DiViNAS-II study (Disease Variability in NOTCH3 Associated Small vessel disease - a two year follow-up study): A study investigating phenotype, progression, biomarkers and disease modifiers of CADASIL and CADASIL-like hereditary cerebral small vessel diseases.

CADASIL is a hereditary cerebral small vessel disease (SVD) that is caused by
characteristic cysteine altering mutations in the NOTCH3-gene, leading to
mid-adult onset ischemic strokes and vascular dementia. In the past four years,
the CADASIL research group of the LUMC has shown that NOTCH3 variants located
in epidermal growth-factor like repeat (EGFr) domains 1-6 of the NOTCH3 protein
are associated with a more severe SVD phenotype than NOTCH3 variants located in
EGFr domains 7-34. To further study the impact of NOTCH3 mutation position on
disease severity, the DiViNAS-I baseline study was performed at LUMC between
May 2019 and December 2020 (CME P18.164). Approximately 200 individuals with a
NOTCH3 mutation participated in DiViNAS, of which approximately half have a
mutation located in one of EGFr domains 1-6 and half in one of EGFr domains
7-34. Participants were fully characterized (including medical history,
neuroimaging, neuropsychological test battery, skin vessel abnormalities). Our
preliminary analysis of DiViNAS-I data validates our hypothesis that the NOTCH3
mutation position is associated with disease severity. Moreover, we find a
novel association between NOTCH3 mutation position and CADASIL vessel wall
abnormalities, which may shed light on the molecular mechanism underlying the
NOTCH3 mutation position effect.
In order to study the effect of NOTCH3 mutation position on disease
progression, we aim to now perform a 2-year follow-up study of the DiViNAS-I
cohort, in order to improve personalized disease prediction, identify potential
disease monitoring biomarkers and discover new (genetic) disease modifiers.
DiViNAS-I and DiViNAS-II are the first prospective studies world-wide assessing
the impact of NOTCH3 mutation position on disease severity and disease
progression in a cohort of CADASIL patients.
The main aims of DiViNAS-II are i) to study differences in 2-year disease
progression between mild and severe genotypes, ii) to identify other major
environmental or genetic disease modifiers and iii) to identify (early-disease
stage) biomarkers. Secondary aims are comparing CADASIL to novel CADASIL-like
small vessel diseases such as CADASIL type 2 and CARASAL, as well as
identifying the underlying genetic cause in families with hereditary SVD of
unknown etiology (hSVD-u).

Aim and objectives
The overall aim of DiViNAS-II is to determine the impact of genotype on 2-year
disease progression, to discover other disease modifiers and to identify
disease monitoring biomarkers in CADASIL.

Primary objectives
1. To determine differences in 2 year disease progression between patients with
NOTCH3 EGFr 1-6 mutations versus 7-34 mutations.
2. To identify biomarkers which are suitable surrogates for monitoring disease
progression (e.g. neuroimaging markers, NOTCH3-score and ultrastructural
analysis of skin vessels, fluid biomarker levels, retinal abnormalities etc.)
3. To identify additional disease modifiers, using a novel and improved
approach by stratifying study participants according to their genotype.

Secondary objectives
1. To create a baseline cohort of patients and families with CADASIL-like
hereditary SVDs, such as Cathepsin-A-related arteriopathy with strokes and
leukoencephalopathy (CARASAL), Cerebral autosomal recessive arteriopathy with
subcortical infarcts and leukoencephalopathy (CARASIL), CADASIL type 2 and
families with hereditary SVD with unknown genetic cause (hSVD-u).
2. To acquire hiPSCs for in vitro modelling of rare hereditary small vessel
diseases, especially to investigate the molecular mechanisms underlying
similarities and differences (in collaboration with Prof. Dr. W.M.C. van
Roon-Mom from the Department of Human Genetics and Dr. Valeria Orlova from the
Department of Anatomy and Embryology). These hiPSCs will be stored in the LUMC
Biobank.

This is a non-intervention 2 year follow-up study including up to circa 200
individuals with cysteine altering NOTCH3 mutations (CADASIL), recruited from
participants from the baseline DiViNAS-cohort at LUMC (CME P18.164), who have
indicated that they want to be informed about follow-up studies of DiViNAS. An
additional small number of individuals (up to max n = 50) with other
CADASIL-like hereditary cerebral small vessel diseases and their family members
will be asked to participate in DiViNAS-II.
The DiViNAS-II study will be performed at the Leiden University Medical
Center (LUMC) at the Department of Clinical Genetics, in collaboration with
Radiology (MRI), Neurosurgery (Lumbar puncture), Dermatology and Pathology
(Skin biopsy), Ophthalmology (OCT), Neurology and Psychiatry
(Neuropsychological test battery and psychiatric inventories). We aim to start
the study in May 2021 and complete the study inclusion 1.5 years later in
November 2022.
The DiViNAS-II study protocol will be largely identical to the study protocol
of DiViNAS-I to ensure compatibility of data (neuroimaging protocol,
neuropsychological test battery, interim medical records, skin biopsies, blood
withdrawal). In addition, to enable maximum potential for biomarker discovery,
a subgroup of individuals with CADASIL will be asked to consent to a lumbar
puncture for obtaining CSF and all individuals will be asked to consent to
retinal imaging. CSF and retinal measurements will therefore be new baseline
measures in DiViNAS-II and will be studied cross-sectionally and serve as novel
baseline measures for future follow-up studies.
Pre-defined disease outcome measures (e.g. clinical, neuroimaging, fluid
biomarker, (semi-) quantification of skin vessel pathology) will be used to
determine 2-year disease progression
Biomarkers identified as promising in DiViNAS, in other LUMC clinical CADASIL
studies or in the medical literature will be selected for further study in
DiViNAS-II and correlation with disease progression will be assessed. Examples
of promising biomarkers are NfL in serum or CSF [10, 26] and neuroimaging
markers such as DTI [16]. We also have recently discovered a novel skin vessel
biomarker which associates with genotype (manuscript in preparation), which we
aim to further assess and validate in DiViNAS-I and DiViNAS-II. Next to
assessment of selected biomarkers, there will also be biomarker discovery
studies, for example in serum and CSF using biomarker discovery technology
platforms such as O-Link. We will also introduce new baseline measures for the
study of biomarkers in cerebrospinal fluid and the retina (optical coherence
tomography (OCT) and optical coherence tomography-angiography (OCT-A)), as well
as introducing new participants with (rare) CADASIL-like hereditary small
vessel diseases.
hiPSCs will be generated from a maximum of 10 patients with a rare hereditary
cerebral small vessel disease seen in this study.

The burden and potential risks of this study are moderate. All examinations
will take place in the LUMC during a single visit of maximally 7 hours,
including breaks. Prior to the examination day, patients will be asked to fill
in questionnaires. During the day the patients will undergo two 4mm punch skin
biopsies, blood withdrawal (maximum of 70 ml), neuropsychological testing,
optical coherence tomography of the retina, an MRI-scan and in a subgroup also
a lumbar puncture.

MRI risks
The risks of MRI are minimal and may include movement of paramagnetic objects
in the body and claustrophobia. Prior to the procedure, patients will be
screened by a physician for the identification of MRI contra-indications. The
MRI-procedure will be described in great detail to reduce possible stress
experienced by the participant. Patients may stop the scanning procedure at any
given time.

Skin biopsy risks
In rare cases, skin biopsies may lead to site infection or post-procedure
bleeding. This risk is minimalized by disinfecting the site prior to the
procedure and observing the biopsy site for excessive bleeding afterwards.

Neuropsychological testing burden
A potential risk of neuropsychological testing is temporary psychological
distress and fatigue during and after the examination.

OCT/OCT-A risks
The examinations will be performed without dilation of the pupil. There are no
health risks associated with OCT and OCT-A without pupil dilatation.

Lumbar puncture
A subgroup of patients will also undergo a lumbar puncture. A lumbar puncture
is a relatively safe procedure, but complications may arise and most commonly
include post-LP headache, back pain and minor neurologic symptoms such as
radicular pain and numbness. Rare complications include infection, bleeding and
cerebral herniation in patients with intracranial tissue displacement or
possible raised intracranial pressure. To minimize the risks of lumbar
puncture, the patient, together with the researcher, will fill in a
questionnaire prior to the research day over the telephone to exclude the
presence of any contra-indications in addition to screening of the medical
history and medication list by the study physician. On the study site, the
study physician will additionally screen the patient with blood work-up. The
procedure will be performed by an experienced consultant Neurosurgeon or senior
Neurosurgery resident.