Response to Letter Regarding Article, "Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro".
Response to Letter Regarding Article, "Cysteine-Sparing CADASIL Mutations in NOTCH3 Show Proaggregatory Properties In Vitro".
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对有关文章“NOTCH3 中保留半胱氨酸的 CADASIL 突变显示体外聚合特性”的信件的回复
DOI：
10.1161/strokeaha.115.009473
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发表时间：
2015
期刊：
影响因子：
8.3
通讯作者：
Düring M
中科院分区：
文献类型：
--
作者：
Wollenweber F;Haffner C;Düring M
我们感谢Rutten等人对我们关于大脑常染色体显性动脉病变伴皮层下梗死和白质脑病（CADASIL）中保留半胱氨酸的NOTCH3突变的工作的评论。2我们完全同意非典型突变患者的诊断检查需要一个经验丰富的CADASIL团队，并且需要纳入所有可用的信息，包括医疗和家族史、临床和神经心理学检查、活检结果、神经影像学数据以及至少所有编码EGF重复序列的NOTCH3外显子的测序。在这方面，大多数先前发表的关于半胱氨酸保留突变的报告是不完整的。因此，这些研究中的CADASIL诊断确实值得质疑。与此一致，我们的体外聚集分析表明，在以前报道的半胱氨酸保留突变之间存在异质性。因此，我们非常小心地为我们家庭中的患者提供全面和高质量的检查。CADASIL的诊断是在经过彻底的临床、分子和病理检查后做出的。结果基本符合Rutten等人推荐的标准。3此外，还纳入了基因组数据库（如Exome Sequencing Project）的信息，以估计所鉴定的D80G突变的群体频率。频率大大超过CADASIL频率的变异不太可能代表致病突变。不幸的是，在我们的研究中无法对NOTCH3聚集体进行免疫染色。然而，应该注意的是，免疫染色也被认为是诊断CADASIL的支持而不是明确的，因为它的灵敏度约为85%至90%。综上所述，我们认为，毫无疑问，我们研究中的患者确实患有CADASIL。有趣的是，颗粒性亲锇物质仅在1例患者中检测到。尽管颗粒状亲渗物质是CADASIL的典型致病物质，NOTCH3聚集被认为是发病的关键因素，但血管壁内的毒性机制尚不清楚。正如我们在文章中所讨论的，可以检测到的颗粒状亲锇物质的大沉积物可能仅仅代表NOTCH3聚集过程的终点，而在疾病发病机制中没有关键作用。正如在阿尔茨海默病等神经退行性疾病中所证明的那样，聚集过程中的中间体，而不是最终产物，可能代表有毒物质。因此，在没有可检测到的颗粒亲锇物质的情况下，CADASIL的发展并非完全不可想象。因此，我们认为我们的体外单颗粒聚集试验在分类新变异及其临床意义方面增加了有价值的信息。值得注意的是，我们建议将单颗粒聚集试验作为评估NOTCH3突变致病性的额外工具，而不是替代皮肤活检。我们同意Rutten等人的观点，即单颗粒聚集分析数据的重要性只能与使用上述工具进行全面和高质量的诊断检查相结合进行评估。最终，在缺乏金标准的情况下，收集临床、神经影像学、活检和单颗粒聚集测定信息的NOTCH3突变（典型和非典型）公共数据库可能有助于进一步阐明保留半胱氨酸突变的作用，并促进诊断决策。
We thank Rutten et al1 for their comments on our work on cysteine-sparing NOTCH3 mutations in cerebral autosomal-dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL). 2 We fully agree that the diagnostic workup of patients with atypical mutations requires an experienced CADASIL team and needs to incorporate all available information including medical and family history, clinical and neuropsychological examination, biopsy results, neuroimaging data, and sequencing of at least all NOTCH3 exons encoding EGF repeats. In that respect, the majority of previously published reports on cysteine-sparing mutations were incomplete. Hence, the CADASIL diagnosis in some of these studies can indeed be questioned. In accord with this, our in vitro aggregation assay suggests heterogeneity among previously reported cysteine-sparing mutations. Therefore, we took great care to provide a comprehensive and high-quality workup of the patients within our family. The diagnosis CADASIL was made only after a thorough clinical, molecular, and pathological examination. Results largely met the criteria recommended by Rutten et al. 3 In addition, information from genome databases such as the Exome Sequencing Project was included to estimate the population frequency of the identified D80G mutation. Variants with a frequency largely exceeding the frequency of CADASIL are unlikely to represent a disease-causing mutation. Unfortunately, immunostaining of NOTCH3 aggregates could not be performed in our study. It should, however, be noted that immunostaining is also considered to be only supportive rather than definite for diagnosing CADASIL owing to its sensitivity of≈ 85% to 90%. 4 In conclusion, in our view, little doubt remains that the patients presented in our study truly have CADASIL. Interestingly, granular osmiophilic material could be detected in only 1 patient. Although granular osmiophilic material is pathognomonic for CADASIL and NOTCH3 aggregation is considered a key factor in the pathogenesis, the mechanisms of toxicity within vascular walls are poorly understood. As discussed in our article, large deposits detectable as granular osmiophilic material might merely represent an end point in the NOTCH3 aggregation process without a key role in disease pathogenesis. As demonstrated in neurodegenerative diseases such as Alzheimer disease intermediates in the aggregation process, not the end product, might represent the toxic agent. 5 Thus, the development of CADASIL in the absence of detectable granular osmiophilic material is not completely inconceivable. Therefore, we feel that our in vitro single particle aggregation assay adds valuable information when categorizing novel variants with respect to their clinical significance. It is important to note that we propose our single particle aggregation assay as an additional tool to assess the pathogenicity of NOTCH3 mutations, not as replacement for a skin biopsy. We agree with Rutten et al3 that the significance of single particle aggregation assay data can only be evaluated in combination with a thorough and high-quality diagnostic workup using the tools discussed above.Ultimately, in the absence of a gold standard, a public database of NOTCH3 mutations (typical and atypical) collecting clinical, neuroimaging, biopsy and perhaps single particle aggregation assay information could help to further elucidate the role of cysteine-sparing mutations and facilitate diagnostic decision-making.
Rutten 等人关于文章“NOTCH3 中保留半胱氨酸的 CADASIL 突变显示体外聚合特性”的信函。
影响因子：
8.3
作者：
J. Rutten;S. V. van Duinen;S. L. Lesnik Oberstein
通讯作者：
S. L. Lesnik Oberstein