University College London, London, UK.
Arthritis Rheumatol. 2014 Jan;66(1):197-202. doi: 10.1002/art.38217.
To identify the genetic cause of chronic infantile neurologic, cutaneous, articular syndrome (CINCA syndrome) using whole-exome sequencing in a child who had typical clinical features but who was NLRP3 mutation negative based on conventional Sanger sequencing.
We performed whole-exome sequencing on DNA from peripheral blood, using Illumina TruSeq Exome capture and the HiSeq sequencing platform. Exome data were analyzed in the Galaxy Web-based suite. Whole-exome sequencing findings were confirmed by massively parallel sequencing.
Analysis of variants in known autoinflammatory genes led to the identification of the pathogenic p.F556L NLRP3 missense mutation in 17.7% of Illumina reads (25 of 141). No new candidate genes were identified. Massively parallel sequencing of DNA from peripheral blood (performed in duplicate) unequivocally confirmed the presence of this mutation in 14.5% of alleles. Reexamination of the original Sanger chromatograms revealed a small peak at nucleotide position c.1698 corresponding to the mutated allele. This had initially been regarded as background noise, but in retrospect is completely consistent with somatic mosaicism for the p.F556L NLRP3 mutation in this child with CINCA syndrome.
This is the first description of somatic NLRP3 mosaicism detected using whole-exome sequencing in a "mutation-negative" patient with CINCA syndrome. Our findings suggest that whole-exome sequencing could be an important diagnostic tool for detecting somatic mosaicism, as well as for the discovery of novel causative gene mutations, in patients with clinical features of cryopyrin-associated periodic syndromes who are NLRP3 mutation negative by conventional sequencing. This approach could also be applicable to patients with other autosomal-dominant autoinflammatory diseases characterized by gain-of-function mutations who are mutation negative by conventional Sanger sequencing.
通过对一名具有典型临床特征但 NLRP3 突变阴性的患儿进行全外显子组测序，以确定慢性婴儿神经皮肤关节综合征（CINCA 综合征）的遗传原因。
我们使用 Illumina TruSeq Exome 捕获和 HiSeq 测序平台对来自外周血的 DNA 进行全外显子组测序。在 Galaxy 基于网络的套件中分析外显子组数据。通过大规模平行测序验证全外显子组测序结果。
对已知自身炎症性疾病基因中的变异进行分析，导致在 17.7%的 Illumina 读段（141 个中的 25 个）中发现致病性 NLRP3 错义突变 p.F556L。未发现新的候选基因。对来自外周血的 DNA 进行大规模平行测序（重复进行两次），明确证实该突变在 14.5%的等位基因中存在。重新检查原始 Sanger 色谱图显示，在核苷酸位置 c.1698 处有一个对应于突变等位基因的小峰。该峰最初被认为是背景噪音，但回想起来，这与该 CINCA 综合征患儿 NLRP3 突变的体细胞镶嵌完全一致。
这是首次在 CINCA 综合征“突变阴性”患者中使用全外显子组测序检测体细胞 NLRP3 镶嵌的描述。我们的发现表明，全外显子组测序可能是一种重要的诊断工具，可用于检测体细胞镶嵌，以及发现 NLRP3 突变阴性的 Cryopyrin 相关周期性综合征患者的新致病基因突变。这种方法也可应用于其他以功能获得性突变为特征的常染色体显性自身炎症性疾病的患者，这些患者的 NLRP3 突变阴性。
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