Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro
Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro
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DOI：
10.1161/strokeaha.114.007472
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发表时间：
2015-03-01
期刊：
影响因子：
8.3
通讯作者：
Duering, Marco
中科院分区：
文献类型：
--
作者：
Wollenweber, Frank Arne;Hanecker, Patrizia;Duering, Marco
背景和目的--NOTCH3基因突变导致常染色体显性遗传性脑动脉病合并皮质下梗塞和白质脑病(CADASIL)，这是导致中风和血管性痴呆的最常见的单基因原因。半胱氨酸影响突变引起的NOTCH3蛋白的错误折叠和聚集被认为是致病的关键机制。方法：通过标准化病史、临床检查、核磁共振、皮肤活检超微结构分析和所有NOTCH3外显子的Sanger测序，我们研究了一个遗传性小血管疾病家系。此外，我们利用重组蛋白片段和单颗粒聚集分析对NOTCH3变异进行了体外鉴定。结果-我们在4个家庭成员中发现了一个新的半胱氨酸节约型NOTCH3突变(D80G)，这在一个健康的兄弟姐妹中是缺失的。所有突变携带者都表现出CADASIL典型的脑成像和临床表型，而皮肤活检结果不一致。与影响半胱氨酸的突变相比，D80G突变体的体外聚集行为相似。这是从先前报道的表型与CADASIL一致的家系中复制的，保留半胱氨酸的突变与CADASIL的表型一致。结论-我们的发现支持这样的观点，即D80G等保留半胱氨酸的突变可能导致CADASIL的表型与半胱氨酸突变基本无法区分。对非典型NOTCH3突变的体外聚集分析为病理机制提供了新的见解，并可能成为评估其临床意义的工具。
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 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.