title: Cysteine-sparing CADASIL mutations in NOTCH3 show proaggregatory properties in vitro.
abstract: OBJECTIVE
  Mutations in NOTCH3 cause cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), the most common monogenic cause of stroke and vascular dementia. Misfolding and aggregation of NOTCH3 proteins triggered by cysteine-affecting mutations are considered to be the key disease mechanisms. However, the significance of cysteine-sparing mutations is still debated.
  
  
  METHODS
  We studied a family with inherited small vessel disease by standardized medical history, clinical examination, MRI, ultrastructural analysis of skin biopsies, and Sanger sequencing of all NOTCH3 exons. In addition, we performed in vitro characterization of NOTCH3 variants using recombinant protein fragments and a single-particle aggregation assay.
  
  
  RESULTS
  We identified a novel cysteine-sparing NOTCH3 mutation (D80G) in 4 family members, which was absent in a healthy sibling. All mutation carriers exhibited a CADASIL typical brain imaging and clinical phenotype, whereas skin biopsy showed inconsistent results. In vitro aggregation behavior of the D80G mutant was similar compared with cysteine-affecting mutations. This was reproduced with cysteine-sparing mutations from previously reported families having a phenotype consistent with CADASIL.
  
  
  CONCLUSIONS
  Our findings support the view that cysteine-sparing mutations, such as D80G, might cause CADASIL with a phenotype largely indistinguishable from cysteine mutations. The in vitro aggregation analysis of atypical NOTCH3 mutations offers novel insights into pathomechanisms and might represent a tool for estimating their clinical significance.
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Frank Arne
authors.initials: FA
authors.lastname: Wollenweber
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Patrizia
authors.initials: P
authors.lastname: Hanecker
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Anna
authors.initials: A
authors.lastname: Bayer-Karpinska
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Rainer
authors.initials: R
authors.lastname: Malik
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Hansjörg
authors.initials: H
authors.lastname: Bäzner
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Fiona
authors.initials: F
authors.lastname: Moreton
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Keith W
authors.initials: KW
authors.lastname: Muir
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Susanna
authors.initials: S
authors.lastname: Müller
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Armin
authors.initials: A
authors.lastname: Giese
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Christian
authors.initials: C
authors.lastname: Opherk
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Martin
authors.initials: M
authors.lastname: Dichgans
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.).
authors.forename: Christof
authors.initials: C
authors.lastname: Haffner
authors.affiliation: From the Institute for Stroke and Dementia Research, Klinikum der Universität München (F.A.W., P.H., A.B.-K., R.M., C.O., M.D., C.H., M.D.), Institute for Pathology (S.M.), and Center for Neuropathology and Prion Research (A.G.), Ludwig-Maximilians-University, Munich, Germany; Department of Neurology, Klinikum Stuttgart, Stuttgart, Germany (H.B.); Institute of Neuroscience and Psychology, University of Glasgow, Glasgow, United Kingdom (F.M., K.W.M.); Department of Neurology, Klinikum am Gesundbrunnen, SLK-Kliniken, Heilbronn, Germany (C.O.); and Munich Cluster for Systems Neurology (SyNergy), Munich, Germany (M.D.). marco.duering@med.uni-muenchen.de.
authors.forename: Marco
authors.initials: M
authors.lastname: Duering
mesh_terms: D000368:Aged; D001706:Biopsy; D046589:CADASIL / Q000235:genetics*; D003545:Cysteine / Q000235:genetics*; D005260:Female; D006801:Humans; D008279:Magnetic Resonance Imaging; D008297:Male; D009154:Mutation; D011485:Protein Binding; D017510:Protein Folding; D000071656:Receptor, Notch3; D051880:Receptors, Notch / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012867:Skin / Q000648:ultrastructure
chemical_list: C499374:NOTCH3 protein, human; D000071656:Receptor, Notch3; D051880:Receptors, Notch; D003545:Cysteine
keywords: CADASIL; NOTCH3 protein, human; cerebral small vessel disease; genetic testing; recombinant proteins