2024 Volume 11 Pages 413-419
Moyamoya disease (MMD) is characterized by distinct histopathological changes in intracranial arteries, such as narrowing of the arterial lumen due to thickening of the tunica intima, waving of the internal elastic membranes, and thinning of the tunica media. Ring finger protein 213 (RNF213) is a susceptibility gene for MMD that affects clinical outcomes. However, little is known about its relationship with histopathology. Herein, we present the postmortem histopathological evaluation of 2 MMD cases with and without RNF213 p.R4810K variant. The first patient was a 39-year-old female, with a history of undergoing bilateral indirect revascularization at the age of 10 years, who died from brainstem ischemic stroke. The second patient was a 32-year-old female who was conservatively treated from the age of 14 years but died from intraventricular hemorrhage. Postmortem analysis revealed that the first patient had a homozygous variant, whereas the second patient lacked this variant. Both cases exhibited similar advanced stages of MMD with posterior artery involvement on magnetic resonance angiography. However, compared with the second patient without the RNF213 p.R4810K variant, the first patient with this homozygous variant showed more prominent histopathological changes, such as narrowing of the arterial lumen due to thickening of the tunica intima, waving of the internal elastic membranes, and thinning of the tunica media. Although an accumulation of cases is required to draw a definite conclusion, these cases suggest that RNF213 p.R4810K variant status may affect the vascular histopathology of MMD and its clinical outcome.
Moyamoya disease (MMD) is a steno-occlusive intracranial arterial disease1,2) characterized by vascular histopathology, such as narrowing of the arterial lumen due to thickening of the tunica intima, waving of the internal elastic membranes, and thinning of the tunica media.3) Ring finger protein 213 (RNF213) is a well-established susceptibility gene for MMD in East Asian population.4) In vivo investigations have demonstrated that the RNF213 variant inhibits angiogenesis in endothelial cells under hypoxic conditions5) and is associated with alterations in the peri-endothelial matrix, potentially leading to endothelial vulnerability to vascular shear stress, and thus, intimal thickening.6) Moreover, clinical investigations revealed that the RNF213 p.R4810K variant may be a biomarker of onset age,6,7) posterior cerebral artery (PCA) involvement,8) and the progression rate of unilateral disease to bilateral disease.9,10) However, the association of this variant with the histopathological findings of MMD has rarely been reported. There are limited autopsy case reports on MMD,11-13) but to our knowledge, the relationship between the RNF213 p.R4810K variant and the histopathological findings of the intracranial arteries has not yet been explored. Herein, we report 2 postmortem MMD cases, one with and one without the RNF213 p.R4810K variant, and examine their histopathological findings in the intracranial arteries.
(Figs. 1 and 2). A 10-year-old female experienced transient weakness of bilateral upper and lower extremities from the age of 8 and developed right hemianopia. She was eventually diagnosed with ischemic stroke in the left occipital lobe resulting from MMD. At 10 years old, the patient was then referred to our institute and underwent bilateral indirect revascularization surgery (encephalo-duro-arterio-synangiosis), and at 18 years old, she underwent additional right revascularization (encephalo-galeo-synangiosis to the occipital area). When the patient was 33 years old, her magnetic resonance angiography (MRA) showed advanced stages of MMD with steno-occlusive changes in the bilateral internal carotid arteries (ICAs) and PCAs with well-developed extracranial arteries. At the age of 39 years, she experienced brainstem and cerebellar ischemic stroke,7,11,12) and her MRA and catheter angiography showed an occluded basilar artery in addition to advanced stages of MMD that remained unchanged from her MRA at the age of 33 years.12) She died 48 days after this stroke onset, and the family consented to a brain autopsy and gene analysis.
Clinical images of Case #1.
Magnetic resonance angiography (MRA, left panel) at the age of 33 years reveals advanced stages of Moyamoya disease involving the bilateral internal carotid arteries (ICAs) and posterior cerebral arteries (PCA). T2 weighted image (T2WI, right upper panel) reveals an old ischemic stroke scar in the bilateral temporo-occipital lobe that had existed from the first image acquired at the age of 10 years. At the age of 39 years, the patient developed ischemic stroke in the brainstem and cerebellum (the left lower panel shows high signal intensity on diffusion-weighted imaging [DWI] acquired at this episode) and died in our hospital.
Histopathological specimens of Case #1.
On macroscopic investigations, all intracranial arteries consisting of the circle of Willis appear thin with white cord-like structures (left lower panel). Numerous thin, tortuous moyamoya vessels extend around the terminal portion of internal cerebral arteries (ICAs, white arrow). Elastica van Gieson staining of arteries demonstrates prominent histopathological characteristics of Mo- yamoya disease, including narrowing of the arterial lumen due to fibrotic thickening of the tunica intima (white arrowheads), waving of the internal elastic membranes (white arrows), and thinning of the tunica media (black arrowheads). These features are the strongest in arteries around the terminal portion of ICAs such as anterior cerebral arteries (ACAs), middle cerebral arteries (MCAs), and posterior communicating arteries (PcomAs). However, posterior cerebral arteries (PCAs) also show similar features. Note that the scales of all vascular specimens are unified throughout. Gene analysis of the formalin-fixed specimen reveals a homozygous variant of the RNF213 p.R4810K (left lower part, red arrow).
On macroscopic investigation, the terminal branches of the ICAs, anterior cerebral arteries (ACAs), middle cerebral arteries (MCAs), posterior communicating arteries (PcomAs), and PCAs appeared as thin white cord-like structures. Numerous thin and tortuous moyamoya vessels extended from the arteries of the circle of Willis. While the PcomAs and PCAs were thin and white, the vertebral and basilar arteries were relatively thick and showed few degenerative changes.
On microscopic evaluation, Elastica van Gieson staining of intracranial artery specimens revealed typical strong histopathological changes-such as thickening of the tunica intima, waving of the internal elastic membranes, and thinning of the tunica media-in bilateral ICAs, ACAs, and MCAs throughout the main trunk to the distal branches. These arterial lumens exhibited significant stenosis due to dense fibrous thickening of the intima, elastic fibers, or smooth muscles on the inner side of the intima. These findings were also evident in the PcomAs and PCAs. In contrast, the vertebral and basilar arteries were relatively preserved, with mild thickening of the tunica intima and waving of the internal elastic membranes. The histopathological findings of the basilar artery and thrombus,12) as well as the histopathology of the neovascularized arterial network after indirect revascularization of this patient have been presented in our previous report.11)
Gene analysis of formalin-fixed histopathological specimens revealed a homozygous RNF213 p.R4810K variant.
Case #2(Figs. 3 and 4). A 13-year-old female, who developed involuntary movements, was diagnosed with MMD and received conservative treatment at another hospital. At the age of 14, here magnetic resonance imaging (MRI) revealed advanced stages of MMD with steno-occlusive changes in the ICAs and PCAs and visible left MCA branches. At 19 years of age, stenosis of the left MCA progressed, with diminished signal intensity in the distal branches. The appearance of all the intracranial arteries remained grossly unchanged until 27 years of age; however, at the age of 32, she became comatose due to an intraventricular hemorrhage arising from the left thalamus. Owing to her critical illness, no vascular images were acquired during this episode. She passed away at our hospital 7 days after onset, and the family consented to a brain autopsy and genetic analysis.
Clinical images of Case #2.
Magnetic resonance angiography (left panel) at the age of 27 years reveals advanced stages of Moyamoya disease with steno-occlusive changes in the internal carotid arteries (ICAs) and posterior cerebral arteries (PCA). The distal branches of the left middle cerebral artery and moyamoya vessels around the top of the basilar artery are observed. T2 weighted image (T2WI) reveals an old ischemic stroke scar in the right central ovale that remained unchanged from the time of diagnosis (right upper panel). At the age of 32, the patient developed an intraventricular haemorrhage arising from the left thalamus and died in our hospital (right lower panel).
Histopathological specimens of Case #2.
On macroscopic investigations, the arteries around terminal portion of the internal carotid arteries (ICAs) such as anterior cerebral arteries (ACAs), middle cerebral arteries (MCAs), and posterior communicating arteries (PcomAs) appear thinner than the basilar artery. Thinning of the artery is most evident around the right ICA terminal (white arrow). Numerous thick, tortuous mo- yamoya vessels extend from these arteries. On microscopic evaluation, Elastica van Gieson staining of intracranial artery specimens reveals thickening of the tunica intima that caused 10–50% stenosis in intracranial arteries (white arrowheads), which is the strongest in MCAs and right ICA adjacent to the PcomA (the left artery in the upper panel named “Rt ICA & PcomA”). Typical waving of the internal elastic membranes (white arrows) is evident in MCAs, ACAs, and the right PcomA and mild in ICAs, PCAs, and left PcomA. Thinning of the tunica media is especially evident in the distal branches of ACAs and MCAs and mild in the right ICA and right PcomA (black arrowheads). Note that the scales of macroscopic and microscopic vascular specimens are unified to that in Fig. 2 with the same horizontal scale, except for right ICA and right PCA, which are too large and difficult to organize with unified scales. Gene analysis of the formalin-fixed histopathological specimens reveals that the RNF213 p.R4810K is the wild type (left lower part, red arrow).
Macroscopic investigations revealed no anastomosis between the extracranial arteries and cortex. The terminal branches of ICAs, ACAs, MCAs, PcomAs, and PCAs appeared thinner than the basilar artery. The arterial shrinkage was the most evident around the terminal portion of the right ICA. Numerous thin and tortuous moyamoya vessels extended from the arteries of the circle of Willis. No microaneurysms were observed in the moyamoya vessels.
On microscopic evaluation, Elastica van Gieson staining of intracranial artery specimens revealed thickening of the tunica intima that caused 10%-50% narrowing of the arterial lumen, which was the strongest in the MCAs. Typical waving of the internal elastic membranes was noted in the MCAs, ACAs, and right PcomA, whereas only mild waving was noted in the ICAs and PCAs. The left PcomA appeared almost normal. Thinning of the tunica media was particularly evident in the distal branches of the ACAs and MCAs and also observed in the ICAs and right PcomA to some degree. Thickening of the tunica intima and tunica media was less pronounced in other arteries compared with that in the MCAs.
Gene analysis of formalin-fixed histopathological specimens revealed that the RNF213 p.R4810K status was wild type.
Comparison of Case #1 and #2When comparing the MRA findings of the 2 patients (Figs. 1 and 3), the intracranial arteries showed similar advanced stages of MMD with bilateral ICA and PCA involvement, except for visible distal branches of the left MCA in Case #2. However, on microscopic and macroscopic evaluation (Figs. 2 and 4), shrinkage of arteries and typical histopathological changes of MMD were more evident in Case #1 with the homozygous RNF213 p.R4810K variant than in Case #2 without this variant.
Herein, we present the autopsy and histopathological data of 2 adult female patients with pediatric-onset MMD who died in their 30s. While some differences exist in the clinical course (previous revascularization for ischemic stroke vs conservative treatment for involuntary movement), the 2 patients showed similar onset ages, advanced stages of MMD with PCA involvement from the time of diagnosis, and similar age at death. The histopathological changes, including narrowing of the arterial lumen due to thickening of the tunica intima, waving of the endoelastic plate, and thinning of the tunica media, were more prominent in Case #1 with the homozygous RNF213 p.R4810K variant than in Case #2 without this variant.
Only a few studies have investigated the relationship between the RNF213 variant and histopathological changes in MMD arteries. In vivo investigations reported that the RNF213 variant inhibits angiogenesis in endothelial cells under hypoxic conditions5) and is associated with alterations in the peri-endothelial matrix, potentially leading to endothelial vulnerability to vascular shear stress, and thus, intimal thickening.6) Additionally, a recent autopsy study of a teenage patient with heterozygous RNF213 p.R4810K variant revealed typical histopathological changes of MMD such as thickening of the intima and irregular waving of the internal elastic lamina in systemic arteries including peripheral pulmonary arteries.13) However, because the patient's family did not consent to a brain autopsy, intracranial arteries were not evaluated. A human study using surgical specimens revealed that the superficial temporal artery in patients with MMD carrying this variant had increased thickness in the tunica intima and internal elastic membrane layers compared to those without the variant.14) While this prior study focused on superior temporal artery specimens and did not assess intracranial arteries, it is reasonable to postulate that the RNF213 p.R4810K variant affects the endothelial cells of intracranial arteries and causes histopathological changes. Consistent with this previous report, we found that thickening of the tunica intima and waving of the internal elastic lamina were more prominent in the patient with the homozygous variant than in the patient without the variant.
In this report, the patient with the homozygous RNF213 p.R4810K variant had severe ischemia and PCA involvement, whereas the patient without this variant had a less severe clinical course before the severe hemorrhagic stroke onset. Patients with homozygous variants are known to have a younger onset age,6,7) more severe onset with ischemic stroke,6) familial cases,15) and PCA involvement;8) these characteristics are also signs of poor social outcome.16,17) In Case #1, the patients with homozygous variants experienced ischemic stroke onset and had PCA involvement. While the patient in Case #2 had PCA involvement since the time of diagnosis, her clinical course was not severe, probably because of the markedly developed moyamoya vessels that may compensate for ischemia. The RNF213 p.R4810K variant may affect intracranial arterial histopathology and lead to different clinical outcomes in patients with MMD. Nevertheless, as the patient's clinical course such as surgical treatment might also affect vascular histopathological changes, this notion remains a hypothesis that needs further validation.
Patients with MMD are known to have shrinkage of the outer diameter of arteries (i.e., negative remodeling) around the terminal portion of ICAs on MRI;18) however, the relationship between the RNF213 p.R4810K variant and these findings remains unknown. A recent study evaluating patients with intracranial atherosclerosis revealed that on MRI, the outer diameters of all intracranial arteries were smaller in RNF213 p.R4810K variant carriers than in noncarriers.19) On macroscopic evaluation, Case #1, with the homozygous variant, showed more prominent shrinkage of the arteries than Case #2, without the variant. Therefore, patients with MMD of this variant may have a smaller outer diameter of the intracranial arteries than those without this variant. Nevertheless, again, we cannot conclude that the difference in the outer diameter in our patients was only due to the presence of this variant. Reduction of antegrade flow from intracranial arteries induced by successful surgical revascularization may have enhanced the shrinkage and thinning of intracranial arteries in Case #1. On the other hand, conservative treatment in Case 2 may have required intracranial arteries and moyamoya vessels to reserve antegrade flow and prevent shrinkage. Future studies should evaluate the relationship between arterial diameter and the RNF213 p.R4810K variant using high-resolution vessel wall imaging in a large number of patients.
This report has limitations. First, the histopathological changes may have been caused by differences in the clinical course (e.g, severe ischemia with ischemic stroke vs involuntary movement and previous surgical vs conservative treatment) rather than genetic status. Additional cases with a more diverse clinical history are required to corroborate how the RNF213 variant affects histopathological findings. Second, because our patients died more than a decade before this report and the raw formalin-fixed vascular specimens were lost, we could not perform a detailed comparison of MRA and vascular specimens. We could no longer identify where exactly the specimen was obtained within each vessel because tissue blocks were randomly sampled from each vessel, as in the common practice of Pathologists. Finally, additional analysis of RNF213 rare variant6,20) and immunohistochemical staining21) that may provide more insights into the histopathology of the RNF213 variant in MMD were not conducted. Additional serum or cerebrospinal fluid analyses to evaluate the markers related to autoimmunity or inflammation were also not possible.22) If we have the opportunity to perform autopsies in the future, we will perform detailed histopathological, genetic, and epigenetic analyses together with other institutions.
We report postmortem vascular histopathology findings of 2 patients with MMD-one with and one without the RNF213 p.R4810K variant. Further accumulation of cases is necessary to explore the relationship between RNF213, intracranial arterial histopathology, and clinical outcomes.
This work was supported by Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research "KAKENHI" (Grant Number # 23K08514), which was awarded to Shoko Hara.
Tomo Kinoshita contributed to the acquisition, interpretation of data, and drafting of the article. Shoko Hara contributed to the conception, design, acquisition, interpretation of data, and drafting of this article. Natsumi Tamada, Maki Mukawa, Hiroshi Shintaku, Motoki Inaji, and Tadashi Nariai contributed to the acquisition and interpretation of data. Yoji Tanaka and Taketoshi Maehara supervised this study. All authors have approved the final manuscript as submitted and agreed to be accountable for all aspects of this work.
The family members of the 2 deceased patients consented to the postmortem histopathological analysis, genetic analysis, and submission of the case report to the journal.
The datasets generated and/or analyzed during the current study are not publicly available due to conditions of ethical approval but are available from the corresponding author upon reasonable request.
The genetic analysis performed in this study was approved by the Ethics Review Committee of Tokyo Medical and Dental University (G2016-009).
All authors have no conflict of interest.
