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A novel cysteine-sparing G73A mutation of NOTCH3 in a Chinese CADASIL family

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Abstract

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common monogenic disease leading to stroke and vascular dementia. CADASIL is an inherited small blood vessel disease caused by mutations in the gene encoding the neurogenic locus notch homolog protein 3 (NOTCH3). NOTCH3 is large type I membrane receptor mainly expressed in vascular smooth muscle cells and pericytes. Most identified mutations result in insert or deletion of a cysteine residue within the EGF-like repeats. To date, some cases with a cysteine-sparing mutant have been described. Genetic analysis revealed a novel mutation in NOTCH3 in a CADASIL family. Molecular analysis revealed its potential pathogenic mechanism in causing CADASIL. In this paper, we present a Chinese family with a novel cysteine-sparing mutation in exon 3 (c.218G>C, p.G73A) of the NOTCH3 gene. Family carriers of the same mutation presented with symptoms and imaging abnormalities characteristic of CADASIL. The location of glycine 73 in between C5-C6 disulfide bond of EGF-like domain 1 shows high conservation from humans to zebra fish. It has previously been suggested that the aggregate-prone property of mutant NOTCH3 contributes to a cytotoxic effect in the pathogenic mechanism underlying CADASIL. Here, we investigated the pathogenic mechanism of the new mutation in vitro using HEK293 cells transfected with either a wild-type (WT) or c.218G>C (p.G73A) NOTCH3ECD plasmids, and we found p.G73A NOTCH3ECD was more prone to form aggregation and resistant to degradation. Moreover, the p.G73A NOTCH3ECD compromised cell viability by promoting apoptosis. Two known CADASIL mutants R133C and R75P showed similar results with G73A mutants. Our study here identified G73A as a new mutation in NOTCH3 to cause CADASIL and revealed that the G73A mutation and two known mutants R75P and R133C decreased NOTCH3 protein turnover and induced cell death.

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Abbreviations

ANOVA :

analysis of variance

CADASIL :

cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

CCK8 :

Cell Counting Kit-8

CHD :

coronary heart disease

CHX :

chlorhexidine

DAPI :

4′,6′-diamino-2-phenylindole

DM :

diabetes mellitus

DSL :

Delta/Serrate/Lag-2

ECD :

extracellular domain

ECM :

extracellular matrix

EGF-like repeats :

epidermal growth factor-like repeats

ER :

endoplasmic reticulum

FCM :

flow cytometry

FITC :

fluorescein isothiocyanate

FLAIR:

fluid-attenuated inversion recovery magnetic resonance imaging

GOM :

granular osmiophilic material

HAMA:

Hamilton Anxiety Scale

HEK293 :

human embryonic kidney 293 cells

ICD :

intracellular domain

IGT :

impaired glucose tolerance

MRI :

magnetic resonance imaging

MMSE :

mini-mental state examination

NOTCH3 :

neurogenic locus notch homolog protein 3

PBS :

phosphate-buffered saline

PI :

propidium iodide

SWI :

susceptibility weighted imaging

UPR :

unfolded protein response

VSMCs :

vascular smooth muscle cells

MH :

T2-weighted white matter hyperintensity

WT :

wild type

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Acknowledgements

We thank Dr. Weihong Song for his instructive suggestions.

Availability of data and materials

All data generated or analyzed during this study are included in this published article (and as supplement original data.pdf).

Funding

This study was supported by grants from NSFC (81771155) to Xiulian Sun and grants from Shandong Key Research Project (2018GSF118104) to Hongchun Wang.

Author information

Authors and Affiliations

  1. Department of Neurology, Qilu Hospital of Shandong University, No. 107 West Wenhua Road, Jinan, 250012, Shandong Province, China

    Liyan Huang, Wei Li & Yi Li

  2. Brain Research Institute, Qilu Hospital of Shandong University, No. 107 West Wenhua Road, Jinan, 250012, Shandong Province, China

    Xiulian Sun

  3. Department of Neurology, Qingdao Municipal Hospital, No.1 Jiaozhou Rd, Qingdao, 266011, Shandong Province, China

    Wei Li

  4. Otolarygology Key Lab of National Health Committee, Qilu Hospital of Shandong University, No. 107 West Wenhua Road, Jinan, 250012, Shandong Province, China

    Pin Wang

  5. Department of Neurology, Secondary Hospital of Shandong University, No. 247 Beiyuanda St, Jinan, 250010, Shandong Province, China

    Chaoyuan Song

  6. Department of Clinical Laboratory, Qilu Hospital of Shandong University, No. 107 West Wenhua Road, Jinan, 250012, Shandong Province, China

    Hongchun Wang

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  1. Liyan Huang
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Contributions

L. H. and X. S. designed the experiments and wrote the manuscript. W. L. collected the clinical data. L. H. performed the molecular studies. Y. L., C. S., P. W., and H. W. helped in preparing the materials. All authors read and approved the final manuscript.

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Correspondence to Xiulian Sun.

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Study protocols were approved by the Medical Ethical Committee of Qingdao Municipal Hospital (2016-002). All persons gave their informed consent prior to their inclusion in the study.

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The authors declare that they have no conflict of interest.

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Huang, L., Li, W., Li, Y. et al. A novel cysteine-sparing G73A mutation of NOTCH3 in a Chinese CADASIL family. Neurogenetics 21, 39–49 (2020). https://doi.org/10.1007/s10048-019-00592-3

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  • DOI: https://doi.org/10.1007/s10048-019-00592-3

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