Neurobiology of Aging 36 (2015) 547.e5e547.e11

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Hypomorphic NOTCH3 mutation in an Italian family with CADASIL features Marcello Moccia a, Lorena Mosca b, Roberto Erro c, Mariarosaria Cervasio d, Roberto Allocca a, Carmine Vitale e, f, Antonio Leonardi g, Ferdinando Caranci h, Maria Laura Del Basso-De Caro d, Paolo Barone i, Silvana Penco b, * a

Department of Neuroscience, Reproductive Science and Odontostomatology, Federico II University, Naples, Italy Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca’Granda Hospital, Milan, Italy Sobell Department of Motor Neuroscience and Movement Disorders, University College London (UCL) Institute of Neurology, Queen Square, London, UK d Department of Advanced Biomedical Sciences, Anatomopathology Unit, Federico II University, Naples, Italy e Department of Motor Sciences, University of Naples “Parthenope,” Naples, Italy f Istituto di Diagnosi e Cura (IDC) Hermitage-Capodimonte, Naples, Italy g Department of Molecular and Biotechnological Medicine, Federico II University, Naples, Italy h Department of Advanced Biomedical Sciences, Neuroradiology Unit, Federico II University, Naples, Italy i Center for Neurodegenerative Diseases (CEMAND), Neuroscience Section, Department of Medicine, University of Salerno, Salerno, Italy b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 June 2014 Received in revised form 31 July 2014 Accepted 22 August 2014 Available online 27 August 2014

The cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is because of NOTCH3 mutations affecting the number of cysteine residues. In this view, the role of atypical NOTCH3 mutations is still debated. Therefore, we investigated a family carrying a NOTCH3 nonsense mutation, with dominantly inherited recurrent cerebrovascular disorders. Among 7 family members, 4 received a clinical diagnosis of CADASIL. A heterozygous truncating mutation in exon 3 (c.307C>T, p.Arg103X) was found in the 4 clinically affected subjects and in one 27-year old lady, only complaining of migraine with aura. Magnetic resonance imaging scans found typical signs of small-vessel disease in the 4 affected subjects, supporting the clinical diagnosis. Skin biopsies did not show the typical granular osmiophilic material, but only nonspecific signs of vascular damage, resembling those previously described in Notch3 knockout mice. Interestingly, messenger RNA (mRNA) analysis supports the hypothesis of an atypical NOTCH3 mutation, suggesting a nonsense-mediated mRNA decay. In conclusion, the present study broadens the spectrum of CADASIL mutations, and, therefore, opens new insights about Notch3 signaling. Ó 2015 Elsevier Inc. All rights reserved.

Keywords: CADASIL NOTCH3 Stroke Dementia Genetic

1. Introduction The cerebral autosomal dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is the most common hereditary small-vessel disease. Its diagnosis may require a multidisciplinary approach to its clinical, radiological, pathologic, and genetic features. Although the age at onset and the clinical picture vary substantially between and within families, CADASIL is characterized

* Corresponding author at: Medical Genetics Unit, Department of Laboratory Medicine, Niguarda Ca’Granda Hospital, Piazza Ospedale Maggiore 3, 20162 Milan, Italy. Tel.: þ39 02 6444 2803; fax: þ39 02 6444 2783. E-mail address: [email protected] (S. Penco). 0197-4580/$ e see front matter Ó 2015 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.neurobiolaging.2014.08.021

by 5 main clinical features: migraine with aura, subcortical ischemic events, mood disturbances, apathy, and cognitive impairment. Migraine with aura may represent the first clinical symptom in up to 20%e40% of patients, with an average age at onset of 30 years. In addition, patients usually have recurrent strokes from the age of 40 years, progressively leading to motor and cognitive impairments. Magnetic resonance imaging (MRI) abnormalities precede the onset of clinical symptoms, involving virtually all the affected subjects by the age of 35 years and increasing as the disease progresses (Chabriat et al., 2009). CADASIL brain pathology reveals severe leukoencephalopathy, vascular smooth muscle cell (VSMC) degeneration, and granular osmiophilic material (GOM) deposits in the tunica media, close to the basal membrane of the VSMC. GOM deposits are also found in extracerebral vessels, for instance, in dermal arterioles; thus, skin biopsy has been suggested as a screening procedure in subjects

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with suspected CADASIL. This diagnostic procedure has a relatively high specificity but presents the risk of false negative patients because of difficulties in observing a sufficient number of vessels with muscular walls in skin biopsies (Malandrini et al., 2007). From a genetic perspective, CADASIL is an autosomal dominant disease caused by mutations in NOTCH3. This gene encodes a single-pass transmembrane receptor that functions at the cell surface as a heterodimer. Its extracellular domain (ECD) needs to be stabilized by the formation of disulfide bonds between cysteine residues within the 34 epidermal growth factor (EGF) repeats. NOTCH3 has 33 exons, but all CADASIL mutations apparently occur in exons 2e24 that encode the ECD, with strong clustering in exons 3 and 4 (EGF repeats 2e5), resulting in an uneven number of cysteine residues in one of the EGF repeats. This change in the number of cysteines seems to be the common denominator in CADASIL mutations and may underlie neomorphic properties with the gain of a toxic function (Chabriat et al., 2009). In particular, GOM formation could be enhanced by the gradual accumulation of the mutated NOTCH3 (Joutel, 2010). However, it is still debated if GOM deposits are part of CADASIL pathogenesis or only represent an epiphenomenon in this disease. In particular, little is known about NOTCH3 additional functions in adults, and animal models suggest that CADASIL subcortical ischemia may be related to an impaired cerebral blood flow autoregulation with consequent white matter hypoperfusion (Joutel, 2010; Joutel et al., 2010). Interestingly, those mutations altering the number of cysteine residues in the ECD of NOTCH3 can be unambiguously classified as pathogenic, whereas all the other NOTCH3 mutations (i.e., hypomorphic mutations) may well be responsible for symptoms that do not present the fully fledged phenotype of CADASIL but instead a syndrome along the spectrum of CADASIL-like disorders (Schmidt et al., 2011). In particular, Rutten et al. (2013) and we recently described a NOTCH3 nonsense mutation (exon 3, c.307C>T, p.Arg103X) in 2 different families (Erro et al., 2014). This is the first described NOTCH3 nonsense mutation. The variant is located in the EGF-like 2 region of exon 3 and could possibly produce a truncated protein lacking of EGF-like, transmembrane, and intracytoplasmic domains or not even be translated because of nonsense-mediated decay. Rutten et al. (2013) reported 2 brothers carrying this mutation and, considering the lack of significant CADASIL features in w50 year olds, suggested this NOTCH3 nonsense mutation to be a neutral variant (Rutten et al., 2013). Conversely, we previously described a

carrier of the same mutation with a clinical picture of vascular parkinsonism and cognitive impairment, an onset after 70 years, and an autosomal dominant stroke family history (Fig. 1, subject 4) (Erro et al., 2014). Considering that there are no previous investigations about the effects of NOTCH3 nonsense mutations and additional efforts are clearly required to explore the relevance of such mutation, we extended our investigations to the entire family and followed it up, to hereby report effects of this mutation on clinical features, neuroradiological imaging, pathologic findings at skin biopsy, and messenger RNA (mRNA) activity. In particular, the present study has the main aim to investigate if this nonsense NOTCH3 mutation is causing CADASIL, CADASIL-like symptoms, or an unknown disease falling into a spectrum of CADASIL-like syndromes. 2. Materials and methods 2.1. Subjects and clinical and radiological assessments The local ethical committee approved this study, and all subjects gave a written informed consent. A nonconsanguineous family from Naples (Italy) was clinically examined because of recurrent cases of cerebrovascular disease. The family consisted of 4 generations with a pattern of possible autosomal dominant inheritance (Fig. 1). All subjects were evaluated by means of the CADASIL scale, a screening tool to select subjects with high suspicion of CADASIL before genetic testing (Pescini et al., 2012). Asymptomatic subjects underwent a multidisciplinary and multistep procedure for presymptomatic diagnostic test to reduce, as much as possible, potentially harmful consequences of genetic testing (Reyes et al., 2012). Additional risk factors for cerebrovascular disease were evaluated by means of clinical records (smoking), physical examination (blood pressure and body weight), and blood tests (cholesterol and glycemia). Brain MRI studies were performed at 1.5 T. Imaging sequences included a 3-dimensional T1-weighted gradient-echo volume with isotropic voxels and axial diffusion-weighted, axial and coronal T2-weighted turbo spin-echo, and FLAIR images. 2.2. Skin biopsy Skin biopsies were performed in at least 2 different body sites by means of 3-mm skin punches after a small injection of a local anesthetic (mepivacaine 3%). Skin biopsy samples were fixed in 2.5% glutaraldehyde/0.1 M cacodylate buffer, rinsed in cacodylate buffer, postfixed in 1% osmium tetroxide/0.1 M cacodylate buffer, and rinsed again in buffer. Tissue samples were gradually dehydrated in a series of ascending concentrations of ethanol and, then, were immersed in propylene oxide before infiltration with the epoxy resin Epon 812. Ultrathin sections double stained with uranyl acetate and lead citrate were examined with Transmission Electron Microscope (Zeiss 900). 2.3. Genetic studies

Fig. 1. Pedigree of the investigated family. Squares represent males and circles females. Subjects with cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy clinical features are represented in black, c.307C>T mutation carriers are asterisked (*), nonmutation carriers are represented by the symbol “-,” the arrow indicates the proband, and the pattern of inheritance is suggestive of autosomal dominant inheritance.

DNA samples from all members were isolated from whole blood using standard procedures (Miller et al., 1988). Subjects involved in the present study gave informed consent for DNA storage and authorized the use of this material for current and possible future diagnostic and research procedures, on request. Considering the mutation (c.307C>T, p.Arg103X) identified in the proband (subject 4) (Erro et al., 2014), the recruited family members were analyzed only for this NOTCH3 mutation. The nucleotide position of mutation refers to the mRNA sequence (NM_000435). Mutation Taster (http://www.mutationtaster.org/) was used to predict the

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apparently healthy woman (71-year-old female [subject 5]); subject 6 died from colon cancer (61-year-old male); subject 7 had recurrent strokes in her 40s, presented a severe cognitive impairment, and was wheelchair dependent because of hemiparesis (67-yearold female, CADASIL scale 18/25); and subject 8 was apparently healthy (64-year-old male, CADASIL scale 2/25). We did not include subjects 3 and 6 in the present report, considering that extensive information was not available because of their death. In the third generation, we investigated siblings from subject 4; in particular, subject 9 had a history of recurrent strokes (42-year-old male, CADASIL scale 15/25) and married an apparently healthy woman, and subject 11 had a stroke at 38 years and had suffered from migraine with aura since juvenile age (39-year-old female, CADASIL scale 18/25). In the fourth generation, subject 12 suffered from migraine with aura since juvenile age (27-year-old female, CADASIL scale 6/25), subject 13 was apparently healthy (23-year-old female, CADASIL scale 3/25), and subject 14 was excluded from the present study because of ethical concerns for subjects T mutation carrier CADASIL scale items Migraine (1) Migraine with aura (3) TIA or stroke (1) TIA or stroke onset T in subjects 4, 7, 9, 11, and 12. The variant is located in the EGF-like 2 region of exon 3 and causes the substitution of arginine with a stop codon at position 103 of the protein (p.Arg103X). Therefore, the formation of such premature stop codon would result in the production of a truncated protein lacking the amino acids encoded by exon 3 and subsequent exons (4/33) and, therefore, characterized by the absence of all EGF-like repeat domains except EGF-like 1. Prediction programs hypothesize a disease-

Skin biopsies for mRNA studies were performed in 2 mutation carriers (subjects 4 and 7) and in 1 nonmutated healthy subject (subject 8). All experiment results would argue against the presence of a mutated mRNA allele that could be translated into a truncated protein and strongly suggests a nonsense-mediated decay, leaving only the wild-type allele to be amplified and sequenced. No exon 3 mutations were detected by direct sequencing of the amplified fragment. Although no qualitative differences were found between cases and control at mRNA level,

Fig. 3. Ultrastructural analysis of dermal small arteries (subject 7). (A) Endothelial cells, showing numerous protrusions bulging into the lumen of the vessels, nuclei filled with dense chromatin, and cytoplasm with electrolucent vacuoles (arrows) (electron microscope [EM] 3000). (B) Smooth muscle cell, showing irregularities in shape and presenting electrolucent vacuoles (arrowheads) within the cytoplasm, as for degenerative changes (EM 20,000). (C) Endothelial cell (detail), showing atypical nucleus and peripheral chromatin condensation (C, EM 20,000). C, collagen fibrils; E, erythrocyte; EC, endothelial cell; L, lumen; N, nucleus; SMC, smooth muscle cell.

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mutated subjects showed a weaker band corresponding to the amplified fragment, whereas a band more evident was present in the sample collected from the nonmutated healthy subject (data not presented because of the bad quality of images). Experiment results were compatible with the presence of exon 3 indicating that the identified nucleotide variant does not affect the splicing mechanism, thus excluding a typical CADASIL mutation. 4. Discussion CADASIL is caused by NOTCH3 dominant mutations leading to an uneven number of cysteine residues in one of the 34 EGF receptor domains constituting the ECD of the receptor. All other mutations should be called unclassified variants at the first observation, but significant efforts have been done to clarify whether such hypomorphic NOTCH3 mutations are neutral polymorphisms or causative for a distinct cerebrovascular entity (Joutel, 2013). In particular, NOTCH3 nonsense mutations have never been reported, although a frameshift deletion leading to a premature stop codon has been previously associated to CADASIL phenotype (Dotti et al., 2004). In this view, we explored, for the first time, clinical, radiological, pathologic, genetic, and molecular findings in a family carrying a NOTCH3 nonsense mutation in exon 3 (c.307C>T, p.Arg103X). First of all, it is important to remember that CADASIL is defined by several specific symptoms and traits, must affect the brain, and be dominantly inherited (Schmidt et al., 2011). In the present family, the clinical picture was highly suggestive of CADASIL, with autosomal dominant inheritance and a typical timeline, clearly shown by the progressive increase of CADASIL scale scores through generations (Table 1) (Chabriat et al., 2009; Pescini et al., 2012). In particular, migraine with aura was reported by subjects 9 and 11 and was the only manifestation in the youngest mutation carrier (subject 9). Moreover, a history of recurrent strokes was present in all mutation carriers after the age of 40 years. Finally, progressive cognitive and motor impairments were recorded in elderly members (parkinsonism with mood disturbances and apathy in subject 4 and dementia and hemiparesis in subject 7) (Erro et al., 2014). Notably, no significant cerebrovascular risk factors were reported. In addition to this, MRI scans of the 4 definitely affected subjects showed cerebrovascular signs in the external capsule, in the anterior part of temporal lobes, in periventricular areas, and in the centrum semiovale, whereas U fibres and cortex were relatively spared, as typically described in CADASIL (Auer et al., 2001; Cirillo et al., 2012; O’Sullivan et al., 2001; Yousry et al., 1999). Although in advanced phases such signs become more diffused, white matter hyperintensities have been described in CADASIL patients since the age of 21 years (Chabriat et al., 2009; Chawda et al., 2000). However, subject 12 (27-year-old female) presented no significant abnormalities on MRI. This is possibly because of CADASIL phenotype variability or, at least, of gender differences with females less prone to larger subcortical infarcts (Gunda et al., 2012). At skin biopsy, in contrast with the typical neuropathology related to NOTCH3 mutations affecting cysteine residues in the ECD, we failed to show any GOM deposits. The risk of false negative has been strongly minimized by the number of subjects receiving 2 different skin biopsies and by the observation of at least 20 vessels per biopsy. Therefore, we may hypothesize that if GOM formation is enhanced by NOTCH3 with uneven cysteine residues, the p.Arg103X nonsense mutation is not associated with GOM because of the absence of unstable Notch3. Indeed, noncanonical NOTCH3 mutations have already been described in subjects with hereditary small-vessel disease of the brain without GOM deposits and Notch3 accumulation (Fouillade et al., 2008),

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suggesting that the absence of GOM deposits in our patients could be related to the specific truncating mutation carried by this family. However, we could identify nonspecific VSMC alterations in all mutation carriers. Interestingly, the structural and functional integrities of small arteries are impaired in knockout mice for Notch3 (Notch3/), with increased susceptibility to ischemic brain injury. These mice are known to present marked defects in distal muscular arteries, particularly in the cerebral ones, including enlarged arteries with a thinner muscular coat and abnormal shape and smaller size of smooth muscle cells, in the absence of GOM accumulation (Joutel, 2010). This pattern might in fact resemble the neuropathologic findings in the present family; thus, a pathogenic mechanism not involving GOM accumulation should be hypothesized, at least for hypomorphic NOTCH3 mutations, and could possibly explain the increased clinical heterogeneity of CADASIL-like syndromes (Malandrini et al., 2007; Wallays et al., 2011). At genetic studies, CADASIL mutations typically result in an odd number of cysteine residues, with 1 residue subsequently unpaired. Even if CADASIL mutations have traditionally been considered to result in a gain of function, the molecular mechanisms leading to the development of NOTCH3-related diseases remain elusive (Joutel, 2010; Joutel et al., 2010). In addition, Notch3 has a noticeable ability to recruit coactivators and/or corepressors and to undergo different conformational changes, and it is also unclear whether the possible relationships between Notch3 and other molecules/pathways are responsible for the pathogenesis of NOTCH-related diseases (Bellavia et al., 2008; Opherk et al., 2006; Penton et al., 2012). In our case, considering our preliminary mRNA analysis and prediction program results, the stop codon introduced at amino acid position 103 causes the substitution of arginine with a stop codon, possibly leading to a nonsense-mediated mRNA decay. Therefore, a loss-of-function mechanism cannot be excluded, in particular for CADASIL-like syndromes with noncanonical NOTCH3 mutations. We must acknowledge some limitations and, in particular, are aware that some loss-of-function alleles are tolerated and not disease causing, and the lack of quantitative studies on mRNA and of functional studies on Notch3 protein activity limits our conclusions. Considering that subjects did not consent to muscle biopsy, we performed our analyses on skin biopsy samples that only provided a limited quantity of material for processing. In addition, prediction software is usually less accurate in the presence of a truncated protein (Schwarz and Seelow, 2013). However, we performed a preliminary mRNA analysis that suggests a possible nonsensemediated mRNA decay. In a recent review, Rutten et al. (2013) stated that variants in NOTCH3 leading to loss of NOTCH3 function (i.e., frameshift and stop mutations) do not cause CADASIL until proven otherwise (Arboleda-Velasquez et al., 2011; Rutten et al., 2014), in line with the family they previously described. However, Rutten et al. (2013) described 2 clinically unaffected siblings in their 50s carrying the NOTCH3 mutation, but these individuals could still develop the disease particularly because this same mutation is associated in the proband of the present study with an age at onset of atypical symptoms of 72 years. By contrast, in our family, there is a strict cosegregation of the mutation with CADASIL features, well explained by clinical and MRI findings, thus indicating that this is a causative mutation. To account both families, we may hypothesize that nontypical NOTCH3 mutations may originate a CADASIL-like syndrome without the full spectrum of signs of CADASIL and with different clinical phenotypes presenting variable penetrance and/or age at onset. It is worthwhile to report that there is a significant variability in the clinical picture of CADASIL also for typical mutations, as recently shown in a subject with clinical Alzheimer disease, carrying a typical

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NOTCH3 mutation (Guerreiro et al., 2012). Clinical variability for different mutations in the same gene is well known for several neurological disorders, and, for instance, it has already been studied for point mutations in PRNP gene that are typically associated with prion diseases but have been reported also in cases of Alzheimer’s disease, systemic amyloidosis, and autonomic dysfunctions, not fitting the typical prion disease phenotype (Guerreiro et al., 2014; Jansen et al., 2010; Jayadev et al., 2011; Mead et al., 2013). 5. Conclusions We described clinical, radiological, and pathological features of an extended family carrying a nonsense NOTCH3 mutation. Considering our findings, the p.Arg103X nonsense mutation seems to be responsible of a CADASIL-like syndrome and may play a greater role in ischemic stroke than previously thought (Erro et al., 2014; Joutel, 2013; Moccia et al., 2014; Ross et al., 2013; Testi et al., 2012). In addition, hypomorphic mutations in NOTCH3 may possibly be responsible for nontypical CADASIL pathology, and further investigations should be specifically addressed. The present study broadens the spectrum of CADASIL mutations and, therefore, potentially enhances our understanding of the Notch3 signaling and of the underlying disease mechanism. Disclosure statement On behalf of all the authors, the corresponding author states that there is no conflict of interest or potential financial disclosures. Funding/support: The present study received no specific support. Acknowledgements Dr Moccia, Dr Cervasio, and Dr Allocca received grants from the “Federico II” University of Naples, Italy. Dr Mosca and Dr Penco received salary from Niguarda Ca’Grande Hospital, Milan, Italy. Dr Erro received grants from the University of Verona, Italy, and honoraria from the University College London Institute, London, UK. Dr Vitale received salary from the University Parthenope, Naples, Italy; honoraria from IDC Hermitage Capodimonte, Naples, Italy; and honoraria for symposia from Boehringer Ingelheim, Lundbeck, Novartis, and Schwarz Pharma/UCB. Dr Leonardi, Dr Caranci, and Dr Del Basso-De Caro received salarium from the “Federico II” University of Naples, Italy. Dr Barone received salarium from the University of Salerno, Italy; honoraria as a Consultant and Advisory Board Memberships for Novartis, Schwarz Pharma/UCB, Merck-Serono, Eisai, Solvay, General Electric and Lundbeck; and research support from Boehringer Ingelheim, Novartis, Schwarz Pharma/UCB, Merck-Serono, Solvay, and Lundbeck. References Arboleda-Velasquez, J.F., Manent, J., Lee, J.H., Tikka, S., Ospina, C., Vanderburg, C.R., Frosch, M.P., Rodríguez-Falcón, M., Villen, J., Gygi, S., Lopera, F., Kalimo, H., Moskowitz, M.A., Ayata, C., Louvi, A., Artavanis-Tsakonas, S., 2011. Hypomorphic Notch 3 alleles link Notch signaling to ischemic cerebral small-vessel disease. Proc. Natl. Acad. Sci. U. S. A 108, E128eE135. Auer, D.P., Pütz, B., Gössl, C., Elbel, G., Gasser, T., Dichgans, M., 2001. Differential lesion patterns in CADASIL and sporadic subcortical arteriosclerotic encephalopathy: MR imaging study with statistical parametric group comparison. Radiology 218, 443e451. Bellavia, D., Checquolo, S., Campese, A.F., Felli, M.P., Gulino, A., Crepanti, I., 2008. Notch3: from subtle differences to functional diversity. Oncogene 27, 5092e5098. Chabriat, H., Joutel, A., Dichgans, M., Tournier-Lasserve, E., Bousser, M.G., 2009. CADASIL. Lancet Neurol. 8, 643e653. Chawda, S.J., De Lange, R.P., Hourihan, M.D., Halpin, S.F., St Clair, D., 2000. Diagnosing CADASIL using MRI: evidence from families with known mutations of Notch 3 gene. Neuroradiology 42, 249e255.

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