Journal of the Neurological Sciences 267 (2008) 170 – 173
www.elsevier.com/locate/jns

Short communication

A pathogenic mutation on exon 21 of the NOTCH3 gene causing
CADASIL in an octogenarian paucisymptomatic patient
Francesca Pescini a , Silvia Bianchi b , Emilia Salvadori a , Anna Poggesi a , Maria Teresa Dotti b ,
Antonio Federico b , Domenico Inzitari a , Leonardo Pantoni a,⁎
a

Department of Neurological and Psychiatric Sciences, University of Florence, Italy
Department of Neurological and Behavioural Sciences, University of Siena, Italy

b

Received 8 August 2007; received in revised form 28 September 2007; accepted 4 October 2007
Available online 19 November 2007

Abstract
CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy) is an inherited small vessel
disease causing migraine, early strokes, cognitive impairment and premature death. The disease is caused by NOTCH3 gene puntiform mutations
on one of the exons coding for the epidermal-growth factor (EGF)-like repeats of the extracellular domain of the NOTCH3 receptor. Mutations have
been reported with higher frequency on some exons, and never on 6 out of a total of 23. We report for the first time a mutation (c.3471CNG) on exon
21 of the NOTCH3 gene that leads to a cysteine substitution (p.1131CNW) in the EGF-like repeat 29 of the NOTCH3 receptor extracellular domain,
and that is responsible for CADASIL in a functionally independent elderly man who came to our attention at the age of 79 because of a minor stroke.
CADASIL suspicion aroused only from the finding of severe white matter changes extended to the temporopolar region on cerebral magnetic
resonance imaging. This case report underlines that, when CADASIL is suspected, molecular analysis should be performed on all the NOTCH3
exons coding for EGF-like repeats and not be limited to those where mutations have been found with higher frequency, and that the disease may be
encountered also in mildly symptomatic elderly patients. The newly reported mutation might sustain a milder expressivity of the disease.
© 2007 Elsevier B.V. All rights reserved.
Keywords: CADASIL; NOTCH3; Aging; Genotype; Phenotype; Leukoencephalopathy; Magnetic resonance imaging; Stroke

1. Introduction
CADASIL is an inherited small vessel disease causing
cerebral periventricular and deep white matter changes
(typically extending to the temporopolar region) and multiple
lacunar infarcts in the basal ganglia and brainstem [1,2]. The
main clinical manifestations are migraine more often with
aura, early onset of stroke (30–50 years) and cognitive decline
(50–60 years), psychiatric disturbances, and epilepsy [3–5].
The disease has a progressive course and severe disability and
dementia develop in about three forth of the patients at the end
stage [6]. Premature death has been reported (at median age of
65 years for men and 71 years for women) [6].
⁎ Corresponding author. Department of Neurological and Psychiatric
Sciences, University of Florence, Viale Morgagni 85, 50134 Florence, Italy.
Tel.: +39 055 7947995; fax: +39 055 4298461.
E-mail address: pantoni@neuro.unifi.it (L. Pantoni).
0022-510X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jns.2007.10.017

The disease is caused by mutations in one of the exons
(from 2 to 24) of the NOTCH3 gene coding for the EGF-like
repeat domains of the extracellular portion of the NOTCH3
receptor. Mainly, these are missense mutations leading to a
cysteine substitution in the receptor [7,8]. Clustering of
mutations on exons 3, 4, 5, 8, and 11 has been reported [8,9],
while until now mutations on 6 exons (12, 13, 16,17, 21, and
24) have never been published.
Here, we describe for the first time a missense mutation
on exon 21 of the NOTCH3 gene responsible for CADASIL
in a minimally affected 82-year-old patient.
2. Materials and methods
Following informed consent, total genomic DNA was
extracted from peripheral blood leucocytes using standard
procedures. Polymerase chain reaction (PCR) was performed
with primers (comprising intron–exon boundaries) specific

F. Pescini et al. / Journal of the Neurological Sciences 267 (2008) 170–173

171

for exons 2–24 of the NOTCH3 gene. PCR was made in 25 μl
reaction volume containing 100 ng genomic DNA, 10 pmol
forward and reverse primers, 100 mM dNTPs, 1 U DNA
polymerase (Red Hot DNA polymerase; ABgene, UK),
1.5 mM MgSO4, 1× Reaction buffer, using a DNA thermal
cycler (PTC-200; MJ Research, Waltham, MA). Following
purification of PCR products, sequencing was performed
using the automated sequencer ABI 3730 (Applied Biosystems, Foster City, CA).
3. Case report
A 79-year-old man came to our observation for an acute
episode of dysarthria and weakness of the right arm and
lower part of his face from which he recovered almost

Fig. 2. Cerebral MRI. Extension of leukoencephalopathy to the external
capsule bilaterally (arrows) on an axial T2-weighted image (a), and to the
anterior temporal poles (arrows) on an axial FLAIR image (b).

Fig. 1. Cerebral MRI. Diffuse, symmetric, and confluent hyperintense lesions
in the periventricular and deep white matter on an axial FLAIR image (a), and
bilateral thalamic lacunar infarcts on an axial T2-weighted image (b, arrows).

completely in a few weeks. He had a 10-year history of
hypertension and had been a cigarette smoker for as long as
62 years (20 packs year). Family history was negative for
cerebro- and cardio-vascular diseases. Laboratory and
instrumental evaluations were performed and revealed an
increased carotid intima–media thickness on neurosonological evaluation, bradycardia on electrocardiogram, and mild
hypertensive cardiac abnormalities on transthoracic echocardiogram. Cognitive deficits were not reported by the
patient or his relatives and he was functionally independent
in the activities of daily living. A neuropsychological
evaluation showed only mild alterations in some cognitive
domains such as attention and executive functions. Cerebral
magnetic resonance imaging (MRI) showed a severe degree
of white matter lesions that appeared confluent in the
periventricular and deep areas and were associated with

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F. Pescini et al. / Journal of the Neurological Sciences 267 (2008) 170–173

multiple subcortical lacunar infarcts in the basal ganglia and
thalami (Fig. 1). These alterations were initially deemed to
represent expression of small vessel disease secondary to
hypertension and aging. Treatment with anti-hypertensive
agents was modified to improve blood pressure control, and
aspirin was initiated. The patient was then followed-up with
yearly clinical and functional assessment, and a cerebral
MRI was repeated after 3 years. At the age of 82, he was still
completely functionally autonomous (no item compromised
in the instrumental activities of daily living scale [10]) and
the neurological examination only showed mild signs of the
previous stroke. No new cerebrovascular events had
occurred and the cognitive and mood measurements remained stable during follow-up (Mini-Mental State Examination [11] score = 27/30 both at baseline and 3-year followup, Geriatric Depression Scale [12] score = 2/15 at baseline
and 1/15 at 3-year follow-up). A better appreciation of the
severity of white matter changes and of their extension to the
external capsule and temporopolar regions (Fig. 2) when the
second MRI was evaluated led to suspect CADASIL. Family
history was then re-investigated under this light but only a
history of migraine in one of the two sons emerged (Fig. 3).
The diagnosis of CADASIL was confirmed when molecular
genetic analysis revealed the heterozygous mutation
c.3471CNG on exon 21 of the NOTCH3 gene responsible
for the replacement of a cysteine at position 1131 with a
tryptophan (p.1131CNW) (Fig. 4). The mutation affects the
EGF-like repeat 29 of the NOTCH3 receptor extracellular
domain. The molecular analysis performed in the son with
migraine according to the protocols for presymptomatic

Fig. 4. Sequence chromatograms of exon 21 (NOTCH3 gene) around
nucleotide 3471 from a control individual (top) and the patient (bottom). The
affected nucleotide is indicated by the arrow.

testing for Huntington disease ruled out the presence of the
same mutation.
4. Discussion

Fig. 3. Family pedigree. Our 82-year-old patient is indicated by the arrow.
Symptomatic subjects for CADASIL typical disturbances are represented in
black filled symbols. When known, age at death or current age (for living
subjects) are reported. It is worthy to note the young age of death of patient's
father, probably for nephritis.

We have described for the first time a pathogenic
mutation responsible for CADASIL on exon 21 of the
NOTCH3 gene. The presence of a mutation leading to a
cysteine substitution, that lies within an EGF-like repeat,
follows the stereotyped nature of mutations typical of
CADASIL, thus supporting the role of this novel mutation
in causing the disease. A skin biopsy was not performed;
however, it should be noted that the reported sensitivity of
this exam in comparison with genetic testing is about 60%
[13], with the consequence that a negative result would have
not ruled out the presence of the disease.
Our report further expands the genetic spectrum of this
disease and confirms the need of analysing all the exons from 2
to 24 when CADASIL is suspected, particularly in patients
from countries where mutations have been found to be not
restricted to a few exons [14,15]. Since the first studies that
reported clustering of mutations in CADASIL [8], genetic
alterations have been described on many of the 23 exons
coding for the EGF-like repeats of NOTCH3 extracellular
domain [9]. However, some of these exons are involved less
frequently than others and up to now 6 had never been reported
as interested by mutations. In addition to a true unbalanced
distribution of mutations across the exons, this could partly
depend on a biased laboratory approach in the past. In fact,

F. Pescini et al. / Journal of the Neurological Sciences 267 (2008) 170–173

being the molecular analysis of the NOTCH3 gene expensive
and time consuming, several laboratories had restricted the
sequencing to the exons reported to have a higher probability
of carrying the mutation. As a consequence, the disease might
have gone undiagnosed in some patients and the hypothesis of
an unbalanced distribution of mutations across the 23 exons
could have been erroneously reinforced.
Our patient was initially considered to be affected by
sporadic cerebral small vessel disease because of his
advanced age, presence of vascular risk factors and absence
of relevant family history. Only a re-evaluation of MRI
findings led to hypothesise a diagnosis of CADASIL. The
mild clinical picture that this octogenarian CADASIL patient
presented, in fact, is not the one expected in a disease in
which symptoms onset is generally in the mid-adult age and
the course is progressively disabling. Few data can be found
in the literature about such mildly affected elderly CADASIL
patients [16,17]. In large European CADASIL series only
few geriatric patients were included [3–6]. The oldest
CADASIL patient (94 years) was reported in Finland, but no
further clinical information was provided [18]. In a German
study on the long-term prognosis of the disease, about 90%
of male patients older than 70 years were bedridden [6]. The
presence of paucisymptomatic CADASIL patients and the
possible association between mild clinical pictures and some
mutations could be another possible cause for the reported
unbalanced pattern of mutations distribution because of a
lack of disease suspicion in these cases. Although a
genotype–phenotype correlation has not been proven in
CADASIL and only few data support its existence [6,19],
one might speculate that the novel mutation found in our
patient has a very mild effect on the expressivity of the
disease. The lack of typical CADASIL symptoms among the
patient ancestors would support this hypothesis. Alternatively, a de novo mutation could have occurred in our patient
or in his father, who died at young age because of a renal
disease. Finally, in the hypothetic presence of a mutation
with mild effect on the phenotype, hypertension and cigarette
smoking could have contributed to the symptoms onset in
our patient. In fact, since clinical features in CADASIL are
highly variable, the role of possible additional modulating
factors has been recently studied and smoking has been
found related to earlier stroke occurrence [5].
5. Conclusions
The reported case contributes to expand the genotypic
spectrum of CADASIL and underlines that the diagnosis of the
disease can be missed if not all the NOTCH3 exons 2–24 are
analysed. Moreover, it confirms the heterogeneity of the
clinical picture in CADASIL, and supports that paucisymptomatic patients can be encountered even in a geriatric age.

173

Whether this reported novel mutation is associated with a mild
clinical picture remains a subject for future investigation.
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