Original paper

What factors determine phenotype of cerebral autosomal
dominant arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL)? Considerations in the context
of a novel pathogenic R110C mutation in the NOTCH3 gene

Dorota Dziewulska1,2, Dorota Sulejczak3, Michalina Wężyk4
1Department of Neurology, Medical University of Warsaw, Warsaw, 2Department of Experimental and Clinical Neuropathology,
Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, 3Department of Experimental Pharmacology,
Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, 4Laboratory of Neurogenetics, Mossakowski Medical
Research Centre, Polish Academy of Sciences, Warsaw, Poland

Folia Neuropathol 2017; 55 (4): 295-300	

DOI: https://doi.org/10.5114/fn.2017.72387

Abstract
We report patients from a Polish family with cerebral autosomal dominant arteriopathy with subcortical infarcts and
leukoencephalopathy (CADASIL) who possess a novel heterozygous R110C mutation in exon 3 of the NOTCH3 gene
leading to stereotypical cysteine loss. The proband had only seizure attacks and her magnetic resonance imaging
(MRI) showed very numerous hyperintense foci in the cerebral white matter in a location characteristic of CADASIL.
Distinctive ultrastructural assessment of vessels from skin-muscle biopsy revealed only mild degenerative changes
but relatively numerous homogeneous deposits of granular osmiophilic material (GOM). In the other symptomatic
family members with the same mutation ischaemic strokes were present but not epilepsy. In the proband’s affected brother at a similar age, the brain MRI was normal but vessels showed pronounced degenerative changes and
irregular GOM deposits. The present report not only extends the list of known pathogenic mutations responsible for
CADASIL but also emphasizes clinical and morphologic variability among family members with the same NOTCH3
mutation, suggesting that probably additional factors, not only mutations, may influence the disease phenotype.
Key words: CADASIL, GOM, microangiopathy, NOTCH3, seizures.

Introduction
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is
due to mutations in the NOTCH3 gene, but its pathomechanism is still unclear in spite of a growing number
of new findings concerning its pathogenesis [3,9,12].

Clinical manifestations of the disease include
migraine with or without aura, recurrent ischaemic
strokes, psychiatric disorders and cognitive decline.
There is no genotype-phenotype correlation between
certain NOTCH3 mutations and CADASIL symptoms,
and therefore it is a challenge for researchers to find

Communicating author
Dorota Dziewulska, MD, PhD, Department of Neurology, Medical University of Warsaw, 1A Banacha St., 02-097 Warsaw, Poland,
phone: +48 22 599 18 63, fax: +48 22 599 18 57, e-mail: dorota.dziewulska@wum.edu.pl

Folia Neuropathologica 2017; 55/4

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Dorota Dziewulska, Dorota Sulejczak, Michalina Wężyk

of the extra- and intracranial arteries, cerebrospinal
fluid analysis, MRI of the cervical spinal cord, ECG
Holter, psychological examination, and routine biochemical tests. Nonspecific changes in brain MR spectroscopy were observed. Brain MRI revealed in T2 projection and FLAIR images numerous hyperintense foci
and confluent hyperintensities in the cerebral white
matter including both anterior temporal lobes (Fig. 2)
and external capsules. In differential diagnosis CADASIL was taken into consideration; therefore skin-muscle
biopsy and genetic examination of the NOTCH3 gene
were performed.

an explanation for CADASIL’s extremely variable symptomatology. The disease is diagnosed on the basis of
genetic examination and/or results of ultrastructural
assessment of vessels in skin/muscle material. Unfortunately, despite progress in neurodiagnostics, CADASIL
is still under‑recognized or diagnosed post mortem [19].
We report a CADASIL patient with epilepsy as a single
and early clinical symptom and other symptomatic
family carriers of the same novel NOTCH3 mutation
with different clinical and ultrastructural phenotypes.

Material and methods
The clinical history of the examined family disclosed symptoms characteristic of CADASIL in 4 members (2 men and 2 women) in three generations (Fig. 1).
Genetic testing for CADASIL performed in the proband,
her father and brother revealed a novel R110C mutation
in exon 3 of the NOTCH3 gene. In silico analysis showed
that the mutation might be pathogenic. The mutation
was probably passed on from the proband’s grandmother (Fig. 1 I-2).

Proband’s brother – a 38-year-old man
(Fig. 1, III-9)
At the age of 33 years he had the first ischaemic
attack with transient hemiparesis and aphasia. His only
brain MRI scan performed at that time was normal. Then
he had a few minor episodes of transient ischaemic
attacks without any permanent neurological deficits.

Proband’s father – a 61-year-old man
(Fig. 1, II-3)

Proband, a 36-year-old woman
(Fig. 1, III-8)

At the age of 44 years he had the first ischaemic
stroke of the left cerebral hemisphere without permanent neurological deficit. The strokes repeated twice
at the ages of 50 and 60 years, resulting in persistent
right-sided spastic hemiparesis, mild aphasia and cognitive disturbances. There were neither migraine, mood
disturbances nor seizure attacks in his medical history.

The previously healthy woman was admitted to hospital at the age of 34 years after the first in her life tonicclonic seizure attack. The EEG record revealed polymorphic theta and delta waves in both temporal lobes.
Neurologic examination of the patient was normal, as
were results of her brain CT scan, Doppler examination

I
1

2

II
4

3

III

8 PROBAND

9

5

10

6

11

12

7

13

14

15

16

IV
17

18

19

20

Fig. 1. Pedigree of the family with R110C mutation in the NOTCH3 gene; generations I-IV.

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What factors determine phenotype of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)?
Considerations in the context of a novel pathogenic R110C mutation in the NOTCH3 gene

Fig. 2. Proband’s MRI scans. In T2 projection there are visible focal and confluent hyperintense changes in
the cerebral white matter, also involving both anterior temporal lobes (arrows).
Biopsy was not performed but genetic examination
showed R110C mutation in the NOTCH3 gene.

Proband’s grandmother (Fig. 1, I-2)
She died at the age of 48 years due to ischaemic
stoke. In her medical history headaches and migraine
were present but not seizure attacks.

Results
The ultrastuctural examination of vessels in skin-muscle biopsies material from proband and proband’s brother
was performed.
In proband ultrastructural examination of vessels
re­vealed pathognomonic for CADASIL deposits of granular
osmiophilic material (GOM) in arterial media and capillary
basal membrane (Fig. 3A-B). Vessels with GOMs were not
numerous but when the deposits were found their number was relatively abundant. GOM deposits were round
or oval, homogenously osmiophilic, and located mainly
in indentations of VSMC/pericyte cell membrane. Except
the presence of GOMs, vessel appearance was generally
normal e.i. neither VSMC/pericyte degeneration or loss
nor thickening of the basal lamina was observed.
In skin-muscle biopsy of proband’s brother, a relatively abundant pathological vessels at advanced stage
of degenerative process and GOM deposits were found.

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The affected vessels were often collapsed, and showed
thickened basal lamina (Fig. 3D-E), endothelial and
myocyte/pericyte vacuolization (Fig. 3C-E), cell shrinkage with abnormal nuclei and lucent enlarged mitochondria. GOM deposits were both regularly shaped
and uniformly electron-dense as well as irregular and
inhomogeneously dense (Fig. 3C-F). The latter were
a bit more numerous. They were situated rather more
distant from the cells within the basement membrane,
and sometimes GOMs were observed even in vessels
without visible VSMC/pericytes.

Discussion
Recently, an increasing number of pathogenic
NOTCH3 mutations have been described. In some of
these reports the authors emphasized phenotypic variability of the disease even among family members with
the same mutation [18] or monozygotic twins [11]. Our
article on a CADASIL family with a novel R110C mutation joins that group of publications. The affected family members, especially the proband and her brother,
who are of similar age, showed different brain MRI
scans, dissimilar clinical symptomatology, and diverse
ultrastructural vascular changes.
In the course of CADASIL, epilepsy is usually secondary to stroke and rarely appears early [2,13]. The aeti-

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Dorota Dziewulska, Dorota Sulejczak, Michalina Wężyk

A

B

C

D

E

F

Fig. 3. Ultrastructural vascular changes in proband’s (A, B) and her brother’s (C-F) biopsy materials. A) Affected
myocyte with pale cytoplasm and irregular nucleus in the arterial tunica media, bar – 2 μm. B) Normal appearance
of capillary vessel with numerous dark, dense, and oval GOM deposits near pericytic processes, bar – 500 nm.
C) Vacuolar degeneration of VSMC (asterisk). In the media irregularly shaped and dense GOM deposits are
visible (arrows), bar – 1 μm. D) Vacuolar degeneration of pericytes. In the thickened basal lamina a single GOM
deposit is visible (arrow), bar – 500 nm. E) Capillary vessel with thickened basal lamina and very narrow vessel
lumen. Visible are clear, enlarged endothelial cells with various-sized vacuoles and a single GOM deposit (arrow),
bar – 500 nm. F) Atypical longitudinal GOM deposits in the wall of capillary vessel (arrows), bar – 500 nm.

ology of epileptic seizures in CADASIL is still a matter
of debate. Epileptogenic changes could be triggered
by ischaemic brain lesion, but in the proband neither
clinical nor neuroimaging findings indicated ischaemic

298

stroke. They may also be associated with brain damage of a different mechanism than an ischaemic stroke.
MRI hyperintensities in the temporal pole of CADASIL
patients can be explained by enlarged perivascular

Folia Neuropathologica 2017; 55/4

What factors determine phenotype of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL)?
Considerations in the context of a novel pathogenic R110C mutation in the NOTCH3 gene

space and degeneration of myelin accompanied by lack
of drainage of the interstitial fluid [20]. Also, epilepsy
in CADASIL may be directly related to altered properties of the Notch 3 signalling system [17]. In temporal
lobe epilepsy, Notch signalling was activated both in an
induced epilepsy model and in human epileptogenic
tissues, and the effects of activated Notch signalling
on seizures can be attributed to its regulatory role in
excitatory synaptic activity in pyramidal neurons [16].
It is also known that some regions of the brain are
more epileptogenic than others, and abnormalities in
the anterior temporal regions are especially associated
with epileptogenesis. Hyperintensities in the anterior
temporal white matter visible in MRI are frequently
observed in patients with a CADASIL diagnosis. Moreover, patients with temporal lobe epilepsy sometimes
also have white matter abnormalities in the anterior
temporal lobe [1]. In the proband, both MRI scans and
the EEG record revealed pathological changes in the
temporal lobes. These findings, in the context of the
above data, can confirm the hypothetical origin of her
epilepsy in the temporal lobes.

Diverse ultrastructural changes
in vessels of skin biopsies
In the proband’s skin-muscle biopsy, the appearance of vessels containing GOM deposits was normal,
and only very few of them exhibited mild pathological changes. In contrast, in the biopsy of the proband’s
brother numerous vessels at an advanced stage of
degeneration and with GOM deposits of various morphology were found. There are two main types of GOM:
uniformly electron-dense deposits and mixed ones with
inhomogeneous morphology [7,8]. Based on our previous studies on CADASIL we have suggested that GOM
deposits can be formed on the surface of VSMC/pericytes, can penetrate from the cell into the basement
membrane, and then can disperse in the extracellular
matrix [7]. Although qualitative or semi-quantitative
changes in GOM do not correlate directly with patients’
age [10], there are some data indicating a possible relationship of the deposits with progression of histopathological changes in CADASIL. GOM deposits are very early alterations whose development precedes detectable
morphological changes in cerebral vessels [4] but not
their dysfunction [14]. In a human fetus with a mutated
NOTCH3 gene, GOM deposits were not found [5], but
they started to be observed in patients at the age of
20 years [6], and then, after a period in which the num-

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ber GOM deposits was relatively constant, their number gradually decreased with duration of the disease.
These dynamics imply that GOM deposits as aberrations secondary to alterations in the Notch 3 signalling pathway may mirror – to a certain extent – activity
of the pathological process in CADASIL. Effects of the
Notch 3 signalling system are context- and cell-dependent, and therefore there are various factors influencing the results of its action. Additionally, the NOTCH3
gene is highly variable. Since these genetic variations
play a role in age-related cerebral small vessel diseases
[15], they may also have some impact on CADASIL.
In summary, since the diversity of clinical and morphological pictures of individuals with CADASIL falls
within the disease spectrum, there are probably other
factors than just NOTCH3 mutations that influence its
phenotype.

Disclosure
Authors report no conflict of interest.
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