The NLRP3 p.A441V Mutation in NLRP3-AID Pathogenesis: Functional Consequences, Phenotype-Genotype Correlations and Evidence for a Recurrent Mutational Event
The NLRP3 p.A441V Mutation in NLRP3-AID Pathogenesis: Functional Consequences, Phenotype-Genotype Correlations and Evidence for a Recurrent Mutational Event
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DOI：
10.1002/acr2.1039
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发表时间：
2019-06-01
影响因子：
3.4
通讯作者：
Amselem, Serge
中科院分区：
文献类型：
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作者：
Awad, Fawaz;Assrawi, Eman;Amselem, Serge
目标。目的：在一个患有Muckle-Wells综合征的多代法国家庭和一个来自葡萄牙的家族性冷自炎综合征患者中，确定自体炎症综合征的分子和细胞基础。方法：研究方法。对所有患者进行NLRP3外显子3测序。微卫星和全基因组单核苷酸多态基因分型用于检测已识别的变异的家庭内分离，以及ii)寻找创始人效应。功能分析包括：i)在HEK293T细胞中瞬时表达野生型或突变的NLRP3蛋白(稳定表达ASC-绿色荧光蛋白和前caspase 1-FLAG)的凋亡相关SPEC样蛋白(ASC)SPECK形成；ii)转THP-1细胞分泌IL-1β的水平；以及iii)从危象患者(来自两个家族的先证者)、相关脱危患者和对照组分离的单核细胞中炎症相关基因的表达和细胞因子的分泌。结果。在两个家系中发现了位于Nacht结构域的相同杂合突变(c.1322C>；T，p.A441V)，与第一个家系中的疾病分离。该突变被发现与不同的核心单倍型有关。NLRP3-A441V导致ASC斑点形成增加，分泌高水平的IL-1β。在脱离危象的患者中，单核细胞炎症体相关基因的表达和细胞因子的分泌在正常范围内，发现两个先证者之间存在差异调节，与其表型状态相关。结论。这些分子和细胞发现表明了一种反复发生的突变事件，清楚地表明了P.A441V错义突变在NLRP3相关自体炎症性疾病中的致病性，并指出了研究患者原代细胞以评估疾病活动性的兴趣。
Objective. To determine the molecular and cellular bases of autoinflammatory syndromes in a multigenerational French family with Muckle-Wells syndrome and in a patient originating from Portugal with familial cold autoinflammatory syndrome. Methods. Sequencing of NLRP3 exon 3 was performed in all accessible patients. Microsatellite and whole-genome single nucleotide polymorphism genotyping was used i) to test the intrafamilial segregation of the identified variant and ii) to look for a founder effect. Functional analyses included the study of i) apoptosis-associated speck-like protein containing a CARD (ASC) speck formation in HEK293T cells (stably expressing ASC-green fluorescent protein and pro-caspase 1-FLAG) transiently expressing the wild-type or mutated NLRP3 protein, ii) levels of IL-1 beta secreted from transfected THP-1 cells, and iii) inflammasome-related gene expression and cytokine secretion from monocytes isolated from patients in crisis (probands from the two families), related patients out of crisis, and from controls. Results. The same heterozygous mutation (c.1322C>T, p.A441V) located in the NACHT domain, segregating with the disease within the first family, was identified in the two families. This mutation was found to be associated with different core haplotypes. NLRP3-A441V led to increased ASC speck formation and high levels of secreted IL-1 beta. Monocyte inflammasome-related gene expression and cytokine secretion, which were within the normal range in patients out of crisis, were found to be differentially regulated between the two probands, correlating with their phenotypic status. Conclusion. These molecular and cellular findings, which indicate a recurrent mutational event, clearly demonstrate the pathogenicity of the p.A441V missense mutation in NLRP3-associated autoinflammatory disease and point to the interest of studying patients' primary cells to assess disease activity.