult patients. Thus, the successful indirect bypass development was observed in 44-47% of adult MMD patients treated with combined direct and indirect bypass [5,28] or indirect bypass [21]. Consistent with these past research, our study showed that only 56% of adult MMD showed excellent indirect bypass development in adult patients without RNF213-founder polymorphism (RNF213-WT group, Please see Fig. 2A). On the other hand, most (78%) of the patients with RNF213-founder polymorphism (RNF213-WT group) demonstrated excellent indirect bypass development. Based on these observations, preoperative genetic analysis for the RNF213 founder mutation might be useful for the clinical management of adult MMD patients, especially when making a decision whether adding indirect bypass on direct bypass procedure for adult MMD.
To further determine key factors responsible for indirect and direct bypass development, respectively, we focused on significant clinical and genetic factors correlated with the excellent postoperative development of surgical collaterals by multivariate logistic regression analyses. We  excellent indirect bypass development. This is partly a current knowledge. Thus, advanced angiographical stage, preoperative trans-dural collateral vessels (i.e., MMA), and heterozygous p.R4810K variant are known radiographic and genetic biomarkers of the increased capacity of postoperative surgical collateral production [8,32]. In addition to the current knowledge, our study suggests significant correlation between p.R4810K and excellent indirect, but not direct bypass development after combined bypass, although its underlying mechanism is unclear based on our study, which is briefly discussed below. Thus, it remains unclear how p.R4810K plays a pathological role in MMD (i.e., why earlier disease onset, higher disease severity [16,27], and prolonged/delayed cerebral hyperperfusion after STA-MCA anastomosis [34] occur more frequently in MMD with the RNF213 founder mutation?). Earlier experimental studies reported that cellular gene expression analysis of RNF213 in adult human tissues revealed markedly high expression in immune tissues such as the spleen and leukocytes [14,25]. Genome-wide plasma/ serum microRNA profiling [6,13,35] revealed a panel of significant MMD-related plasma/serum microRNAs whose target genes were involved in inflammatory or angiogenesis-related molecular pathways. Bidirectional major pathways that are influential in the inflammatory response potentially causing collateral formation in MMD are (1) the anti-inflammatory cytokine pathway and (2) proinflammatory cytokine pathway activating RNF213. Fujimura et al. (2018) recently showed increased serum production of soluble CD163 and CXCL5 in MMD patients, suggesting the involvement of intrinsic M2 macrophage-related immune reactions [7]. The immune responses associated with angiogenesis are promoted by M2 macrophages and angiogenic mediators are activated through these anti-inflammatory cytokines [25]. Ohkubo et al. (2015) reported that pro-inflammatory cytokines activated transcription of RNF213 both in vitro and in vivo. p.R4810K variant was more likely linked to the functional deficiency of the RNF213 gene based on markedly high matrix metalloproteinase production upon experimental silencing of RNF213 [29]. Bang et al. (2016) reported a marked increase in the blood caveolin-1 level in RNF213 founder mutation carriers [3]. As caveolin-1 negatively regulates proliferation of endothelial cells, but positively regulates endothelial angiogenic function such as tube formation [22], the increased caveolin-1 levels may accelerate angiogenesis in MMD patients. In vivo experimental study demonstrated increased angiogenesis in mice lacking RNF213 after chronic hind-limb ischemia [12], suggesting a role of RNF213 abnormality in the development of pathological vascular networks in chronic ischemia. Taken together, our observation of marked angiogenesis represented by excellent indirect bypass development in adult MMD patients with P.R4810K variant can be explained bidirectionally by the loss-of-function (i.e., marked angiogenesis by lacking RNF213 gene function) and gain-of-function (i.e., marked angiogenesis through RNF213 mutation) mechanisms of the RNF213 gene, which is the next question to be addressed. Another novel finding demonstrated in this study is that the comorbid condition of dyslipidemia was negatively correlated with excellent direct bypass development. In this study, all the thirteen patients with dyslipidemia in this series were treated with statin and had well-controlled preoperative blood LDL level before surgery. There was no significant difference in the preoperative blood LDL level between dyslipidemia and non-dyslipidemia group (125.5 ± 32.8 mg/dl and 122.6 ± 27.3 mg/dl, P = 0.73, unpaired t-test). Recently, Church et al. reported an association between hyperlipidemia and radiological progression of unilateral type to bilateral type in MMD, with possible explanations including synergistic effects of increased lipids in the underlying Moyamoya vasculopathy and the inadvertent inclusion of cerebral atherosclerotic disease in the study population [4]. One possible explanation for our finding may also be the inadvertent involvement of atherosclerosis in our MMD patients, which leads to a poorer condition of both the donor and recipient arterial wall, resulting in poor long-term showing the caliber change ratios for the superficial temporary artery (STA), deep temporal artery (DTA), and middle meningeal artery (MMA) from quantitative analysis of the source images of MR angiography before and after combined bypass in adult MMD. ns, not significant; ***P < 0.001, two-way ANOVA followed by Bonferroni multiple comparison patency. We should further validate this issue in a larger cohort with optimal stratification by the different (well-or poorly controlled blood LDL level).

Limitation
Our study is limited by the following several points. First, our study did not include pediatric subgroup of MMD. As we previously reported that almost all (95%) pediatric MMD exhibited effective indirect revascularization after combined bypass [36], we only investigated adult MMD in this study. Second, catheter angiography follow-up was not available in the most bypass surgeries in this study, although measurement of vessel calibers by catheter angiography is optimal. Last, consistent hemodynamic evaluations were not available for all patients using single-photon emission computed tomography or positron emission tomography. We do not consider these limitations to affect the interpretation of the results, but further studies are warranted.

Conclusions
We found that the excellent development of postoperative indirect pial synangiosis after combined direct and indirect bypass occurs more frequently in adult MMD patients with the RNF213 founder mutation (p.R4810K) allele. This confirms a novel clinical role of the RNF213 founder polymorphism in the marked angiogenesis via indirect pial synangiosis in adult patients with MMD, and suggests the utility of preoperative genetic analysis for RNF213 polymorphism in MMD.