CADASIL mutations enhance spontaneous multimerization of NOTCH3

Opherk, Christian; Duering, Marco; Peters, Nils; Karpińska, Anna; Rosner, Stefanie; Schneider, Edgar W.; Bader, Benedikt; Giese, Armin; Dichgans, Martin

doi:10.1093/hmg/ddp211

Cited by 74 publications

(70 citation statements)

References 31 publications

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“…It has therefore been proposed that mutant proteins may promote aggregate formation via their unpaired sulfhydryl groups. 8 We were recently able to reproduce the spontaneous multimerization of mutant NOTCH3 in vitro using recombinant NOTCH3 proteins and a singleparticle detection assay. 9 This technique allows studying the propensity to form multimers on a molecular level in a highly controlled setting.…”

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confidence: 99%

Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro

Wollenweber

Hanecker

Bayer-Karpinska

et al. 2015

Stroke

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Background and Purpose-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

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confidence: 99%

Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro

Wollenweber

Hanecker

Bayer-Karpinska

et al. 2015

Stroke

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“…Typical CADASIL mutations involve the addition or elimination of a cysteine residue in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, resulting in mismatched disulphide bridging and altered protein function, a hypothesis which has been borne out by observational and functional studies [37, 38]. Under the current American College of Medical Genetics and Genomics (ACMG) guidelines for variant classification, functional studies supporting a damaging effect for a variant on gene function constitute strong evidence for pathogenicity.…”

Section: Discussionmentioning

confidence: 99%

Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients

et al. 2016

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BackgroundCerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic, hereditary, small vessel disease of the brain causing stroke and vascular dementia in adults. CADASIL has previously been shown to be caused by varying mutations in the NOTCH3 gene. The disorder is often misdiagnosed due to its significant clinical heterogeneic manifestation with familial hemiplegic migraine and several ataxia disorders as well as the location of the currently identified causative mutations. The aim of this study was to develop a new, comprehensive and efficient single assay strategy for complete molecular diagnosis of NOTCH3 mutations through the use of a custom next-generation sequencing (NGS) panel for improved routine clinical molecular diagnostic testing.ResultsOur custom NGS panel identified nine genetic variants in NOTCH3 (p.D139V, p.C183R, p.R332C, p.Y465C, p.C597W, p.R607H, p.E813E, p.C977G and p.Y1106C). Six mutations were stereotypical CADASIL mutations leading to an odd number of cysteine residues in one of the 34 NOTCH3 gene epidermal growth factor (EGF)-like repeats, including three new typical cysteine mutations identified in exon 11 (p.C597W; c.1791C>G); exon 18 (p.C977G; c.2929T>G) and exon 20 (p.Y1106C; c.3317A>G). Interestingly, a novel missense mutation in the CACNA1A gene was also identified in one CADASIL patient. All variants identified (novel and known) were further investigated using in silico bioinformatic analyses and confirmed through Sanger sequencing.ConclusionsNGS provides an improved and effective methodology for the diagnosis of CADASIL. The NGS approach reduced time and cost for comprehensive genetic diagnosis, placing genetic diagnostic testing within reach of more patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s40246-016-0093-z) contains supplementary material, which is available to authorized users.

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“…When the multimerization behavior of NOTCH3-EGF 1-5 a fragment encompassing the mutational hot spot for disease-associated mutations, was analyzed, mutant NOTCH3 showed a much stronger tendency to self-aggregate than the wild-type protein. 89 Moreover, mutant NOTCH3 was shown to form mixed multimers with wild-type NOTCH3 and Thrombospondin-2, a known interactor of NOTCH3, providing direct evidence for a pathologic co-aggregation mechanism. 90 These findings were recently extended by the demonstration of a co-aggregation between mutant NOTCH3 and latent TGF--binding protein (LTBP-1), a key regulator of the TGF-pathway (see below).…”

Section: Cerebral Autosomal Dominant Arteriopathy With Subcortical Inmentioning

confidence: 98%

Genetic factors in cerebral small vessel disease and their impact on stroke and dementia

Haffner

Malik

Dichgans

2015

J Cereb Blood Flow Metab

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Cerebral small vessel disease (SVD) is among the most frequent causes of both stroke and dementia. There is a growing list of genes known to be implicated in Mendelian forms of SVD. Also, genome-wide association studies have identified common variants at a number of genetic loci that are associated with manifestations of SVD, among them loci for white matter hyperintensities, small vessel stroke, and deep intracerebral hemorrhage. Driven by these discoveries and new animal models substantial progress has been made in elucidating the molecular, cellular, and physiologic mechanisms underlying SVD. A major theme emerging from these studies is the extracellular matrix (ECM). Recent findings include a role of structural constituents of the ECM such as type IV collagens in hereditary and sporadic SVD, the sequestration of proteins with a known role in ECM maintenance into aggregates of NOTCH3, and altered signaling through molecules known to interact with the ECM. Here, we review recent progress in the identification of genes involved in SVD and discuss mechanistic concepts with a particular focus on the ECM.

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CADASIL mutations enhance spontaneous multimerization of NOTCH3

Cited by 74 publications

References 31 publications

Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro

Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro

Targeted next generation sequencing identifies novel NOTCH3 gene mutations in CADASIL diagnostics patients

Genetic factors in cerebral small vessel disease and their impact on stroke and dementia

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