DiViNAS study (Disease Variability in NOTCH3 Associated Small vessel disease): the NOTCH3 mutation position effect and other disease modifiers in CADASIL

CADASIL is a hereditary dementia and stroke syndrome, caused by archetypal
cysteine altering NOTCH3 mutations. LUMC is the national CADASIL expertise
centre and next to patient care, the research group has set up a CADASIL
registry and has been studying disease natural history and genetic intervention
therapies amongst others. CADASIL has an estimated minimal prevalence of
2-5:100.000. However, we have recently discovered that archetypal
CADASIL-causing NOTCH3 mutations have a prevalence of 1:300 in the general
population worldwide (i.e. approximately 100 times more frequent than the
estimated minimal CADASIL prevalence), suggesting that CADASIL is likely much
more prevalent than previously suspected, implying that the disease spectrum
must be considerably broader than recognised to date, with the majority of
individuals not having a CADASIL diagnosis. Our hypothesis is that the NOTCH3
associated phenotype may have an attenuated form which may be much more common
than *classical* severe CADASIL. We were indeed able to show that even within
CADASIL samples, there are significant differences in disease severity, and
that this partially explained by a hitherto unknown effect of the position of
the mutation along the 34 epidermal growth factor-like repeat (EGFr) domains of
the NOTCH3 protein. These new findings have important implications in our
understanding of the NOTCH3/CADASIL disease spectrum, disease pathomechanism,
disease modifiers and potential protective measures and future therapeutic
targets. Firstly, our objective is to capitalize on our recent findings by
performing the first prospective study of the NOTCH3 mutation position effect
in a large (n=200) sample of CADASIL patients with specific inclusion criteria
for mutation position. Secondly, our objective is to study a potential
attenuated form of CADASIL by phenotyping elderly undiagnosed individuals
harbouring NOTCH3 mutations in known CADASIL pedigrees. Thirdly, our objective
is to investigate potential surrogate markers for disease severity, such as the
NOTCH3 score we developed in our transgenic NOTCH3 mice, NOTCH3 levels in
plasma, BOLD response with fMRI, neurofilament light chain levels in serum and
endostatin levels in plasma.

Aim and Objectives
The overall aim is to define the broad disease spectrum of cysteine altering
NOTCH3 mutations, specifically the as yet unexplored milder end of this
spectrum, and to determine to what extent the mutation position predicts
disease severity.

Primary Objectives
1. To assess to what extent mutation position determines NOTCH3 disease
severity and what the additional effect is of other known modifiers, such as
vascular risk profile.
2. To delineate the mildest end of the NOTCH3 disease spectrum by assessing
clinical severity, the MRI and cognitive profile of 60+ year old individuals
with a cysteine altering NOTCH3 mutation, but without a clinical CADASIL
diagnosis.

Secondary objectives
1. Create a large baseline cohort with symptomatic and asymptomatic CADASIL
patients that can be used in future follow-up studies, including biobanking.
2. Determine the correlation between candidate surrogate markers (e.g. vascular
reactivity, skin NOTCH3 score and blood biomarkers) and CADASIL disease
severity.
3. To acquire hiPSCs for in vitro modelling of CADASIL (vessel-on-a-chip),
especially to investigate the molecular mechanisms underlying the NOTCH3
mutation position effect (collaboration with Prof. C. Mummery, dep. Of Anatomy
and Embryology).

This is a non-intervention cross-sectional study including approximately 250
individuals with cysteine altering NOTCH3 mutations (CADASIL), recruited from
the Dutch CADASIL registry.

Two cohorts will be included:
1. The NOTCH3 spectrum cohort
This is a cohort including CADASIL-patients aged 20 years and older; n=100
patients with an EGFr 1-6 mutation and n=100 individuals with an EGFr 7-34
mutation.

2. Elderly NOTCH3 sample
This is a sample of maximally 50 individuals, aged 60 years or older,
harbouring a NOTCH3 mutation but without a prior CADASIL diagnosis.

The study will be performed in the LUMC at the departments of clinical
genetics, radiology, neurology and dermatology. We aim to start the study in
January 2019 and to complete the study in June 2020.

The main procedures the subjects will undergo are:
- neuropsychological test battery
- MRI
- Two skin punch biopsy
- Blood withdrawal

Patients will be invited for a half-day visit to the LUMC, all measurements
will be performed during this visit. Prior to the research day, patients will
be asked to fill in a questionnaire about their medical history. During the day
the patients will undergo two 4 mm punch skin biopsy, blood withdrawal (maximum
70 ml), neuropsychological testing and a MRI scan.
The risks of MRI are minimal. Potential risks from the MRI study include
movement of paramagnetic objects in the body. Furthermore, some subjects may
feel claustrophobic in the restricted space of the MR scanner. Claustrophobia
from the MRI scan will be reduced by explaining the nature of the scanner in
detail before enrollment. At all times, the subjects can request to be removed
from the scanner. The physician will assess whether there are risk factors for
MRI, and decide whether or not the participant is allowed to go into the
scanner.
During and after the neuropsychological examination, there may be temporary
distress and fatigue. Skin biopsy is generally a safe procedure, and will be
performed under local anesthesia. In rare cases, local inflammation at the
biopsy site can occur. The biopsy site will be sterilized with alcohol to
minimize the risk of infection. In conclusion, the overall risk of this study
is minimal.

The study will not be directly beneficial for the patient, however through
this study we will obtain important knowledge about CADASIL, where the whole
population of CADASIL-patients can benefit from in the future. Firstly,
validating and further delineating the mutation position effect and finding
other disease modifiers will be important for predicting disease course in
diagnosed CASASIL patients as well as in patients with a chance finding of a
cysteine altering NOTCH3 mutation in whole exome sequencing or whole genome
sequencing analysis for another indication. Secondly, delineating the mildest
end of the NOTCH3 disease spectrum will possibly reveal important differences
between severe and mild patient groups, and thus direct future research into
protective measures and therapeutic targets. Thirdly, development of surrogate
markers is crucial for novel CADASIL therapeutic development.