doi: 10.2169/internalmedicine.6096-20
Intern Med 60: 2479-2482, 2021
http://internmed.jp

【 CASE REPORT 】

Heterozygous Cysteine-sparing NOTCH3 Variant
p.Val237Met in a Japanese Patient with Suspected Cerebral
Autosomal Dominant Arteriopathy with
Subcortical Infarcts and Leukoencephalopathy
Yuya Kano 1,2, Ikuko Mizuta 3, Akihiko Ueda 4, Hiroaki Nozaki 5, Keita Sakurai 6,
Osamu Onodera 5, Yukio Ando 4,7, Kentaro Yamada 2, Hiroyuki Yuasa 1 and Toshiki Mizuno 3

Abstract:
A 64-year-old Japanese man with recurrent cerebral ischemic events and cognitive impairment was suspected of having cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy
(CADASIL) because of a family history and brain magnetic resonance imaging findings of cerebral white
matter hyperintensities. The cysteine-sparing variation p.Val237Met was identified in NOTCH3. An intensive
skin biopsy showed negative results (no granular osmiophilic material or positive NOTCH3 immunostaining),
suggesting that the patient’s definite diagnosis and pathogenicity of p.Val237Met were uncertain. We additionally reviewed previous reports of two Japanese families with p.Val237Met.
Key words: cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy
(CADASIL), p.Val237Met, granular osmiophilic material (GOM), pathogenicity
(Intern Med 60: 2479-2482, 2021)
(DOI: 10.2169/internalmedicine.6096-20)

Introduction
Cerebral autosomal dominant arteriopathy with subcortical
infarcts and leukoencephalopathy (CADASIL), clinically
characterized by migraine, recurrent ischemic events, mood
disturbance, apathy, and cognitive impairment, is the most
common form of hereditary small-vessel disease caused by
mutations in the NOTCH3 gene (1, 2). CADASIL can be
definitively diagnosed by genetic testing. Thus far, more
than 200 mutations localized to the domain of the 34 epidermal growth factor repeats (EGFrs) encoded by exons 2-24 in
NOTCH3 have been reported (3, 4). The typical pathogenic
mutations are heterozygous missense ones altering the number of cysteine amino acids in a certain EGFr (1).
If a cysteine-sparing missense variant of unknown signifi-

cance is identified, the diagnosis of CADASIL can be confirmed by detecting granular osmiophilic material (GOM) or
positive NOTCH3 immunostaining in skin biopsy specimen (5). GOM, the pathological marker of CADASIL, is an
abnormal protein aggregation containing the NOTCH3 extracellular domain (N3ECD), localizing around the basement
membrane of vascular smooth muscle cells (6). We recently
identified a cysteine-sparing NOTCH3 variant, p.Val237Met,
in a patient with suspected CADASIL. Thus far, two groups
have reported Japanese patients with this mutation. However,
their interpretations of its pathogenicity were contradictory,
with one concluding it to be pathogenic (7, 8) while the
other concluded that it does not have certain pathogenicity (9).
In this study, we evaluated the pathogenicity of NOTCH3
p.Val237Met based on the findings in our present patient

１

Department of Neurology, Tosei General Hospital, Japan, ２ Department of Neurology, Nagoya City East Medical Center, Japan, ３ Department of
Neurology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Japan, ４ Department of Neurology, Graduate School
of Medical Science, Kumamoto University, Japan, ５ Department of Neurology, Brain Research Institute, Niigata University, Japan, ６ Department
of Radiology, National Center for Geriatrics and Gerontology, Japan and ７ Department of Amyloidosis Research, Nagasaki International University, Japan
Received: August 12, 2020; Accepted: January 14, 2021; Advance Publication by J-STAGE: March 8, 2021
Correspondence to Dr. Toshiki Mizuno, mizuno@koto.kpu-m.ac.jp

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A

DOI: 10.2169/internalmedicine.6096-20

B

C

E

F

L

R
D

Figure. Brain MRI and electron micrograph of a dermal artery. Brain fluid-attenuated inversionrecovery imaging shows multiple hyperintense lesions in the bilateral subcortical white matter, basal
ganglia, and thalami. Despite small nonspecific signal changes in the external capsules, no abnormal
signal changes are detected in the subcortical white matter of the temporal poles (A, B, C). Susceptibility-weighted imaging shows many micro bleeds, mainly in the basal ganglia (D, E). Granular osmiophilic material (GOM) deposition is not detected around the basement membrane of the vascular
smooth muscle cells of small arterioles (Bar=2 μm) (F).

and those from the two previous reports.

Case Report
A 64-year-old Japanese man who experienced two episodes of lacunar infarction at 59 and 62 years old and an
episode of transient ischemic attack of weakness in the left
leg at 64 years old was admitted to our hospital. The patient
experienced difficulty moving his right leg and was administered cilostazol (200 mg/day) for cerebral infarction. He had
no vascular risk factors other than smoking and had no history of migraine. His brother and sister had suffered from
stroke; however, little medical information was available regarding his deceased parents.
On admission, his blood pressure was 140/92 mmHg, and
his pulse was regular. A neurological examination showed
right hemiplegia, and his score on the Mini-Mental State
Examination (10 of a total of 30: disorientation, memory
disturbance, and executive function impairment were noticeable) indicated cognitive impairment. His laboratory parameters, including blood counts, lipid profile, and blood glucose

level, were within normal ranges. All of the following
coagulation-related test results were also within normal
ranges: protein S, protein C, anti-DNA antibodies, lupus anticoagulant, anti-cardiolipin β2-glycoprotein I complex antibodies, and anti-cardiolipin antibodies.
Stenoses and plaques in the internal carotid artery were
not observed on carotid ultrasound, and echocardiography
revealed normal wall motion without thromboses. No arrhythmias were detected on the 24-hour Holter electrocardiogram. However, diffusion-weighted brain magnetic resonance imaging (MRI) demonstrated a hyperintense area in
the left basal ganglia, indicating the presence of an acute infarct. Additionally, fluid-attenuated inversion recovery
(FLAIR) imaging showed bilateral multiple hyperintense lesions in the subcortical white matter, basal ganglia, thalami,
and brainstem. Slight nonspecific FLAIR signal changes
were detected in the external capsules, whereas no abnormal
changes were detected in the subcortical white matter of the
temporal poles (Figure). Susceptibility-weighted imaging
showed multiple microbleeds in the bilateral basal ganglia,
subcortical white matter, and brainstem; however, no abnor-

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DOI: 10.2169/internalmedicine.6096-20

Table. Carrier Frequencies of NOTCH3 c.709G>A (p.Val237Met) in General Populations of Different Ethnicities.

Number of individuals
Number of carriers
(frequency)

East Asian

South Asian

European
(Non-Finnish)

Finnish

Latino

Ashkenazi
Jewish

African

9,973
47
(0.47%)

15,307
1
(0.0065%)

64,265
2
(0.0031%)

12,559
2
(0.016%)

17,711
2
(0.011%)

5,163
0
(0%)

12,471
0
(0%)

Values were downloaded on July 29, 2020 from gnomAD (https://gnomad.broadinstitute.org). All carriers were heterozygous.

malities were observed on MR angiography. These abnormalities and brain atrophy gradually progressed over the
next five years.
We suspected a diagnosis of hereditary cerebral small vessel disease from the patient’s clinical course, family history,
and detection of severe small-vessel diseases on brain MRI.
The cause of cognitive impairment appeared to be vascular.
We ruled out the possibility of Alzheimer’s disease, idiopathic normal pressure hydrocephalus, and chronic subdural hematoma based on the MRI features. In addition, his
blood tests showed a normal thyroid function, ammonia levels, and drug resistance; thus, they were also ruled out.
Genetic testing was performed after receiving written informed consent, and approval for the genetic study was obtained from the University Ethical Committee. CADASIL
was first suspected, as it is the most common hereditary
small-vessel disease. Sanger sequencing of whole exons 133 of NOTCH3 identified the heterozygous cysteine-sparing
variation c.709G>A (p.Val237Met in EGFr 6) in exon 5 of
the NOTCH3 gene.
Because the pathogenicity of p.Val237Met had not yet
been established, we next performed a skin biopsy. Immunoreactivity for the NOTCH3 extracellular domain was
not detected in the vessels of the skin specimens (data not
shown). Electron microscopy did not detect GOM in the
small arterioles in the deep dermis, which is where this material preferentially localizes (Figure).

Discussion
Based on the negative findings of the skin biopsy (no
GOM or NOTCH3 immunostaining), the patient’s definite
diagnosis and pathogenicity of p.Val237Met remain uncertain.
The diagnostic test is limited by the sensitivity of the skin
biopsy. Although the specificity of GOM is 100%, its sensitivity has ranged between 44.4% and 100% in previous reports (10). The successful detection of GOM depends on the
number of vessels included in the sample as well as technical factors. Light microscopic observation of NOTCH3 immunostaining has the advantage of evaluating a broad area,
including multiple vessels. The sensitivity and specificity of
NOTCH3 immunostaining are reported to be 90.2-85.4%
and 95.2-100%, respectively (11). To overcome these technical issues, an analysis of the skin biopsy specimen was performed by the expert (A.U.) in this study group who spe-

cializes in the pathological diagnosis of CADASIL (12, 13).
Another limitation is the lack of research on other causative genes of hereditary cerebral small vessel diseases. We
next focused on high-temperature requirement A serine peptidase 1 (HTRA1), a gene that plays a causative role in cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL) (14). Heterozygous mutations in HTRA1 have occasionally been identified
among NOTCH3-negative patients with cerebral small-vessel
disease, establishing the concept of cerebral small-vessel disease (CSVD) with a heterozygous HTRA1 mutation (15, 16).
Genetic testing of HTRA1 in our patient revealed a negative
result. Further diagnostic approaches with this patient should
involve analyses of other causative genes of hereditary
CSVDs, including COL4A1, COL4A2, TREX1, GLA, and
CTSA (5), as well as CSVD-associated genes, including
ACE and MTHFR (17).
The p.Val237Met variant was previously reported in two
Japanese families. The first family was reported by Adachi
et al. The two affected siblings developed gait disturbance in
their sixth decade of life, and one of them had dementia and
headache. The MRI findings included periventricular hyperintensities and multiple small infarctions in the subcortical
white matter and basal ganglia (7). They were diagnosed
with CADASIL because GOM was detected in the skin biopsy obtained from one of the patients. However, a typical
cysteine-related mutation could not be completely excluded,
as the regions they analyzed were limited to NOTCH3 exons
3-4, 11, and 19 as well as the neighboring regions (8).
The second patient with p.Val237Met was identified by
Kunii et al. through a genetic analysis of 60 adult patients
with leukoencephalopathy by next-generation sequencing of
all exons of 55 genes, including NOTCH3, associated with
leukoencephalopathy (9). The reported patient was a 34year-old man without any family history of stroke. He had
no symptoms except for a history of headache from 31
years old. They noted that the MRI findings were atypical
for CADASIL and concluded that the pathogenicity of p.Val
237Met was uncertain, mainly due to the relatively high carrier frequency in their in-house database consisting of
exome data obtained from 575 Japanese individuals (3/575,
0.52%) (9).
The p.Val237Met carrier frequency in the controls described by Kunii et al. is comparable to that in East Asian
populations (47/9,973; 0.47%) reported in the Genome Aggregation Database (gnomAD, https://gnomad.broadinstitute.

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DOI: 10.2169/internalmedicine.6096-20

org), a large-scale genome variation database from exome
and genome sequencing data of the general population. The
p.Val237Met carrier frequency in other ethnic populations is
extremely rare or absent, suggesting that p.Val237Met may
be a rare polymorphism accumulating in East Asians (Table).
The pathogenicity of p.Val237Met remains unclear, as we
did not note GOM or NOTCH3 immunostaining on a skin
biopsy in our patient with p.Val237Met. Our results will
contribute to the accurate assessment of p.Val237Met in the
genetic testing of CADASIL and aid in the performance of a
comprehensive genomic analysis. Cumulative evidence from
further analyses is needed to validate our findings.
The authors state that they have no Conflict of Interest (COI).
Financial Support
This study was supported by a grant-in-aid for Research on
Intractable Disease from the Japanese Ministry of Health, Labor,
and Welfare, Japan (H30-Nanchitou(Nan)-Ippan-006).

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