Cerebral small vessel disease (SVD) is an important cause of stroke, cognitive impairment, and mood disorders in the elderly. Besides the common sporadic forms, mostly related to age and hypertension, a minority of SVD has a monogenic cause, among which the most common and best known is cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). CADASIL provides a unique model for the study of the most prevalent forms of sporadic SVD. CADASIL is caused by mutations in the NOTCH3 gene, which maps to the short arm of chromosome 19 and encodes the NOTCH3 receptor protein, predominantly expressed in adults by vascular smooth muscle cells and pericytes [1]. Thousands of families with CADASIL have now been diagnosed worldwide in many different ethnic groups. The disorder is often overlooked and misdiagnosed. Its minimum prevalence has been estimated between 2 and 5 in 100,000 but may vary between populations [2–5].

In this article, we discuss the main clinical, genetic, neuroimaging, neuropathological, and management aspects of the disease.

The main clinical features include migraine usually with aura presenting in early adulthood [6], recurrent subcortical ischemic events, mood disturbances [7], progressive cognitive impairment mostly affecting executive function, and acute encephalopathy [8, 9]. Pescini et al. [10] developed a score which may be helpful in confirming the clinical suspicion of CADASIL and identifying patients in whom mutation screening has a reasonable yield.

CADASIL is characterized by a large phenotypic variability both across and within families. No clear genotype–phenotype correlation has been found to date [43] and both modulating genetic and risk/environmental factors probably play a role.

Among the conventional risk factors, hypertension and smoking are associated with early age of onset of stroke [9, 43, 44] and with clinical worsening towards dementia [45, 46]. In a recent longitudinal study, smoking was associated with a three-fold increase in stroke risk [46]. Homocysteine levels have been found to be higher in patients with CADASIL who have migraine, but this finding needs replicating [43].

Opherk et al. [47, 48] found that white matter lesion volumes are influenced by genetic factors distinct from the causative NOTCH3 mutation, but the nature of the responsible modulating genetic factors is currently unknown. The role of Apolipoprotein E (APOE) is debated – Gesierich et al. [49] suggested a modifying influence of APOE ɛ2 on the volume of white matter hyperintensities, while Singhal et al. [43] detected no relationship between phenotype and APOE ɛ4 genotype.

Gunda et al. [50] hypothesized on a possible role of ovarian hormones in sex-related differences in CADASIL, data that need further confirmation.

Recently, Pescini et al. [51] evaluated a possible role of plasma levels of von Willebrand factor, blood levels of endothelial progenitor cells, and endothelial impairment in the pathogenesis of the disease.

Life expectancy is reduced in CADASIL patients. An age at death in men of 64.6 years and in women of 70.7 years has been reported in a large study of 411 subjects [16]. Pneumonia in patients with disability was the major cause of death (38%), but a high number of sudden unexpected deaths were also observed, accounting for up to 26% [16]. Whether cardiac arrhythmias, QT variability index, and myocardial infarction are more frequent in CADASIL than in the general population remains to be confirmed [52–55].

Another point of interest is the finding of a non-dipper pattern of nocturnal blood pressure [56]. A lower nocturnal blood pressure fall may be associated with incidence and/or worsening of deep white matter lesions in CADASIL. The pathogenesis of abnormal blood pressure profile in CADASIL remains to be clarified. Central and peripheral mechanisms controlling blood pressure variations may be involved.

The association between CADASIL and right-to-left cardiac shunt has been reported but remains under debate [57, 58].

Cerebral Magnetic Resonance Imaging (MRI) in CADASIL shows age- and disease stage-dependent extensive white matter abnormalities, seen as large symmetrical hyperintense signals on T2-weighted and FLAIR images [59], and lacunes, i.e., fluid-filled cavities with cerebrospinal fluid-isointense signal. Diffusion-weighted imaging may show acute and subacute infarctions, and can also be used to quantify chronic white matter changes through diffusion tensor metrics. White matter changes often involve the anterior temporal lobe, the external capsule and the superior frontal gyrus [60]. Anterior temporal pole changes have been shown to have a high sensitivity and specificity for the disease (approximatively 90% for each) and are useful in diagnosis [61]. In Asian populations, anterior temporal lobe involvement is less common [62]. External capsule changes also have a high sensitivity (approximatively 90%) but a lower specificity (approximately 50%). In fact, a recent systematic analysis showed a similar involvement of the external capsule in CADASIL and sporadic SVD [63]. Corpus callosal signal abnormalities, rarely occurring in sporadic SVD, are described in CADASIL [59] (Fig. 2); such abnormalities are also a feature of multiple sclerosis, which is one reason for the misdiagnosis of CADASIL as multiple sclerosis. Cerebral microbleeds, which are shown by Gradient-echo images with dot-like hypointense lesions, occur in a variable proportion of cases (30–70%) and usually increase with age and risk factors such as high blood pressure, and intracerebral hemorrhage have also been described in a number of patients [64–66], especially those of Asian origin [67]. Nevertheless, the clinical significance of the cerebral microbleeds observed in CADASIL has not been clearly elucidated.

MRI white matter signal abnormalities are found in presymptomatic CADASIL mutation carriers aged 20–30 years or older [59, 68].

The MRI findings that have been most strongly related to clinical deficit are number and volume of lacunes, brain atrophy, and white matter damage as assessed by diffusion tensor imaging [69, 70]. CADASIL has primarily been defined as a subcortical disease, but recent studies with high-field 7-Tesla MRI, which allows higher resolution imaging, have detected primary involvement of the cortex, including the demonstration of cortical microinfarcts and early diffuse cortical alterations [23] in frontal and parietal regions most often following a symmetrical pattern in both hemispheres. These changes were not related to cortical thinning or to subcortical lesions and have been hypothesized to be secondary to venous vascular density or intramyelin edema [71]. Similar data have also been reported in experimental models [72]. Moreover, subcortical changes might induce secondary cortical changes [69, 72–74]. In a longitudinal study, incident lacunes were followed by cortical thinning specifically in connected brain regions [31]. Another recent development is the use of diffusion tensor imaging to characterize tissue damage [75, 76]. The typical change in diffusion metrics observed in CADASIL patients is a reduction in fractional anisotropy (a measure for the directionality of diffusion) and an increase in the apparent diffusion coefficient or mean diffusivity (a measure for the extent of diffusion) [69, 77]. Histogram analysis has been proposed as a sensitive tool to measure CADASIL-related changes cross-sectionally as well as longitudinally [78–80].

The pathological hallmark of CADASIL is deposition of granular osmiophilic material (GOM) in close relation to vascular smooth muscle cells, seen on electron microscopy. GOM can also be detected in vessels of extracerebral tissues, including skin and muscle. Consequently, skin biopsy has been considered an important diagnostic tool [81], with a specificity of 100%, and variable sensitivity from 45% to 100% according to the different reports [82–84]. The reasons for this discrepancy may be related to different factors, including different stages of the disorder, the size of the biopsy and the number of arteriolar and precapillary vessels in the specimen, or the focality of lesions. It is also important to differentiate GOM from dense bodies and non-specific granular debris present in degenerated smooth muscle cells. The reason why the small-to-medium sized cerebral arteries are most severely affected and why there are no extra-cerebral clinical disease manifestations is still not fully understood. Further, the composition of GOM remains partly unclear. Previous immunoelectron microscopy studies reported the accumulation of the NOTCH3 ectodomain in proximity of GOM formation [85]. More recent investigation by post-embedding immunoelectron microscopy analysis using antibodies against the extra- and intracellular portions of NOTCH3, showed the NOTCH3 ectodomain to be a major component of GOM [86–88]. Immunohistochemistry analysis using a NOTCH3 monoclonal antibody was proposed as a further reliable tool for CADASIL diagnosis [89]. Although high specificity and sensitivity, ranging from 90% to 100%, were reported, this high level of accuracy has not been reported by any other group and the technique is not currently widely used in diagnosis [90].

In conclusion, preferably, skin biopsy analysis should include both NOTCH3 immunohistochemistry and electron microscopy. For electron microscopy analysis at least 4–6 arteriolar vessels for each patient should be examined. In most CADASIL centers, skin biopsy is only reserved in those cases with suspected CADASIL, but who did not have a NOTCH3 mutation or who had an unclassified variant in NOTCH3. Genetic testing is becoming an increasingly accessible diagnostic tool and, in patients with a clinical suspicion of CADASIL, NOTCH3 Sanger sequencing of EGFr encoding exons, if available, is the first choice for confirming the clinical diagnosis. This has the added advantage that testing of family members (both symptomatic and asymptomatic) is feasible once the mutation is known in the index patient.

There are few brain neuropathological analyses published in CADASIL, and these have mainly focused on cerebral vessel changes that are reported to be consistently narrowed with intimal thickening and degeneration of vascular smooth muscle cells and GOM deposition [91]. In 11 patients, Craggs et al. [92] reported disruption of either cortico-cortical or subcortico-cortical networks in the white matter of the frontal lobe, explaining motor deficits and executive dysfunction present in CADASIL.

NOTCH3 is the causative gene for CADASIL, which maps to the short arm of chromosome 19 (19p13.2-p13.1) [1, 93]. It is a large gene containing 33 exons [94]. The NOTCH3 gene codes for a single-pass transmembrane receptor protein, with a large extracellular domain, with 34 EGFRs (tandem epidermal growth factor-like repeat domains) and 3 LNR repeats, a transmembrane portion and an intracellular domain containing ankyrin-repeat motifs and a PEST domain [95]. NOTCH3 protein is expressed in vascular smooth muscle cells, whose degeneration results in progressively impaired cerebrovascular autoregulation, hypoperfusion, and ischemia [84]. More than 200 different NOTCH3 gene CADASIL-associated mutations have been reported, the majority of them consisting of stereotyped missense substitutions of a single base leading to loss or gain of a cysteine residue within one of the EGFRs of NOTCH3 extracellular domain [96]. Initially, most mutations were found to cluster in exons 3 and 4, but there are now many reports of mutations in other exons encoding the extracellular portion of the protein [2, 39, 97, 98]. Therefore, screening should include all EGFR encoding exons (exon 2–24). A few patients are reported with homozygous or compound heterozygous (Dotti unpublished data, [99]) NOTCH3 mutations; in these patients, the phenotype is almost always within the classical CADASIL spectrum [100–105].

Whether non-cysteine variants can cause CADASIL is debated. Small NOTCH3 deletions, duplications, splice site mutations, and deletion/insertions associated with CADASIL have been described. Most of these mutations lead to a numerical cysteine acid change [106]. In one study, a small intronic deletion leading to intron 3 retention was found to be associated with a GOM-positive CADASIL patient; the 25 additional amino acids in the EGFR likely disturb the disulphide bond formation [107]. Several studies have reported atypical, cysteine-sparing, NOTCH3 mutations associated with a familial CADASIL phenotype. These variants may be interpreted as causative mutations only if the complete screening of the EGFR-encoding exons excludes other classic mutations and GOM are present in skin biopsy [106, 108, 109]. However, one study suggested that GOM analysis might be less sensitive in patients with non-cysteine involving mutations [110].

Mutations causing loss of function (frame-shift deletion or nonsense mutations) are rarely reported in CADASIL patients, and their role is still debated [106, 111, 112]; they should be carefully analyzed to determine their pathogenicity. NOTCH3 mutations located outside of the EGFR encoding exons, reported in few cases, are not associated with a CADASIL phenotype [113–115].

A simple flow chart for CADASIL diagnosis is shown in Fig. 3.

Genetic counseling should be offered to all patients diagnosed with CADASIL and should always precede predictive genetic testing of family members. For predictive genetic testing, the Huntington’s disease guidelines can be used [116, 117]. It is important to note that a MRI scan in an unaffected family member can have a similar impact as a genetic test if it shows the characteristic MRI hallmarks of the disease. Genetic counselling also plays an important role in family planning and questions about options for prenatal diagnosis or pre-implantation genetic diagnosis may be discussed in countries where these techniques are available [118, 119].

In view of the evidence of a more severe disease course in individuals with vascular risk factors, particularly smoking and hypertension, control of vascular risk factors is an important part of CADASIL management. Concerning the use of antiplatelet drugs, such as aspirin or clopidogrel, most neurologists apply the guidelines used for sporadic stroke when treating CADASIL patients. However, the appropriateness of this approach is undetermined. In fact, the thrombotic genesis of ischemic events in this disease has not been proven so far. On the other hand, many reports have stressed the presence of microhemorrhages (microbleeds) in a considerable percentage of CADASIL patients [121]. For these reasons, the safety of antiplatelet drugs in this disease remain to be clarified [65]. Similarly, the benefit of thrombolysis is uncertain, although it has been suggested that it is of benefit in sporadic lacunar stroke [122].

Following the data reported by Keverne et al. [121] showing a cholinergic neuronal impairment in CADASIL, a multicenter trial in 168 patients with donepezil was performed; this showed no improvement in the primary outcome of the Vascular Dementia Assessment scale – cognitive subscale. Improvements were noted on several measures of executive function, but the clinical relevance of these findings is not clear [123]. Complications of CADASIL, such as depression and migraine, appear to respond to similar treatments to those used in sporadic disease, although further studies are required to determine the efficacy of these approaches in CADASIL patients.

A differential diagnosis in some cases may be hard. However, in CADASIL, in comparison to multiple sclerosis, temporo-polar MRI lesions are present in a high percentage, the optic nerve and spinal cord are generally not involved, and oligoclonal bands in the cerebrospinal fluid are absent. Corpus callosum hyperintensities are described both in CADASIL and in multiple sclerosis [124, 125]. In relation to cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CARASIL), in CADASIL, extraneurological findings (early-onset spondylosis and early-onset alopecia) are absent, clinical onset is generally later, MA is a typical sign, and inheritance is autosomal dominant [126]. Other hereditary leukoencephalopathies to take into account for a correct differential diagnosis are MELAS, Krabbe disease, Fabry disease, CARASAL, hereditary diffuse leukoencephalopathy with axonal spheroids, and other more rare monogenic forms of SVD [127].

CADASIL is the most frequent monogenic cerebral SVD, and has received a lot of attention in recent years as a model for SVD, with an increased number of cases. However, more work remains to be done in order to better explore the underlying mechanism of its pathogenesis. The studies on animal models (see elsewhere in this issue) will improve the understanding of the pathogenic mechanisms and may open new therapeutic strategies.