INTRODUCTION ERAP1 has been recently proposed as risk marker of Behçet syndrome (BS). Gene single nucleotide polymorphisms (SNPs) could affect the enzymatic activity and the conserved active site is pivotal for the aminopeptidase function. This study aims to characterize the ERAP1 active site in a cohort of BS patients vs healthy controls (HC) integrating genomics, transcriptomics and bioinformatics approach. MATERIALS AND METHODS We recruited 109 consecutive Italian BS patients (63M:46 F; mean age: 45.07 ± 12.28 years) and 106 matched HC (55M:51 F; mean age: 42.57 ± 12.29 years). DNA was isolated and amplified using PCR with home made-primer pairs. PCR products were directly sequenced and computational analyses were performed to search active site SNPs (NCBI-BlastN tool), to predict SNPs functional effect (PolyPhen-2 software) and to obtain protein 3D modelling (Protean3D software). In a second phase of analysis, RNA was extracted and reverse transcribed. Quantitative Real-Time PCR (qPCR) was performed to assess ERAP1 mRNA level in presence (target) and in absence (control) of gene polymorphisms. The Fold change was calculated for the relative quantification of gene expression. RESULTS A novel coding variation (NG_027839.1:g.25637 T > G; NP_057526.3:p.Phe360Cys, HGSV nomenclature) was found in heterozygosity state in 5/109 BS patients (4.59 % of cases) and none of HC. It was recognized in association with rs2287987, rs30187, rs17482078, and rs27044 BS-related polymorphisms for 4 out of 5 patients. All patients carrying the novel SNP were HLA-B*51-positive. The novel SNP was released in GenBank database with MK140632.1 ID. The SNP was predicted to be damaging and resides within the Zn-binding HEXXH(X)18E region of the active site, changing the structurally conserved region for the amminopeptidase function. In fact, the change in energy (ΔE) score between wild-type and SNP-containing protein showed a less stable protein in presence of p.Cys360 (ΔE:3.584) (Protean3D prediction). Preliminary qPCR results underlined a significant difference in fold change value when target and control values were compared (p < 0.05), suggesting a reduced expression of ERAP1 mRNA in presence of the novel SNP. CONCLUSIONS Our study strengthens the association between ERAP1 and BS. The most significant point was the localization of the novel p.Phe360Cys SNP within the Zn-binding region of protein active site that was predicted to affect its function, causing protein destabilization. Our findings need to be tested in larger genetic studies.

中文翻译：

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从结构到功能来表征Behçet综合征的ERAP1活性位点。与已知基因变异相关的新型多态性。

引言ERAP1最近被提出作为Behçet综合征（BS）的危险标志。基因单核苷酸多态性（SNPs）可能会影响酶的活性，保守的活性位点对于氨肽酶的功能至关重要。这项研究旨在结合基因组学，转录组学和生物信息学方法，对BS患者与健康对照（HC）队列中的ERAP1活性位点进行表征。材料与方法我们招募了109名连续的意大利BS患者（63M：46 F；平均年龄：45.07±12.28岁）和106名相匹配的HC（55M：51 F；平均年龄：42.57±12.29岁）。使用自制引物对使用PCR分离并扩增DNA。对PCR产物进行直接测序，并进行计算分析以搜索活性位点SNP（NCBI-BlastN工具），预测SNP的功能效果（PolyPhen-2软件）并获得蛋白质3D建模（Protean3D软件）。在第二阶段的分析中，提取RNA并进行反转录。进行定量实时PCR（qPCR）以评估在基因多态性存在（目标）和不存在（对照）的情况下ERAP1 mRNA的水平。计算倍数变化以用于基因表达的相对定量。结果在5/109 BS患者（4.59％的病例）中杂合性状态下发现了新的编码变异（NG_027839.1：g.25637 T> G； NP_057526.3：p.Phe360Cys，HGSV命名法），但无HC。5位患者中有4位与rs2287987，rs30187，rs17482078和rs27044 BS相关的多态性相关联。所有携带新型SNP的患者均为HLA-B * 51阳性。新型SNP已在GenBank数据库中以MK140632.1 ID发行。预计SNP会损坏，并位于活动位点的Zn结合HEXXH（X）18E区域内，从而改变了氨肽酶功能的结构保守区域。实际上，在存在p.Cys360的情况下，野生型蛋白和含SNP的蛋白之间的能量（ΔE）得分变化显示蛋白质稳定性较差（ΔE：3.584）（Protean3D预测）。初步的qPCR结果强调了当比较目标值和对照值时倍数变化值的显着差异（p <0.05），表明在存在新型SNP时ERAP1 mRNA的表达降低。结论我们的研究加强了ERAP1和BS之间的联系。最重要的一点是小说p的本地化。蛋白活性位点锌结合区内的Phe360Cys SNP预计会影响其功能，导致蛋白不稳定。我们的发现需要在更大的遗传研究中进行检验。