VIEWS AND REVIEWS OPEN ACCESS Cerebral autosomal …ng.neurology.org/content/nng/6/3/e434.full.pdfdefects, mainly in the attention and recall ability and a Frontal AssessmentBattery(6of18)thatrevealedaseriousdisorderin

VIEWS AND REVIEWS OPEN ACCESS

Cerebral autosomal dominant arteriopathy withsubcortical infarcts and leukoencephalopathyrevisitedGenotype-phenotype correlations of all published cases

Georgia Xiromerisiou MD PhD Chrysoula Marogianni MD MSc Katerina Dadouli MSc

Christina Zompola MD Despoina Georgouli MD MSc Antonios Provatas MD PhD

Aikaterini Theodorou MD Paschalis Zervas MD Christina Nikolaidou MD Stergios Stergiou MD

Panagiotis Ntellas MD Maria Sokratous MD MSc Pantelis Stathis MD PhD

Georgios P Paraskevas MD PhD Anastasios Bonakis MD PhD Konstantinos Voumvourakis MD PhD

Christos Hadjichristodoulou MD PhD Georgios M Hadjigeorgiou MD PhD and

Georgios Tsivgoulis MD PhD

Neurol Genet 20206e434 doi101212NXG0000000000000434

Correspondence

Dr Xiromerisiou

georgiaxiromerisiougmailcom

AbstractObjectiveThe aim of this study was to evaluate the correlation between the various NOTCH3mutationsand their clinical and genetic profile along with the presentation of a novel mutation ina patient

MethodsHere we describe the phenotype of a patient with cerebral autosomal dominant arteriopathywith subcortical infarcts and leukoencephalopathy (CADASIL) harboring a novel mutationWe also performed an extensive literature research forNOTCH3mutations published since theidentification of the gene and performed a systematic review of all published cases withNOTCH3 mutations We evaluated the mutation pathogenicity in a great number of patientswith detailed clinical and genetic evaluation and investigated the possible phenotype-genotypecorrelations

ResultsOur patient harbored a novel mutation in theNOTCH3 gene the c3084 G gt C correspondingto the aminoacidic substitution pTrp1028Cys presenting with seizures as the first neurologicmanifestation We managed to find a correlation between the pathogenicity of mutationsseverity of the phenotype and age at onset of CADASIL Significant differences were alsoidentified between men and women regarding the phenotype severity

ConclusionsThe collection and analysis of these scarce data published since the identification of NOTCH3qualitatively by means of a systematic review and quantitatively regarding genetic profile andpathogenicity scores highlight the significance of the ongoing trend of investigating phenotypicgenotypic correlations

From the Department of Neurology (GX CM DG AP MS GMH) University Hospital of Larissa Faculty of Medicine School of Health Sciences University of Thessaly LarissaGreece Second Department of Neurology (CZ AT PZ AB KV GT) ldquoAttikonrdquo University Hospital School of Medicine National and Kapodistrian University of Athens 12462Athens Greece Department of Neurology (GMH) Medical School University of Cyprus Nicosia Cyprus Department of Hygiene and Epidemiology (KD CH) Faculty of MedicineUniversity of Thessaly Larissa Greece Department of Medical Oncology (PN) University Hospital of Ioannina Ioannina Greece Department of Neurology (PS) MediterraneoHospital Glyfada Athens Greece Histopathological Department (CN SS) Hippokration General Hospital Thessaloniki and Department of Neurology (GPP) School of MedicineNational and Kapodistrian University of Athens Eginition Hospital Athens Greece

Go to NeurologyorgNG for full disclosures Funding informaton is provided at the end of the article

The Article Processing Charge was funded by the authors

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License 40 (CC BY-NC-ND) which permits downloadingand sharing the work provided it is properly cited The work cannot be changed in any way or used commercially without permission from the journal

Copyright copy 2020 The Author(s) Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology 1

Cerebral autosomal dominant arteriopathy with subcorticalinfarcts and leukoencephalopathy (CADASIL) is the mostcommon heritable cause of stroke in adults younger than 65years old1 Although the description of the first case was madearound 19552 the official characterization of the disorder wasdefined in 1993 after the discovery of the responsible geneNOTCH3 on chromosome 193 Although the clinical mani-festations of the disease are directly linked to the brain lesionsthere is a systemic arteriopathy that affects the skin the spleenthe liver the kidneys and the aorta apart from the brain4

CADASIL consists of 4 basic clinical characteristics which aremigraine with aura relapsing episodes of transient ischemicattacks (TIAs) and ischemic strokes psychiatric symptoms asapathy and severe mood swings and gradual cognitive im-pairment which eventually lead to severe dementia3

Another fundamental element of this disorder is the leu-koencephalopathy and the subcortical infracts identified onthe brain MRI especially in the external capsules and anteriorpole of temporal lobes5

Pathologic findings have confirmed the extensive changes inthe brain parenchyma compatible with chronic small arterydisease mainly affecting the white matter in the periventricularareas and the region of basal ganglia It is worthmentioning thatthe cortex which appeared unaffected in neuroimaging ina macroscopic examination displays extended neuronal apo-ptosis Furthermore in microscopic testing of the brain lesionsa specific arteriopathy has been revealed in which there isa thickening of the arterial wall of small penetrating cerebraland leptomeningeal arteries leading to lumen stenosis6 At thesame time with an electronic microscope in a specimen froma pathologic skin biopsy deposits of granular osmiophilic ma-terial (GOM) located in the basement membrane of smoothmuscle cells can be identified7 CADASIL is the only disorderwhose GOM has been identified However some reports onthe sensitivity of detecting GOM in skin biopsy of patients withgenetically proven CADASIL have been contradictory89

CADASIL is an autosomal dominant inherited arteriopathycaused by mutations in the NOTCH3 The NOTCH3 geneencodes a single pass transmembrane protein with receptorproperties This receptor is mainly expressed in the smoothmuscle cells of blood vessels and pericytes10 After ligandbinding the intracellular part translocates to the nucleus andactivates transcription factors NOTCH3 has 33 exons but allthe mutations found are located in exons 2ndash24 which areresponsible for encoding 34 epidermal growth factor repeats

EGFR Most of these mutations are missense mutations al-though there are only a few in-frame deletions or splice-sitemutations11 The gene mutation analysis of NOTCH3 is thegold standard to diagnose this genetically inherited diseaseand there are more than 230 different mutations located in 20different exons reported in patients with CADASIL12

In this review we report a patient with CADASIL with a novelheterozygousNOTCH3mutation and epileptic seizures as thevery first manifestation of the disorder In silico analysisrevealed the pathogenicity of the mutation However weproceeded to the skin biopsy to detect deposits of GOM

In addition we performed a systematic review of all publishedcases with NOTCH3 mutations We evaluated the mutationpathogenicity in all previously reported cases to investigatepossible phenotype-genotype correlations

MethodsCase description clinical findingsA 62-year-old woman visited the outpatient stroke clinic of a ter-tiary care stroke center inAthensGreece The patient experiencedepisodes of loss of consciousness some of them accompanied bytonic contraction of the upper arms and bite of her tongue for thepast 40 years She has been receiving an antiepileptic drug (leve-tiracetam 1250 mg daily) for the past 2 years because of repeatepisodes of tonic-clonic seizures She had also experienced anischemic lacunar stroke in the distribution of the left middle ce-rebral artery 4 months before her visit to the outpatient clinic ofour department that resulted in right hemiparesis The patientrsquoshusband and children reported changes in her personality andcognitive decline gradually worsening over the past 5 years Herfamily history revealed a brother with epilepsy and a father withposttraumatic epilepsy who died at the age of 60 years old

The patient had no history of hypertension or diabetes or otherknown cardiovascular risk factors She was receiving escitalo-pram (20 mg) for depression levetiracetam (1250 mg) forseizures clopidogrel (75 mg) and atorvastatin (20 mg) forsecondary stroke prevention

The patient was admitted in our department for further di-agnostic workupWe repeated brainMRI that revealed extensivebilateral white matter lesions located mainly in the frontal lobesthe anterior temporal lobe the external capsules the centrumsemiovale and the basal ganglia (figure 1 AndashC) There was noevidence of acute infarction Furthermore multiple cerebralmicrobleeds (CMBs) located predominantly in the thalami were

GlossaryCADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathyCADD = combinedannotation-dependent depletion CI = confidence interval CMB = cerebral microbleed ECD = extracellular domain EGFr =epidermal growth factor-like repeat GOM = granular osmiophilic material TIA = transient ischemic attack

2 Neurology Genetics | Volume 6 Number 3 | June 2020 NeurologyorgNG

documented on susceptibility-weighted imaging The patientunderwent an extensive workup for stroke (including trans-esophageal echocardiography neck and brain MR angiographyfull coagulation disorder panel repeat 24-hour holter ECGrecordings molecular genetic screening for Fabry disease im-munologic screening for autoimmune disorders) that was un-remarkable We also found no evidence for demyelinating CNSdisorder on CSF analysis (normal results with absent oligoclonalbands) and cervical as well as thoracic spinal cord MRI

Bedside neuropsychologic examination showed moderatecognitive decline (Mini-Mental State Examination 2030) withdefects mainly in the attention and recall ability and a FrontalAssessment Battery (6 of 18) that revealed a serious disorder inconceptualization and mental flexibility

The patientrsquos daughter complained of chronic tension-typeheadaches and was further investigated with brain MRI thatrevealed multiple hypertense subcortical white matter lesions(figure 1 DndashF) The patientrsquos son was asymptomatic and re-fused to undergo brain MRI despite our suggestions

Case description genetic analysisGiven the aforementioned extended workup the relevant clinicalcourse the symptoms and the compatible neuroimaging thesuspicion of CADASIL was raised so we proceeded toNOTCH3genetic analysis Based on the mode of inheritance the age atonset and the phenotype we suspected a genetic disorder Allstudy participants provided written informed consent for furthergenetic analysis Our study was approved by the Ethics Com-mittee of theUniversityHospital of Attikon GenomicDNAof all

available family members was extracted from the peripheral whiteblood cells according to the standard protocols

The NOTCH3 analysis led to the identification of a heterozy-gous mutation in the exon 19 NOTCH3 gene c3084 G gt Ccorresponding with the amino acid substitution pTrp1028CysThis is a novel mutation compatible with CADASIL syn-drome leading to the gain of a cysteine residue in one of the 34epidermal growth factor-like repeat (EGFr) domains of theprotein encoded by NOTCH3 (1312) This results in an un-even number of cysteine residues in the given EGFr domainmost likely modifying the tertiary structure of the protein

According to the American College of Medical Genetics andGenomics and the Association for Molecular Pathology 2015guidelines the pathogenicity potential of the pTrp1028Cysvariant is ldquopathogenicrdquo based on the following criteria (1) nullvariant (PVS1)mdasha missense mutation leading to a gain ofa cysteine residue (2) the absence of the variant from the con-trols in the Exome Sequencing Project 1000 Genomes Projector Exome Aggregation Consortium (PM2) (3) in silico bio-informatics tools (Homologene GEPR Varsome) predictedthat the variant causes a deleterious effect on the gene (PP3)because it occurs in a highly conserved area across multiplespecies accordingly (ncbinlmnihgovhomologene) and (4)the patientrsquos phenotype is highly specific for the disease (PP4)

Case description histopathologic stainingFor immunohistochemistry we used 10-lm tissue specimensEndogenous peroxidase activity was eliminated by treatmentwith 05 periodic acid solution for 10 minutes The sections

Figure 1 Brain MRI scan (AndashC) of the patient showing extensive leukoencephalopathy mainly in the frontal lobes theanterior temporal lobe the external capsules and the basal ganglia in the FLAIR sequences (DndashF) of the daughtershowing multiple hyperintensities in the FLAIR sequences

NeurologyorgNG Neurology Genetics | Volume 6 Number 3 | June 2020 3

were incubated in a blocking buffer (1 bovine serum albu-min and 5 rabbit serum in phosphate-buffered saline) Amonoclonal antibody against the extracellular domain ofNOTCH3 diluted 1100 in blocking buffer served as theprimary antibody The sections were counterstained withVictoria blue To observe the internal elastic lamina of vas-cular walls we used Victoria bluendashhematoxylin and eosinstaining No specific immunoreactive signal was detected inthe vessels therefore the specimen was scored as negative

Review of published CADASIL data

Systematic searchWe conducted a systematic review of the literature in-vestigating all known mutations of NOTCH3 and their phe-notypic characteristics We performed searches on PubMedCochrane Library and MEDLINE (via PubMed) to identifyall published studies before August 2019 A combination ofthe termsldquoCADASILrdquo ldquoCerebral autosomal dominant arterio-pathy with subcortical infarcts and leukoencephalopathyrdquoldquoNOTCH3rdquo and ldquoNOTCH3 MUTATIONSrdquo were used Allstudies presenting original data that reported the clinicalgenetic and radiologic characteristics of patients withCADASIL were included for further review English languagewas the only filter used initially References of selected articlesand reviews were also searched for additional records

Data extracted from each study were title author year ofpublication the exon the mutation and the exact amino acidchange the total cases screened carrying the mutant allele andsociodemographic characteristics (origin age and sex) age atonset family history clinical features of the disease (migrainestroke psychiatric disorders cognitive decline acute en-cephalopathy and atypical findings) and findings from di-agnostic procedures (MRI findings and skin biopsy)

Data collection and eligibility criteriaStudies were selected when they met the following criteria (1)diagnosis of CADASIL was confirmed by a mutation analysis(2) clinical findings of CADASIL were described (3) theidentified mutation was described (4) when the same patientpopulation was presented in more than one publication theone with the largest sample size or most recent publication datewas used as the primary study for data extraction (5) languagesbeing limited to English and (6) Full study

Evaluation of mutationsFor the evaluation of the pathogenicity of the published muta-tions computational prediction analysis was used A combinedannotation-dependent depletion (CADD) algorithm was usedfor scoring the deleteriousness of single nucleotide variants andinsertiondeletions on the function of NOTCH3 Prediction isbased on empirical rules applied to the sequence phylogeneticand structural information characterizing the amino acid sub-stitution1314 A qualitative characterization of the mutations wasbased on the score the CADD score is above gt30 was highlypathogenic above gt20 was pathogenic between 15 and 20 waslikely pathogenic below lt15 was likely benign and finally belowlt10 was benign The Human Splicing Finder tool37 was used toevaluate mutations that potentially affect splicing15

Statistical analysisWeused descriptive statistics to present demographic clinical andother characteristics of the patients overall Data were checked fordeviation from normal distribution (Shapiro-Wilk normality test)Categorical data were analyzed with the use of a χ2 test Student ttest Mann-Whitney U test analysis of variance Kruskal-Wallistest and were performed for continuous data as appropriateSpearman correlation analyses were used to estimate the corre-lations between quantitative variables A multivariate analysis wasperformed in the form of multiple linear regression and

Figure 2 Flow chart depicting our literature search and study selection for the systematic review


multinomial regression and multinomial (polytomous) logisticregression The cutoff value of age at onset was calculated usingthe receiver operating characteristic curve For all the analysesa 5 significance level was set The Bonferroni method was usedto correct the significance level where it was necessary1617 Theanalysis was carried out with SPSS version 250

ResultsLiterature reviewWe screened a total of 695 articles yielded by our literaturesearch we excluded 476 that evaluated animal models lackedclinical and genetic data and that were duplicates After full-text retrieval and review we included the 224 remainingarticles because they met the predefined eligibility criteria andwe proceeded in the qualitative analysis (Supplementary 1linkslwwcomNXGA262) The selected studies consistedof noncontrolled case series and case reports (figure 2)

Demographics clinical phenotypeA total of 752 patients (50 men) were included in our sys-tematic review Most of the patients were Caucasians of Euro-pean origin (45) and Asians followed with 43 Only a fewpatients were included originating from North and SouthAmerica Africa and AustraliaNew Zealand Most case reportsand small case series report patients originated from Japan

China and Italy The frequencies of CADASILmutations acrossvarious countries according to our reviewed cases are presentedin a world map shown in figure 3 However the largest caseseries with CADASIL that were excluded from our analysisbecause of the lack of detailed clinical and genetic informationoriginated from the Netherlands Germany UK and Japan

Detailed clinical informationwas available for 752 patients Theoverall mean age of the reported cases was 52 plusmn 14 yearswhereas the mean age at onset was 43 plusmn 14 years Apprimately64 of the patients that have been included in the study hada positive family history 10 had no family history and noinformation was available for the remaining 25

Migraine was reported in 23 of patients Most of all reportedcases presented with stroke (52) followed by cognitive de-cline (46) while psychiatric disorders had a prevalence of24 Psychiatric disorders included predominantly depressivesymptoms apart from a minority of cases with bipolar disorder(2 cases) and psychosis (1 case) Epileptic disorders werereported in 4 of the patients

There are certain clinical characteristics mentioned in the in-cluded studies which are atypical or in some cases could beexplained from the recurrent stroke episodes that the patientssuffered For example we have found sensory deficits of the upperarms and findings of polyneuropathy in 10 cases Furthermore

Figure 3 A world map showing the frequencies of CADASIL mutations across various countries according to our reviewedcases


several cases (approximately 7) with intracerebral hemorrhagehave been described An interesting fact is the presence ofmovement disorders such as ataxia and parkinsonism in ap-proximately 4 of our included patients Symptoms such asdizziness vertigo gait disturbance and dysarthria could probablybe consequences of the recurrent strokes At some point in thecourse of their disease approximately 2 of patients presenteda confusional state andor encephalopathy (CADASIL coma)after severe headache that lasted several days1819

We divided the patients according to the presentation of oneor a combination of 2 or all of the above major clinicalcharacteristics [phenotype 1 = patients with migraine phe-notype 2 = patients with migraine and stroke or stroke onlyand phenotype 3 = (1) patients with migraine and stroke andcognitive disorder andor psychiatric disorder and (2)patients with stroke and cognitive decline andor psychiatricdisorder] We noticed that most patients 34 presented withphenotype 3 followed by phenotype 2 (32) Phenotype 1was documented in 7 of the patients included in this sys-tematic review

Neuroimaging and skin biopsyThe predominant radiologic manifestations of CADASIL onthe brain MRI include hyperintense located in the whitematter of the anterior temporal poles centrum semiovaleexternal capsule basal ganglia and pons Most of the patientshad radiologic signs of leukoencephalopathy in their brainMRI scans (72) where the precise distribution of the whitematter lesions was not mentioned in most of them We did

not find any information in 25 of the patients regarding thepresence of white matter lesions Furthermore CMBs havebeen reported in 7 of cases and were absent in 28 of thebrain MRI scans However in most cases (65) the presenceof microbleeds was not reported

Skin biopsy data a sensitive diagnostic tool in the diagnosis ofCADASIL were not available for more than half of thereported cases Approximately 39 of patients had beentested with a skin biopsy Approximately 14 of the patientshad compatible findings with the diagnosis of CADASILwhereas 24 were tested negative for the characteristicgranular deposits in the basal lamina of blood vessels In moststudies GOM was confirmed with electron microscopywhereas in the rest of them with immunohistochemistry

MutationsMost mutations described were located in the exon 4 (29)followed by exon 3 (14) exon 11 (8) and exon 19 (6)(figure 4) Most of the mutations are predicted to result ina gain or loss of at least one of a cysteine residue 14 of themutations described did not involve a cysteine residue Mostmutations were missense mutations (74) whereas a fewsplicing mutations and deletions were also reported In total85 of these mutations were pathogenic and 12 were foundto be highly pathogenic whereas approximately 1 corre-sponded to benign mutations Regarding quantitative patho-genicity scores the mean score was 26 (max = 44 min = 06SD = 4) Themost commonmutation was pR169C in exon 4followed by pR182C in the same exon

Figure 4 The distribution of the most common mutations across the NOTCH3 gene


Genotype-phenotype correlationInitially we performed several univariate analyses concerningthe associations of all clinical characteristics and phenotypeswith genotypic characteristics and pathogenicity scores Wefound that the age at onset of CADASIL was significantly as-sociated with the pathogenicity score of the mutation (rho =minus0165 p lt 0001) Highly pathogenic and pathogenic muta-tions appear to induce an earlier age at onset compared withlikely pathogenic benign and likely benign variants The higherthe pathogenicity score the earlier the onset of the disease(figure 5)

Multivariable analyses taking into account several clinical andgenetic factors further indicate that the age of disease onsetwas independently (B = minus04 95 confidence interval [CI] =minus07 to 01 p lt 0004) associated with pathogenicity score(table e-1 linkslwwcomNXGA263)

Regarding phenotype severity women appear to have a differ-ent phenotype compared withmen (p = 0003) as presented infigure 6 Migraine is more common by 44 in women thanmen while women usually present a less severe phenotype

Phenotype severity is also highly correlated with age (p lt0001) (table e-2 linkslwwcomNXGA264) Migraine isprevalent at the age of 40 years whereas a more severe phe-notype that contains stroke or stroke and migraines is moreprevalent in older patients (50ndash60 years) Cognitive decline orother psychiatric disorders manifest at the same age range(50ndash60 years) as stroke (figure 7) This indicates a cumulativeeffect of neurologic deficits with the progression of the disor-der More specifically the mean age at onset in patients pre-senting with migraine stroke and cognitive decline or otherpsychiatric disorders is 40 52 and 55 years respectively

Pathogenicity score appears to be solely affected by the lo-cation of the mutation (p lt 0001) and is not associated withphenotype severity (figure 8) All 3 phenotypes describedharbor the same pathogenic mutations in qualitative andquantitative analysis

The clinical manifestations and phenotype severity did notvary significantly between patients harboring cysteine andcysteine-sparing mutations However the age at onset wassignificantly earlier (mean difference = 78 years 95 CI = 22135) in patients with cysteine-sparing mutations (p = 0009)The mean age at onset in patients with cysteine and cysteine-sparing mutations was 51 and 43 years old respectively

Regarding the clinical profile and the phenotypic characteristicsamong Asian and Caucasian patients there are certain signifi-cant differences In particular phenotypes 1 (patients with mi-graine) and 2 (patients with migraine and stroke or stroke only)of CADASIL were more prevalent in Caucasians than in Asians(figure 3) Regarding distribution of mutations there are severaldifferences between Asians and Caucasians because cysteinemutations are more prevalent in Asian populations (figure 4)

DiscussionThe present review summarizes the clinical and neuroimagingfindings of a novel mutation in exon 19 of the NOTCH3 genethe c3084 G gt C corresponding to the aminoacidic sub-stitution pTrp1028Cys We have also documented that type

Figure 5 Univariate analysis showing the effect of patho-genicity score of the mutations on the age at on-set of the disorder

Figure 6 Bar chart showing the differences between menand women regarding phenotype severity

Phenotype severity-sex p value = 0003


and pathogenicity of themutation are independently associatedwith the age of disease onset Finally phenotypic and genotypiccharacteristics differ significantly between Asians andCaucasians

Most mutations in the NOTCH3 gene are located within thelarge extracellular domain (ECD) of the transmembrane re-ceptor This domain consists of 34 epidermal growth factor(EGF)-like repeats that contain cysteines The pathogenicmutations are associated with changes in the number of cys-teines leading to a misfolding of the receptor This misfoldingcontributes significantly to the formation of oligomers andECD aggregation which is considered to be the pathogenicmechanism of the disease2021

A few mutations have been found that do not affect the numberof cysteines The interesting fact is that these mutations result inan uneven number of cysteine residues The pathogenic role ofthese mutations is controversial22 Several of these atypicalmutations appear to be associated with conformational changesin the protein similar to the changes observed for typical cysteineinvolving mutations However these mutations seem to causea similar phenotype without any significant differences regardingthe age at onset or phenotype severity21 Our findings clearlydemonstrate this concentration ofmutations in the ECDdomainespecially in exon 4 and 323 Furthermore they highlight thesignificant association of the location of the mutations withpathogenicity score and phenotype severity We have not foundany differences in the age at onset and the clinical phenotypeoverall regarding the cysteine and cysteine-sparing mutationsThese results are consistent with most previous studies in Cau-casian or Asian populations However there are small regionalstudies in Taiwan and Korea that presented different resultsindicating that the most commonmutations were in exon 11 and18 These differences may simply reflect a founder effect2425

Our study investigated several possible associations betweenthe genetic profile and clinical manifestations in all previouslypublished cases within the parameters stated earlier Severalstudies have thoroughly described the phenotypic manifes-tations and the natural history of the disorder evaluating thefrequency and age distribution of several characteristics26

There are also other studies with a limited number ofpatients investigating possible phenotypic-genotypic corre-lation by comparing patients carrying mutations involvingcysteine residues with patients with cysteine-sparing muta-tions without showing any significant differences27

It has also been found that genetic variants located in theEGFr domain 7ndash34 are identified in the general populationwhereas in patients with CADASIL the variants are pre-dominately located in the EGFR 1 This must be furtherinvestigated with wider population studies to elucidate therole of many variants in small vessel disease phenotype

Skin biopsy is considered an important diagnostic tool forCADASIL diagnosis highly specific but with variable sensitivity

Many technical factors related to the actual biopsy or the severityof the disordermay be themain reasons for the discrepancy acrossstudies regarding the sensitivity of the method According to ourstudy it seems that the implementation of this tool is not widelyrecognized A small percentage of studies reported electron mi-croscopy studies on skin biopsies or immunohistochemistryanalysis using a NOTCH3 monoclonal antibody28 The studiesthat did not show the typical depositions presented similar phe-notype with themost of all the other described cases and involvedpatients with typical and cysteine sparing mutations as well Inmost centers investigating and examining patients with CADASILphenotype it seems that skin biopsy is reserved for patients withunclassified variants Genetic testing is the core diagnostic tool forthe disorder Our described case failed to manifest these charac-teristic findings with immunochemistry Electron microscopystudies are more sensitive than immunochemistry29 Technicalreasons the location of the biopsy and the severity of the disordercould be possible explanations for the lack of typical findings30

Furthermore we noticed in our study that leukoencephalopathyin MRI scans is reported in most patients with geneticallyconfirmed CADASIL and CMBs in a minority of them How-ever a detailed description of MRI findings is not universallyfollowed and so certain clinicoradiological associations cannotbe drawn There are several other radiologic focused studiesshowing that the MRI lesion load and pattern can vary quitesignificantly across patients A consistent finding is the presenceof symmetrical white matter hyperintensities on T2-weightedand fuid-attenuated inversion recovery images31 Anteriortemporal pole changes have been associated with high sensi-tivity and specificity for the disorder External capsule changesalso have a high sensitivity but low specificity The occurrence ofCMBs is disproportionate across published cases32 Dilatedperivascular spaces and subcortical and cortical atrophy havealso been detected33 The only significant finding that was

Figure 7 Box plot depicting the distribution of phenotypesof CADASIL across age

Phenotype severity-age p lt 0001


highlighted during our study with the limited data available wasthe fact thatMRICMBs seem to be an age-related phenomenonassociated with the progression of the disorder

Regarding clinical presentation migraine is often the earliestfeature of the disease According to the previous studies itseems that migraine is the first clinical symptom in 41 ofsymptomatic patients and an isolated symptom in 123435

Migraine is also reported in approximately 55ndash75 of Cauca-sian cases although it is less frequent in Asian populationsTIAs and stroke are reported in approximately 85 of symp-tomatic individuals35 Several studies have shown that the totallacunar lesion load is strongly associated with the developmentof disability4 Cognitive impairment in CADASIL involves in-formation processing speed and executive functions mostly Itis also often associated with apathy and depression Our studypresents findings consistent with previous research Howeverwe managed to investigate the effect of several factors onphenotype severity according to the presence of several clinicalsymptoms At some point the results clearly indicated thatphenotype severity is an age-related phenomenon so the dis-order seems progressive with the accumulation of new lacunarinfarcts that affect cognition Pathogenicity score does notsignificantly affect the combination of symptoms that a patientpresents but it appears to be related to the age at onset Wecannot make assumptions based on the pathogenicity of themutation for the severity of the disorder so genetic adviceshould be limited to the presence of the disorder and not to theappearance of specific phenotypic characteristics Another im-portant clinical conclusion that we drew was the fact thatphenotype severity is also affected by sex withmenmanifestingmore severe symptoms36

Regarding cognitive decline we should also mention thatcognitive performance was either not measured with the sameassessments across all studies or it was not performed at all37

Therefore the presence of impairment is possibly under-estimated Longitudinal studies are also needed to investigatechanges related to disease progression and possible associationwith MRI changes This will shed light to the pathogenicprocesses underlying these symptoms38

Other atypical clinical characteristics have also been reported inpatients with CADASIL39 A very important manifestationseems to be acute encephalopathy or coma40 This is a rathermisleading manifestation that has been reproduced several timesin the literature but is extremely rare Acute encephalopathy hasbeen reported in 2 cases as the initial manifestation of the dis-order in our reviewed cases However an acute encephalopathicpresentation of the disease was previously described in 10 ofpatients who were part of a the British CADASIL prevalencestudy41 All these patients had a history of migraine The severesymptoms that they presented were episodes of migraine orepileptic seizures that lasted longer than usual resulting inconfusion and disorientation and were self-limited

Several small cohort studies have previously described specificphenotypic and genotypic characteristics of patients withCADASIL in detail (Supplementary data linkslwwcomNXGA262) They have also focused on specific findingssuch as ophthalmologic manifestations cognitive profile andMRI features or the effect of other factors such as cardio-vascular risk factors on phenotype4243 Some of these studiesinclude a large number of patients gt200 However the clinicaland genetic information for every patient separately is not

Figure 8 Bar chart showing the distribution of mutations across the exons of the gene according to their pathogenicityscore

Εxon-pathogenicity score p valuelt 0001


publicly available This fact prevented us from including thesepatients in our analysis and posed a bias concerning the fre-quency of mutations across several countries and more de-tailed genotypic phenotypic correlations

However we strongly believe that the collection and analysis of allthese scarce data published since the identification of NOTCH3qualitatively by means of a systematic review and quantitatively interms of genetic profile and pathogenicity scores highlight thesignificance of the ongoing trend of investigating phenotypic ge-notypic correlations

Study fundingNo targeted funding

DisclosureAll authors declare that they have no conflicted interests Goto NeurologyorgNG for full disclosures

Publication historyReceived by Neurology Genetics January 7 2020 Accepted in final formApril 2 2020

References1 Tournier-Lasserve E Joutel A Melki J et al Cerebral autosomal dominant arterio-

pathy with subcortical infarcts and leukoencephalopathy maps to chromosome19q12 Nat Genet 19933256ndash259

2 Van Bogaert L Encephalopathie sous corticale progressive (Binswanger) aevolutionrapide chez des soeurs Med Hellen 195524961ndash972

3 Federico A Bianchi S Dotti MT The spectrum of mutations for CADASIL diagnosisNeurol Sci 200526117ndash124

4 Tan RYY Markus HS CADASIL migraine encephalopathy stroke and their interrelationships PLoS One 201611e0157613

5 OrsquoSullivan M Jarosz JM Martin RJ et al MRI hyperintensities of the temporal lobeand external capsule in patients with CADASIL Neurology 200156628ndash634

6 Craggs L Yamamoto Y IharaM et al White matter pathology and disconnection in thefrontal lobe in cerebral autosomal dominant arteriopathy with subcortical infarcts andleukoencephalopathy (CADASIL) Neuropathol Appl Neurobiol 201440591ndash602

7 Goebel HH Meyermann R Rosin R Schlote W Characteristic morphologic mani-festation of CADASIL cerebral autosomal-dominant arteriopathy with subcorticalinfarcts and leukoencephalopathy in skeletal muscle and skin Muscle Nerve 199720625ndash627

8 Markus HS Martin RJ Simpson MA et al Diagnostic strategies in CADASILNeurology 2002591134ndash1138

9 Tikka S Mykkanen K Ruchoux M-M et al Congruence between NOTCH3 muta-tions and GOM in 131 CADASIL patients Brain 2009132933ndash939

10 Joutel A Corpechot C Ducros A et al Notch3 mutations in CADASIL a hereditaryadult-onset condition causing stroke and dementia Nature 1996383707ndash710

11 Monet-Lepretre M Bardot B Lemaire B et al Distinct phenotypic and functionalfeatures of CADASIL mutations in the Notch3 ligand binding domain Brain A JNeurol 2009132(pt 6)1601ndash1612

12 Muintildeo E Gallego-Fabrega C Cullell N et al Systematic review of cysteine-sparingNOTCH3 missense mutations in patients with clinical suspicion of CADASIL Int JMol Sci 2017181964

13 Hack R Rutten J Lesnik Oberstein SA (1993) CADASIL 2000 Mar 15 [Updated2019 Mar 14] In AdamMP Ardinger HH Pagon RA et al (editors) GeneReviewsreg[Internet] Seattle (WA) University of Washington Seattle 1993-2020 Availablefrom httpswwwncbinlmnihgovbooksNBK1500

Appendix Authors

Name Location Contribution

GeorgiaXiromerisiou MDPhD

University ofThessaly LarissaGreece

Design andconceptualized the studyanalyzed the data draftedthe manuscript forintellectual content

ChrysoulaMarogianni MDMSc

University Hospitalof Larissa Greece

literature search

Katerina DadouliMSc


Statistical analysis

Christina ZompolaMD

ldquoAttikonrdquo UniversityHospital AthensGreece

Case description

Georgouli DespoinaMD MSc


literature search

Antonios ProvatasMD PhD


Data extraction

AikateriniTheodorou MD


Case description

Paschalis ZervasMD


Case description

ChristinaNikolaidou MD

HippokrationGeneral HospitalThessalonikiGreece

Histopathologicexamination

Stergios StergiouMD



Appendix (continued)


Panagiotis NtellasMD

University Hospitalof IoanninaIoannina Greece


Maria SokratousMD MSc


Data extraction

Pantelis Stathis MDPhD

MediterraneoHospital AthensGreece

Revised the manuscriptfor intellectual content

Georgios PParaskevas MDPhD

National andKapodistrianUniversity ofAthens Greece


Anastasios BonakisMDPhD



KonstantinosVoumvourakis MDPhD



ChristosHadjichristodoulouMD PhD



Georgios MHadjigeorgiou MDPhD

University ofCyprus NicosiaCyprus

Interpreted the datarevised the manuscriptfor intellectual content

Georgios TsivgoulisMD PhD


Interpreted the datarevised the manuscriptfor intellectual contentcase description


14 Rentzsch P Witten D Cooper GM Shendure J Kircher M CADD Predicting thedeleteriousness of variants throughout the human genome Nucleic Acids Research201947D886ndashD894

15 Human Splicing FindermdashVersion 31 (nd) Available at umdbeHSF AccessedOctober 13 2019

16 Berkeleyrsquos stats website Statistics for Bioinformatics Available at statberkeleyeduusersmgoldman Accessed September 2019

17 Benjamini Y Hochberg Y Controlling the false discovery rate a practical and pow-erful approach to multiple testing J R Stat Soc Ser B (Methodological) 199557289ndash300

18 Eswaradass VP Ramasamy B Kalidoss R Gnanagurusamy G Cadasil coma unusualcause for acute encephalopathy Ann Indian Acad Neurol 201518483ndash484

19 Ragno M Pianese L Morroni M et al ldquoCADASIL comardquo in an Italian homozygousCADASIL patient comparison with clinical and MRI findings in age-matched het-erozygous patients with the same G528C NOTCH3 mutation Neurol Sci 2013341947ndash1953

20 Lackovic V Bajcetic M LackovicM et al Skin and sural nerve biopsies ultrastructuralfindings in the first genetically confirmed cases of CADASIL in Serbia UltrastructuralPathol 201236325ndash335

21 Santa Y Uyama E Chui DH et al Genetic clinical and pathological studies ofCADASIL in Japan a partial contribution of Notch3 mutations and implications ofsmooth muscle cell degeneration for the pathogenesis J Neurol Sci 200321279ndash84

22 Wollenweber FA Hanecker P Bayer-Karpinska A et al Cysteine-sparing CADASILmutations in NOTCH3 show proaggregatory properties in vitro Stroke 201546786ndash792

23 Matsushima T Conedera S Tanaka R et al Genotypendashphenotype correlations ofcysteine replacement in CADASIL Neurobiol Aging 201750169e7ndash169e14

24 Ueda A Ueda M Nagatoshi A et al Genotypic and phenotypic spectrum ofCADASIL in Japan the experience at a referral center in Kumamoto University from1997 to 2014 J Neurol 20152621828ndash1836

25 Lee YC Liu CS Chang MH et al Population-specific spectrum of NOTCH3mutations MRI features and founder effect of CADASIL in Chinese J Neurol 2009256249ndash255

26 Rutten JW Dauwerse HG Gravesteijn G et al Archetypal NOTCH3 mutationsfrequent in public exome implications for CADASIL Ann Clin Translational Neurol20163844ndash853

27 Liu X Zuo Y Sun W et al The genetic spectrum and the evaluation of CADASILscreening scale in Chinese patients with NOTCH3 mutations J Neurol Sci 201535463ndash69

28 DichgansMMayer M Uttner I et al The phenotypic spectrum of CADASIL clinicalfindings in 102 cases Ann Neurol 199844731ndash739

29 Gustavsen WR Reinholt FP Schlosser A Skin biopsy findings and results of neu-ropsychological testing in the first confirmed cases of CADASIL in Norway Eur JNeurol 200613359ndash362

30 Morroni M Marzioni D Ragno M et al Role of electron microscopy in the diagnosisof cadasil syndrome a study of 32 patients PLoS One 20138e65482

31 Dziewulska D Sulejczak D Wezyk M What factors determine phenotype of cerebralautosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy(CADASIL) Considerations in the context of a novel pathogenic R110Cmutation inthe NOTCH3 gene Folia Neuropathologica 201755295ndash300

32 Shi Y Li S Li W et al MRI lesion load of cerebral small vessel disease and cognitiveimpairment in patients with CADASIL Front Neurol 20189862

33 Puy L De Guio F Godin O et al Cerebral microbleeds and the risk of incidentischemic stroke in CADASIL (cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy) Stroke 2017482699ndash2703

34 Wang Z Yuan Y Zhang W et al NOTCH3 mutations and clinical features in 33mainland Chinese families with CADASIL J Neurol Neurosurg Psychiatry 201182534ndash539

35 Guey S Mawet J Herve D et al Prevalence and characteristics of migraine inCADASIL Cephalalgia 2016361038ndash1047

36 Burkett JG Dougherty C Recognizing CADASIL a secondary cause of migraine withaura Curr Pain Headache Rep 20172121

37 Gunda B Herve D Godin O et al Effects of gender on the phenotype of CADASILStroke 201243137ndash141

38 Yoon CW Kim YE Seo SW et al NOTCH3 variants in patients with subcorticalvascular cognitive impairment a comparison with typical CADASIL patients Neu-robiol Aging 2015362443ndash2447

39 Brookes RL Hollocks MJ Tan RYMorris RGMarkus HS Brief screening of vascularcognitive impairment in patients with cerebral autosomal-dominant arteriopathy withsubcortical infarcts and leukoencephalopathy without dementia Stroke 2016472482ndash2487

40 Spinicci G Conti M Cherchi MV Mancosu C Murru R Carboni N Unusual clinicalpresentations in subjects carrying novel NOTCH3 gene mutations J Stroke Cere-brovasc Dis 201322539ndash544

41 Feuerhake F Volk B Ostertag C et al Reversible coma with raised intracranialpressure an unusual clinical manifestation of CADASIL Acta Neuropathologica2002103188ndash192

42 Schon F Martin RJ Prevett M Clough C Enevoldson TP Markus HS ldquoCADASILcomardquo an underdiagnosed acute encephalopathy J Neurol Neurosurg Psychiatry200374249ndash252

43 Cumurciuc R Massin P Paques M et al Retinal abnormalities in CADASIL a ret-rospective study of 18 patients J Neurol Neurosurg Psychiatry 2004751058ndash1060


DOI 101212NXG000000000000043420206 Neurol Genet

Georgia Xiromerisiou Chrysoula Marogianni Katerina Dadouli et al leukoencephalopathy revisited Genotype-phenotype correlations of all published cases

Cerebral autosomal dominant arteriopathy with subcortical infarcts and

This information is current as of May 11 2020

reserved Online ISSN 2376-7839Published by Wolters Kluwer Health Inc on behalf of the American Academy of Neurology All rightsan open-access online-only continuous publication journal Copyright Copyright copy 2020 The Author(s)

is an official journal of the American Academy of Neurology Published since April 2015 it isNeurol Genet

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httpngneurologyorgcontent63e434fullhtmlincluding high resolution figures can be found at

References httpngneurologyorgcontent63e434fullhtmlref-list-1

This article cites 40 articles 7 of which you can access for free at

Subspecialty Collections

httpngneurologyorgcgicollectionmriMRI

httpngneurologyorgcgicollectionmigraineMigraine

httpngneurologyorgcgicollectionall_geneticsAll Genetics

okehttpngneurologyorgcgicollectionall_cerebrovascular_disease_strAll Cerebrovascular diseaseStrokefollowing collection(s) This article along with others on similar topics appears in the

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Cerebral autosomal dominant arteriopathy with subcorticalinfarcts and leukoencephalopathy (CADASIL) is the mostcommon heritable cause of stroke in adults younger than 65years old1 Although the description of the first case was madearound 19552 the official characterization of the disorder wasdefined in 1993 after the discovery of the responsible geneNOTCH3 on chromosome 193 Although the clinical mani-festations of the disease are directly linked to the brain lesionsthere is a systemic arteriopathy that affects the skin the spleenthe liver the kidneys and the aorta apart from the brain4

CADASIL consists of 4 basic clinical characteristics which aremigraine with aura relapsing episodes of transient ischemicattacks (TIAs) and ischemic strokes psychiatric symptoms asapathy and severe mood swings and gradual cognitive im-pairment which eventually lead to severe dementia3

Another fundamental element of this disorder is the leu-koencephalopathy and the subcortical infracts identified onthe brain MRI especially in the external capsules and anteriorpole of temporal lobes5

Pathologic findings have confirmed the extensive changes inthe brain parenchyma compatible with chronic small arterydisease mainly affecting the white matter in the periventricularareas and the region of basal ganglia It is worthmentioning thatthe cortex which appeared unaffected in neuroimaging ina macroscopic examination displays extended neuronal apo-ptosis Furthermore in microscopic testing of the brain lesionsa specific arteriopathy has been revealed in which there isa thickening of the arterial wall of small penetrating cerebraland leptomeningeal arteries leading to lumen stenosis6 At thesame time with an electronic microscope in a specimen froma pathologic skin biopsy deposits of granular osmiophilic ma-terial (GOM) located in the basement membrane of smoothmuscle cells can be identified7 CADASIL is the only disorderwhose GOM has been identified However some reports onthe sensitivity of detecting GOM in skin biopsy of patients withgenetically proven CADASIL have been contradictory89

CADASIL is an autosomal dominant inherited arteriopathycaused by mutations in the NOTCH3 The NOTCH3 geneencodes a single pass transmembrane protein with receptorproperties This receptor is mainly expressed in the smoothmuscle cells of blood vessels and pericytes10 After ligandbinding the intracellular part translocates to the nucleus andactivates transcription factors NOTCH3 has 33 exons but allthe mutations found are located in exons 2ndash24 which areresponsible for encoding 34 epidermal growth factor repeats

EGFR Most of these mutations are missense mutations al-though there are only a few in-frame deletions or splice-sitemutations11 The gene mutation analysis of NOTCH3 is thegold standard to diagnose this genetically inherited diseaseand there are more than 230 different mutations located in 20different exons reported in patients with CADASIL12

In this review we report a patient with CADASIL with a novelheterozygousNOTCH3mutation and epileptic seizures as thevery first manifestation of the disorder In silico analysisrevealed the pathogenicity of the mutation However weproceeded to the skin biopsy to detect deposits of GOM

In addition we performed a systematic review of all publishedcases with NOTCH3 mutations We evaluated the mutationpathogenicity in all previously reported cases to investigatepossible phenotype-genotype correlations

MethodsCase description clinical findingsA 62-year-old woman visited the outpatient stroke clinic of a ter-tiary care stroke center inAthensGreece The patient experiencedepisodes of loss of consciousness some of them accompanied bytonic contraction of the upper arms and bite of her tongue for thepast 40 years She has been receiving an antiepileptic drug (leve-tiracetam 1250 mg daily) for the past 2 years because of repeatepisodes of tonic-clonic seizures She had also experienced anischemic lacunar stroke in the distribution of the left middle ce-rebral artery 4 months before her visit to the outpatient clinic ofour department that resulted in right hemiparesis The patientrsquoshusband and children reported changes in her personality andcognitive decline gradually worsening over the past 5 years Herfamily history revealed a brother with epilepsy and a father withposttraumatic epilepsy who died at the age of 60 years old

The patient had no history of hypertension or diabetes or otherknown cardiovascular risk factors She was receiving escitalo-pram (20 mg) for depression levetiracetam (1250 mg) forseizures clopidogrel (75 mg) and atorvastatin (20 mg) forsecondary stroke prevention

The patient was admitted in our department for further di-agnostic workupWe repeated brainMRI that revealed extensivebilateral white matter lesions located mainly in the frontal lobesthe anterior temporal lobe the external capsules the centrumsemiovale and the basal ganglia (figure 1 AndashC) There was noevidence of acute infarction Furthermore multiple cerebralmicrobleeds (CMBs) located predominantly in the thalami were

GlossaryCADASIL = cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathyCADD = combinedannotation-dependent depletion CI = confidence interval CMB = cerebral microbleed ECD = extracellular domain EGFr =epidermal growth factor-like repeat GOM = granular osmiophilic material TIA = transient ischemic attack



























































































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints





























































































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints



















































































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints










































































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints

































































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints

























































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints













































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints

































Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints























Appendix Authors







literature search

Katerina DadouliMSc



Christina ZompolaMD


Case description



literature search



Data extraction



Case description

Paschalis ZervasMD


Case description




Stergios StergiouMD










Data extraction







































































Reprints




















































Reprints





















Reprints















Reprints




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VIEWS AND REVIEWS OPEN ACCESS Cerebral autosomal …ng.neurology.org/content/nng/6/3/e434.full.pdfdefects, mainly in the attention and recall ability and a Frontal AssessmentBattery(6of18)thatrevealedaseriousdisorderin