On the Diagnosis of CADASIL - IOS Press

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Journal of Alzheimer’s Disease 17 (2009) 787–794 DOI 10.3233/JAD-2009-1112 IOS Press

On the Diagnosis of CADASIL Israel Ampueroa,∗, Javier Alegre-Abarrateguia,b, Izaskun Rodal a, Antonio Espan˜ aa, Raquel Rosa , ´ Cervello´ d , Ana Belén Camineroe , José Luis Lopez Send o´ nb , Eva Garc´ıa Gallowayc, Angeles f g g Antxon Zabala , Elena Erro , Fernando Jarauta , Lorenzo Morlánh , Eva Lo´ pez-Valdésh, Yolanda Aladroh , Manuel Seijoi , Guillermo Garc´ıa Rivasb , David G. Mun˜ oza and Justo Garc´ıa de Yébenesa,b a

Banco de Tejidos para Investigaciones Neurológicas (BTIN), Facultad de Medicina, Universidad Commplutense de Madrid, Madrid, Spain b Servicio de Neurolog´ıa, Hospital Ramo´ n y Cajal, Madrid, Spain c Servicio de Genética, Hospital Ram o´ n y Cajal, Madrid, Spain d Hospital General de Valencia, Valencia, Spain e ´ Hospital Nuestra Sen˜ ora de Sonsoles, Avila, Spain f Cl´ınica Puerta de Hierro, Madrid, Spain g Hospital de Navarra, Pamplona, Spain h Hospital de Getafe, Madrid, Spain i Complexo Hospitalario de Pontevedra, Pontevedra, Spain

Accepted 25 January 2009

Abstract. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), a genetic arteriopathy related to Notch3 mutations, is difficult to diagnosis. The goal of this study was to determine the value of clinical, immunohistochemical, and molecular techniques for the diagnosis of CADASIL. Clinical features and the immunohistochemical and molecular findings in 200 subjects with suspected CADASIL in whom 93 biopsies and 190 molecular studies are reported. Eighteen pathogenic mutations of the Notch3 gene, six of them previously unreported, were detected in 67 patients. The clinical features did not permit differentiation between CADASIL and CADASIL-like syndromes. The sensitivity and specificity of the skin biopsies was 97.7% and 56.5%, respectively, but increased to 100% and 81.5%, respectively, in cases with proven family history. In conclusion, a clinical diagnosis of CADASIL is difficult to determine and confirmatory techniques should be used judiciously. Keywords: CADASIL, familial stroke with dementia, Notch3, NOTCH3 immunostaining

INTRODUCTION Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a NOTCH3 related cerebral vasculopathy. Its features have been recently delineated though similar cas∗ Corresponding author: Israel Ampuero, Banco de Tejidos para Investigaciones Neurológicas, Facultad de Medicina, Universidad Commplutense de Madrid, Avda Complutense, s/n. Madrid 28040, Spain. Tel.: +34 91 3941326; Fax: +34 91 3941329; E-mail: [email protected].

es were reported in the past [1] including the philosopher Friedrich Nietzsche [2]. The diagnosis is not easy since the clinical features (recurrent strokes, often lacunes, in young adults without cardiovascular risk factors, vascular dementia, or recurrent migraine) and magnetic resonance imaging (MRI) findings (cerebral infarcts and white matter hyperintensities, typically in the anterior temporal lobe and external capsule) occur in other diseases [3–12]. Furthermore, a genetic pattern not always is apparent. The vasculopathy of CADASIL involves the brain, skin, and muscle [3,13–15] and, therefore, biopsies

ISSN 1387-2877/09/$17.00  2009 – IOS Press and the authors. All rights reserved

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I. Ampuero et al. / On the Diagnosis of CADASIL

of these tissues allow for intra vitam diagnosis. The pathological hallmark, present only in about 45% of cases [16], is a dense, darkly stained granular osmiophilic material (GOM) in the arterial walls, identified by electron microscopy (EM). Notch3 immunohistochemistry is considered an alternative to EM in skin biopsies [17]. The definitive diagnosis of CADASIL requires the identification of the pathogenic mutation in the Notch3 gene, a large gene with 33 exons. Numerous mutations have been reported, mostly located within the 23 exons that encode the 34 epidermal-growth-factor-like (EGFlike) repeats [18]. 70% of the mutations are clustered in exons 3 and 4, coming to 90% if we analyze the exons from 2 to 6 [19]. Almost all these mutations lead to loss or gain of a cysteine. Small deletions [6], splice site mutation causing an in-frame deletion [20], or a frameshift deletion [21] have also been reported. The molecular diagnosis of CADASIL is, therefore, difficult, expensive, time consuming, and at times perturbed by false negative results [19]. We analyze here the clinical features, NOTCH3 immunohistochemistry, and molecular findings in 200 Spanish patients with suspected CADASIL. We hope to provide useful keys for the diagnosis of CADASIL and CADASIL-like syndromes.

PATIENTS AND METHODS We studied 200 Caucasian patients from Spain with aclinical diagnosis of CADASIL, referred to the BTIN, from 1 January 2002 through 30 June 2008. Sixty seven subjects, including 15 asymptomatic, belong to 19 kindreds with proven family history of CADASIL, by evaluation of two or more of their members. The other 133 cases had suspected, but not proven, family history – no relatives could be examined. The clinical diagnosis of CADASIL was made according to the following criteria: 1) clinical history of unexplained recurrent strokes or transient ischemic attacks (TIA) under 55 years, vascular dementia [22], or dominant inheritance; and 2) MRI compatible with CADASIL [23]. Supporting clinical features included migraine, mood or behavioral disturbances, pseudobulbar palsy, and epilepsy. All subjects were evaluated using a purpose-built questionnaire by a neurologist. MRI of the brain was performed (T1 and T2 weighted images) and informed by neuroradiologists. High blood pressure or dyslipemia were diagnosed according to international criteria [24,25]. High alcohol consumption was consid-

ered if greater than 50 grams per day. Blood samples for DNA analysis and skin biopsies for immunohistochemistry against Notch3 were obtained, after informed consent, from 190 and 90 subjects, respectively. The patients were classified as “proven CADASIL” and “CADASIL-like” according to the presence or absence of Notch3 mutations, respectively. Histological studies Skin biopsies were obtained by a 4-mm punch, fixed in 4% neutral-buffered formalin, and embedded in paraffin. Immunohistochemistry was performed as described [17] using the Notch3 1E4 monoclonal antibody (raised against EGF-like extracellular repeats 17– 21, kindly donated by Dr. A. Joutel). Skin biopsies were considered typical of CADASIL when a pure granular pattern of immune staining, characterized by separated and intensely stained rounded granules in the media of vessels, was observed; and negative for CADASIL when there was no stain, linear stain of structures in the media, or the adventitia or mixed granular and linear stain [17]. Molecular analysis DNA from leukocytes was purified according to standard procedures. Genotyping was done by polymerase chain reaction (PCR) with primers which allowed analysis of the exon/intron boundaries, followed by sequencing of exons 2, 3, 4, 5, 6, 8, 11, 14, 18, 19, 22, and 23 of the Notch3 gene, which contain 98.6% of the mutations described in the Human Gene Mutation Database (HGMD) and Leiden Open Variation Database (LOVD) for CADASIL and in previous reports [26]. These include small deletions and most splicing abnormalities of the exons analyzed. We also searched for the polymorphisms C381T (exon 3), G684A (exon 4) and G1803A (exon 11) of uncertain clinical relevance. The frequency of these polymorphisms was also assessed in 100 chromosomes of healthy controls from Spain in order to test for linkage disequilibrium in CADASIL. Statistical analysis The prevalence of categorical variables was analyzed by Chi square test, with Fisher’s correction. Comparisons between scale parametric variables were performed by Student’s t-test. Differences were consid-


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Table 1 Clinical features of patients with “proven CADASIL” and CADASIL-like syndromes

Age (years) Age at onset (years) Family history of stroke at young age (n / %) Family history of vascular dementia (n / %) Family history of migraine (n / %) Personal history of hypertension (n / %) Personal history of dyslipemia (n / %) Personal history of diabetes mellitus (n / %) Personal history of smoking (n / %) Personal history of alcoholism (n / %) Personal history of hyper-homocysteinemia (n / %) Main neurological problem at disease onset Stroke (including TIA) Cognitive impairment Migraine Parkinsonism Other

Mutation + (n = 65) 53.0 + 14.2 45.0 + 13.2 46 / 70.7*# 35 / 53.8*# 13 / 20 3 / 4.6*# 6 / 9.2 1 / 1.5 8 / 12.3 4 / 6.2 0/0

Mutation − (n = 70) 52.0 + 13.2 48.0 + 14.0 22 / 33.3 21 / 31.8 8 / 12.3 11 / 16.9 9 / 13.8 3 / 4.6 9 / 13.8 5 / 7.7 1 / 1.5

Polymorphisms + (n = 45) 56.0 + 11.2 51.0 + 12.0 14 / 31.1 10 / 22.2 5 / 11.1 10 / 22.2 7 / 15.6 2 / 4.4 5 / 11.1 0/0 2 / 4.4

28 / 43.1 12 / 18.5 5 / 7.7 4 / 6.2 2 / 3.1

23 / 35.4 18 / 27.7 11 / 16.9 1 / 1.5 1 / 1.5

22 / 48.8 11 / 24.4 11 / 24.4 1 / 2.2 1 / 2.2

*: p < 0.05 with respect to mutation negative cases; #: 0.05 with respect to mutation negative cases but with one or more polymorphisms of Notch-3.

ered significant when p < 0.05. Bonferroni correction was applied when several comparisons were performed. For each statistical comparison, individuals without accurate information of the relevant item were excluded for each analysis. This missing information did not follow any systematic pattern.

The most frequent clinical onset in proven CADASIL was stroke, including TIA, followed by vascular dementia (Table 1). Parkinsonism was the initial symptom in 6.2% of patients with “proven CADASIL” and less frequently in CADASIL-like cases. Four patients were initially diagnosed with multiple sclerosis and one of them, with a Notch3 mutation, Arg332Cys (Table 1), had oligoclonal bands in the cerebrospinal fluid (CSF).

RESULTS Clinical features of patients with “proven CADASIL” The mean age and age at onset were, respectively, 53 ± 14.2 (mean ± standard deviation) and 45 ± 13.2 for patients with “proven CADASIL”; 52 ± 13.2 and 48 ± 14, respectively, for those with “CADASIL-like” syndromes without Notch3 polymorphisms; and 56 ± 11.2 and 51 ± 12, respectively, for patients with one or more of the G381T and G684A polymorphisms (Table 1). These data were not statistically different. Gender distribution was also similar in all groups. Patients with proven CADASIL had a familial history of stroke in 70.8% (46/65), vascular dementia in 53.8% (35/65), and migraine in 20% (13/65) of the cases. These familial antecedents were much less frequent in patients with CADASIL-like syndromes (Table 1). Hypertension, dyslipemia, diabetes mellitus, and hyperhomocysteinemia were more prevalent in CADASIL-like than in CADASIL-proven patients (Table 1), but there was no difference in the prevalence of smoking or chronic alcoholism.

EM and immunohistochemical findings Two EM studies were performed, one in a cerebral biopsy and the other in a skin biopsy. The cerebral biopsy was performed in 1988, before CADASIL was fully recognized. The pathological report described “osmiophilic deposits”, which are now considered characteristic of CADASIL. The patient was obviously not tested for Notch3 mutations but 20 years later we identified him as the father of two of our patients and the obligate carrier of the Arg110Cys mutation present in that family. The other EM study was performed in a skin biopsy. It was considered diagnostic for CADASIL. The molecular analysis failed to reveal any Notch3 mutations. Ninety-three skin biopsies were performed and 90 were analyzed for NOTCH immunoreactivity. Forty-six showed granular immunostaining typical of CADASIL. Twenty-six of these patients had Notch3 mutations (Table 2). Eleven patients with positive biopsies had one or more Notch3 polymorphisms and 9 patients lacked any variation.

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I. Ampuero et al. / On the Diagnosis of CADASIL Table 2 Histological results in patients with proven CADASIL and CADASIL like syndromes Histological studies: 93. Cerebral biopsy: 1. EM+. Arg110Cys present in two of his children. Skin biopsies: 93. 1: Insufficient blood vessels. 1: EM+, not IHC. 1: EM+, molecular analysis negative. 90 cases with IHC in skin biopsy + molecular analysis 44 skin biopsies with negative IHC. 20 cases: No staining. 19: molecular analysis negative. 1: Cys194Tyr mutation. 13 cases: Non specific weak linear staining. Molecular analysis negative. 11 cases: Non specific granulo-linear staining. Molecular analysis negative. 46 skin biopsies with positive IHC staining (typical granular inclusions). 26 cases. Molecular analysis positive. 11 cases. One or more Notch3 polymorphisms, no mutations detected. 9 cases. Molecular analysis negative

Of the 44 patients with negative skin biopsies one had a Cys194Tyr mutation. The sensitivity and the specificity of the immunohistochemistry, with respect to the molecular analysis, were 97.7% and 56.5%, respectively. In patients with proven family history of CADASIL, however, the sensitivity of the biopsy rose to 100% and the specificity to 81.5% Molecular findings Eighteen mutations, 6 of which previously unreported, were found in 65 subjects (Table 3). The molecular analysis was not performed in 10 patients, most of them fully symptomatic members of families with known mutations, and it was negative in 125. Fortyfive of the patients without mutations of the Notch3 gene presented the polymorphisms C381T (27 cases), the G684A (15 cases), both (2 cases) in heterozygosis and the C381T polymorphism in homozygosis (1 case). The frequency of these polymorphisms in these subjects with CADASIL-like syndromes is similar to that in the general population in Spain (data not shown). The new mutations discovered in this study are the following: G428A (Cys117Tyr), C529G (Gln151Glu), T541A (Cys155Ser), T562C (Cys162Arg), A587G (His170Arg), and G3089A (Cys1004Tyr). Sixteen of these 18 mutations changed the number of cysteines in a EFG-like repeat [17] but two, Gln151Glu and His170Arg, did not involve cysteine residues. Exon 4 was the hotspot where most mutations were found. The most prevalent mutation in this study was, however, the Arg607Cys, exon 11, present in 22 subjects from three families and one isolated case.

DISCUSSION We analyzed the clinical features, NOTCH3 immunostaining in skin biopsy, and Notch3 mutations in 200 patients with suspected CADASIL. By sequencing exons 2–6, 8, 11, 14, 18–19, and 22–23, which covers the hot spot areas (around 98.6% of the mutations already described in the 33 exons of the Notch3 gene), we found pathogenic mutations, six of them previously unreported, in 65 patients. Forty-five additional patients, 22.5% of the cases of this series, had one or more polymorphisms of the Notch3 gene. These polymorphisms are unlikely to be pathogenic since their frequency was similar to that found in the control population of Spain and since their presence did not modify the clinical features. The term CADASIL describes a combination of clinical features (an autosomal dominant disease with subcortical infarcts, dementia, and migraine) with MRI findings (a subcortical leucoencephalopathy, with early predominant involvement of the anterior temporal lobes and the external capsule and subcortical lacunar infarcts) [23]. However, none of these features is specific. Familial aggregates of subcortical infarcts could appear in subjects with common sporadic vascular risk factors, such as hypertension and smoking [27], and in patients with genetic disorders such as familial dyslipemias or hereditary hyperhomocysteinemias. And the pattern of lesions in the MRI studies could be confused with that of many other disorders. In this study we found 3 patients with CADASILlike pictures and hyperhomocysteinemia due to mutations of the methyl-tetra-hydro-folate reductase gene. We also found 2 patients with Notch3 mutations, previously diagnosed as multiple sclerosis, one of them


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Table 3 Notch3 mutations found in Spanish referred patients with clinical suspicion of CADASIL Exon

3 4

6 8 11 19

Patients with this mutation 5 4 1 4 1 4 3 1 1 4 3 1 1 2 3 1 25 1

AA Exchange

Nucleotide Exchange

EFG repeat

Cys

Ca

Arg90Cys Arg110Cys Cys117Tyr* Arg133Cys Gln151Glu* Arg153Cys Cys155Ser Cys162Arg* Gly165Cys* Arg169Cys His170Arg* Cys194Tyr Arg207Cys Tyr220Cys Arg332Cys Arg449Cys Arg607Cys Cys1004Tyr*

C346T C406T G428A C475T C529G C535T T541A T562C G571T C583T A587T G659A C697T A737G C1072T C1423T C1897T G3089A

2 2 2 3

+1 +1 +1 +1 = +1 −1 −1 +1 +1 = −1 +1 +1 +1 +1 +1 −1

− − − −

with oligoclonal bands in the CSF. Oligoclonal bands were previously reported in 2 out of 12 patients with CADASIL [4]. Two additional patients, out of 135 with CADASIL-like syndromes, were considered to have multiple sclerosis. In patients with suspected “familial multiple sclerosis”, molecular testing for CADASIL is warranted.

3 3 4 4 4 4 4 5 5 8 11 15 26

Cases with mutation per exon, N (%) 9 (13.85) 26 (40.00)

− − + + + + − − + −

3 (4.62) 1 (1.54) 25 (38.45) 1 (1.54)

CADASIL could be related to subcortical vascular lesions of the nigrostriatal dopamine system, pre- or postsynaptically. The good response to L-DOPA in one patient and slightly positive to selegiline in two suggests that, in these subjects, the mechanism is presynaptic. As in previous studies [4,5,8,27], we found wide phenotypic variability in patients with the same Notch3 mutations.

Clinical features Immunohistochemical findings The clinical features of the patients with “proven CADASIL” are similar to those reported in previous studies. These patients had strokes, including TIAs, at a younger age and with fewer cardiovascular risk factors than patients with CADASIL-like syndromes [28, 29]. The prevalence of migraine, clinical cerebrovascular events, and cognitive decline was slightly lower in this study than in others [4,5,8,9], perhaps because the diagnosis of CADASIL is now made at an earlier stage than in the past decade. We found 4 cases (6%) of parkinsonism, one with a good response to L-DOPA, in patients with proven CADASIL. Parkinsonism has been reported so far in one patient with CADASIL [30] but, since parkinsonism is frequent in the general population, it is difficult to know its significance. In this study, however, the prevalence of parkinsonism in proven CADASIL is greater than the expected 2.5% of the population in subjects over 65 years of age in Spain, and greater than the observed prevalence in the CADASIL-like groups, thus suggesting a pathogenic link. Parkinsonism in

NOTCH3 immunostaining in skin biopsies of nonselected patients with suspected CADASIL has a sensitivity of 97.7% and a specificity of 56.5% with respect to the molecular analysis, but both parameters increased to 100% and 81.5%, respectively, in patients with clinically confirmed family history. The real specificity of the procedure could slightly increase with complete molecular analysis of the Notch3 gene [17,31] but that approach is not cost effective since the gene is large and the probability of mutations outside the hot spot areas rare. The specificity encountered, lower than that reported by Joutel and colleagues [17], could relate to interobserver variations in interpretation of staining patterns, since no single individual was responsible for the performance of this test throughout the duration of the study. We believe this result is more representative of real-world as opposed to ideal conditions situations. False positive NOTCH3 immunostaining has been reported in other diseases, such as in familial cerebral amyloidosis [31]. False negative immunohistochemi-

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I. Ampuero et al. / On the Diagnosis of CADASIL Criteria for clinical suspicion (3 or more of the following): - Stroke below 55, w/out cardiovascular risk factors. - Autosomal dominant transmission. - Compatible MRI findings - Additional clinical findings (vascular dementia, persistent migraine)

NOTCH3 immunohistochemistry in skin biopsy Molecular analysis of Notch3 hot spot areas

Mutation +: true CADASIL

Mutation -, biopsy -: No CADASIL

Mutation -, biopsy +

3 or more family members available: Linkage analysis to 19q12. If positive, full sequentiation of Notch3. If negative no further studies

Less than 3 family members available: No further studies

Fig. 1. Diagnostic procedures proposed for CADASIL.

cal studies have been reported in cases with exon 11 mutations, which could disrupt the recognition epitope of the antibody around the nearby exon 14. We only found one symptomatic, unexplained, false negative NOTCH3 immunostaining, in a patient with an exon 4, Cys194Tyr mutation. Additional antibodies against other epitopes could further improve this diagnostic tool. We observed that weak linear staining is associated with hypertension. It is unknown whether the stained antigens in the vessels with linear and the granularlinear patterns are altered NOTCH3 or cross reactingproteins, such as components of the extracellular matrix (collagen, elastin), or if the stain is related to a fixation-derived artifact. True NOTCH3 immunostaining could happen in subjects with undetected Notch3 mutations, or in subjects with other genetic or environmental, as yet unknown, mechanisms of enhancing NOTCH3 deposition. Molecular findings We found 6 previously unreported mutations: four involved a cysteine, two of them (Cys117Tyr, Cys162 Arg) in codons where previous different mutations were described, and the other two (Cys155Ser, Cys1004Tyr) at new sites. Two new additional mutations, Gln151Glu and His170Arg, did not change the number of cysteines

but took place at codons very close to others where changes in cysteines have been reported. Most of the previously recognized pathogenic mutations in Notch3 change the number of cysteines in areas with EGF repeats implying that this number is essential for protein conformation. Our results suggest that changes of amino acids close to critical cysteines could have the effects. The proportion of patients with mutations in exon 4 is similar to that found in other studies in continental Europe [17,31] but not in the United Kingdom [16]. As in other Mediterranean studies [21,32], we found a high prevalence of exon 11 mutations. The most frequent was the Arg607Cys, though that was due to the presence of a large family with this change. In order to make the diagnoses of CADASIL costefficient, we propose that the molecular analysis, due to its cost, should be performed in the following cases: 1) symptomatic or asymptomatic members of families with an already identified mutation of Notch3 gene; 2) patients with a clinical disorder strongly suggestive of CADASIL, including family history, early onset cerebral infarcts without other identifiable risk factors, and typical MRI findings; and 3) patients with positive NOTCH3 immunoreactivity in skin biopsies. The initial molecular analysis should be restricted to the hot spot areas of the gene where most mutations have been identified so far. When there are three or more af-


fected family members available for molecular testing, we suggest that linkage analyses to the locus 19q12 is performed in cases with positive biopsy and negative molecular findings in the hot spot areas. Confirmation of linkage to this locus should warrant further molecular investigation. A diagram of diagnostic procedure is presented in Fig. 1. We hope that this study contributes to better management of CADASIL and CADASIL-like disorders.

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