
==== Front
Neurol Genet
Neurol Genet
nng
NNG
Neurology: Genetics
2376-7839
Wolters Kluwer Baltimore

NXG-2024-100109DN
10.1212/NXG.0000000000200176
00010
3
11
2
27
32
91
Clinical/Scientific Note
Blended Phenotype of NOTCH3 and RNF213 Variants With Accelerated Large and Small Artery Crosstalk
A Case Report and Literature Review
https://orcid.org/0000-0001-8608-058X
Saito Satoshi MD, PhD *
https://orcid.org/0000-0002-4241-3677
Hosoki Satoshi MD, PhD *
Yamaguchi Eriko MD
https://orcid.org/0000-0003-0071-7338
Ishiyama Hiroyuki MD
https://orcid.org/0000-0001-9528-8702
Abe Soichiro MD
https://orcid.org/0000-0003-3178-8171
Yoshimoto Takeshi MD, PhD
https://orcid.org/0000-0002-6023-236X
Tanaka Tomotaka MD, PhD
https://orcid.org/0000-0002-1495-4433
Hattori Yorito MD, PhD
Liao Yi Chu MD, PhD
https://orcid.org/0000-0003-0102-164X
Lee Yi-Chung MD, PhD
Mizuta Ikuko MD, PhD
Mizuno Toshiki MD, PhD
https://orcid.org/0000-0002-7102-4048
Ihara Masafumi MD, PhD
From the Department of Neurology (S.S., S.H., E.Y., H.I., S.A., T.Y., T.T., Y.H., M.I.), National Cerebral and Cardiovascular Center, Suita, Japan; Department of Neurology (Y.C.L., Y.-C.L.), Taipei Veterans General Hospital, Taipei, Taiwan; and Department of Neurology (I.M., T.M.), Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Correspondence Dr. Ihara ihara@ncvc.go.jp
Go to Neurology.org/NG for full disclosures. Funding information is provided at the end of the article.

The Article Processing Charge was funded by AMED.

Submitted and externally peer reviewed. The handling editor was Associate Editor Suman Jayadev, MD.

* These authors contributed equally to this work as co-first authors.

10 2024
6 9 2024
6 9 2024
10 5 e20017618 4 2024
03 6 2024
Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.
2024
American Academy of Neurology
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND), which permits downloading and sharing the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.

Objectives

Recent advancements in genome research have revealed not only the importance of variants associated with cerebrovascular diseases but also a notably high frequency of carriers harboring multiple variants, presenting with an elusive blended phenotype. In this study, we report the case of a 66-year-old man who experienced 3 stroke episodes over a 4-year period, starting at the age of 62 years. The patient presented with isolated infarcts in the left temporal pole with progressive stenosis in the ipsilateral middle cerebral artery based on large and small artery crosstalk.

Methods

Exons 2–24 of the NOTCH3 gene were analyzed by direct genomic DNA sequencing. The presence of the p.Arg4810Lys variant of the ring finger protein 213 (RNF213) gene was evaluated using real-time PCR.

Results

Diagnoses of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy and RNF213-related vasculopathy were made based on the early-onset recurrent stroke episode, progressive intracranial artery stenosis, and presence of the heterozygous NOTCH3 p.Cys1250Arg and RNF213 p.Arg4810Lys variants.

Discussion

Temporal pole infarcts could represent a blended phenotype of both variants. This case highlights the importance of large and small artery crosstalk and the pivotal role of genetic analysis in determining the pathogenesis of stroke and dementia.

OPEN-ACCESSTRUE
==== Body
pmcIntroduction

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common hereditary cerebral small vessel disease. Cysteine-altering NOTCH3 variants associated with CADASIL occur at a frequency of 3.4 individuals per 1,000 in the general population worldwide, with a significantly higher rate of 9.0 individuals per 1,000 in East Asia.1 Moyamoya disease is another hereditary cerebrovascular disorder characterized by occlusion of the internal carotid artery. The ring finger protein 213 (RNF213) p.Arg4810Lys variant (c.14429G>A) is found in approximately 1.5% of healthy individuals in Japan and Korea, 0.5% of healthy individuals in China, 80–90% of patients with familial moyamoya disease in Japan and Korea, and 20–30% of patients with familial moyamoya disease in China.2 However, the p.Arg4810Lys variant has rarely been found in White individuals, with an allele frequency of less than 0.0006. Nevertheless, distinct RNF213 variants, such as p.Asn4013Asp or p.Arg4062Glu, have been reported in White patients with moyamoya disease.2 The RNF213 p.Arg4810Lys variant is also common in patients with large artery atherosclerosis and is regarded as a strong risk factor of intracranial artery stenosis (ICAS).3 Based on the high frequencies of NOTCH3 and RNF213 variants in East Asia, it is estimated that the double heterozygous variants of the 2 genes occur in approximately 1 of 7,000 individuals, at most, in East Asia. In fact, RNF213 p.Arg4810Lys variant carriers were found in 6 of 124 (4.8%) Japanese patients with CADASIL, which is a considerably higher prevalence than the frequency of the RNF213 p.Arg4810Lys variant (1.5%) in the general population. The presence of both the NOTCH3 and RNF213 variants may prompt a diagnosis of CADASIL because of an accelerated clinical course or a blended phenotype.4 However, the longitudinal course of double heterozygous variant carriers remains unknown. In this study, we report a case of recurrent stroke caused by coexisting large and small artery diseases based on both the NOTCH3 and RNF213 variants.

Case Description

A 62-year-old man with mild dyslipidemia and type 2 diabetes mellitus was admitted to our hospital because of a sensorimotor stroke. Magnetic resonance diffusion-weighted imaging (MR-DWI) showed a high-intensity lesion in the right thalamus (eFigure 1). Despite effective management of dyslipidemia and diabetes, he had a second stroke episode presenting with aphasia at the age of 63. MRI and digital subtraction angiography revealed isolated acute infarcts in the left temporal pole with ipsilateral middle cerebral artery stenosis (Figure 1, A–C). At 66 years, he experienced a third stroke episode, presenting with dysarthria and mild hemiplegia in the right limbs. MR-DWI revealed a high-intensity lesion in the pons (eFigure 2). Over the course of 4 years of monitoring by longitudinal MR angiography, progressive stenosis of the left middle cerebral artery was observed (Figure 1, D–F). Fluid-attenuated inversion recovery images showed cortical atrophy, multiple lacunae in the subcortical white matter and basal ganglia, and white matter hyperintensities (eFigure 3). Multiple cerebral microbleeds were observed on T2*-weighted images (eFigure 4). Montreal Cognitive Assessment score was 14. The completion times for the trail-making tests were 100 seconds for part A and 171 seconds for part B, suggesting a delayed processing speed. Given the family history of ischemic stroke (eFigure 5), a genetic disorder was suspected. NOTCH3 genetic analysis revealed a heterozygous cysteine-altering p.Cys1250Arg variant (c.3748T>C), leading to the diagnosis of CADASIL. Furthermore, progressive ICAS, with genetic confirmation of the heterozygous RNF213 p.Arg4810Lys variant (c.14429G>A), led to the diagnosis of RNF213-related vasculopathy. In the present case, exons 2–24 of the NOTCH3 gene were analyzed by direct sequencing of genomic DNA, and genotyping of the p.Arg4810Lys variant of the RNF213 gene was conducted using real-time PCR. The patient provided written informed consent, and the study, including gene analysis, was approved by the National Cerebral and Cardiovascular Center ethics review board (M27-071 and M29-003).

Figure 1 Clinical Images of Cerebrovascular Injuries in Patients With NOTCH3 and RNF213 Variants

Diffusion-weighted MRI performed when the patient was 63 years illustrates a high-intensity lesion in the left temporal pole (A) with a low apparent diffusion coefficient (B). The hyperintense lesion in the left temporal pole on the T2-weighted image persisted after recovery from clinical symptoms (C, 2 years later than A or B). Progressive stenosis in the left middle cerebral artery is demonstrated on magnetic resonance angiography at the ages of 62 years (D, white arrowheads), 63 years (E), and 66 years (F).

Discussion

In this case, progressive stenosis in the left middle cerebral artery, early-onset recurrent stroke, and cognitive impairment could be considered a blended phenotype resulting from heterozygous variants of NOTCH3 p.Cys1250Arg and RNF213 p.Arg4810Lys. Dyslipidemia and type 2 diabetes mellitus might have altered the clinical course. However, even with the best medical treatment, progressive stenosis and recurrence of ischemic stroke were observed in this case, suggesting the possibility of factors other than vascular risk factors.

The NOTCH3 gene variant position is correlated with differences in the age of the first stroke.5 The p.Cys1250Arg variant located within the epidermal growth factor-like repeat 32 of the NOTCH3 protein is classified as a medium-risk variant, resulting in a high probability (approximately 70% free of stroke) of being stroke-free at the age of 62 years, the age of the first stroke in this case.5 According to the literature, this case represents the second report of CADASIL with the NOTCH3 p.Cys1250Arg variant, following our previous report, in which the age of the first stroke was 73 years.6 These reports suggest the possibility of an early-onset ischemic stroke in this case due to the blended phenotype. However, an analysis of more cases is necessary.

RNF213 encodes a large protein containing dual AAA+ ATPase domains and an E3 ligase domain, with an important role in regulating vascular remodeling, endothelial barrier functions, angiogenesis, and anatomical variations in the circle of Willis.2 Accumulating evidence has shown that large and small artery crosstalk exacerbates arterial damage, establishing a vicious circle.7 For example, chronic cerebral hypoperfusion induced by bilateral common carotid artery stenosis results in endothelial damage, blood-brain barrier disruption, and white matter damage in mice.7 In this case, the presence of double heterozygous variants of NOTCH3 and RNF213 genes would substantially affect the arterial system, damaging both large and small arteries through crosstalk between large and small arteries,8 as the NOTCH3 variants contribute to the loss of arteriolar and capillary pericytes as well as the degeneration of smooth muscle cells of the small penetrating arterioles.9,10 Compared with small infarcts, large infarcts are rare in CADASIL,11 whereas ICAS and ICAS-related cerebral infarcts, which are classified as large artery atherosclerosis, have been predominantly reported in patients with CADASIL in East Asia (Table).4,12-14 This may, at least partially, be attributed to the high prevalence of the RNF213 p.Arg4810Lys variant in East Asians, which is notably rare in other regions.2 However, without other factors, the high frequency (13.7%–52.2%) of ICAS in patients with CADASIL in East Asia cannot be explained. Therefore, further studies examining other genetic and environmental factors are warranted.

Table Frequency of Intracranial Artery Stenosis in Patients With Cerebral Autosomal Dominant Arteriopathy With Subcortical Infarcts and Leukoencephalopathy (CADASIL)

Reference	N	Female (%), mean age	ICAS (%)	ICAS-related cerebral infarcts (%)	RNF213 p.R4810K variant (+)	Country	
Yeung et al.4	124	10 (59), 57a	17 (13.7)	4 (3.3)b	6 (4.8)	Japan	
Choi et al.12	13	4 (31), 55	4 (30.8)	1 (7.7)	NA	Korea	
Kang and Kim13	49	27 (55), 51	12 (52.2)c	7 (14.3)	NA	Korea	
Zhang et al.14	37	20 (54), 48	8 (21.6)	1 (2.7)	NA	China	
Data are presented as number or percentage.

a The report only provides the number (%) of female patients and the mean age among 17 patients with CADASIL with ICAS.

b The data on ICAS-related cerebral infarcts were missing for 3 patients with CADASIL.

c The report only provides the frequency of ICAS (n = 12) in patients with CADASIL with ischemic stroke (n = 23). ICAS, intracranial artery stenosis; NA, not available.

Another notable aspect of this case was the occurrence of an isolated temporal pole infarction. The left temporal pole is involved in the networks underlying the naming and comprehension of objects.15 The presence of white matter hyperintensities in the anterior temporal pole on MRI serves as an indicator of CADASIL.6 The white matter hyperintensities reflect various pathologic changes, including enlarged perivascular spaces and demyelination.10 However, isolated temporal infarction is uncommon,15 and CADASIL presenting with temporal pole infarction has not been reported. In this case, temporal pole infarcts may have resulted from large and small artery crosstalk based on RNF213 and NOTCH3 variants (Figure 2). Temporal pole infarcts could represent a blended phenotype of the 2 variants.

Figure 2 Proposed Mechanism Explaining Isolated Temporal Pole Infarcts Associated With NOTCH3 and RNF213 Variants

The temporal pole infarction may have resulted from degenerated small vessels and negative remodeling of the left middle cerebral artery based on the NOTCH3 and RNF213 variants, respectively. Coexisting large artery disease may have exacerbated small vessel disease changes that would normally be confined to white matter changes, which are more severe after large and small artery crosstalk.

In conclusion, this case highlights the importance of large and small artery crosstalk and the pivotal role of genetic analysis in determining the pathogenesis of stroke and dementia. Common and frequent variants are reflected in the heterogeneous presentation and broad spectrum of CADASIL and RNF213-related vasculopathy. However, further investigations are warranted to enhance our understanding of the phenomena.

Study Funding

This study was supported by the Japan Agency for Medical Research and Development (JP22ek0109516) and the Japan Society for the Promotion of Science KAKENHI Grant (21K16944).

Disclosure

The authors report no relevant disclosures. Go to Neurology.org/NG for full disclosures.

Appendix Authors

Name	Location	Contribution	
Satoshi Saito, MD, PhD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data; study concept or design; analysis or interpretation of data	
Satoshi Hosoki, MD, PhD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content; major role in the acquisition of data	
Eriko Yamaguchi, MD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Hiroyuki Ishiyama, MD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Soichiro Abe, MD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Takeshi Yoshimoto, MD, PhD	Department of Neurology, National Cerebral and Cardiovascular CenterSuita, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Tomotaka Tanaka, MD, PhD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Yorito Hattori, MD, PhD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Yi Chu Liao, MD, PhD	Department of Neurology, Taipei Veterans General Hospital, Taipei, Taiwan	Drafting/revision of the manuscript for content, including medical writing for content	
Yi-Chung Lee, MD, PhD	Department of Neurology, Taipei Veterans General Hospital, Taipei, Taiwan	Drafting/revision of the manuscript for content, including medical writing for content	
Ikuko Mizuta, MD, PhD	Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Toshiki Mizuno, MD, PhD	Department of Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto, Japan	Drafting/revision of the manuscript for content, including medical writing for content	
Masafumi Ihara, MD, PhD	Department of Neurology, National Cerebral and Cardiovascular Center, Suita, Japan	Drafting/revision of the manuscript for content, including medical writing for content; study concept or design; analysis or interpretation of data
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References

1. Rutten JW, Dauwerse HG, Gravesteijn G, et al. Archetypal notch3 mutations frequent in public exome: implications for CADASIL. Ann Clin Transl Neurol. 2016;3 (11 ):844-853. doi:10.1002/acn3.344 27844030
2. Ihara M, Yamamoto Y, Hattori Y, et al. Moyamoya disease: diagnosis and interventions. Lancet Neurol. 2022;21 (8 ):747-758. doi:10.1016/S1474-4422(22)00165-X 35605621
3. Okazaki S, Morimoto T, Kamatani Y, et al. Moyamoya disease susceptibility variant RNF213 p.R4810K increases the risk of ischemic stroke attributable to large-artery atherosclerosis. Circulation. 2019;139 (2 ):295-298. doi:10.1161/CIRCULATIONAHA.118.038439 30615506
4. Yeung WTE, Mizuta I, Watanabe-Hosomi A, et al. RNF213-related susceptibility of Japanese CADASIL patients to intracranial arterial stenosis. J Hum Genet. 2018;63 (5 ):687-690. doi:10.1038/s10038-018-0428-9 29500468
5. Hack RJ, Gravesteijn G, Cerfontaine MN, et al. Three-tiered EGFr domain risk stratification for individualized NOTCH3-small vessel disease prediction. Brain. 2023;146 (7 ):2913-2927. doi:10.1093/brain/awac486 36535904
6. Yamamoto Y, Liao YC, Lee YC, Ihara M, Choi JC. Update on the epidemiology, pathogenesis, and biomarkers of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. J Clin Neurol. 2023;19 (1 ):12-27. doi:10.3988/jcn.2023.19.1.12 36606642
7. Ihara M, Yamamoto Y. Emerging evidence for pathogenesis of sporadic cerebral small vessel disease. Stroke. 2016;47 (2 ):554-560. doi:10.1161/STROKEAHA.115.009627 26742799
8. Laurent S, Briet M, Boutouyrie P. Large and small artery cross-talk and recent morbidity-mortality trials in hypertension. Hypertension. 2009;54 (2 ):388-392. doi:10.1161/HYPERTENSIONAHA.109.133116 19546376
9. Fang C, Magaki SD, Kim RC, Kalaria RN, Vinters HV, Fisher M. Arteriolar neuropathology in cerebral microvascular disease. Neuropathol Appl Neurobiol. 2023;49 (1 ):e12875. doi:10.1111/nan.12875 36564356
10. Rajani RM, Ratelade J, Domenga-Denier V, et al. Blood brain barrier leakage is not a consistent feature of white matter lesions in CADASIL. Acta Neuropathol Commun. 2019;7 (1 ):187. doi:10.1186/s40478-019-0844-x 31753008
11. Zhang R, Chen C-H, Tezenas Du Montcel S, et al. The CADA-MRIT. Neurology. 2023;101 (17 ):e1665-e1677. doi:10.1212/WNL.0000000000207713 37652700
12. Choi EJ, Choi CG, Kim JS. Large cerebral artery involvement in CADASIL. Neurology. 2005;65 (8 ):1322-1324. doi:10.1212/01.wnl.0000180965.79209.50 16247072
13. Kang HG, Kim JS. Intracranial arterial disease in CADASIL patients. J Neurol Sci. 2015;359 (1-2 ):347-350. doi:10.1016/j.jns.2015.11.029 26671140
14. Zhang C, Li W, Li S, et al. Intracranial large artery abnormalities and association with cerebral small vessel disease in CADASIL. Front Neurol. 2020;11 :726. doi:10.3389/fneur.2020.00726 33013610
15. Tsapkini K, Frangakis CE, Hillis AE. The function of the left anterior temporal pole: evidence from acute stroke and infarct volume. Brain. 2011;134 (Pt 10 ):3094-3105. doi:10.1093/brain/awr050 21685458
