Acta Neurol Scand 2015: 131: 30–36 DOI: 10.1111/ane.12284
© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd ACTA NEUROLOGICA SCANDINAVICA
Clinical, familial, and neuroimaging features of CADASIL-like patients Nannucci S, Pescini F, Bertaccini B, Bianchi S, Ciolli L, Valenti R, Dotti MT, Federico A, Inzitari D, Pantoni L. Clinical, familial, and neuroimaging features of CADASIL-like patients. Acta Neurol Scand 2015: 131: 30–36. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
S. Nannucci1, F. Pescini4, B. Bertaccini2, S. Bianchi3, L. Ciolli1, R. Valenti1, M. T. Dotti3, A. Federico3, D. Inzitari1, L. Pantoni4
Objectives – Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease caused by NOTCH3 mutations. There are no clinical and neuroimaging findings pathognomonic of the disease. The aim of this paper was to provide a description of a group of NOTCH3-negative patients with a phenotype closely resembling that of CADASIL. Materials and methods – We performed NOTCH3 analysis (exons 2-23) in 117 probands because of a clinician’s suspicion of CADASIL. The CADASIL scale, a recently developed tool that allows to better select patients for NOTCH3 analysis, was retrospectively applied to NOTCH3-negative patients; the patient subgroup that scored higher than the screening cutoff for CADASIL was defined as CADASIL-like. Results – Thirty-four CADASIL-like patients (mean age at onset 57.8 years [52.1–63.4], 50% males) were identified. Compared with 25 patients with CADASIL for clinical, familial, and neuroimaging features, only the following variables were significantly (a level 14 [16]. Definition of clinical and neuroimaging variables used for the comparison
For all patients, information on clinical features commonly associated with CADASIL, vascular risk factors, and family history (of transient ischemic attack (TIA), stroke, cognitive deficits, psychiatric disorders, headache, and seizures) was collected by means of a structured interview and previous medical records. To collect neuroimag-
ing data, we used magnetic resonance scans performed for clinical purposes. Most of the scans were performed on 1.5 Tesla magnets. For the aim of this study, we used only data available at the time of the disease suspicion when blood was withdrawn for genetic test. In patients, stroke and TIA were defined according to current criteria [17,18]. Psychiatric disorders were recorded as present in the case of any of the following: (1) previous diagnosis of a psychiatric disease by a certified specialist (psychiatrist, geriatrician, or neurologist); (2) previous or current use of antipsychotic or antidepressant drugs, or psychotherapy (the sole use of anxiolytics/benzodiazepines was not regarded as sufficient); and (3) mood or behavior disorders referred by the patient or his or her family, not immediately related to bereavement, and that had interfered for at least 6 months with daily or work activities. Cognitive disorders were recorded as present if one of the following instances was present: (1) previous diagnosis of cognitive impairment (mild cognitive impairment or dementia) by a certified specialist; (2) overt cognitive impairment emerged during the first evaluation at our center; and (3) presence of cognitive decline from a previously normal status referred by a next of kin or by the patient and confirmed by neuropsychological testing. Neuropsychological cognitive performances were judged impaired if the patient scored 1.5 standard deviations below the age- and education-corrected means in at least one cognitive test. Headache and migraine with and without aura were defined according to the Headache Classification Committee of the International Headache Society [19] without using any structured questionnaire. Seizures were defined according to the International League Against Epilepsy Commission Report [20]. The presence of hypertension was defined based on a previous diagnosis or according to the World Health Organization Guidelines [21] as a systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure ≥90 mmHg in patients who were not taking antihypertensive medication based on multiple blood pressure measurements, taken on several separate occasions. Diabetes mellitus was defined according to the American Diabetes Association criteria [22]. Hyperlipidemia was defined according to the National Cholesterol Education Program AdultTreatment Panel II [23]. Hypercholesterolemia included total cholesterol >200, low-density lipoprotein >130, and high-density lipoprotein 200 mg/dL (in at least two measurements). Smoking was considered as 31
Nannucci et al. present in case of current or previous history. Renal dysfunction was defined when creatinine levels were superior to normal values according to local laboratory. Presence of heart failure and previous myocardial infarction was registered according to clinical charts. A neurologist experienced in brain imaging evaluation (L.P.) and blind to genetic diagnosis reviewed all the available scans and assessed the following features: 1. Presence and severity of white matter changes on fluid-attenuated inversion recovery (FLAIR) or T2-weighted images according to a modified Fazekas scale [24]: grade 1: single lesions must be below 10 mm; areas of ‘grouped’ lesions must be smaller than 20 mm in any diameter; grade 2: single lesions between 10 and 20 mm; areas of ‘grouped’ lesions more than 20 mm in any diameter; no more than ‘connecting bridges’ between individual lesions; grade 3: single lesions or confluent areas of hyperintensity 20 mm or more in any diameter. 2. Presence of hyperintense lesions (FLAIR and T2-weighted images) in the anterior temporal lobe white matter. 3. Presence of hyperintensity in the external capsule. 4. Presence of lacunar infarcts defined as focal hyperintensities on T2-weighted images, 3 mm in size or larger, and with a corresponding hypointensity on T1-weighted images [25]. Lacunar infarcts were coded as single or multiple. Pons involvement was distinguished into pontine leukoaraiosis and presence of lacunar infarcts. The disturbances used to define the age of disease onset were the following: TIA/ischemic stroke (27 probands), psychiatric disturbances (14), cognitive deficits (6), hemorrhagic stroke (3), gait disturbances (3), seizures (3), retinal thrombosis (1), migraine aura (1), transient unilateral oculomotor deficit (1). Migraine was not considered in this regard unless other disturbances were absent because of the difficulty in exactly defining its age of onset. Data about headache were missing for one CADASIL-like patient. In the CADASIL group, involvement of external capsule was not assessable for two patients and magnetic resonance scan quality did not allow to assess the presence of pontine leukoaraiosis and pontine lacunar infarcts for four and eight patients, respectively. Pons involvement was not assessable for two CADASIL-like patients. In one CADASIL-like patient and two patients with CADASIL, family history information was completely or partially not obtainable. Other two patients with CADASIL were found to belong to the same family after the genetic screening. Therefore, we included in the analysis the data deriving from this family only one time. 32
NOTCH3 gene analysis
Total genomic DNA was extracted from peripheral blood leukocytes using standard procedures. PCR was performed with primers (comprising intron–exon boundaries) specific for exons 2-23 of the NOTCH3 gene. PCRs were performed in 25 lL reaction volumes containing 100 ng genomic DNA, 10 pmol forward and reverse primers, 100 mM dNTPs, 1 U DNA polymerase (Red Hot DNA polymerase; ABgene, UK), 1.5 mM MgSO4, 19 Reaction buffer, using a DNA thermal cycler (PTC-200; MJ Research, Waltham, MA, USA). Following purification of PCR products, sequencing was performed using the automated sequencer ABI 3730 (Applied Biosystems, Foster City, CA). Genetic analysis was performed at the Department of Neurological and Behavioral Sciences, University of Siena, Italy. Statistical analysis
To test differences between patients with CADASIL and CADASIL-like patients, we firstly used Fisher’s exact test for all the binary variables. The test uses a combinatorial approach to compute the exact probability that a particular observed pattern or more extreme patterns may occur by chance. Although in practice it is employed for all sample sizes, Fisher’s exact test is a statistical significance test particularly useful when sample sizes are small or when data are heavily unequally distributed inside the two groups. Because the Fisher’s exact test tends to be slightly conservative (the rejection rate is lower than the nominal significance level) [26,27], to confirm the findings, we repeated the tests applying the Wilson score methods (with and without continuity correction) for those variables which gave a Fisher’s exact test Pvalue just slightly higher than the significant level (P-value between 0.05 and 0.11). Wilson testing procedures are based on interval estimation for the difference of proportions, free from aberrations that could arise in particular patterns (interval estimations exceeding the parameter space), and achieve better coverage properties than most of the other methods [28]. To test differences in quantitative variables between patients with CADASIL and CADASILlike patients, we used the traditional t-test. Results
Of the 92 patients who underwent NOTCH3 gene analysis and in whom no pathogenic mutation was found, 24 were excluded because they had
CADASIL-like patients missing data and did not achieve a score >14 on the CADASIL scale. Therefore, we applied the scale on the remaining 68 patients identifying 34 CADASIL-like probands. There were no statistically significant differences between the patients with CADASIL and CADASIL-like patients considering age of disease onset and age of first cerebral ischemic event, gender, and a number of clinical variables with the exception of hypertension that was significantly more frequent in CADASIL-like patients (79 vs 48%) (Table 1). Although not at a statistically significant level, a history of migraine with or without aura was more frequent in patients with CADASIL, but this difference greatly decreased when considering only migraine with aura (Table 1). No patient suffered from heart failure. A history of myocardial infarction Table 1 Comparison of clinical features, gender, and vascular risk factors between patients with CADASIL and CADASIL-like patients
Mean age at disease onset, years Mean age at first ischemic stroke/TIA, years Male gender, n (%) Clinical features, n (%) TIA Ischemic stroke TIA/ischemic stroke ≤50 years Recurrent TIA/ ischemic stroke Hemorrhagic stroke Cognitive deficits Psychiatric disturbances Headache (no migraine) Migraine (with or without aura) Migraine with aura Seizures Neurological findings, n (%) Gate disturbances Urinary disturbances Dysphagia Dysarthria Vascular risk factors, n (%) Hypertension Diabetes mellitus Hyperlipidemia Smoking
Patients with CADASIL (n = 25)
CADASIL-like patients (n = 34)
P
59.0 [54.0–64.0]
57.8 [52.1–63.4]
0.833a
59.4 [52.8–66.0]
61.9 [57.2–66.5]
0.789a
8/25 (32)
17/34 (50)
0.132b
8/25 (32) 13/25 (52) 4/25 (16)
5/34 (15) 16/34 (47) 4/34 (12)
0.103b 0.738b 0.804b
9/25 (36)
8/34 (24)
0.225b
3/25 (12) 12/25 (48) 14/25 (56)
2/34 (6) 22/34 (65) 22/34 (65)
0.354b 0.155b 0.341b
2/25 (8)
6/33 (18)
0.236b
16/25 (64)
13/33 (39)
0.055b
5/33 (15) 5/34 (15)
b
7/25 (28) 5/25 (20)
was present in only 3/34 CADASIL-like patients, and 1/34 CADASIL-like patient had a mild renal dysfunction without significant differences in comparison with patients with CADASIL (P = 0.184 and P = 0.576, respectively). No statistically significant difference was found between the two groups concerning the use of antithrombotic drugs (19/25, 76%, in patients with CADASIL and 27/33, 82%, in CADASIL-like patients, P = 0.412) and statin (10/25, 40%, in patients with CADASIL and 6/33, 18%, in CADASILlike patients, P = 0.062). Instead, the use of antihypertensive drugs was more frequent in CADASIL-like patients (26/34, 76%, vs 12/25, 48%, P = 0.024). Regarding neuroimaging characteristics, presence and severity of leukoencephalopathy, temporal poles, external capsules, pons involvement, and lacunar infarcts number were equally distributed in patients with CADASIL and CADASILlike patients (Table 2). Almost 90% of patients of both groups showed a severe degree of leukoencephalopathy. The external capsule was involved in all patients and, also in the CADASIL-like group, a considerable number of patients showed white matter changes in temporal poles. Some examples of neuroimaging features of CADASIL-like patients are shown in the Fig. 1. All patients had a positive family history in at least one generation and most of them also in at least two generations (Table 3). The only one difference we found concerned a positive family history of TIA or stroke before the age of 60 that was significantly more frequent, both in 1 and 2 generations, in patients with CADASIL. To note, we did not find any difference in the Table 2 Comparison of neuroimaging findings between patients with CADASIL and CADASIL-like patients Patients with CADASIL (n = 25), n (%)
0.192 0.813b
15/25 12/24 5/24 8/25
(60) (50) (21) (32)
16/33 16/33 3/34 10/33
(48) (48) (9) (30)
0.872b 0.648b 0.179b 0.666b
12/25 3/25 16/25 9/25
(48) (12) (64) (36)
27/34 7/34 15/34 16/34
(79) (21) (44) (47)
0.013b 0.307b 0.106b 0.281b
CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; TIA, transient ischemic attack; Bold, statistically significant. a t-test. b Fisher’s exact test.
Leukoencephalopathy Grade 1 Grade 2 Grade 3 Temporal pole involvement External capsule involvement Lacunar infarcts (any number) Single lacunar infarct Multiple lacunar infarcts Pontine leukoaraiosis Pontine infarcts
25/25 0/25 3/25 22/25 22/25 23/23 25/25 2/25 23/25 13/21 10/17
(100) (0) (12) (88) (88) (100) (100) (8) (92) (62) (59)
CADASIL-like patients (n = 34), n (%) 34/34 1/34 3/34 30/34 27/34 34/34 31/34 2/34 29/34 16/32 13/32
(100) (3) (9) (88) (79) (100) (91) (6) (85) (50) (41)
Pa 1 0.576 0.798 0.641 0.890 1 1 0.800 0.886 0.872 0.935
CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. a Fisher’s exact test.
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Nannucci et al. A
personal history of stroke/TIA in young age (Table 1).
B
Discussion
C
D
Figure 1. Brain magnetic resonance images of some CADASIL-like patients. Three axial fluid-attenuated inversion recovery (FLAIR) images showing a severe leukoencephalopathy (A), a continuous involvement of the external capsules (B), and a bilateral temporal pole involvement (C). A coronal T1-weighted image showing multiple subcortical lacunar infarcts in a context of a severe leukoencephalopathy (D).
Table 3 Comparison of family history between patients with CADASIL and CADASIL-like patients
Family history in at least 1 generation Family history in at least 2 generations One generation Stroke/TIA any age Stroke/TIA ≤60 years Stroke/TIA >60 years Cognitive deficits Psychiatric disturbances Headache Seizures Two generations Stroke/TIA any age Stroke/TIA ≤60 years Stroke/TIA >60 years Cognitive deficits Psychiatric disturbances Headache Seizures
Patients with CADASIL (n = 25), n (%)
CADASIL-like patients (n = 34), n (%)
24/24 (100)
34/34 (100)
1
23/24 (96)
32/34 (94)
0.806
20/23 14/22 11/23 20/23 15/23 20/23 4/23
(87) (64) (48) (87) (65) (87) (17)
27/34 10/33 23/34 23/34 21/34 30/34 5/34
(79) (30) (68) (68) (62) (88) (15)
0.863 0.015 0.111 0.979 0.706 0.596 0.742
13/23 6/22 2/22 7/23 10/23 11/23 0/23
(57) (27) (9) (30) (43) (48) (0)
11/34 1/33 5/33 8/34 9/34 14/34 1/34
(32) (3) (15) (24) (26) (41) (3)
0.062 0.013 0.411 0.813 0.147 0.779 0.596
Pa
CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; TIA, transient ischemic attack; Bold, statistically significant. a Fisher’s exact test.
34
After our initial report on NOTCH3-negative patients [10], we have refined the selection of patients to be subjected to NOTCH3 analysis. Based on the experience collected and on the review of selected international CADASIL series, we developed the CADASIL scale, a screening tool to identify patients with a high suspicion of the disease [16]. Applying retrospectively the scale to a group of NOTCH3-negative patients (part of whom included in the previous paper [10]), we have been able to select a group of CADASILlike patients with a phenotype nearly overlapping that of CADASIL, also when comparing features not included in the CADASIL scale. The differences between patients with CADASIL and CADASIL-like patients were few. The first one was that a family history of stroke before the age of 60 was more frequent in patients with CADASIL; this is in agreement with the fact that the disease is characterized by the occurrence of acute cerebrovascular events in middle age [2]. However, we did not find any difference relative to a personal history of juvenile stroke between patients with CADASIL and CADASIL-like patients. Of note, most of our patients with CADASIL presented with a TIA or ischemic stroke at a mean age relatively higher than that reported in the literature [2]. The higher frequency of hypertension in the CADASIL-like group is difficult to interpret. One could hypothesize that, at least in some of these patients, the small vessel disease is caused by hypertension. Unfortunately, we lack more precise data hinting at a possible role of hypertension in this CADASIL-like group, for example, about duration and efficacy of treatment. However, the very frequent involvement of external capsule and temporal pole, a finding quite uncommon in hypertension-related small vessel disease, would suggest that hypertension is not able to explain entirely the presence of white matter changes in these patients. Finally, the role of a possible interaction between hypertension and other so-far unknown genes in this group remains unexplored. Our study has some limitations. The first one is its retrospective nature. Furthermore, the use of the CADASIL scale to select CADASIL-like patients obviously decreased the chance of finding differences between the two groups of patients. However, as a natural evolution of our previous report [10], we were interested in defining a group of patients with the highest suspicion of
CADASIL-like patients CADASIL in whom performing further genetic tests such as expanding the NOTCH3 gene analysis to all the remaining exons and introns [29–32] and evaluating other recently described genetic forms of cerebral small vessel disease [33–36]. We did not perform skin biopsy in any but 2 CADASIL-like patients in whom we found NOTCH3 alterations not leading to cysteine substitution with unclear pathogenic significance [10]. In neither of them, GOM deposits were found. However, the sensitivity of this investigation is today questioned [14,15]. Finally, with the number of patients analyzed, strong differences between the two groups would almost certainly reject the null hypothesis. However, a greater number of patients would strengthen the power of the statistical tests. We think that the report of our experience in the diagnosis of CADASIL could be useful to physicians involved in the study of cerebral small vessel disease by highlighting the growing number of patients presenting with a phenotype closely similar to CADASIL and a high suspicion of an inherited form of small vessel disease but without NOTCH3 mutations. This group of patients might be heterogeneous from the genetic point of view and, therefore, requires a more extensive genetic study. Acknowledgments None.
Conflict of interest and sources of funding statement The study was supported by MIUR (Ministero dell’Istruzione, dell’Universita e della Ricerca), programmi di ricerca cofinanziati-2006 (MIUR 2006-prot. 2006065719), MIUR (Ministero dell’Istruzione, dell’Universit a e della Ricerca), programmi di ricerca cofinanziati-2009 (MIUR 2009-prot. 20095JPSNA), Regione Toscana, programma per la Ricerca Regionale in Materia di Salute, 2009 (project ‘Evaluation of NOTCH3 mutations and correlation with clinical phenotypes’ Prot. reg. AOO GRT 190899/Q.20.70.20). S. Nannucci, F. Pescini, B. Bertaccini, S. Bianchi, L. Ciolli, R. Valenti, and M. T. Dotti report no disclosure. A. Federico is Editor-in-Chief of Neurological Sciences. D. Inzitari has served on a scientific advisory board for SERVIER, serves on the editorial board of Stroke, and has received speaker honoraria from Bayer Schering Pharma, Novartis, Pfizer Inc., and Sanofi-Aventis. L. Pantoni serves on the editorial boards of Acta Neurologica Scandinavica and Cerebrovascular Diseases and as Vascular Cognitive Impairment Section Editor for Stroke.
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© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd ACTA NEUROLOGICA SCANDINAVICA
Clinical, familial, and neuroimaging features of CADASIL-like patients Nannucci S, Pescini F, Bertaccini B, Bianchi S, Ciolli L, Valenti R, Dotti MT, Federico A, Inzitari D, Pantoni L. Clinical, familial, and neuroimaging features of CADASIL-like patients. Acta Neurol Scand 2015: 131: 30–36. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
S. Nannucci1, F. Pescini4, B. Bertaccini2, S. Bianchi3, L. Ciolli1, R. Valenti1, M. T. Dotti3, A. Federico3, D. Inzitari1, L. Pantoni4
Objectives – Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is an inherited small vessel disease caused by NOTCH3 mutations. There are no clinical and neuroimaging findings pathognomonic of the disease. The aim of this paper was to provide a description of a group of NOTCH3-negative patients with a phenotype closely resembling that of CADASIL. Materials and methods – We performed NOTCH3 analysis (exons 2-23) in 117 probands because of a clinician’s suspicion of CADASIL. The CADASIL scale, a recently developed tool that allows to better select patients for NOTCH3 analysis, was retrospectively applied to NOTCH3-negative patients; the patient subgroup that scored higher than the screening cutoff for CADASIL was defined as CADASIL-like. Results – Thirty-four CADASIL-like patients (mean age at onset 57.8 years [52.1–63.4], 50% males) were identified. Compared with 25 patients with CADASIL for clinical, familial, and neuroimaging features, only the following variables were significantly (a level 14 [16]. Definition of clinical and neuroimaging variables used for the comparison
For all patients, information on clinical features commonly associated with CADASIL, vascular risk factors, and family history (of transient ischemic attack (TIA), stroke, cognitive deficits, psychiatric disorders, headache, and seizures) was collected by means of a structured interview and previous medical records. To collect neuroimag-
ing data, we used magnetic resonance scans performed for clinical purposes. Most of the scans were performed on 1.5 Tesla magnets. For the aim of this study, we used only data available at the time of the disease suspicion when blood was withdrawn for genetic test. In patients, stroke and TIA were defined according to current criteria [17,18]. Psychiatric disorders were recorded as present in the case of any of the following: (1) previous diagnosis of a psychiatric disease by a certified specialist (psychiatrist, geriatrician, or neurologist); (2) previous or current use of antipsychotic or antidepressant drugs, or psychotherapy (the sole use of anxiolytics/benzodiazepines was not regarded as sufficient); and (3) mood or behavior disorders referred by the patient or his or her family, not immediately related to bereavement, and that had interfered for at least 6 months with daily or work activities. Cognitive disorders were recorded as present if one of the following instances was present: (1) previous diagnosis of cognitive impairment (mild cognitive impairment or dementia) by a certified specialist; (2) overt cognitive impairment emerged during the first evaluation at our center; and (3) presence of cognitive decline from a previously normal status referred by a next of kin or by the patient and confirmed by neuropsychological testing. Neuropsychological cognitive performances were judged impaired if the patient scored 1.5 standard deviations below the age- and education-corrected means in at least one cognitive test. Headache and migraine with and without aura were defined according to the Headache Classification Committee of the International Headache Society [19] without using any structured questionnaire. Seizures were defined according to the International League Against Epilepsy Commission Report [20]. The presence of hypertension was defined based on a previous diagnosis or according to the World Health Organization Guidelines [21] as a systolic blood pressure ≥140 mmHg and/or a diastolic blood pressure ≥90 mmHg in patients who were not taking antihypertensive medication based on multiple blood pressure measurements, taken on several separate occasions. Diabetes mellitus was defined according to the American Diabetes Association criteria [22]. Hyperlipidemia was defined according to the National Cholesterol Education Program AdultTreatment Panel II [23]. Hypercholesterolemia included total cholesterol >200, low-density lipoprotein >130, and high-density lipoprotein 200 mg/dL (in at least two measurements). Smoking was considered as 31
Nannucci et al. present in case of current or previous history. Renal dysfunction was defined when creatinine levels were superior to normal values according to local laboratory. Presence of heart failure and previous myocardial infarction was registered according to clinical charts. A neurologist experienced in brain imaging evaluation (L.P.) and blind to genetic diagnosis reviewed all the available scans and assessed the following features: 1. Presence and severity of white matter changes on fluid-attenuated inversion recovery (FLAIR) or T2-weighted images according to a modified Fazekas scale [24]: grade 1: single lesions must be below 10 mm; areas of ‘grouped’ lesions must be smaller than 20 mm in any diameter; grade 2: single lesions between 10 and 20 mm; areas of ‘grouped’ lesions more than 20 mm in any diameter; no more than ‘connecting bridges’ between individual lesions; grade 3: single lesions or confluent areas of hyperintensity 20 mm or more in any diameter. 2. Presence of hyperintense lesions (FLAIR and T2-weighted images) in the anterior temporal lobe white matter. 3. Presence of hyperintensity in the external capsule. 4. Presence of lacunar infarcts defined as focal hyperintensities on T2-weighted images, 3 mm in size or larger, and with a corresponding hypointensity on T1-weighted images [25]. Lacunar infarcts were coded as single or multiple. Pons involvement was distinguished into pontine leukoaraiosis and presence of lacunar infarcts. The disturbances used to define the age of disease onset were the following: TIA/ischemic stroke (27 probands), psychiatric disturbances (14), cognitive deficits (6), hemorrhagic stroke (3), gait disturbances (3), seizures (3), retinal thrombosis (1), migraine aura (1), transient unilateral oculomotor deficit (1). Migraine was not considered in this regard unless other disturbances were absent because of the difficulty in exactly defining its age of onset. Data about headache were missing for one CADASIL-like patient. In the CADASIL group, involvement of external capsule was not assessable for two patients and magnetic resonance scan quality did not allow to assess the presence of pontine leukoaraiosis and pontine lacunar infarcts for four and eight patients, respectively. Pons involvement was not assessable for two CADASIL-like patients. In one CADASIL-like patient and two patients with CADASIL, family history information was completely or partially not obtainable. Other two patients with CADASIL were found to belong to the same family after the genetic screening. Therefore, we included in the analysis the data deriving from this family only one time. 32
NOTCH3 gene analysis
Total genomic DNA was extracted from peripheral blood leukocytes using standard procedures. PCR was performed with primers (comprising intron–exon boundaries) specific for exons 2-23 of the NOTCH3 gene. PCRs were performed in 25 lL reaction volumes containing 100 ng genomic DNA, 10 pmol forward and reverse primers, 100 mM dNTPs, 1 U DNA polymerase (Red Hot DNA polymerase; ABgene, UK), 1.5 mM MgSO4, 19 Reaction buffer, using a DNA thermal cycler (PTC-200; MJ Research, Waltham, MA, USA). Following purification of PCR products, sequencing was performed using the automated sequencer ABI 3730 (Applied Biosystems, Foster City, CA). Genetic analysis was performed at the Department of Neurological and Behavioral Sciences, University of Siena, Italy. Statistical analysis
To test differences between patients with CADASIL and CADASIL-like patients, we firstly used Fisher’s exact test for all the binary variables. The test uses a combinatorial approach to compute the exact probability that a particular observed pattern or more extreme patterns may occur by chance. Although in practice it is employed for all sample sizes, Fisher’s exact test is a statistical significance test particularly useful when sample sizes are small or when data are heavily unequally distributed inside the two groups. Because the Fisher’s exact test tends to be slightly conservative (the rejection rate is lower than the nominal significance level) [26,27], to confirm the findings, we repeated the tests applying the Wilson score methods (with and without continuity correction) for those variables which gave a Fisher’s exact test Pvalue just slightly higher than the significant level (P-value between 0.05 and 0.11). Wilson testing procedures are based on interval estimation for the difference of proportions, free from aberrations that could arise in particular patterns (interval estimations exceeding the parameter space), and achieve better coverage properties than most of the other methods [28]. To test differences in quantitative variables between patients with CADASIL and CADASILlike patients, we used the traditional t-test. Results
Of the 92 patients who underwent NOTCH3 gene analysis and in whom no pathogenic mutation was found, 24 were excluded because they had
CADASIL-like patients missing data and did not achieve a score >14 on the CADASIL scale. Therefore, we applied the scale on the remaining 68 patients identifying 34 CADASIL-like probands. There were no statistically significant differences between the patients with CADASIL and CADASIL-like patients considering age of disease onset and age of first cerebral ischemic event, gender, and a number of clinical variables with the exception of hypertension that was significantly more frequent in CADASIL-like patients (79 vs 48%) (Table 1). Although not at a statistically significant level, a history of migraine with or without aura was more frequent in patients with CADASIL, but this difference greatly decreased when considering only migraine with aura (Table 1). No patient suffered from heart failure. A history of myocardial infarction Table 1 Comparison of clinical features, gender, and vascular risk factors between patients with CADASIL and CADASIL-like patients
Mean age at disease onset, years Mean age at first ischemic stroke/TIA, years Male gender, n (%) Clinical features, n (%) TIA Ischemic stroke TIA/ischemic stroke ≤50 years Recurrent TIA/ ischemic stroke Hemorrhagic stroke Cognitive deficits Psychiatric disturbances Headache (no migraine) Migraine (with or without aura) Migraine with aura Seizures Neurological findings, n (%) Gate disturbances Urinary disturbances Dysphagia Dysarthria Vascular risk factors, n (%) Hypertension Diabetes mellitus Hyperlipidemia Smoking
Patients with CADASIL (n = 25)
CADASIL-like patients (n = 34)
P
59.0 [54.0–64.0]
57.8 [52.1–63.4]
0.833a
59.4 [52.8–66.0]
61.9 [57.2–66.5]
0.789a
8/25 (32)
17/34 (50)
0.132b
8/25 (32) 13/25 (52) 4/25 (16)
5/34 (15) 16/34 (47) 4/34 (12)
0.103b 0.738b 0.804b
9/25 (36)
8/34 (24)
0.225b
3/25 (12) 12/25 (48) 14/25 (56)
2/34 (6) 22/34 (65) 22/34 (65)
0.354b 0.155b 0.341b
2/25 (8)
6/33 (18)
0.236b
16/25 (64)
13/33 (39)
0.055b
5/33 (15) 5/34 (15)
b
7/25 (28) 5/25 (20)
was present in only 3/34 CADASIL-like patients, and 1/34 CADASIL-like patient had a mild renal dysfunction without significant differences in comparison with patients with CADASIL (P = 0.184 and P = 0.576, respectively). No statistically significant difference was found between the two groups concerning the use of antithrombotic drugs (19/25, 76%, in patients with CADASIL and 27/33, 82%, in CADASIL-like patients, P = 0.412) and statin (10/25, 40%, in patients with CADASIL and 6/33, 18%, in CADASILlike patients, P = 0.062). Instead, the use of antihypertensive drugs was more frequent in CADASIL-like patients (26/34, 76%, vs 12/25, 48%, P = 0.024). Regarding neuroimaging characteristics, presence and severity of leukoencephalopathy, temporal poles, external capsules, pons involvement, and lacunar infarcts number were equally distributed in patients with CADASIL and CADASILlike patients (Table 2). Almost 90% of patients of both groups showed a severe degree of leukoencephalopathy. The external capsule was involved in all patients and, also in the CADASIL-like group, a considerable number of patients showed white matter changes in temporal poles. Some examples of neuroimaging features of CADASIL-like patients are shown in the Fig. 1. All patients had a positive family history in at least one generation and most of them also in at least two generations (Table 3). The only one difference we found concerned a positive family history of TIA or stroke before the age of 60 that was significantly more frequent, both in 1 and 2 generations, in patients with CADASIL. To note, we did not find any difference in the Table 2 Comparison of neuroimaging findings between patients with CADASIL and CADASIL-like patients Patients with CADASIL (n = 25), n (%)
0.192 0.813b
15/25 12/24 5/24 8/25
(60) (50) (21) (32)
16/33 16/33 3/34 10/33
(48) (48) (9) (30)
0.872b 0.648b 0.179b 0.666b
12/25 3/25 16/25 9/25
(48) (12) (64) (36)
27/34 7/34 15/34 16/34
(79) (21) (44) (47)
0.013b 0.307b 0.106b 0.281b
CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; TIA, transient ischemic attack; Bold, statistically significant. a t-test. b Fisher’s exact test.
Leukoencephalopathy Grade 1 Grade 2 Grade 3 Temporal pole involvement External capsule involvement Lacunar infarcts (any number) Single lacunar infarct Multiple lacunar infarcts Pontine leukoaraiosis Pontine infarcts
25/25 0/25 3/25 22/25 22/25 23/23 25/25 2/25 23/25 13/21 10/17
(100) (0) (12) (88) (88) (100) (100) (8) (92) (62) (59)
CADASIL-like patients (n = 34), n (%) 34/34 1/34 3/34 30/34 27/34 34/34 31/34 2/34 29/34 16/32 13/32
(100) (3) (9) (88) (79) (100) (91) (6) (85) (50) (41)
Pa 1 0.576 0.798 0.641 0.890 1 1 0.800 0.886 0.872 0.935
CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy. a Fisher’s exact test.
33
Nannucci et al. A
personal history of stroke/TIA in young age (Table 1).
B
Discussion
C
D
Figure 1. Brain magnetic resonance images of some CADASIL-like patients. Three axial fluid-attenuated inversion recovery (FLAIR) images showing a severe leukoencephalopathy (A), a continuous involvement of the external capsules (B), and a bilateral temporal pole involvement (C). A coronal T1-weighted image showing multiple subcortical lacunar infarcts in a context of a severe leukoencephalopathy (D).
Table 3 Comparison of family history between patients with CADASIL and CADASIL-like patients
Family history in at least 1 generation Family history in at least 2 generations One generation Stroke/TIA any age Stroke/TIA ≤60 years Stroke/TIA >60 years Cognitive deficits Psychiatric disturbances Headache Seizures Two generations Stroke/TIA any age Stroke/TIA ≤60 years Stroke/TIA >60 years Cognitive deficits Psychiatric disturbances Headache Seizures
Patients with CADASIL (n = 25), n (%)
CADASIL-like patients (n = 34), n (%)
24/24 (100)
34/34 (100)
1
23/24 (96)
32/34 (94)
0.806
20/23 14/22 11/23 20/23 15/23 20/23 4/23
(87) (64) (48) (87) (65) (87) (17)
27/34 10/33 23/34 23/34 21/34 30/34 5/34
(79) (30) (68) (68) (62) (88) (15)
0.863 0.015 0.111 0.979 0.706 0.596 0.742
13/23 6/22 2/22 7/23 10/23 11/23 0/23
(57) (27) (9) (30) (43) (48) (0)
11/34 1/33 5/33 8/34 9/34 14/34 1/34
(32) (3) (15) (24) (26) (41) (3)
0.062 0.013 0.411 0.813 0.147 0.779 0.596
Pa
CADASIL, cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy; TIA, transient ischemic attack; Bold, statistically significant. a Fisher’s exact test.
34
After our initial report on NOTCH3-negative patients [10], we have refined the selection of patients to be subjected to NOTCH3 analysis. Based on the experience collected and on the review of selected international CADASIL series, we developed the CADASIL scale, a screening tool to identify patients with a high suspicion of the disease [16]. Applying retrospectively the scale to a group of NOTCH3-negative patients (part of whom included in the previous paper [10]), we have been able to select a group of CADASILlike patients with a phenotype nearly overlapping that of CADASIL, also when comparing features not included in the CADASIL scale. The differences between patients with CADASIL and CADASIL-like patients were few. The first one was that a family history of stroke before the age of 60 was more frequent in patients with CADASIL; this is in agreement with the fact that the disease is characterized by the occurrence of acute cerebrovascular events in middle age [2]. However, we did not find any difference relative to a personal history of juvenile stroke between patients with CADASIL and CADASIL-like patients. Of note, most of our patients with CADASIL presented with a TIA or ischemic stroke at a mean age relatively higher than that reported in the literature [2]. The higher frequency of hypertension in the CADASIL-like group is difficult to interpret. One could hypothesize that, at least in some of these patients, the small vessel disease is caused by hypertension. Unfortunately, we lack more precise data hinting at a possible role of hypertension in this CADASIL-like group, for example, about duration and efficacy of treatment. However, the very frequent involvement of external capsule and temporal pole, a finding quite uncommon in hypertension-related small vessel disease, would suggest that hypertension is not able to explain entirely the presence of white matter changes in these patients. Finally, the role of a possible interaction between hypertension and other so-far unknown genes in this group remains unexplored. Our study has some limitations. The first one is its retrospective nature. Furthermore, the use of the CADASIL scale to select CADASIL-like patients obviously decreased the chance of finding differences between the two groups of patients. However, as a natural evolution of our previous report [10], we were interested in defining a group of patients with the highest suspicion of
CADASIL-like patients CADASIL in whom performing further genetic tests such as expanding the NOTCH3 gene analysis to all the remaining exons and introns [29–32] and evaluating other recently described genetic forms of cerebral small vessel disease [33–36]. We did not perform skin biopsy in any but 2 CADASIL-like patients in whom we found NOTCH3 alterations not leading to cysteine substitution with unclear pathogenic significance [10]. In neither of them, GOM deposits were found. However, the sensitivity of this investigation is today questioned [14,15]. Finally, with the number of patients analyzed, strong differences between the two groups would almost certainly reject the null hypothesis. However, a greater number of patients would strengthen the power of the statistical tests. We think that the report of our experience in the diagnosis of CADASIL could be useful to physicians involved in the study of cerebral small vessel disease by highlighting the growing number of patients presenting with a phenotype closely similar to CADASIL and a high suspicion of an inherited form of small vessel disease but without NOTCH3 mutations. This group of patients might be heterogeneous from the genetic point of view and, therefore, requires a more extensive genetic study. Acknowledgments None.
Conflict of interest and sources of funding statement The study was supported by MIUR (Ministero dell’Istruzione, dell’Universita e della Ricerca), programmi di ricerca cofinanziati-2006 (MIUR 2006-prot. 2006065719), MIUR (Ministero dell’Istruzione, dell’Universit a e della Ricerca), programmi di ricerca cofinanziati-2009 (MIUR 2009-prot. 20095JPSNA), Regione Toscana, programma per la Ricerca Regionale in Materia di Salute, 2009 (project ‘Evaluation of NOTCH3 mutations and correlation with clinical phenotypes’ Prot. reg. AOO GRT 190899/Q.20.70.20). S. Nannucci, F. Pescini, B. Bertaccini, S. Bianchi, L. Ciolli, R. Valenti, and M. T. Dotti report no disclosure. A. Federico is Editor-in-Chief of Neurological Sciences. D. Inzitari has served on a scientific advisory board for SERVIER, serves on the editorial board of Stroke, and has received speaker honoraria from Bayer Schering Pharma, Novartis, Pfizer Inc., and Sanofi-Aventis. L. Pantoni serves on the editorial boards of Acta Neurologica Scandinavica and Cerebrovascular Diseases and as Vascular Cognitive Impairment Section Editor for Stroke.
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