Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) leading to inflammatory lesion in brain and spinal cord. In contrast, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary NOTCH3-related vasculopathy causing ischemic white matter lesions. Magnetic Resonance Imaging (MRI) is the gold standard to evaluate CNS lesion in both diseases. However, achieving the correct diagnosis is not always easy. Indeed, several CADASIL cases were reported as MS misdiagnosis. Adding complexity is the co-occurrence of the two diseases in some cases. To date, a systematic investigation about frequency and clinical features of NOTCH3 mutation carriers among MS cases is still lacking. We will select a cohort of fifty MS patients with family history of MS, migraine, strokes or dementia or with MRI brain lesion compatible with MS and CADASIL. Each patient will undergo NOTCH3 molecular analysis. In those with variants of unknown significance or new mutations, deposits of granular osmophilic material on hypodermal vessels will be searched by electronic microscopy. Moreover, the impact of all detected mutations on the Notch3-receptor activity will be predicted by specific bioinformatics tools. Family history, neurologic and retinal evaluation, cerebrospinal fluid (CSF) profile and MRI features will be assessed, comparing NOTCH3 carriers and non-carriers among the above defined cohort.
This study aims at detecting the NOTCH3 mutations frequency in patients with MS and the clinical, MRI and CSF features able to discern MS-CADASIL co-occurrence from ¿inflammatory CADASIL¿. The results of this study would improve clinical management of our patients and their relatives at risk of developing the disease. DNA, plasma and fibroblasts from patients and carriers will be stored for further studies aimed at investigating the role of inflammation in the pathogenetic cascade related to specific NOTCH3 variants.
With this study the PI and the collaborators aim at defining the frequency of pathogenic, hypomorphic and new NOTCH3 mutations in patients with MS diagnosis, as well as at outlining possible genotype-phenotype correlations. The ascertainment of the pathogenicity of ¿not canonical¿ variants by electron microscopy will allow to widen the spectrum of CADASIL causative mutations. The prediction of the impact that each mutation has on the protein by bioinformatic tool, will allow to distinguish mutations that abrogate the activity of the Notch3 receptor from those that do not change or, conversely, improve it. To date, it is unknown if the increase of Notch3 receptor activity by specific NOTCH3 mutations may predispose to an inflammatory CADASIL. Nevertheless, it has been proven that the Notch3 activity favors the pro-inflammatory responses of activated T-cells and macrophages (Long Long, 2020) and that its abrogation reduces inflammatory responses (Jurynczyk 2008; Ling, 2019).
A detailed phenotypic characterization of each patient could highlight which clinical, biochemical and/or neuroradiological findings may lead to request the sequencing in MS patients. Moreover, among mutated cases, the identification of possible associations between peculiar sub-phenotypes and specific mutations, such as those reducing and those not changing or increasing the Notch3 activity, could be a useful aid to discriminate an inflammatory form of CADASIL from the co-occurrence of the two diseases, as well as to disclose possible contribution of NOTCH3-related pathways to MS etiopathogenesis.
Although to date the genetic diagnosis has no substantial consequences on therapeutic choices for cases with such inflammatory diseases, the management of the patients and, mostly, of their families will be improved. In particular, the extension of molecular analysis to relatives will allow to identify healthy NOTCH3 carriers at risk of developing the disease. Healthy carriers could benefit from preclinical surveillance and could represent informative cases for discovering endophenotypes and/or prodromal signs that would lead to an early diagnosis. This could open new hopes for very early or even preventive therapeutic interventions.
The prediction of the impact that each mutation has on the protein and the identification of possible genotype-phenotype correlations may also have innovative implications. The detection of a NOTCH3 pathogenic variant predisposing to the inflammatory form of CADASIL in a paucisymptomatic patient may help clinicians to better plan the clinical management and to promptly evaluate the most appropriate therapeutic strategies, even from the very early occurrence of the first symptoms. Moreover, an interesting hypothesis that should be investigated in future studies is whether treatments able to reduce inflammation may have consequences also on the ischemic lesions and their progression.
To understand the role of the inflammation in the pathogenesis of CADASIL, and possible different roles for specific mutations might open new hopes for precision therapy. The inhibition of the Notch3 receptor activity in CADASIL patients could be a promising target for new therapies able to reduce the progression of the disease. Indeed, inhibitors of Notch3 receptor pathway, such as GSI, have been suggested to reduce proinflammatory responses on animal and cellular models of CADASIL (Jurynczyk 2008; Ling 2019). Further studies are needed to investigate these hypotheses. Biological samples and cells from patients and healthy relative with NOTCH3 pathogenic variants could be useful for such studies.