**3. Genetics**

NOTCH3 gene mutations are causative of the disease. This gene, consisting of 33 exons spanning roughly 7 kb and located on chromosome 19p13 [48], encodes a single-pass transmembrane heterodimer receptor Notch3 of 2321 amino acids involved in cellular signaling and fate during embryonic development [49, 50]. Notch3 protein comprising an N-terminal extracellular domain (NECD) involved in ligand binding, a transmembrane domain (NTMD), and an intracellular domain (NICD), which contains seven ankyrin repeats is required for downstream signal transduction (**Figure 1**) [51, 52]. More specifically, the NECD is non-covalently associated with the membrane-tethered intracellular domain, and it is composed of 34 epidermal growth factor (EGF)-like repeats, followed by 3 Notch/lin12 repeats [53]. Each EGF-like repeat encompasses six cysteine residues, forming three pairs of disulfide bonds [54, 55]. The receptor is synthesized as single precursor protein which is cleaved during transport to the cell surface (S1 cleavage), where it is expressed as heterodimer. Upon binding of its ligand (a protein of the delta/jagged family) [56] at EGF repeats 10–11, Notch3 receptor undergoes two other proteolytic cleavages: at first, N3 is cleaved (S2 cleavage) in its extracellular domain by a TNF-α-converting enzyme (TACE), subsequently in its transmembrane domain (S3 cleavage) in a presenilin-dependent manner. These proteolytic events, mutually dependent, generate the NICD fragment, which released from the NTMD enters the nucleus for activating the transcription of its target genes [53, 57–59]. Although the mutations are highly stereotyped, atypical phenotypes have been recognized, and the disease is probably underdiagnosed in most of the stroke population. Most CADASIL-associated mutations result in a gain or loss of cysteine residue in one of the 34 EGF-like repeats in the extracellular domain of the Notch3 protein, thus sparing the number of cysteine residues within the domain [60–62]. The alteration of the 3-D structure of the Notch3 protein, which is due to an aberrant dimerization of Notch3 through an abnormal disulfide bridging with another Notch3 molecule or with another protein, may play a central role in the pathogenesis of CADASIL [63–65]. A founder effect has been documented for the Finnish population but

**Figure 1.** *Schematic structure of Notch3 protein: Notch3 domains are differently colored.*

not for other countries [66]. To date, more than 200 different mutations in the NOTCH3 gene have been reported in CADASIL patients, of which 95% are heterozygous missense point mutations [67]. The remaining consist of small deletions, duplications, in frame [68–71] and frame shift mutations, splice site mutations [36], and a small deletion not directly involving a cysteine residue [72]. Moreover, a three-nucleotide insertion has been described as the first pathogenic insertion [73]. Recent studies have found that mutations that do not affect the number of cysteines (unlike the typical mutations) seem to be associated with clinical CADASIL syndrome. However, the pathogenic role of these mutations is uncertain. Although it has been suggested that some mutations may be associated with a milder or more severe phenotype, so far no clear genotype-phenotype correlation has been found [7]. Moreover, only a few cases in the literature reported homozygous mutations of NOTCH3 [74–78]. Many polymorphisms have also been identified in the NOTCH3 coding sequence [67], some of them leading to amino acid substitutions [79]. However, it is unknown whether these polymorphisms affect Notch signaling or whether they are involved in cerebrovascular disease.
